Pudney 1995 PDF

MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995)
Spermatogenesis in Nonmammalian Vertebrates

JEFFREY PUDNEY
Fearing Research Laboratory, Brigham and Women's Hospital, Haruard Medical School, Boston, Massachusetts 02115
KEY WORDS Anamniotes, Spermatocysts, Amniotes
ABSTRACT Spermatogenesis appears to be a fairly conserved process throughout the verte-

brate series. Thus, spermatogonia develop into spermatocytes that undergo meiosis to produce
spermatids which enter spermiogenesis where they undergo a morphological transformation into
spermatozoa. There is, however, variation amongst the vertebrates in how germ cell development
and maturation is accomplished. This difference can be broadly divided into two distinct patterns,
one present in anamniotes (fish, amphibia) and the other in amniotes (reptiles, birds, mammals).
For anamniotes, spermatogenesis occurs in spermatocysts (cysts) which for most species develop
within seminiferous lobules. Cysts are produced when a Sertoli cell becomes associated with a
primary spermatogonium. Mitotic divisions of the primary spermatogonium produce a cohort of
secondary spermatogonia that are enclosed by the Sertoli cell which forms the wall of the cyst. With
spermatogenic progression a clone of isogeneic spermatozoa is produced which are released, by
rupture of the cyst, into the lumen of the seminiferous lobule. Following spermiation, the Sertoli
cell degenerates. For anamniotes, therefore, there is no permanent germinal epithelium since
spermatocysts have to be replaced during successive breeding seasons. By contrast, spermatogen-
esis in amniotes does not occur in cysts but in seminiferous tubules that possess a permanent
population of Sertoli cells and spermatogonia which act as a germ cell reservoir for succeeding bouts
of spermatogenic activity. There is, in general, a greater variation in the organization of the testis
and pattern of spermatogenesis in the anamniotes compared to amniotes. This is primarily due to
the fact there is more reproductive diversity in anamniotes ranging from a relatively unspecialized
condition where gametes are simply released into the aqueous environment to highly specialized
strategies involving internal fertilization. These differences are obviously reflected in the mode of
spermatogenesis and this is particularly true of the stage of spermiogenesis where the morphology
of the species-specific spermatozoon is determined. Moreover, unlike amniotes, many anamniotes
display a spermatogenic wave manifest, depending upon the species, either at the level of the cyst
or seminiferous lobule.
This variation in the organization of the testis makes certain anamniotes perfect models for
investigating germ cell development and maturation. For instance, the presence of a spermatogenic
wave provides an opportunity to manually isolate discrete germ cell stages for analysis of specific
Sertoli/germ cell interactions. Furthermore, for many anamniotes, germ cells mature in association
with a morphologically poorly developed Sertoli cell. This seeming independence of Sertoli cell
regulation allows the in vitro culture of isolated germ cells of some species of anamniotes through
several developmental stages. Thus, due either to the anatomical organization of the testis, or
structural simplicity of the germinal units, nonmammalian vertebrates can provide excellent ex-
perimental animal models for investigating many basic problems of male reproduction.
0 1995 Wiley-Liss, Inc.
INTRODUCTION spermatogenesis depend primarily on several parame-

Throughout the vertebrate series it would appear ters which are not necessarily mutually exclusive; (1)
that germ cell development and maturation proceeds in the organization of the testis, i.e., the anatomical dis-
a similar fashion. Thus, spermatogonial stem cells di- position of the primary germinal compartment; (2) the
vide to produce generations of spermatogonia which relationship between germ cells and Sertoli cells dur-
enter the spermatogenic cycle. Spermatogonia develop ing the spermatogenic cycle; (3) whether fertilization is
into spermatocytes which undergo meiosis to produce external or internal; and (4) whether the species is
spermatids. These spermatids then proceed through a
morphological metamorphosis during spermiogenesis
to form spermatozoa. When, however, spermatogenesis
is compared between the different classes of verte- Received May 31, 1994; accepted in revised form August 12, 1994.
brates it is obvious this process can be accomplished in Address reprint requests to Dr. Jeffrey Pudney, Fearing Research Laboratory,
a variety of ways. These differences in the pattern of Brigham and Women's Hospital, 250 Longwood Avenue, Boston, MA 02115.
0 1995 WILEY-LISS, INC.

460 J. PUDNEY
cold-blooded (poikilotherm) or warm-blooded (homeo-

therm).
Since the structural association between germ cells
and Sertoli cells during spermatogenesis largely dic-
tates the morphological appearance of the germinal
compartment, these two factors will be discussed to-
gether.
THE ORGANIZATION OF THE TESTIS AND

SERTOLUGERM CELL RELATIONSHIPS
It is now well established that Sertoli cells play a
pivotal role during spermatogenesis. A recent review is
available dedicated to the Sertoli cell in nonmamma-
lian vertebrates (Pudney, 1993). For this present re-
port, therefore, information on the Sertoli cell will be
restricted to aspects of this cell which affect or impinge
on the pattern of spermatogenesis in different classes
of nonmammalian vertebrates.
THE VERTEBRATE TESTIS Fig. 1. Amphiorus testes develop from coelomic pouches. This tes-
tis is filled with mature spermatozoa.
In the phylum, chordata, nonmammalian verte-
brates include the jawless fishes, lampreys and hagfish
(Agnatha); cartilaginous fishes, sharks, rays, and chi-
meras (Chondrichthys); the bony fishes (Osteichthys);
newts, salamanders, frogs, toads, and caecilians (Am-
phibia); turtles, snakes, lizards, crocodiles, alligators
(Reptilia), and the birds (Aves).
In the protochordate Amphioxus the testes are
formed from metamerically arranged coelomic pouches
(Fig. 1).As vertebrates evolved, however, these rows of
testes tended to form localized compact organs. In most
vertebrates the testes are paired except for the Ag-
natha in which fusion of the testes has occurred and
there are several species of bony fishes which possess a
single testis. The shape of the vertebrate testis varies
from extremely elongated, as in most cartilaginous and
bony fishes, to round or oval as in many amphibia,
reptiles, and birds. For some urodelean amphibia and
bony fishes, however, the testes are composed of dis-
tinct lobes. In nonmammalian vertebrates the testes lie
within the body attached by a mesorchium to the body
wall. Usually the testes are isolated structures but can
be associated with other tissue elements such as he-
mopoietic tissue, the epigonal organ which invests the
testes in selachians or the fat body attached to the tes-
tes of anuran amphibia. An interesting feature in bony Fig. 2. Ambisexual teleost containing an ovary (0)and testis (T).
fishes is the occurrence of ambisexual species which
possess both an ovary and a testis (Fig. 2). These go-
nads may be present simultaneously or develop se- ary conserved feature of the vertebrate testis and even
quentially. Those species in which the testis develops occurs in the testis of Amphioxus (Pudney, 1993).
first are termed protandrous, while those where the
ovary develops first are protogynous. ORGANIZATION OF THE TESTIS
The vertebrate testis is composed of two distinct com- Within the vertebrate series the general structural
partments. One consists of the interstitial tissue, con- appearance of the testis can be divided into two distinct
taining blood vessels, lymphatics and Leydig cells patterns, one present in the anamniotes (fish, am-
which secrete the male hormones or androgens. The phibia) the other in amniotes (reptiles, birds, mam-
second is the seminiferous compartment which con- mals).
tains the germinal epithelium defined as a tripartite
structure consisting of an acellular basement mem- Anamniotes
brane, germ cells and Sertoli cells (Grier, 1993). The In these nonmammalian vertebrates, the unit of
association of germ cells with a somatic element, i.e., spermatogenesis is a spermatocyst, usually referred to
Sertoli cell of the germinal epithelium, is an evolution- as a cyst (von La Valette, St. George, 1876). Incipient
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 46 1
B Studies on the morphology of the testis of chondrich-
thyan fishes have also described the germinal compart-
ment as consisting of cysts contained within lobules (or
follicles or ampullae). This organization of the testes in
these species, however, has been recently shown to be
anatomically incorrect (Grier, 1992). This study was
based on the observation that the germinal comDart-
dG $. ment in the vertebrate testis is composed of a i e p i -
thelium (germinal) which by definition rests on a
basement membrane that sequesters it from the sur-
rounding interstitial tissue. Called the extracellular
matrix hypothesis, it was shown that, chronologically,
+@ sG--@ @ the production of a basement membrane by the germi-
nal compartment differed in the testes of vertebrate
groups.
Utilizing this hypothesis, it was reported that a ma-
jor factor in how the anamniote testis is organized was
when the germinal elements became isolated from the
interstitial tissue, by the secretion of a basement mem-
brane, to form a separate germinal compartment.
Thus, in the cartilaginous fishes, when germ cells (pri-
mary spermatogonia) and precursor somatic (Sertoli)
cells initially come together they lie free within the
+ interstitial tissue. These cells are not enclosed by a
basement membrane and so do not represent a germi-
nal compartment. Then the basement membrane is se-
creted t o surround and isolate the Sertoli cell and germ
cells (now secondary spermatogonia) from the intersti-
tial tissue to produce a distinct germinal compartment,
the spermatocyst. In the chondrichthyan testis, there-
fore. cvsts are not Dresent within lobules. but occur as
Fig. 3. Schematic diagram showing general features of cyst for- isolatid structure; Since the chondrichthyan testis is
mation in anamniotes. A: Initially a Sertoli cell (SC) becomes associ- composed of a mass of cysts, it is called polyspermato-
ated with a primary spermatogonium (PG). B: The Sertoli cell com- cystic (Grier, 1992).
pletely surrounds the germ cell to produce a nascent cyst. C: The
primary spermatogonium undergoes repeated mitotic divisions to pro- Similarly, the agnathan testis is also made up of nu-
duce secondary spermatogonia (SG) enclosed by Sertoli cytoplasm merous individual cysts, i.e., polyspermatocystic. The
which forms the wall of the cyst. D Germ cell maturation then pro- composition of cysts, however, differs between the two
ceeds with the eventual production of a n isogeneic clone of spermato- vertebrate classes. Cysts in the agnathan testis all con-
zoa which are often orientated with their heads towards the Sertoli
cell nucleus. E: Spermiation is accomplished by rupture of the cyst. F: tain a single generation of isogeneic germ cells. By
The Sertoli cell then undergoes degeneration. (Reproduced from Pud- contrast, the chondrichthyan cysts contain many (al-
ney, 1993, with permission of Cache River Press.) though fixed in number) Sertoli cells, each of which is
associated with a clone of germ cells. These individual
Sertoli cells plus their cohort of germ cells present
cysts are formed when a primary spermatogonium be- within a cyst have been called spermatoblasts (Callard,
comes engulfed by a Sertoli cell (Fig. 3). The primary 1991). The term spermatoblast was initially used to
spermatogonium then undergoes numerous mitotic di- define a functional unit of spermatogenesis consisting
visions to produce secondary spermatogonia. The cyst of a Sertoli cell with its associated germ cells within
is now composed of a mass of germ cells surrounded by the mammalian seminiferous epithelium (Von Ebner,
Sertoli cytoplasm which forms the wall of the cyst. 1871). It should be noted that in the chondrichthyan
Germ cell maturation then occurs with formation of an testis, each cyst is composed of spermatoblasts which
isogeneic clone of spermatozoa which are released by all contain germ cells at roughly the same stage of
rupture of the cyst wall. Following spermiation, in development.
most anamniotes, Sertoli cells usually degenerate. For the agnatha, most species of bony fishes, and
In bony fishes and amphibia, cysts develop within anuran amphibia, the cysts are stationary within the
larger structures that develop from sperm collecting testis. By contrast, in the chondrichthyes, several spe-
ducts. In the literature, these blind-ending pouches cies of bony fishes and urodelean amphibia, cysts move
have been referred to as either lobules, tubules, ampul- through the testis as they mature during spermatogen-
lae, or follicles. Recently, a consistent and unifying ter- esis. The direction of this cystic migration, however,
minology has been proposed for describing the anam- differs amongst these nonmammalian vertebrates. In
niote testis (Grier, 1993). In this study, the lobule was urodeles, cysts form in the region of the lobule proxi-
defined as the anatomically correct term for applying mal to the central sperm collecting duct. During sper-
to the germinal compartment of the testis in most bony matogenesis, as cysts mature they move distally to-
fishes and all amphibia. wards the blind end of the lobule. Spermiation occurs
462 J. PUDNEY
when these cysts rupture and the released spermatozoa ment, in the amniote testis there is a stratification of
must then pass down the lobule, through the immature germ cells at different phases of maturation within the
region, to enter the sperm duct. For certain species of seminiferous epithelium. Furthermore, in anamniotes,
bony fishes, however, the reverse occurs with nascent following spermiation, Sertoli cells undergo degenera-
cysts located at the blind end of the lobule which then tion, whereas in amniotes, Sertoli cells represent a per-
migrate, as they mature, down towards the sperm duct. manent feature of the seminiferous epithelium. There
On reaching the sperm duct these cysts break down to is, in general, less variation in the organization of the
release spermatozoa either as free entities or as naked testis and pattern of spermatogenesis in the amniotes
(spermatozeugmata) or encapsulated (spermatophores) compared to the anamniotes.
bundles. In the chondrichthyes it is the cyst which mi-
grates through the testis. Developing cysts originate in
a germinal zone, the location of which varies in the SEASONAL CYCLES
testes of different chondrichthyan species (Pratt, 1988). In vertebrates the pattern of spermatogenesis varies
For example, in the spiny dogfish Squalus acanthias, according to whether: (1) species are cold-blooded
the germinal zone is found on the dorsolateral surface (poikilotherm), e.g., fish, amphibia, reptiles, or warm-
of the gonad. During spermatogenesis, as cysts mature, blooded (homeotherm), e.g., birds, mammals; and (2)
they migrate radially through the testis t o the opposite reproduction is seasonal. Poikilotherm vertebrates un-
side by which time they are filled with spermatozoa. dergoing reproductive seasonal cycles exhibit postnup-
Spermiation then occurs and the cysts degenerate. This tial spermatogenesis. This is because spermatogenesis
pattern of cyst development has been called diametri- begins soon after the conclusion of the breeding season.
cal since it occurs across the diameter of the testis In contrast, spermatogenesis essentially stops in ho-
(Pratt, 1988). meothermic vertebrates following the breeding season.
It should be mentioned that the migration of cysts in For these species, therefore, spermatogenesis is pren-
lobules of bony fishes and urodeles or testes of chon- uptial because recrudescence does not occur until the
drichthyans does not occur by movement of the cysts start of the next breeding season.
themselves. Cysts are passive structures and it is pre- In general, all feral vertebrates exhibit a seasonal
sumably the continual formation of cysts and/or growth cycle of reproductive activity. This is more pronounced
and elongation of sperm ducts which cause their mi- for species that inhabit temperate zones than those liv-
gration either along the lobule or through the testis. ing in more stable habitats, e.g., tropical, or benthic
Also, during spermatogenesis, in some anamniotes, environments. For temperate poikilotherms which
e.g., urodeles, although all cysts contain synchro- practice postnuptial spermatogenesis, germ cell devel-
nously developing germ cells, the lobule itself could opment begins in the summer and then proceeds either
consist of different regions composed of cysts at slowly throughout winter, or ceases completely during
different stages of maturation, i.e., an immature zone winter to be reinitiated in the spring. In homeotherms,
with spermatogonial cysts and a mature zone with following the breeding season, there is complete testic-
spermatogenically active cysts. Furthermore, the ular involution and regression of the germinal epithe-
stage of maturation reached by cysts in the mature lium with spermatogonia (usually) as the remaining
zone may vary from lobule to lobule within the same germ cells. It should be pointed out that few descriptive
testis, resulting in production of a spermatogenic studies have been carried out on the changes the testes
wave. A spermatogenic wave also occurs in the of nonmammalian vertebrates undergo throughout
chondrichthyes that involves the progressive matura- their annual reproductive cycles.
tion of cysts as they migrate through the testis. This All amniotes possess a permanent seminiferous epi-
results in a distinct zonation of spermatogenesis in the thelium which contains a resident population of Sertoli
testis of these species. cells and a constant population of germ cells, i.e., pri-
mary spermatogonia which act as stem cells and are
Amniotes responsible for annual reinitiation of spermatogenesis.
In amniotes there is no cystic form of spermatogen- By contrast, the only anamniotes reported to possess a
esis and therefore the testis has been defined as permanent germinal epithelium are anurans (Burgos
postspermatocystic (Grier, 1993). Instead, the seminif- and Vitale-Calpe, 196713; Lofts, 1984). This is because,
erous epithelium is composed of a stable population of although the apical Sertoli cytoplasm is shed at sper-
Sertoli cells associated with successive stages of germ miation, the basal portion remains intact and regener-
cell maturation. For all amniotes the germinal com- ates from one breeding season to the next. For other
partment is composed of seminiferous tubules which anamniotes there is degeneration of cysts following
are single structures except for birds in which exten- spermiation and so these germinal units have to be
sive branching may occur. The number and length of constantly replenished. Recent observations, however,
these tubules vary greatly among anamniotes. The im- suggest that a permanent germinal epithelium occurs
mature germ cells, the spermatogonia, are located a t in some teleosts with persistence of a few Sertoli cells
the base of the seminiferous epithelium while sper- and germ cells (spermatogonia) from one breeding sea-
matocytes and spermatids occur at successively higher son to the next (Grier, 1993).
levels. Mature, elongate spermatids border the lumen Several reviews are available on the organization of
of the tubules into which they are released during sper- the testis in nonmammalian vertebrates (Callard,
miation. Therefore, unlike the anamniote testis where 1991; Grier, 1993; Pilsworth and Setchell, 1981; Pud-
cysts contain germ cells a t the same stage of develop- ney, 1987; Roosen-Runge, 1977).
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 463
Fig. 4. Amphioxus testis. A Germinal epithelium with a layer of matozoa appear primitive and possess a small acrosome (arrowhead),
immature germ cells at the periphery (arrowheads) and mature sper- and a single large mitochondrion (MI at the base of the nucleus and
matozoa in the lumen. B: Spermatogonia are poorly differentiated surrounding the flagellum.
with few organelles except for several large mitochondria. C: Sper-
EXTERNAL VERSUS trioles orientated roughly a t 90" to each other, one of

INTERNAL FERTILIZATION which (presumably the distal centriole) gives rise to
In general, nonmammalian vertebrates that practice the long flagellum. The flagellum contains a typical
external fertilization produce a larger number of axoneme consisting of a 9 + 2 arrangement of doublets.
poorly differentiated spermatozoa compared t o those A single large mitochondrion is located a t the base of
which engage in internal fertilization. The spermato- the nucleus and appears to partially surround the cen-
zoa of external fertilizing species may be considered triole region (Fig. 4). It would appear that spermato-
primitive, but a number also produce gametes with genesis proceeds until the testis is filled with mature
highly specialized features such as the acrosomal fila- spermatozoa (see Fig. 1)at which time the gonad (pre-
ment of petromyzonids (Nicander and Sjoden, 1971). sumably) ruptures to release the gametes to the exter-
Also, spermatozoa of teleosts could be considered prim- nal environment.
itive since they lack an acrosome. The absence of this
structure, however, is due to the method of fertilization AGNATHA
in which spermatozoa gain access to the ovum via a The agnatha consist of the lampreys (Petromyo-
micropyle, thus making the presence of an acrosome nidae) and hagfish (Myxinoidae). Presumably due to
obsolete. the difficulty in obtaining specimens and lack of com-
mercial interest in these fish, few studies are available
PROTOCHORDATES on spermatogenesis in this group of vertebrates.
In Amphioxus, the testes occur as a metamerically Lampreys spend a number of years as larvae, called
arranged series along the length of the body (Fig. 1). ammocetes (which is a nonparasitic feeding stage) be-
Each testis contains a typical germinal epithelium fore they metamorphose into mature animals. Al-
with layers of developing germ cells present a t the pe- though gender is established in the larval stage, go-
riphery and mature spermatozoa occupying the central nadal growth is postmetamorphosis and in some
cavity of the testis (Fig. 4A). Germ cells develop in species is accomplished in a short period of time, i.e.,
association with Sertoli cells (see Pudney, 1993). Sper- 6-9 months. Mitotic activity of germ cells begins fol-
matogonia appear poorly differentiated with nuclei lowing metamorphosis, and cysts develop (Larsen,
containing finely dispersed chromatin, apparently 1965; Weisbart et al., 1978). As spermatogenesis pro-
lacking a distinct nucleolus and few cytoplasmic or- ceeds, the testis becomes a mass of cysts containing
ganelles except for several large mitochondria with spermatozoa. The mature testis eventually fills the
lamelliform cristae (Fig. 4B). The spermatozoa posses a body cavity since, as lampreys spawn once and then
rounded nucleus with clumps of condensed chromatin die, other body organs atrophy.
and a small, cap-shaped acrosome located on the ante- Most myxinoid species do not possess a discrete
rior pole (Fig. 4C). This acrosome appears to be sepa- breeding season since they live in a constant environ-
rated from the nucleus by a clear zone containing a ment in the sea at 50-100 meters or more. Hagfish
flocculent, homogenous substance. The posterior pole juvenile males are progynous because during sexual
of the nucleus contains a fossa which houses two cen- differentiation the anterior portion of the gonad under-
464 J. PUDNEY
the nucleus and into the flagellum. This endonuclear

canal contains an undulating central fiber that extends
into the flagellum where it is bound by apposing inner
and outer nuclear membranes. Apparently, during fer-
tilization, an acrosome reaction occurs, resulting in the
extrusion of the long, preformed central fiber. This fi-
ber, enclosed by the plasma membrane of the sperma-
tozoon, has been referred to as the acrosomal tubule or
filament (Nicander and Sjoden, 1971).The extension of
the central fiber is thought to be involved in sperma-
tozoal penetration of the egg. Lamprey spermatozoa do
not possess a discernible midpiece. The nuclear im-
plantation fossa contains 2 centrioles both aligned
nearly parallel with the longitudinal axis of the sper-
matozoon. One centriole forms the basal body, from
which the axoneme of the flagellum develops, while
the other remains as an accessory centriole. The fla-
gellum contains a typical axoneme consisting of a 9 -t2
arrangement of doublets. The doublets possess coarse
Fig. 5. Polyspermatocystic testis of Myxine sp. fibers apposed to their outer surfaces. A number of
elongate mitochondria are located along the proximal
region of the flagellum.
goes early stages of oogenesis. The posterior region of
the gonad remains sexually indifferent until, a t a late CHONDRICHTHYES
stage in growth, cysts develop containing spermato- The cartilaginous fishes are composed of two
gonia. At this phase the anterior ovarian structures unequal subclasses, the Elasmobranchii (sharks, dog-
generally undergo degeneration. fish, skates, rays) with approximately 600 species and
In both myxinoids and petromyzonids the testis is the Holocephali (ratfish) with only 28 known species.
composed of a mass of cysts, i.e., polyspermatocystic As for agnathans this group of vertebrates has
(Grier, 1992; Fig. 5). Sperm ducts are lacking in ag- received little attention from reproductive biologists.
nathans and spermatozoa are shed into the body cavity The detailed process of spermatogenesis is, therefore,
where they exit to the outside via the abdominal pore. only known for a few species of elasmobranchs and
In myxinoids, germ cell development is synchronous even less for holocephalans.
within individual cysts although different stages of It is apparent from the literature that cyst develop-
spermatogenesis can be found between cysts (Dodd and ment and spermatogenesis, at least as reported at the
Sumpter, 1984). An early light microscope study on light microscope level, is fairly similar for several dif-
spermatogenesis in Myxine glutinosa is available (Cun- ferent species of chondrichthyans: Cetorhinus maximus
ningham, 1891). It has been reported that the most (Matthews, 1950); it should be mentioned that in this
primitive of polyspermatocystic testis occurs in the account an error in observation was made by Matthews
hagfish Eptatretus stouti (Grier, 1993). This is a me- in that cells described as “spermatogonia” represent
senteric type of testis with regions containing sperma- Sertoli cells while the “primary spermatocytes” corre-
tocysts which are interconnected by bridges of tissue spond to spermatogonia: Hydrolagus colliei (Stanley,
identical to the mesorchium. Nascent cysts are derived 1963);Scyliorhinus caniculus (Dobson and Dodd, 1977;
from isolated spermatogonia and Sertoli cells lying free Mellinger, 1965; Stanley, 1966); Torpedo marmorata
within the interstitial tissue (Tsuneki and Gorbman, (Stanley, 1966); Squalus acanthias (Callard et al.,
1977). Prior to meiosis a basement membrane is pro- 1985; Holstein, 1969) and Heterodontus portusjacksoni
duced to isolate Sertoli cells and spermatogonia from (Jones and Jones, 1982). The nuclear changes associ-
the interstitial tissue, resulting in the formation of ated with germ cell development and maturation have
cysts. been described for several species of elasmobranchs
Few electron microscopic studies have been carried (Moore, 1895) In S. acanthias, gonocytes and undiffer-
out on the agnathan testis and so information on the entiated Sertoli cell precursors are located in the ger-
ultrastructure of spermatogenesis is lacking. Several minal zone on the rostra1 aspect of the testis. Nascent
reports, however, are available on the fine structure of cysts form when these Sertoli cells engulf primary
spermatozoa in a few lamprey species, Lampetra fluui- spermatogonia within cytoplasmic processes (Fig. 6A).
atilis (Nicander and Sjoden, 1971) and Lampetra plan- According to the extracellular matrix hypothesis
eri (Follenius, 1965; Stanley, 1967). These descriptions (Grier, 19921,spermatocysts are not produced until the
are extremely similar for both species. The long nu- basement membrane is formed which isolates the Ser-
cleus is rod-shaped and possesses an acrosomal com- toli/germ cell unit from the interstitial tissue. By the
plex. This complex consists of a simple acrosomal ves- time this happens both cell types have undergone sev-
icle positioned on the tip of the nucleus, a dense eral mitotic divisions resulting in the formation of a
subacrosomal ring and an axial nuclear canal lined by spermatocyst consisting of a single layer of Sertoli cells
invaginated nuclear membrane that runs the length of and secondary spermatogonia (Fig. 6B). Cysts then be-
Fig. 6. Germinal zone of S . acunthias testis showing cysts a t var- formed cyst with a lumen and basal layer of secondary spermatogonia
ious stages of development. A: Cysts form when a Sertoli cell (arrow) and Sertoli cells. One cyst (C) has become attached to the system of
engulfs a gonocyte (arrowhead). Nascent cysts are composed of pri- sperm excurrent ducts (arrowhead).
mary spermatogonia (P) surrounded by Sertoli cells (S).B: Fully
come attached to the system of sperm ducts in the testis During spermiogenesis there is a great deal of trans-
(Fig. 6B). location of germ cells within the spermatoblast. This
Sertoli and germ cells undergo repeated mitotic di- eventually results in a single layer of spermatids lining
visions to form a mass of cells within the cyst, which the inner face of the spermatoblast with their tails pro-
now contains a lumen. These cells eventually become jecting into the spermatoblast cavity (Fig. 7D,E). How
organized into a peripheral layer of secondary sper- this movement of germ cells within the spermatoblast
matogonia surrounded by Sertoli cytoplasm with Ser- is accomplished is unknown. The cyst still possesses a
toli nuclei bordering the lumen of the cyst (Fig. 7A). patent lumen which is bound by the apical margins of
For T. marmorata, however, Sertoli and germ cells the Sertoli cells. During spermiogenesis, spermatids
form less conspicuous concentric layers (Stanley, 1966). elongate and begin to form loose bundles within the
At this stage the number of secondary spermatogonia spermatoblast with their tails now projecting into the
approximates the number of Sertoli cells in the cyst. lumen of the cyst (Fig. 7F). Spermatids progressively
Sertoli cells now cease dividing, which heralds the ap- form tighter bundles as they mature, until a t the end of
pearance of spermatoblasts within the cyst. This occurs spermiogenesis they have aggregated into a closely
when each Sertoli cell encompasses a single sper- packed mass located in a pocket of apical Sertoli cyto-
matogonium with cytoplasmic processes (or possibly a plasm (Fig. 7G-I). Spermiation involves release of the
clone of spermatogonia connected by intercellular mature gametes along with the apical portion of Sertoli
bridges) which effectively isolates germ cells from each cytoplasm. Spermatozoa exit the cyst via the sperm
other. The spermatogonia continue to divide, causing duct which has now acquired a patent opening. Follow-
the cyst to increase in size. Identifiable spermatoblasts, ing spermiation the Sertoli cells forming the cyst un-
consisting of a Sertoli cell plus its cohort of isogeneic dergo fatty degeneration. There are species-specific
germ cells, can now be identified in cysts (Fig. 7B). variations in chondrichthyan testicular organization
During this period, Sertoli nuclei enlarge and move (Hoar, 1969). This includes major differences in the
from their adlumenal position towards the periphery of location of the germinal zone and consequently migra-
the cyst wall. By the time this migration of Sertoli tion of spermatocysts through the testis for different
nuclei has been accomplished meiosis has occurred and species (Pratt, 1988).
spermiogenesis proceeds. Quantitative estimates of spermatogenesis are facil-
It is interesting to note that during the initial devel- itated in chondrichthyans since germ cells can be eas-
opment of cysts, mitotic divisions appear to occur ily identified and hence counted in cysts as they ma-
within groups of spermatogonia (Fig. 7A). These ture. The number of divisions spermatogonia undergo
groups could possibly represent clones of germ cells are species-specific. Thus, S. acanthias has 13 sper-
connected by cytoplasmic bridges. During meiosis, matogonial divisions, two of them type A and eleven
however, division appears to be initiated by a group of involving type B spermatogonia (Holstein, 1969). The
germ cells and then proceeds as a wave to involve other number of mitotic divisions carried out by both Sertoli
germ cells present in the cyst (Fig. 7C). cells and spermatogonia are fixed, resulting in cysts
466 J. PUDNEY
Fig. 7.
containing a specific number of germ cells and Sertoli through the testis. Since the zone represents an ab-
cells. Initially, mitotic divisions are synchronized be- sence of germ cells, when the next breeding season oc-
tween spermatogonia and Sertoli cells through the first curs there is a short arrest in spermatogenic activity.
9 divisions (Stanley, 1966). Sertoli cells then cease di- In contrast, a zone of degeneration is absent from S.
viding, at which time the cyst contains approximately tiburo (Parson and Grier, 1992). For this shark there is
500 of these cells. Spermatogonia continue to undergo widespread complete regression of the testis which only
four more cycles of mitoses before they enter meiosis. contains spermatogonial cysts during the period of tes-
The end product of all these divisions is 64 spermatids ticular involution.
associated with each Sertoli cell (Stanley, 1966). This Few electron microscopic studies have been carried
results roughly in 32,000 spermatozoa per mature cyst. out on the chondrichthyan testis. Ultrastructurally,
For Sphyrna tiburo, it has been estimated that a ma- the primary spermatogonia of S. acanthias are
ture spermatocyst contains approximately 460 sperma- characterized by large, spherical, pale-staining nuclei
toblasts, each possessing 64 spermatozoa resulting in with few chromatin clumps and a paucity of organelles
29,000 spermatozoa per spermatocyst (Parsons and in the cytoplasm. Secondary spermatogonia appear
Grier, 1992). smaller in size with irregularly shaped, dark staining
A rare case of germ line chromatin diminution has nuclei containing many clumps of chromatin and
been reported to occur during spermatogenesis in H . possess many mitochondria, plus profiles of both
colliei (Stanley et al., 1984). At metaphase I in meiosis smooth (SER) and rough (RER) endoplasmic reticulum
of primary spermatocytes, a mass of heterochromatin (Fig. 8). Cytoplasmic bridges were observed connecting
accumulates at the side of the metaphase plate. This groups of secondary spermatogonia (Fig. 8). These
clump of heterochromatin is eventually passed into one connections are maintained by clones of germ cells
of the secondary spermatocytes during cytokinesis at throughout spermatogenesis (Fig. 9). Small desmo-
anaphase I. As the nuclear membrane is restored, a some-like junctions were often detected between
double membrane (possibly of nuclear origin) forms spermatogonia and Sertoli cells. At later stages of
around the mass of heterochromatin which is then spermatogenesis, however, no junctional specializa-
termed the chromatin diminution body. By the end of tions were observed between Sertoli cells and germ
the second meiotic division this body ends up in the cells. Primary spermatocytes appear cytologically
cytoplasm of one of the four spermatids where it re- more differentiated than spermatogonia. They possess
mains unchanged until midspermiogenesis. At this large spherical nuclei with finely dispersed chromatin
time the chromatin body is discarded by means of apo- and the cytoplasm contains numerous mitochondria,
crine exocytosis and is engulfed by the Sertoli cell. cisterns of SER, a well-developed Golgi apparatus as
Seasonal changes in spermatogenesis in elasmo- well as an assortment of vacuoles and dense bodies
branchs have been reviewed (Dodd and Sumpter, 1984; (Fig. 9). An occasional lipid droplet and centriole were
Parsons and Grier, 1992; Wourms, 1977). Due to the observed in some spermatocytes. An excellent detailed
difficulty in obtaining sufficient numbers of specimens study of the fine structure of spermiogenesis has been
throughout the reproductive cycle, few species, mainly carried out for S. suckleyi (Stanley, 1971a,b).
restricted to sharks, e.g., S. acanthias (Simpson and Round spermatids contain spherical nuclei with fine
Wardle, 1967)S. tiburo (Parsons and Grier, 1992),have granular chromatin. A number of mitochondria and a
been completely and adequately studied. For many spe- well-developedGolgi apparatus as well as an extensive
cies a seasonal cycle of spermatogenesis manifests it- system of SER is present in the cytoplasm (Fig. 10A).
self by the development of a zone of breakdown. This There appears to be a distinct polarization of some or-
zone, consisting of degenerating cysts, was first de- ganelles such as the Golgi apparatus which appear to
scribed in S. acanthias (Simpson and Wardle, 1967). be located adjacent to the developing flagellum (Fig.
This zone develops annually, in the spring, due to sper- 10B). A bundle of juxtanuclear filaments is commonly
matogonial degeneration and proceeds to migrate detected in round spermatids (Fig. lOC). Interestingly,
the nuclear envelope next to the Golgi apparatus
exhibits structural modifications, related to adhesion
of the acrosome to the nucleus, before the appearance of
Fig. 7. Cyst development in the testis of S. acanthias. A: Mitotic
the acrosomal vesicle (Stanley, 1971a). In this region
activity of both germ cells and Sertoli cells results in growth of the the nuclear membranes become closely apposed and
cyst which is now composed of several basal layers of secondary sper- hence appear dense in appearance under the electron
matogonia surrounded by Sertoli cytoplasm. Nuclei of the Sertoli cells microscope (Fig. 10D). This membrane modification,
(arrowheads) form a distinct layer bordering the lumen. Note: mitotic therefore, is the initial event that gives nuclear polar-
divisions appear t o occur amongst distinct groups of germ cells. B:
Spermatoblast formation occurs when the cytoplasm of a single Ser- ity and is unusual since normally this results from the
toli cell (arrowheads) surrounds and isolates a cohort of secondary formation of the acrosome. The acrosomal vesicle de-
spermatogonia. C Meiosis occurs as a wave that progresses around velops from the Golgi apparatus located close to the
the cyst. D: and E: Spermatoblasts sectioned longitudinally (D) and developing flagellum and becomes attached to the
transversely (E) containing a cohort of isogeneic spermatids in the
lumen. Arrowheads indicate the Sertoli cytoplasm forming the walls dense nuclear membrane (Fig. 11A). As the acrosome
of the spermatoblast. F-I: These micrographs illustrate the progres- vesicle enlarges to form the acrosome it actually in-
sive condensation of elongating spermatids (F)into loosely organized dents upon the nucleus which accommodates by form-
bundles with their heads orientated toward the basement membrane ing a shallow depression (Fig. 11B). An acrosomal
of the cyst (G) that coalesce into closer packed bundles (HI to even-
tually form a tight mass of mature spermatids embedded in Sertoli granule does not appear to develop (Fig. 11B). The nu-
cytoplasm (I). clear envelope continues to condense over the surface of
468 J. PUDNEY
Fig. 8. Secondary spermatogonia possess irregularly shaped nuclei with clumps of chromatin and
contain numerous organelles and are connected by cytoplasmic bridges (arrowhead) S . acunthias.
the nucleus while at the same time fibrous material this stage two filamentous bundles, one striated and
becomes deposited along the outer nuclear membrane one nonstriated, become associated with both distal
to form the fibrous nuclear sheath (Fig. 1lC). Eventu- and proximal centrioles (Fig. 12A). These fibrous bun-
ally this sheath will cover most of the nucleus except dles are precursors of the axial midpiece rod (a charac-
for the posterior pole. There also appears to be a dis- teristic feature of chondrichthyan spermatozoa) that
tinctive pattern of chromatin condensation as sperma- will eventually connect the spermatid nucleus to the
tids mature. This begins with aggregation of the chro- developing flagellum. A dense material is also depos-
matin into coarse granules (see Fig. 10A) which ited around the flagellum where it projects from the
possibly is initiated in the anterior pole of the nucleus spermatid that presumably represents the ring centri-
beneath the acrosome. This process continues but ole or annulus (Fig. 12A). For S. acanthias, however, it
seems to be limited to the central region of the nucleus, appears that nuclear rotation occurs at a later stage of
resulting in a peripheral zone of nucleoplasm virtually spermatid differentiation since the fibrous bundles,
devoid of chromatin granules (see Figs. 10A and 1 1 0 . plus a developing flagellum, could still be detected in a
At a later stage of nuclear condensation, the chromatin lateral position (with respect to the nucleus) within the
granules become transformed into a mass of randomly cell (Fig. 12A). Following nuclear rotation, these fi-
orientated short chromatin fibers (see Fig. 14A). brous bundles increase in length to eventually insert in
Initially the centrioles lie close to the Golgi appara- a deep fossa located at the posterior pole of the nucleus
tus and near the periphery of the cell (Fig. 11A). For S. (Fig. 12B). With further elongation of the spermatid
suckleyi it was reported that as the acrosome develops, the bundles of filaments forming the axial component
the nucleus undergoes a rotation of approximately 180" of the midpiece become twisted upon each other. This
that results in the acrosome becoming located at spiraling, however, appears to only affect the nonstri-
roughly the opposite pole within the cell in respect to ated fibrous bundle which wraps itself around the stri-
the developing flagellum (Stanley, 1971a). This now ated bundle of filaments (Fig. 12C). A characteristic
defines the anterior pole of the cell, containing the ac- feature of round spermatids is the formation of a long
rosome, and posterior pole containing the flagellum. At cytoplasmic projection (Fig. 13A).This process contains
Fig. 9. Primary spermatocytes have round nuclei with evenly dispersed chromatin, abundant or-
ganelles and are connected by cytoplasmic bridges (arrow). Also present in this micrograph is a portion
of the Sertoli cytoplasm (arrowheads) forming the walls of the spermatoblast S. acunthius.
a system of fine filaments with an accumulation of nuclear pores (see Fig. 12B). This development is rem-
dense material a t the tip. Often these projections can iniscent of the redundant nuclear envelope present in
be seen to indent the Sertoli cytoplasm of the sperma- mammalian spermatozoa.
toblast (Fig. 13B). These structures, therefore, would With further maturation of the spermatid, the elon-
seem to act as some kind of anchoring device. gate nucleus assumes a helical shape and a t this time
As the spermatid develops the nucleus becomes elon- the fibrous nuclear sheath disappears. This spiralling
gated. The acrosome now forms an elongated cap over of the nucleus also includes the chromatin which be-
the pointed tip of the nucleus (Fig. 14A). Interestingly, comes orientated into a helical configuration (Fig.
the outer acrosomal membrane is closely associated 14B). This helical pattern begins posteriorly to ad-
with what appears to be a number of electron-dense vance anterially and is accompanied by a transient pro-
bodies that are evenly spaced along this membrane. A gressive loosening of the nuclear membrane adjacent to
short, subacrosomal rod (perforatorium) develops to oc- the chromatin just acquiring a spiral pattern (Fig. 14B;
cupy the space between the nucleus and acrosome (Fig. Stanely, 1971b).
14A). The subacrosomal rod is attached at the base of As nuclear elongation and chromatin condensation is
the acrosome and extend towards, and slightly beyond occurring, the two fibrous elements of the axial mid-
the tip of the nucleus. The nuclear chromatin has now piece rod fuse. At the same time elongate mitochondria
condensed to form a mass of coarse strands orientated aggregate around the axial midpiece rod to form a mi-
along the longitudinal axis of the nucleus (Fig. 14A). tochondrial sheath that now delineates the midpiece.
During nuclear condensation the nuclear volume de- These mitochondria are associated with glycogen gran-
creases from about 180 p3 to about 10 p3 and the sur- ules and are enclosed by a fibrous midpiece sheath. The
face area from around 154 p.' to 58 p2 (Stanley, 1971b). posterior end of the midpiece rod inserts on the basal
It was proposed that the excess nuclear envelope was body (distal centriole) of the flagellum. At doublets 3
discarded by the release of vesicles from the posterior and 8 the fibrous material surrounding the centrioles
margin of the nuclear membrane. For S. acanthias the extends down as two longitudinal columns and accom-
posterior region of the nucleus is dilated and devoid of panies the axoneme the length of the flagellum (Fig.
chromatin and encloses the proximal portion of the fi- 15). As spermatids elongate, the cytoplasm is displaced
brous bundles (see Fig. 12B). Moreover, the inner nu- posteriorly and is reflected to form a sleeve of cyto-
clear membrane facing the fibrous bundle is rich in plasm surrounding the proximal portion of the tail. For
470 J. PUDNEY
Fig. 10. Spermiogenesis in testis of S . acanthias. A Round sper- of filaments present in the cytoplasm of round spermatids. D Nuclear
matids with spherical nuclei and abundant organelles present in lu- membrane adjacent the Golgi apparatus condenses (arrowheads) and
men of the spermatoblast. Note the pattern of chromatin condensa- becomes modified for attachment of the acrosome. Arrows point to
tion which occurs centrally within the nucleus leaving a peripheral what appear to be acrosomal vesicles budding from the Golgi appa-
ring of nucleoplasm virtually devoid of chromatin granules. B: Golgi ratus S. acanthias.
apparatus associated with the developing flagellum. C : A long bundle
Fig. 11. Spermiogenesis in S. acanthias. A: Acrosome initially develops in the vicinity of the devel-
oping flagellum. B Acrosomal vesicle attached to nucleus lacks an acrosomal granule. C:Fibrous sheath
(arrowheads) deposited along outer surface of nucleus. Note the pattern of chromatin condensation into
a mass of coarse granules that occurs centrally producing a clear peripheral zone of nucleoplasm.
some species this remnant cytoplasm is discarded dur- tous sheath contained both contractile proteins. This
ing spermiation, e.g., S. suckleyi (Stanley, 1971b) suggested that during the formation of spermatid bun-
whereas for others it is retained, e.g., H . portusjacksoni dles the system of acto-myosin represented a contrac-
(Jones et al., 1984). During the process of spermiogen- tile structure involved in the orientation of spermatid
esis the early round spermatids are located in the lu- nuclei towards the periphery of the spermatoblast. It
men of the spermatoblast. As spermatids undergo elon- was postulated these filaments were analogous to those
gation, however, they become embedded within developed between Sertoli cells and spermatids in the
individual recesses formed by Sertoli cytoplasm. At mammalian testis. Initially described by Brokelmann
this stage the 64 spermatids are loosely arranged but (1961) and called junctional specializations (Flickinger
as the germ cells mature they move closer and closer and Fawcett, 19671,they are now commonly referred to
together to eventually form a compact bundle that ap- as ectoplasmic specializations (Russell, 1977). Compac-
parently resides in a single pocket formed by Sertoli tion of the spermatids into tight bundles was thought
cytoplasm (Fig. 15). to occur by a mechanism of endocytosis of Sertoli
The mechanisms involved in bundle formation of plasma membrane located between the germ cells. This
chondrichthyan spermatozoa has been investigated in uptake of Sertoli plasma membrane would cause the
the ratfish H . colliei (Stanley and Lambert, 1985). It spermatids to move closer together, resulting in their
was shown that when elongate spermatids indent the coalescence. Stability of the individual spermatids, as
Sertoli cell to occupy a cytoplasmic recess, filaments this process occurred, was maintained by an extracel-
present in Sertoli cytoplasm become concentrated lular dense material present between the tip of the
around the developing germ cell. As spermatids elon- acrosome and Sertoli plasma membrane which was
gate further, these filaments condense into bundles thought to act as an adhesive substance.
which become aligned parallel to the long axis of each A recent study has reported on the fate of mitochon-
spermatid and extend down to the base of the Sertoli dria during spermiogenesis in H . colliei (Stanley and
cell. Eventually a filamentous sheath is formed imme- Lambert, 1990). Apparently there is fragmentation of
diately surrounding the acrosome and anterior nuclear elongate mitochondria into smaller mitochondria.
region of the spermatid. It was suggested that a close These mitochondria then either migrate to surround
transmembrane adherence existed between the Sertoli the midpiece of the developing spermatid or become
cell filaments and tip of the spermatid acrosome. enclosed in large vacuoles. These vacuoles are limited
Through the use of NBD-Phallacidin and specific anti- by a double membrane (with ribosomes along the inner
bodies to actin and myosin it was shown the filamen- membrane). The vacuoles containing excess mitochon-
472 J. PUDNEY
Fig. 12. Development of filamentous bundles in spermatids of S. the nucleus. B The striated (arrowheads) and nonstriated bundles of
acanthias. A Fibrous bundles associated with developing flagellum. filaments insert in a deep fossa at the posterior pole of the nucleus.
The dense material (arrowheads) deposited at the cell membrane in Note that the posterior region of the nucleus that surrounds the fila-
the vicinity of the flagellum represents the ring centriole or annulus. mentous bundles is dilated, devoid of chromatin with numerous nu-
Note that in this spermatid, nuclear rotation has not yet occurred clear pores in the inner membrane that faces the bundles of filaments.
since although the acrosome is well-developed the flagellum projects C: This micrograph shows the nonstriated bundle of filaments (arrow-
laterally from the cell and has not yet moved to the posterior pole of heads) is twisted around the striated bundle of filaments.
Fig. 13. A characteristic feature of spermatids is the formation of a long cytoplasmic process. A: This
process contains an array of fine filaments. B: Often these processes indented the Sertoli cytoplasm
forming the wall of the spermatoblast and may represent an anchoring device S. ucanthius.
dria are eliminated and phagocytosed by the Sertoli oneme and two equal-sized, oval longitudinal columns
cell. a t doublets 3 and 8. Spermatozoa of rays also have two
The morphology of chondrichthyan spermatozoa is longitudinal columns but these are round in cross-sec-
conservative and appears similar for those species tion. For chimeric species, however, the development of
which have been studied, e.g., the sharks S. suckleyi the longitudinal column adjacent doublet 8 is sup-
(Stanley, 1965a, 1964,1971b);H . portusjacksoni (Jones pressed, appearing only as a short rod, whereas the one
et al., 1984); Prionace glauca, Carchurhinus falci- associated with doublet 3 extends the length of the fla-
formis, Centroscymnus owstoni and Chlamydoselachus gellum (Stanley, 1983; Hara and Tanaka, 1990). This
anguineus (Hara and Tanaka, 1990); rays Dasyatis ku- arrangement results in an asymmetric axoneme in
hlii and Dasyatis garouaensis (Hara and Tanaka, 1990) spermatozoa of the chimera. Glycogen granules appar-
and the chimaera H . colliei (Stanley, 1965a, 1983) and ently are commonly present in chondrichthyan sper-
Chimaera phantasma (Hara and Tanaka, 1990). The matozoa. For elasmobranchs this occurs as a small
acrosome tends to be small and elongated with a deep number of granules dispersed between the mitochon-
posterior indentation that fits loosely over the pointed dria (Stanley, 1971b). In H. colliei, however, abundant
tip of the nucleus. The long nucleus is attached to the glycogen granules were detected along the length of
flagellum via the midpiece which contains the mid- the flagellum (Stanley, 1983).
piece rod surrounded by a small number of mitochon- A characteristic feature of chondrichthyan sperma-
dria. The anterior of the midpiece rod inserts into the tozoa is their helical shape. The long, pointed head ap-
nuclear implantation fossa while the centriole complex pears spirally twisted giving it a corkscrew appear-
is positioned at the posterior end of the midpiece rod. ance. This helical twisting also involves the acrosome,
Unlike many other vertebrates, therefore, flagellar el- and elements of both the midpiece, and flagellum. In S.
ements are not present in the midpiece of chondrich- acanthias the axoneme is straight but the microtubules
thyan spermatozoa. The flagellum is composed of a typ- of the doublets follow a helical course (Stanley, 1971b).
ical axoneme consisting of a 9 + 2 array of doublets plus By contrast, in H . colliei both axoneme and microtu-
a characteristic feature of chondrichthyan spermato- bules exhibit a spiral pattern along the length of the
zoa, the longitudinal columns (Fig. 15). flagellum (Stanley, 1983). How spiralization of these
Species differences in spermatozoon morphology pri- components of the spermatozoa is accomplished is un-
marily involve flagellar structures. Thus, in H . colliei known. The helical configuration of the head and
it was reported that the proximal centriole is absent flagellar elements in chondrichthyan spermatozoa are
from the basal body (Stanley, 1965a, 1983).Major vari- related to motility. Forward progression in these sper-
ations, however, involve the number and appearance of matozoa is not achieved by lateral bending of the fla-
the longitudinal columns (Hara and Tanaka, 1990). In gellum but by rotating around their long axis. The
sharks the flagellum is symmetrical with a central ax- function of the longitudinal columns in chondrichthyan
474 J. PUDNEY
Fig. 14. Spermiogenesis in S. acanthias. A Acrosome forms a loose cap over the elongated tip of the
nucleus and a perfortorium (arrowhead) develops in the subacrosomal space. Note chromatin is organized
as a mass of fibers orientated parallel to the long axis of the nucleus. B Elongate spermatid showing
helical pattern of chromatin fibers and loosening of the nuclear membrane adjacent regions of chromatin
acquiring a spiral configuration.
spermatozoa is unknown although it has been sug- al., 1980; Grier, 1981, 1993). The teleost testis is orga-
gested they may restrict lateral movement of the fla- nized as a system of either anastomosing tubules, pos-
gellum allowing the spermatozoa to progress by revolv- sibly restricted to lower fish or lobules present in other
ing about its long axis (Stanley, 1971b). species (Grier, 1993). Furthermore, both types of testes
can be further defined by the distribution of spermato-
OSTEICHTHYES gonia within the tubules or lobules (Grier et al., 1980;
The osteichthyes are composed of two classes of bony Grier, 1981). First, there is the unrestricted sper-
fishes, the Crossopterygii and Actinopterygii. The matogonial testis type characterized by the presence of
Crossopterygii include the Dipnoi (lung fishes) and Co- spermatogonia along the entire length of the tubule or
elacanthini, represented by a living fossil, the coela- lobule (Fig. 16A). During the nonbreeding season, pri-
canth (Latimeria). The Actinopterygii consist of the mary spermatogonia and Sertoli cells occur as solid
Chondrostei (sturgeons), Holostei (bowfin, garpike) cords within the tubule or lobule which contains a ves-
and the Teleostei. Of all these groups, the Teleostei are tigial lumen. With the onset of spermatogenesis, cysts
by far the most important and extensive with more develop and line the tubules or lobules which now have
than 20,000 extant species. Despite the economic im- a patent lumen. Spermiation results in the breakdown
portance of teleosts and their ready availability as re- of the Sertoli cells which form the walls of the cyst to
search subjects, the reproductive biology of relatively release spermatozoa into the lumen. This unrestricted
few species has been adequately studied. spermatogonial testis type is found in most species of
The bony fishes are a very diverse group of verte- teleost.
brates with extremes of reproductive strategies rang- The second pattern of organization found in teleosts
ing from external to internal fertilization. This results is the restricted spermatogonial testis type. This de-
in great variation in the organization of the testis. Due scribes the situation where primary spermatogonia are
to this, the nomenclature describing the different confined to the distal region of the lobule only (Fig.
structural arrangements of the teleost testis has been 16B). During spermatogenesis the primary spermato-
somewhat confusing and inconsistent (Grier, 1981). To gonia associate with Sertoli cells to form cysts. As germ
rectify this condition a more uniform nomenclature has cells mature, cysts migrate down the lobule toward the
been proposed, based on the distribution of spermato- efferent sperm ducts. By the time spermatozoa have
gonia within the seminiferous compartment (Grier et formed, the cyst is located near the sperm duct. Sper-
Fig. 16. Schematic diagrams illustrating spermatogenesis in the

Osteichthyes. A: Unrestricted spermatogonial testis type character-
ized by the presence of spermatogonia (PG) along the length of the
lobule. Cysts form when a Sertoli cell (SC) surrounds a primary sper-
matogonium. With the onset of spermatogenesis, cysts (C) mature and
Fig. 15. Cross-section through a bundle of mature spermatids lo- line the lobule. Spermiation occurs with rupture of the cyst and lib-
cated in a pocket formed by the Sertoli cytoplasm. This section passes eration of spermatozoa (SP)into the lobule lumen. B: Restricted sper-
through the axoneme illustrating the presence of the longitudinal
columns (arrowheads) located at doublet 3 and 8. S. acunthias. matogonial testis type where spermatogonia (PG) are confined to the
distal end of the lobule. Cysts form when a Sertoli cell (SC) surrounds
a primary spermatogonium. As cysts (C) mature they migrate down
the lobule towards the efferent ducts (ED). By the time cysts reach the
efferent duct they contain spermatozoa. Spermiation results when the
miation ensues with release of spermatozoa into the cyst fuses with the efferent duct. (Adapted from Grier, 1993.)
sperm duct. Sertoli cells, depending on the species, ei-
ther degenerate or become incorporated into the epi-
thelium of the efferent ducts. The restricted sper-
matogonial testis type is only found in one group of strategy is the production of spermatozoa packaged ei-
teleosts, the Atheriniformes. Although Atheriniforme ther in naked (spermatozeugmata) or encapsulated
species all possess a common testicular structure, there (spermatophores) bundles which are introduced into
is great diversity in their reproductive biology depend- the female at insemination. This results in complex
ing upon whether fertilization is external or internal. morphological relationships developing between sper-
Atherinimorph teleosts practicing external fertiliza- matids and Sertoli cells not seen in atherinimorph spe-
tion include Fundulus heteroclitus (Selman and Wal- cies with external fertilization, e.g., Lebistes reticula-
lace, 1986) and Oryzias latipes (Gresik et al., 1973). For tus (Vaupel, 1929) Poecilia reticulata (Billard, 1970a)
these species, spermatogenesis is a fairly simple pro- Poecilia latipinna (Grier, 1975a) Mollienisia latipinna
cess. Primary spermatogonia occur at the blind end of (Van den Hurk et al., 1975). Only one atherinimorph
the lobule and undergo several mitotic divisions before species, Horaichthys setnai, produces true spermato-
becoming associated with Sertoli cells to form cysts. As phores, whereas all others develop spermatozeugmata
cysts mature they move down the lobule towards the (Grier et al., 1981). Spermatophores develop within
efferent sperm ducts. Unlike other teleost species, dur- cysts and the walls of these structures, which encapsu-
ing maturation of these cysts, there is little differenti- late the spermatozoa, are formed by a Sertoli cell se-
ation of Sertoli cells which remain remarkably unde- cretion. The process of spermatophore formation has
veloped through the process of spermatogenesis. At the been described for H . setnai (Grier, 1984).Briefly, sper-
completion of spermatogenesis, cysts are located in the matogenesis is initiated and proceeds in a similar fash-
lobular region proximal to the efferent ducts. The Ser- ion to that occurring in species with external fertiliza-
toli cells, forming the cyst, then fuse with epithelial tion until the stage of spermiogenesis is reached. At
cells lining the efferent ducts which results in release this time the Sertoli cell hypertrophies, progressively
of the contained spermatozoa. acquiring a columnar shape, and becomes highly secre-
Those atherinimorph species which engage in inter- tory. The released secretion then self-assemblesto form
nal fertilization demonstrate a more complex process of the capsule of the spermatophore which surrounds the
spermatogenesis. An adaptation for this reproductive spermatozoa within the cyst. Rupture of the cyst occurs
476 J. PUDNEY
with transfer of spermatophores to the efferent ducts.

The remaining Sertoli cells either become incorporated
into the efferent ducts or are sloughed and degenerate.
Spermatozeugonata formation has been reported for
Lebistes reticulatus (Winge, 1922; Vaupel, 1929) Po-
ecilia latipinna (Grier, 1975a) and Mollienisia latipinna
(Van den Hurk et al., 1975). As for other atherini-
morphs, spermatogenesis proceeds normally up to sper-
miogenesis. During this phase the Sertoli cells hyper-
trophy, acquire a columnar shape and in these poeciliid
species spermatids align themselves with their heads
abutting the apical Sertoli cytoplasm to form the sper-
matozeugmata. For goodeide, however, i t is the devel-
oping flagellum that becomes associated with Sertoli
cells, e.g., Ameca splendens, Ataenobius toweri, Chara-
codon lateralis, Xenotoca eiseni (Grier et al., 1978).
Spermatozoa are not held together by junctional spe-
cializations with Sertoli cells. Instead, Sertoli cell pro-
jections anchor spermatozoa at the lumenal margin of
the cyst. When the mature cyst contacts the efferent
duct, spermiation occurs and the contained spermato-
zeugmata are released.
Seasonal reproduction is common for teleosts and the
testis may exhibit large variations in size and histolog-
ical appearance. The length of time following comple-
tion of spermatogenesis and spawning may vary from
species to species (Borg, 1982; Ruby and McMillan
1970) or may occur directly following production of
spermatozoa (Henderson, 1962). For some tropical spe-
cies of teleosts there is no apparent seasonal fluctua-
tion in spermatogenesis. There is diversity in the germ
cells composing the regressed teleost testis. For many
species the quiescent testis contains mainly spermato-
gonia (Shrestha and Khanna, 1978); for others either
primary spermatocytes (Ahsan, 19661, spermatids
(Rosenblum et al., 1987) or spermatozoa (Borg 1982; Fig. 17. Unrestricted spermatogonial testis type seminiferous lob-
ule during the breeding season with a patent lumen and lined by cysts
Ruby and McMillan, 1970). containing maturing germ cells in the testis of the teleost Pugellus sp.
At the light microscope level the process of spermato-
genesis has been described for a number of teleost spe-
cies, e.g., Ictalurus nebulosus (Rosenblum et al., 1987);
Couesius plumbeus (Ahsan, 1966); Gasterosteus aculea- lobule to describe lobules containing spermatozoa with
tus (Borg, 1982); Eucalia inconstans (Ruby and Mc- few scattered cysts; and storage lobule to describe lob-
Millan, 1970); Tandanus tandanus (Davis, 1977); Sci- ules packed with spermatozoa and containing no cysts
aenops ocellatus (Grier et al., 1987). (Grier et al., 1987).
In general, spermatogenesis is very similar for all Germ cell renewal in the lobules has been investi-
these species of teleosts. For most teleosts spermato- gated in several species. It is generally accepted that a
gonia are the prevalent germ cell present in the re- permanent population of primordial stem germ cells
gressed testis. As recrudescence ensues, spermatogonia exist in the lobule wall. During each annual cycle of
undergo repeated mitotic divisions and eventually spermatogenesis these cells undergo mitosis to produce
become associated with Sertoli cells to form cysts. Sper- spermatogonia. The remaining stem cells provide a res-
matogenesis then proceeds and cysts containing matur- ervoir for future spermatogenic cycles. Other studies,
ing germ cells are distributed along the length of the however, have suggested there is a n annual migration
lobule (Fig. 17). of stem cells into the lobules from a n interstitial loca-
Eventually the cysts become filled with spermatozoa tion. Migratory germ cells have been described in the
which are released into the lumen at spermiation re- interstitial tissues of C. plumbeus (Ahsan, 1966); E .
sulting from rupture of the cyst. Few cysts can now be inconstans (Ruby and McMillan, 1970); and Salmo
detected and the lobules contain scattered spermato- gairdneri (Hurk et al., 1978). None of these studies
gonia which will serve as stem cells for the next cycle were able to identify the original source of these germ
of spermatogenesis. To reflect these changes in sper- cells and the morphological evidence (light micros-
matogenesis, the following terms have been proposed copy), illustrating the entry of germ cells into the lob-
to describe the lobules: production lobules to describe ules is not convincing.
the lobule or part that contains maturing cysts, with or A number of reports are available describing differ-
without mature spermatozoa in the lumen; transitional ent aspects of spermatogenesis in various teleost spe-
cies a t the electron microscope level, e.g., Lebistes re- nuclear pores are distinct in young spermatids but dis-
ticulatus (Asai, 1971; Gronberg and Telkka, 1968; appear when the flagellum begins to develop (Grier,
Gronberg and Wartiovaara, 1972; Mattei and Boisson, 1975b; Gronberg and Wartiovaara, 1972).
1966; Mattei et al., 1967);Oligocottus maculosus (Stan- As round spermatids mature there occurs a distinct
ley, 1969); Upeneus prayensis (Boisson et al., 1969); spatial arrangement of organelles that will eventually
Pimephales notatus (Schjeide et al., 1972); Albula define the structural appearance of the resultant sper-
vulpes (Mattei and Mattei, 1973); Gambusia affinis matozoon. Thus, the centrally located nucleus moves to
(Grier, 1975b);Poecilia latipinna (Grier, 1973, 1975a); occupy a position close to the cell surface that (usually)
Trichurus lepturus (Mattei and Mattei, 1976a); An- is adjacent to the Sertoli cell cytoplasm. Then the two
guilla australis schmidtii and A. dieffenbachii (Todd, centrioles plus the developing flagellum migrate from
1976); Oryzias latipes (Grier, 1976; Hamaguchi, 1987); their position at the periphery of the cell to become
Lepodogaster lepadogaster (Mattei and Mattei, 1978a); located close to the nucleus. During this transloction
Liza aurata (Brusle, 1981); Lepomis macrochirus (Sp- the distal centriole carries the flagellum through the
rando et al., 1988); Salmo gairdneri (Billard, 1972, cytoplasm, resulting in the formation of a cytoplasmic
1983);Poecilia reticulatus (Billard and Flechon, 1969); canal (Fig. 18C). The flagellum is covered by reflected
Platichthys flesus (Jones and Butler, 1988a,b); Thy- plasma membrane and is therefore separated from the
mallus thymallus (Lahnsteiner et al., 1991); Rhodeus rest of the cell body (Fig. 18C). In some teleost species
sericeus sinensis (Guan and Afzelius, 1991); Blennius the nucleus undergoes approximately a 90” rotation
pholis (Silveira et al., 1990);Ophidion sp (Mattei et al., and centrioles become located within a nuclear depres-
1989);8 species of blennidae (Lahnsteiner and Patzner, sion. For other species, this nuclear rotation does not
1990);Paroncheilus sp (Mattei and Mattei, 1984);Zcta- occur and centrioles do not become associated with a
lurus punctatus (Poirier and Nicholson, 1982);Brachy- nuclear depression. At this time the previously dis-
danio rerio (Kessel et al., 1983); a large number of persed mitochondria also move to become preferen-
cyprinids (Clerot, 1976; Thiaw et al., 1986; Thiaw and tially associated with the developing flagellum. Due to
Mattei, 1989); several species of gobies (Fishelson et the presence of the cytoplasmic canal these mitochon-
al., 1990). dria do not, as in many other vertebrates, come into
In general, the fine structure of germ cell develop- close physical contact with the flagellum. For some
ment is very similar for all species studied. The sper- species a submitochondrial net, consisting of filaments
matogonia are characterized by large regular nuclei enclosed between two membranes, develops between
usually containing a distinct nucleolus. In P. latipinna the mitochondria and plasma membrane of the cyto-
and P. notatus, spermatogonia contain annulate lamel- plasmic canal (Dadone and Narbaitz, 1967; Grier,
lae and the mitochondria are associated with a dense 1975b). A different location of mitochondria has been
material of nuclear origin (Grier, 1975a; Schjeide et al., described where these organelles surround the base of
1972). In several species of cyprinids, groups of mito- the nucleus in P. flesus (Jones and Butler, 1988a) and
chondria were located at specific sites in the cytoplasm a number of species of blennidae (Lahnsteiner and
of spermatogonia (Clerot, 1976). These mitochondria Patzner, 1990). The mitochondria occupied recesses de-
were related with a dense material (cement). Annulate veloped by the nucleus. In several species of gobies it
lamellae were also often detected associated with this has been reported that small mitochondria present in
intermitochondrial cement. Nuage has been reported round spermatids disappear to be replaced by 2 large
to occur in spermatogonia of B . pholis (Silveira et al., mitochondria (Fishelson et al., 1990). Also in these te-
1990)and 0. latipes (Hamaguchi, 1987).Spermatocytes leosts a peculiar system of alternating RER and SER
possess a large oval nucleus with coarse chromatin and was described that formed rings in the cytoplasm. As
a distinct Golgi apparatus (Fig. 18A). Abundant micro- the spermatids matured, however, these rings of ER
tubules occur in spermatocytes of some cyprinodonts disappeared. A stranger location of mitochondria oc-
(Thiaw and Mattei, 1989) Centriolar division occurs in curs in B . pholis with several of these organelles lo-
spermatocytes. Round spermatids contain a number of cated at the anterior pole of the nucleus, resulting in
mitochondria dispersed throughout the cytoplasm with the absence of a midpiece (Silveira et al., 1990). In T .
two centrioles located a t the periphery of the cell (Fig. thymallus the mitochondria of the midpiece becomes
18B). Annulate lamellae have been described in sper- fused to form a chondriosome which is wound around
matids of B. pholis (Silveira et al., 1990), and glycogen the proximal region of the axoneme (Lahnsteiner et al.,
granules and mitochondria1 cement in spermatids of 1991).
blennidae (Lahnsteiner and Patzner, 1990). The distal In L . lepadogaster it has been reported that a re-
centriole lies closer to the plasma membrane than the gional differentiation occurs at the anterior surface of
proximal centriole which is usually aligned at roughly spermatid nuclei (Mattei and Mattei, 1978a). In this
90” with respect to the distal centriole. It has been re- area a number of lamellae occur dispersed between the
ported that the distal centriole appears to be anchored thickened inner and outer nuclear membranes which
to the plasma membrane by a number of fibers (Mattei were termed “lamelles perinucleaires intermem-
and Mattei, 1976a, 1978a). Usually a well-developed branaires.” It was suggested this represented an ata-
Golgi apparatus is associated with the centrioles. The vistic development of the acrosome which has been lost
flagellum then begins to develop from the distal cen- in the evolution of the teleosts. Also in S. gairdneri, an
triole. Flagella, however, have been detected in sper- abortive acrosomal vesicle seems to develop that dis-
matogonia and spermatocytes (Billard and Flechon, appears later in spermiogenesis (Billard, 1983).
1969). In several teleost species it has been shown that The association of the developing flagellum with the
478 J. PUDNEY
Fig. 18. Various stages of spermatogenesis in L.macrochrius. A (arrow) can be seen in this spermatid. C: Flagellum located in the
Cyst containing primary spermatocytes. B: Round spermatids possess cytoplasmic canal. (Micrographs courtesy of R.L. Sprando and L.D.
bullet-shaped nuclei with a deep implantation fossa (arrowhead) at Russell.)
the posterior pole. A cross-sectional profile of the proximal centriole
nucleus signals chromatin condensation as well as ini- cent condensed chromatin remains intact. Eventually
tiating morphological changes, e.g., elongation, that the nuclear membrane reforms to preferentially en-
eventually result in the species-specific shape of the close only the condensed chromatin, thus eliminating
head of the mature spermatozoon. It has been shown, in excess membrane.
several species, that the condensation of chromatin in Concomitant with changes in nuclear volume, as
the nuclei of spermatids, as they mature, occurs in a spermatids mature, is also a reduction in cell volume.
specific pattern that often begins in the region adjacent For many teleosts this excess cytoplasm is sloughed,
to the developing flagellum (Boisson et al., 1968b;Rus- towards the end of spermiogenesis, as a residual body
sell, 1988; Sprando et al., 1988; Stanley, 1969). A fine which is usually engulfed by the Sertoli cell. For L.
structural study of nuclear differentiation during sper- macrochirus, however, the process of elimination of ex-
miogenesis has been undertaken for Oncorhynchus tra cytoplasm is accomplished by the formation of
tshawytscha (Zirkin, 1975).The nuclei of early sperma- membrane-bound cytoplasmic packets which migrate
tids contain randomly distributed chromatin fibers to the cell surface and are extruded (Sprando et al.,
similar to other (somatic) cells. As spermatids mature 1988). It was estimated that during spermiogenesis in
the chromatin progresses to highly orientated thick fi- L. macrochirus, both nuclear and cytoplasmic volumes
bers and unusual, irregular ribbon-like elements. Nu- of spermatids were reduced some 80% and 92%, respec-
clei of mature spermatozoa were extremely dense and tively, resulting in a total reduction of 87% in cell vol-
showed little substructure. It was suggested these ume.
structural changes were related to the process of re- Following division of the centriole and initiation of
placement of nuclear histones by protamines. flagellum development, a transitory intercentriolor
As the chromatin condenses there is obviously a lamellated body appears in many species, e.g., L. retic-
great reduction in the volume of the nucleus. This re- ulatus (Asai, 1971; Gronberg and Telkka, 1968; Mattei
sults in a large area of redundant nuclear membrane. and Mattei, 1966); U. prayensis (Boisson et al., 1969);
The removal of this excess membrane has been de- P. Zatipinna (Grier, 1973, 1975a); 0. latipes (Grier,
scribed in several teleost species. This can involve the 1976). This structure has been suggested to be derived
release of small vesicles from either the entire surface, from satellites associated with the proximal centriole
e.g., 0. maculosus (Stanley, 1969)or base, e.g.,P. flesus (Grier, 1973, 1976). The intercentriolar lamellated
(Jones and Butler, 1988a) of the nucleus as the chro- body accompanies the centrioles as they migrate from
matin condenses. A more interesting process, however, the cell periphery to the nucleus. The implantation
has been described for L. macrochirus (Sprando et al., fossa, therefore, houses a centriolar complex consisting
1988). Apparently the nuclear membrane associated of: (1)a proximal centriole from which, for various spe-
with clear, chromatin-free nucleoplasm undergoes in- cies, microtubules radiate towards the nucleus to at-
termittent breakdown. The nuclear membrane adja- tach to the nuclear membrane of the implantation fossa
and in some cases to surround the periphery of the composed of a typical 9 + 2 arrangement of doublets.
nucleus; (2) an intercentriolar lamellated body dis- Along the entire length of the flagellum two broad ex-
posed between the proximal and distal centriole; and tensions or “wings” are present which contain a dense
(3) a distal centriole (basal body) which forms the ax- material.
oneme of the flagellum. It has been reported that the For Actinopterygii, the fine structure of spermatozoa
microtubles emanating from the proximal centriole has been studied for the Holostein Lepisosteus osseus
serve to stabilize the nucleus prior to chromatin con- (Afzelius, 1978).In this species the spermatozoa are not
densation as well as the relationship between the cen- highly differentiated and may be considered primative,
triolar complex and nucleus during nuclear morpho- since they lack an acrosome, have a short midpiece
genesis and development of the implantation fossa with a single ring-shaped mitochondrion and lateral
(Grier, 1973).The microtubles are transient and disap- ridges or “wings” on the flagellum that are roughly in
pear as soon as the implantation fossa has formed but the plane of the two central doublets of the axoneme.
before chromatin condensation has ceased. The two centrioles are aligned perpendicular to each
For most species the intercentriolar lamellated body other and the proximal centriole is joined to a striated
appears during spermiogenesis. In a few other species, fibrous body which projects into the posterior fossa of
it has been reported to arise in spermatocytes (Grier, the nucleus. A short cytoplasmic sleeve surrounds the
1975b; Gronberg and Telkka, 1968). The proximal cen- intital portion of the flagellum which contains a 9 + 2
triole and intercentriolar lamellated body are, for arrangement of doublets.
many species, temporary structures and degenerate a t The spermatozoa of the Teleostei, however, have
a later stage of spermiogenesis. A few species, however, been best studied at the electron microscope level, e.g.,
retain either the lamellated body, e.g., Pantodon Cnesterodon decemmaculatus (Sotelo and Trujillo-
buchholzi (VanDeurs and Lastein, 1973) or proximal Cenoz, 1958);L . reticulatus (Mattei and Boisson, 1966);
centriole, e.g., 0. latipes (Grier, 1976). The exact J . lineata (Dadane and Narbaitz, 1967); Oligocottus
function of the intercentriolar lamellated body is maculosus (Stanley, 1969); Dactylopterus uolitans
unknown. (Boisson et al., 1968a);P. reticulata (Billard, 1970b);F .
As yet, ectoplasmic specializations have not been ob- heteroclitus, Cyprinodon variegatus (Yasuzumi, 1971);
served between spermatids and Sertoli cells in the te- 0. latipes (Grier, 1976).
leost testis (Sprando and Russell, 1987a). It was sug- Spermatozoa of species which practice external fer-
gested that since spermatids did not enter into close tilization, e.g., 0. latipes are not highly developed mor-
physical contact with Sertoli cells, ectoplasmic special- phologically and consist of either a round, bullet-shaped
izations are not developed. Occasional “desmosome- or slighly elongated head; a simple centriolar complex;
like” structures have been reported between spermato- few mitochondria; a poorly differentiated midpiece; and
cytes and Sertoli cells in L. macrochirus (Sprando and usually a lateral head to tail articulation. By contrast,
Russell, 198713). species with internal fertilization produce more mor-
A rather remarkable observation has been reported phologically complex spermatozoa, e.g., P. reticulatus
for L . lepadogaster during spermatogenesis (Mattei and which have elongate heads, a highly modified centriolar
Mattei, 1978a). It was reported that mature spermato- complex, a well-developed midpiece associated with a
zoa were commonly seen penetrating spermatocytes large number of mitochondria, and caudal articulation
and spermatids. Usually the spermatozoa penetrated of the head with the tail.
the cytoplasm but occasionally also passed through the The number of mitochondria associated with the mid-
nucleus. A total of 6 spermatozoa were found penetrat- piece of teleost spermatozoa varies from species to spe-
ing a single spermatid. Apparently the embedded sper- cies, e.g., 4 in L. aurata (Brusle, 1981), and 6-10 for L .
matozoa did not interfere with maturation of the germ lepodogaster (Mattei and Mattei, 1978a). In some spe-
cells and were released when the residual cytoplasm cies a submitochondrial net is present, e.g., J . lineata
was shed. Another unusual report concerns a number of (Dadone and Narbaitz, 1967). Species variation also
species of blennidae where immature spermatids were exists in the morphology of the midpiece. For instance,
released from the cyst to mature in the testicular gland in L. lepadogaster a portion of the midpiece is devoid of
and/or spermatic ducts (Lahnsteiner and Patzner, mitochondria (Mattei and Mattei, 197813). The flagel-
1990). lum of most species contains a typical 9 + 2 axoneme of
The fine structure of spermatozoa of many species of doublets and often develops lateral ridges (“wings”)
Osteichthyes has been reported. For the Crossoptery- along the entire length. The axoneme of some other
gii, information is available on the ultrastructure of species, however, is composed of atypical 9 + 0 organi-
several species of lungfish, e.g., Protopterus annectans zation of doublets, e.g., Lampanyctus sp. (Mattei and
(Boisson and Mattei, 1965a) and Neoceratodus forsteri Mattei, 1976b); Anguilla anguilla (Billard and Gins-
(Jespersen, 1971). The head of the spermatozoon con- burg, 1973; Ginsburg and Billard, 1972);Albula vulpes
sists of a very elongated nucleus with a small anterior (Mattei and Mattei, 1973);A. australis schmidtii and A.
acrosome. Two rod-shaped structures extend from the dieffenbachii (Todd, 1976); Lycodontis afer (Boisson et
tip of the acrosome, through the nucleus, to terminate al., 1967). Species variation also exists in the number of
in the cytoplasm. Each rod is enveloped by nuclear flagella developed by spermatozoa. Biflagellated sper-
membrane. The midpiece contains two perpendicularly matozoa are produced by Porichthys notatus (Stanley,
aligned centrioles. The proximal centriole is attached 1965b);I . punctatus (Yasuzumi, 1971);Lampanyctus sp.
to the posterior margin of the nucleus, while the distal (Mattei and Mattei, 1976b);L . lepadogaster (Mattei and
centriole gives rise to the flagellum. The axoneme is Mattei, 1974,1978b);Paroncheilus sp. (Mattei and Mat-
480 J. PUDNEY
tei, 1984) and I . punctatus (Poirier and Nicholson, the tip of the spermatozoon, gives a bulbous protubur-
1982). The centrioles lie parallel to one another and ance to the gamete.
each participates in the development of an individual Patches of regular particles have been detected by
flagellum. Although a large number of teleosts, repre- freeze-fracture in the cell membrane covering the an-
senting different families, produce biflagellated sper- terior head of spermatozoa in B . rerio (Kessel et al.,
matozoa, the evolutionary significance (if any) of this 1983) and R. sericeus sinensis (Guan and Afzelius,
feature is unknown. A more bizarre condition exists in 1991). It was suggested these regions may act as rec-
Gymnarchus niloticus (Mattei et al., 1967b)and a num- ognition andlor adhesion sites allowing spermatozoa t o
ber of species of mormyres (Mattei et al., 1972b) which fuse with the ova.
produce aflagellated spermatozoa. Spermiogenesis ap- AMPHIBIA
pears normal but neither of the two centrioles, which lie
between the nucleus and mitochondria, develops a fla- The amphibia are composed of three extant orders:
gellum. It was suggested these gametes carry out the Anura (frogs, toads) is the largest and more than
ameoboid movement. two-thirds of amphibians belong to this group; Urode-
In a large number of different cyprinodonts it was lia (newts, salamanders) and Apoda (caecilians) a
reported there was great variation in the presence and small group of legless amphibians. Amphibians are the
absence of the dynein arms of axonemal doublets most primitive of terrestrial vertebrates and most spe-
(Thiaw et al., 1986). Despite these differences all sper- cies need to return to an aqueous environment to
matozoa were found to be motile. A number of teleost breed.
species produces morpholgoically bizzare spermatozoa. Morphologically, the amphibian testis is similar to
In L. afer (Boisson et al., 1967) the proximal centriole that of teleosts. Spermatogenesis occurs in cysts
develops to produce an intracellular flagellum. Fur- present in a seminiferous compartment called lobules.
thermore, one of the proximal centriole microtubles be- Based on spermatogonial distribution, three types of
comes closely associated with the nuclear membrane to testes have been described for amphibia (Grier, 1993):
extend along the periphery of the nucleus. A similar one, in urodeles, in which the testis is divided into lobes
condition occurs in A. uulpes (Mattei and Mattei, 1973) and new lobules form from primary spermatogonia and
where the nuclear membrane penetrates the proximal their associated Sertoli cells within connections of the
centriole, causing disruption and dislocation of the mi- testicular lobes, e.g., Taricha granulosa. The other oc-
crotubular triplets which become associated with the curs in urodeles that do not have lobed testes and here
nuclear membrane. These then extend through the cy- primary spermatogonia and Sertoli cells are located in
toplasm, associated with a projection of the nuclear the lobular region proximal to the sperm duct, e.g.,
membrane, to form what has been termed a pseudofla- Necturus maculosus (Fig. 19A). The third is found in
gellum. Furthermore, a large mitochondrion is not as- anuran species in which the primary spermatogonia
sociated with the centrioles but is located at the ante- are present along the length of the lobule (Fig. 24).
rior of the spermatozoon towards the lateral aspect of Most amphibian species undergo a postnuptial sper-
the nucleus. The spermatozoa of P. buchholzi are ex- matogenic cycle. Initiation of spermatogenesis occurs
tremely aberrant (Van Deurs and Lastein, 1973). The soon after spawning and is completed quickly, result-
centriolar complex consists of a single centriole and a ing in spermatozoa being retained within the testis
lamellated cap-shaped body situated between the cen- prior to the next breeding season. Temperate amphib-
triole and the nucleus. The midpiece is composed of 9 ians undergo an annual spermatogenic cycle with mat-
helical mitochondria1 threads, formed by end-to-end fu- uration of spermatozoa occurring in the winter fol-
sion of small, single mitochondria that alternate with 9 lowed by spermiation in the spring breeding season.
helical dense fibers. Posterior to the midpiece is a
structure called the fenestrated sheath composed of in- Urodeles
ner and outer membranes penetrated by pores. This Many urodeles exhibit a spermatogenic wave in the
sheath surrounds the initial portion of the axoneme. testis during the breeding season. This results in a spa-
Many species of anguillidae also produce extremely tial and temporal segregation of germ cells. Spermato-
morphologically exotic spermatozoa, e.g., A. anguilla genesis begins at the caudal end of the testis and
(Billard and Ginsburg, 1973; Ginsburg and Billard, progresses towards the cephalic end. Lobules in se-
1972); A. australis schmidtii and A. diefenbachii, quential stages of maturation, therefore, can be found
(Todd, 1976). The spermatozoa have a sickle- or cres- along the length of the testis with the least mature
cent-shaped head tapering to a narrow neck region and located in the cephalic region. Furthermore, spermato-
a long flagellum. A short striated rod or appendage genesis is confined to regions of the seminiferous lobule
projects from the posterior region of the head near the distal t o the main sperm duct. Lobular regions proxi-
origin of the flagellum. The proximal centriole devel- mal to the sperm duct contain primary spermatogonia
ops and divides into two groups of 4 and 5 microtubular which act as a reservoir for future waves of spermato-
triplets which then course anteriorly along either the genesis (Fig. 19B). In many urodeles, therefore, there is
inner concave or outer concave aspects of the nucleus. a maturational wave of spermatogenesis occurring lon-
A midpiece is not present because the single mitochon- gitudinally in a caudacephalic direction, plus a proxi-
drion becomes displaced (in these species) to the ante- mal-to-distal cycle of differentiating germ cells which
rior pole of the nucleus. The nucleus may partly sur- eventually form the maturing segments of the seminif-
round the mitochondrion that lies in an indentation of erous lobules which initiate seasonal testicular recru-
its concave side. The presence of this mitochondrion, at descence. Spermatogenesis and cyst development has
Fig. 19. Organization of N . maculosus testis. A Seminiferous lob- section of seminiferous lobule containing cysts filled with secondary
ules contain an immature proximal segment composed of cysts filled spermatogonia. Arrowheads point to Sertoli cytoplasm that forms the
with spermatogonia. Arrowheads point to sperm duct. B: Primary walls of the cysts. Note absence of a lumen in the lobule. E: As sper-
spermatogonia are large cells with irregularly shaped nuclei and un- matogenesis proceeds, cysts migrate towards the distal end of the
dergo mitotic activity t o produce secondary spermatogonia. C: Mitotic lobule where spermiation occurs by rupture of the cysts resulting in
divisions of spermatogonia result in growth of the cysts. D: Cross- the lumen of the lobule containing bundles of spermatozoa.
been described a t the light microscope level €or several develop when a primary spermatogonium becomes as-
species of urodeles, e.g., Desmognathus fuscus (Kings- sociated with a Sertoli cell (Fig. 19B). Subsequent mat-
bury, 1901); Taricha torosa (Miller and Robbins, 1954); urational divisions of spermatogonia results in an
Cynops pyrrohogaster pyrrohogaster (Tanaka and Iwa- increase in size of the cysts (Fig. 19C). During sper-
sawa, 1979); N . maculosus (Pudney et al., 1983). matogenesis, cysts migrate toward the distal end of
In general, spermatogenesis proceeds in a similar the seminiferous lobule. By the time this occurs, the
fashion for most urodeles and resembles that occurring cysts contain mature spermatozoa. Spermiation then
in teleosts. For instance, in N . maculosus, nascent cysts occurs, exposing Sertoli cells with isogeneic bundles of
482 J. PUDNEY
spermatozoa embedded in their apical cytoplasm (Fig. posterior pole of the nucleus, resulting in this region
19E). Sertoli cells are either sloughed with the sper- being mostly enclosed by a shell of chromatin. At the
matozoa or undergo degeneration. caudal end of the neck lie the two centiroles. The pres-
A number of studies have reported on the fine struc- ence of a ring centriole (annulus) has been reported for
ture of different aspects of spermatogenesis in several P. waltlii (Picheral, 1972b) and D. fuscus (Lommen,
species of urodels, e.g., Pleurodeles wnltlii (Picheral, 1970). The spermatozoa of urodeles are unusual since
1967, 1972a,b,c, 1979); D. fuscus (Lommen, 1970); they develop an undulating membrane that extends
Salamandra salamandra (Bergmann et al., 1982; almost the length of the flagellum (Fig. 23B). The fla-
Schindelmeiser et al., 1985); Ambystoma mexicanum gellum consists of an axial fiber or rod (also called SUP-
(Keyhani and Lemanski, 1981; Miltner and Arm- porting filament) that extends from the neck region
strong, 1983). and the attached thin undulating membrane with the
Primary spermatogonia are large cells characterized axoneme located a t the free edge along with a dense
by highly irregularly shaped nuclei and with the cyto- structure called the marginal filament (Fig. 2 3 0 . The
plasm appearing relatively devoid of organelles except midpiece contains the mitochondria which are associ-
for a number of mitochondria, several small Golgi re- ated only with the axial fiber and so do not surround
gions and an occasional lipid droplet (Fig. 20A). The the axoneme (Fig. 23D). In A. mexicanurn the mito-
secondary spermatogonia appear smaller with large chondria are smaller than in other species and so
spherical nuclei, numerous mitochondria, long cister- closely packed as to exhibit an almost crystalline ar-
nae of SER, several Golgi areas (Fig. 20B) and profiles rangement (Keyhani and Lemanski, 1981). The flagel-
of annulate lamellae (Fig. 21B). An interesting feature lum of Hynobius nebulosus lacks mitochondria which
of these spermatogonia is that during the early stage of are present in a protoplasmic bead located around the
cyst formation, both develop abundant blunt projec- nucleus (Picheral, 1979).
tions a t their periphery that invaginate the surround-
ing Sertoli cytoplasm (Fig. 21A). It is assumed these Anura
represent anchoring devices that keep individual sper- Like urodeles, anuran spermatogenesis occurs in
matogonia associated with their corresponding Sertoli cysts located in seminiferous lobules (Fig. 24A). Ini-
cell during development of the cysts. At a later stage of tially cyst development progresses similarly to that oc-
cyst development the germ cells have a smooth surface curring in urodeles and teleosts (Fig. 24B), e.g., Rana
(see Fig. 20B). temporaria (Witschi, 1915); Rana esculenta (Lofts,
It has been reported that as cysts mature the germ 1964);Rana pipiens (Burgos, 1955; Rugh, 1939). There
cells lose close physical contact with Sertoli cells (Pud- is a distinct difference, however, in the process of sper-
ney, 1993). Furthermore, at this time, germ cells de- matogenesis that distinguishes these amphibia from
velop filopodia and the intercellular space between Ser- urodeles. During spermiogenesis, as elongate sperma-
toli cells and germ cells widens (Fig. 22). It was tids become embedded in a single Sertoli cell, the for-
suggested this occurred to allow movement and/or mation of the spermatid flagellum signals rupture of
translocation of cells as the cysts become organized the cyst. These open cysts now form the wall of the
within the seminiferous lobule. Spermatocytes of S. seminiferous lobule which is lined by highly differen-
salamandra have been reported to contain annulate tiated Sertoli cells with bundles of spermatids deeply
lamellae and an occassional lipid droplet (Schindel- embedded in their apical cytoplasm (Fig. 24C). At this
meiser et al., 1985). Furthermore, in this species, mi- stage the seminiferous lobule resembles morphologi-
tochondria were located at the periphery of spermato- cally the germinal epithelium present in amniotes.
cytes and this disposition of these organelles was also During this period, at the periphery of the lobule, pri-
observed in early spermatids. Proacrosomal granules mary spermatogonia can be detected surrounded by
were also reportedly detected in secondary spermato- Sertoli cells that are preparing to form cysts for the
cytes. As yet, it would appear that junctional special- next round of spermatogenesis (Fig. 24A). Spermiation
izations have not been described between germ cells then ensues and in some species, e.g., R . esculenta only
and Sertoli cells at any stage of spermatogenesis. the apical portion of the Sertoli cell is shed along with
The fine structure of urodelean spermatozoa has the spermatozoa. The basal portion of the Sertoli cell,
been best studied in P. waltlii (Picheral, 1967, containing the nucleus, remains to develop the next
1972a,b,c, 1979). The spermatozoa possess an acroso- generation of cysts. In these amphibian species, there-
ma1 cap that ends in either a sharp point or rounded fore, a permanent germinal epithelium can be said to
knob and in some species develops a hooked barb, e.g., be present.
P. waltlii, Euproctus aspes. An elongated, conical sub- An unusual mode of spermatogenesis has been re-
acrosomal space contains the perforatorium which con- ported for Bombina uariegata (Obert, 1976).In this spe-
sists of an axial rod that extends deep into the nucleus cies, apparently germ cells develop in the absence of
within an endonuclear canal. The nucleus has promi- Sertoli cells and spermatozoa do not aggregate into
nent ridges composed of nonchromatin material con- bundles but appear as a mass of single cells. It was
sisting of minute tubules packed together (Fig. 23A). unknown whether this represents a primitive condition
Many species of urodeles display this ridge on the sper- or is a peculiarity restricted to discoglossid amphibia.
matozoal nuclei but which differs in composition rang- Electron microscope studies are available on sper-
ing from a single bundle, e.g., P. waltlii to several, e.g., matogenesis in several species of anuran amphibians,
S. salamandra. The neck, or connecting piece appears e.g., Bufo arenarum (Burgos and Fawcett, 1956;
as a long cylinder that fits into a deep fossa on the Cavicchia and Moviglia, 1982); Rana clamitans (Poir-
Fig. 20. Low power electron micrographs of N. maculosus testis merous filopodia at periphery of the germ cell. B Secondary sper-
showing (A) Primary spermatogonia (SP)with irregularly shaped nu- matogonia possess large spherical nuclei. Note that at this stage of
clei which are poorly differentiated and possess a number of mito- development the cells exhibit a smooth outline with no formation of
chondria and a n occasionally lipid droplet. Note development of nu- filopodia.
484 J. PUDNEY
Fig. 21. A: Primary spermatogonia (PG) produce numerous filopodia that invaginate the surrounding
Sertoli cytoplasm. B: Annulate lamellae (arrowheads) present in secondary spermatogonia. N . maculo-
sus.
ier and Spink, 1971); R. pipiens (Poirier and Spink,

1971; Zirkin, 1971); Xenopus laevis (Bernardini et al.,
1986; Reed and Stanley, 1972); Discoglossus pictus
(Sandoz, 1973, 1974); B. variegata (Folliot, 1979);
Megophyrs montana (Asa and Phillips, 1988); Rana
catesbeiana (Sprando and Russell, 1988a); Rhacopho-
rus arboreus and R. schlegelii (Mizuhira et al., 1986).
Morphologically the early stages of spermatogenesis
resemble those occurring in urodeles. Primary sper-
matogonia have large, irregular nuclei with a promi-
nent nucleolus. In X . Zaeuis, spermatocytes possess
abundant SER (Reed and Stanley, 1972). At the
beginning of spermiogenesis the two centrioles are
located close to the Golgi apparatus. Later the
centrioles migrate towards the plasma membrane and
a flagellum develops from the distal entriole. The
centrioles then move towards the nucleus and in doing
so cause retraction of the plasma membrane, resulting
in the formation of a cytoplasmic canal containing the
developing flagellum. The centrioles initially reside in
a small indentation on the anterior pole of the nucleus
adjacent to the acrosome. Then the centrioles become
relocated toward the posterior pole of the nucleus
either by active movement or nuclear rotation. During
spermiogenesis the Golgi apparatus appears poorly
developed and this has been related to the fact that an
acrosomal granule is not produced during formation of
the acrosome, e.g., B. arenarum (Burgos and Fawcett,
1956).X . leavis (Reed and Stanley, 1972);B. uariegata Fig. 22. During cyst formation, secondary spermatogonia lose
(Fo&t, 1979). It has been that distinct close physical contact with Sertoli cells and develop filopodia (arrow-
heads). Furthermore, the intercellular space between germ cells and
modifications of the nuclear envelope occur during Sertoli cells widens. It is assumed this occurs to allow movement of
spermiogenesis of D. pictus (Sandoz, 1974). In early germ cells as cysts mature. N . maculosus.
Fig. 23. N. mucolosus spermatozoa. A: Bundle of tightly packed located next to the marginal filament (small arrow) attached do the
tubules (arrowheads) associated with nucleus. B Scanning electron axial rod (large arrow) by the undulating membrane (large thick ar-
micrograph showing undulating membrane (arrowheads) along row). D: Cross-section through midpiece showing mitochondria asso-
length of flagellum. C: Cross-section through axoneme (arrowhead) ciated with axial rod (arrowheads).
spermatids, nuclear pores are not evenly distributed of spermiogenesis, mitochondria aggregate in the
over the nuclear surface but are preferentially grouped cytoplasmic canal surrounding the flagellum. A
in front of the Golgi apparatus. As the acrosomal single, large mitochondrion, however, has been re-
vesicle becomes associated with the nucleus, however, ported to surround the centrioles at the base of the
and spreads over its surface, these pores gradually nucleus in X . laevis (Reed and Stanley, 1972). A
become displaced towards the posterior pole of the perforatorium, which develops in the space between
nucleus. A manchette has been described during the acrosome and tip of the nucleus and consists of
spermiogenesis in D. pictus (Sandoz, 1974). For X . coarse strands, has been described in spermatids of B .
laevis, as chromatin condenses and nuclear elongation arenurum (Burgos and Fawcett, 1956). During spermi-
occurs in maturing spermatids, excess nuclear and ogenesis of R. catesbeiana, the round spermatids are
plasma membrane appears to be pinched off into the penetrated by processes of Sertoli cytoplasm which
intercellular space between these germ cells and disappear later in development (Sprando and Russell,
Sertoli cells (Reed and Stanley, 1972). At later stages 1988a). Studies carried out to specifically identify
486 J. PUDNEY
Fig. 24. Spermatogenesis in R a m sp. A: Primary spermatogonia (arrowheads) occur along the length
of the seminiferous lobule. B Maturing cysts present in seminiferous lobule. C: Open cysts containing
bundles of spermatids embedded in Sertoli cell cytoplasm line the length of the lobule.
cellular junctions in the anuran germinal epithelium before condensation of nuclear chromatin begins.
have reported desmosome-likejunctions between sper- Moreover, a unique pattern of nuclear morphogenesis
matocytes and Sertoli cells in R. catesbeiana (Sprando takes place. Initially, the chromatin condenses into a
and Russell, 1987b).Furthermore, ectoplasmic special- long, cylindrical coil within the spherical nucleus.
izations also have been described between elongate Later the nuclear membrane conforms to the contours
spermatids and Sertoli cells in this species. (Sprando of the cylinder as it uncoils to form the long, tapering,
and Russell, 1987a). These structures, however, ap- helical nucleus of the mature spermatozoon. Also, the
peared morphologically primitive compared to mam- flagellum has a double axoneme which is connected by
malian ectoplasmic specializations. The layer of cross-bridges extending from the region between sub-
filaments (5-7 nm in diameter) was unorganized, tubules a and b of doublets 3 and 8. During spermio-
poorly developed and often not associated with genesis a manchette does not develop in this species. It
cisternae of ER. Microtubules were often detected was suggested, therefore, that nuclear shaping may be
related to the filaments. controlled by the pattern of DNA and protein aggrega-
Spermatozoa of unusual morphology have been de- tion, indicating that microtubles are not necessarily
scribed for B . variegatu (Folliot, 1979). Helical-shaped universally required to achieved nuclear morphogene-
spermatozoa are produced that cannot be divided into sis.
distinct head and tail regions. This is due to the fact The mechanism of spermation has been described in
that the motile apparatus, including the undulating anurans (Burgos and Vitale-Calpe, 1967a,b; DeRober-
membrane, effectively extends along the entire length tis et al., 1946; Van Oordt et al., 1954) This is accom-
of the head. Thus, the origin of the flagellum, i.e., the plished by distension of the apical Sertoli cell cyto-
centriolar apparatus, is located at the anterior region plasm containing the bundles of spermatozoa. The
of the spermatozoa close to the acrosome. Furthermore, swelling of the Sertoli cytoplasm is due to an influx of
the nuclear chromatin does not fully condense and ma- fluid and results in displacement of spermatozoa to-
ture spermatozoa still retain a portion of residual cy- wards the lumen. Continual expansion of the apical
toplasm. Even stranger spermatozoa have been de- cytoplasm causes the Sertoli cell recesses containing
scribed for R . arboreus and R. schlegelii (Mizuhira et the spermatozoa to unfold and subsequently flush the
al., 1986). These species produce spiral spermatozoa in gametes into the lumen of the seminiferous lobule. Fi-
the shape of a counter clockwise corkscrew. The poste- nally, the swollen apical region of the Sertoli cell is
rior region of the nucleus is surrounded by mitochon- sloughed into the lumen. Further loss of Sertoli cyto-
dria and the flagellum has two axonemes enclosed by a plasm is prevented by apposition of residual, overlap-
mass of microtubules. ping processes which preserve the integrity of the re-
Normally, nuclear condensation and elongation oc- maining nucleated portion of the Seroli cell attached to
curs concurrently with cytoplasmic changes during the basement membrane.
spermiogenesis (Phillips, 1974). This does not, how-
ever, appear to occur in M. montana (Asa and Phillips, REPTILIA
1988). In this species, development of the flagellum With the reptiles, the amniote organization of the
and shedding of residual cytoplasm is almost complete seminiferous compartment is attained with a perma-
nent germinal epithelium contained in tubules. The distal centriole and anteriorly in a ring of granular
reptilia consists of the Squamata (snakes, lizards), material around the base of the acrosome. These mi-
Chelonia (tortoises, turtles) and Crocodilia (crocodiles, crotubules were apparently linked to each other by
alligators). For temperate zone reptiles reproduction is short arms. The manchette disappears at a late stage of
cyclical. The breeding season starts in spring/early spermiogenesis. A distinctive tubular body of as yet
summer followed by a period of testicular regression unknown origin and formation has been described in
with the germinal epithelium reduced to a layer of Ser- spermatids of several reptilian species (Fig. 26B). This
toli cells and spermatogonia. Spermatogenic recrudes- structure has also been called a membranous body
cence is initiated in the late summedfall. For some (Clark, 1967).
species spermatogenesis is completed before winter During spermiogenesis of turtles, the mitochondria
with spermatozoa, for the next breeding season, stored become highly modified with the disappearance of
in the epididymides. This is the typical postnuptial pat- lamelliform cristae which are replaced by a series of
tern of spermatogenesis. In other species, spermato- concentric membranes within the organelles (Fig.
genesis undergoes arrest in the winter (or proceeds at a 26B). This transformation of mitochondria produce
very slow rate) to resume again in the spring, and is structures that have been termed mitochondria1 lamel-
completed just before the breeding season. This pattern lar bodies (Yasuzumi and Yasuda, 1968). In C . picta,
of spermatogenesis, therefore, has been referred to as spermatids possess numerous lipid droplets and a well
prenuptial (see Licht, 1984). Spermatogenesis has been developed system of SER (see Fig. 26B). As spermatids
described a t the light microscope level for several rep- mature the acrosome develops to spread down over the
tilian species, e.g., turtles Terrapene carolina (Altland, nucleus; as this occurs it is accompanied by a thin cy-
1951)Sternotherus odoratus (Risley, 1938); lizards An- lindrical projection of Sertoli cytoplasm. This Sertoli
olis carolinensis (Fox, 1958); Cnemidophorus tigris cytoplasm which envelops the acrosome is in turn sur-
(Goldberg and Lowe, 1966); snakes Naja naja (Lofts et rounded by spermatid cytoplasm (see Fig. 27) and has
al., 1966); Thamnophis elegens terrestris (Fox, 1952). been variously referred to as the spermatid mantle,
Electron microscopic analysis has been carried out hood, or accessory cap (Butler and Gabri, 1984; Del
on several aspects of spermatogeneis in a variety of Conte, 1976; Sprando and Russell, 1988b).
reptilian species, e.g., for lizards Liolaemus weigmani In some species, several intranuclear tubules have
and Amphisbaena darwinii (Sotelo and “rujillo-Cenoz, been described in developing spermatids, e.g., C .japon-
1958);Agama agama (Charnier et al., 1967); Tropidu- ica (Yasuzumi and Yasuda, 1968; Fig. 2 6 0 . Specialized
rus torquatus (da Cruz-Hofling and da Cruz-Landim, cellular junctions have been described between germ
1978;da Cruz Landim and da Cruz-Hofling, 1977);An- cells and reptilian Sertoli cells. Desmosome-like junc-
olis carolinensis (Clark, 1967); Cnemidophorus lemnis- tions occur between spermatocytes and Sertoli cells in
catus (Del Conte, 1976)Lacerta uiuipara (Courtens and P. scripta (Sprando and Russell, 1987b). Ectoplasmic
Depeiges, 1985)Podarcis (-Lacerta) taurica (Butler and specializations are produced by Sertoli cells associated
Gabri, 1984). Turtles, e.g., Clemmys japonica (Yasu- with elongate spermatids for several reptilian species
zumi and Yasuda, 1968); Chelydra serpentina (Mah- (Baccetti et al., 1980; Sprando and Russell, 1987a).
moud et al., 1985) Chrysemys picta (Dubois et al., 1988; These structures are characterized by a prominent
Hess et al., 1991); Pseudemys scripta (Kaplan et al., layer of filaments plus deeper elements of ER. These
1966;Sprando and Russell, 1988b);snakes, e.g., Python ectoplasmic specializations project for some distance
sebae (Boisson and Mattei, 1965b); Lampropeltis getu- beyond the head of the spermatid in C. picta (Fig.
lus (Austin, 1965; Hamilton and Fawcett, 1968); Con- 26D).
strictus constrictus (Hamilton and Fawcett, 1968). A perforatorium has been reported to develop during
In general, the main features of spermatogenesis are spermiogenesis of several reptilian species (Fig. 26D;
similar for most reptilian species. At the beginning of Boisson and Mattei, 196513; Butler and Gabri, 1984;
the breeding season the seminiferous tubules contain Clark, 1967; Courtens and Depeiges, 1985; da Cruz-
spermatogonia which are large cells characterized by Landim and da Cruz-Hofling, 1977; Hess et al., 1991;
irregularly shaped nuclei. The cytoplasm contains a Yasuzumi, 1974; Yasuzumi and Yasuda, 1968). The
large number of organelles including mitochondria, perforatorium in reptiles has been shown to contain
SER and vacuoles as well as a number of lipid droplets actin (Baccetti, 1979). Changes in nuclear chromatin
(Fig. 25). Further maturation and development of the have been reported during reptilian spermiogenesis
germ cell is, in general, morphologically unremarkable (Butler and Gabri, 1984; Clark, 1967; da Cruz-Hofling
for most reptilian species. Several salient points of and da Cruz-Landim, 1978). Nuclei of round sperma-
spermiogenesis are the formation of a manchette, the tids contain dispersed chromatin. As the nucleus elon-
appearance of a tubular body, unusual morphology of gates this chromatin becomes organized into a system
the mitochondria, and formation of the mantle. Ini- of fibers that course parallel to the long axis of the
tially the manchette surrounds the elongating nucleus nucleus. For L . uiuipara, however, it has been sug-
as a loose helix of microtubules. As the nucleus contin- gested the chromatin fibers initially follow a helical
ues to elongate and the chromatin condenses, however, pattern before assuming a longitudinal orientation
the helix of microtubles unwinds to become orientated with respect to the elongating nucleus (Courtens and
parallel to the long axis of the nucleus (Fig. 26A). In A. Depeiges, 1985).With further condensation of the chro-
carolinensis (Clark, 1967) it has been reported that mi- matin, the fibers become thicker and tightly packed to
crotubules of the manchette (caudal sheath) insert cau- produce the fully condensed nuclei of mature sperma-
dally in a ring of granular material associated with the tozoa. It is assumed that chromatin condensation re-
488 J. PUDNEY
Fig. 25. Primary spermatogonia (PG) present in germinal epithelium of C . pictu at beginning of
testicular recrudescence.
sults from dehydration of nucleoprotein and nucleo- to be associated with each pouch. It was proposed these
plasm elimination (Yasuzumi, 1974). pouches transport perinuclear material from the ac-
A study of cell volume changes and cytoplasmic elim- rosomal region to deposit it in the region close to the
ination as spermatids mature has been carried out for centrioles. Structures termed lamellar plates composed
P. scripta (Sprando and Russell, 1988b). The elimina- of two sheets of double membranes containing dense
tion of excess cytoplasm during spermiogenesis is ac- material were also formed. These lamellar plates ap-
complished by several mechanisms. There is a n inva- pear at the anterior region of the nucleus and eventu-
sion of spermatids by Sertoli processes which sequester ally fuse with the nuclear membrane. Both the nuclear
small portions of cytoplasm that are then removed from pouches and lamellar plates disappear towards later
the germ cell. Budding of small mitochondrial-rich cy- stages of spermiogenesis. As spermatids mature follow-
toplasmic fragments also occurs from the midpiece re- ing regression of the manchette, numerous lipid drop-
gion of elongating spermatids. Finally, residual bodies lets have been described surrounding the nucleus of
of varying sizes also function to eliminate excess cyto- some species (Hamilton and Fawcett, 1968). In L. vzui-
plasm. para these have been called tergosomes which have
Some unusual morphological findings have been ob- been reported to fuse with the nuclear membrane to
served during spermiogenesis of L. vivipara (Courtens discharge their contents into the intramembranous
and Depeiges, 1985). Nuclear pouches develop which space (Courtens and Depeiges, 1985). For several spe-
are formed by invaginations of the nuclear envelope. cies of snakes, however, it was reported these lipid
These pouches initially appear close to the developing droplets are discarded with the residual body (Hamil-
acrosome and proceed to migrate down towards the pos- ton and Fawcett, 1968).
terior pole of the nucleus. This movement could be ac- Although reptiles are amniotes, several structural
complished by a system of microtubules that were seen features distinguish their spermatozoa from that of
Fig. 26. Spermiogenesis in C. pictu. A Manchette (arrowheads) matids contain intranuclear tubules (arrowhead). D Elongate sper-
composed of microtubules orientated parallel to long axis of spermatid matid illustrating presence of the perforatorium (arrowhead) and ec-
nucleus. B: Spermatids contain a distinctive tubular body (arrow- toplasmic specialization produced by the Sertoli cell that surrounds
heads), many lipid droplets (small arrows) and highly modified mito- acrosomal region.
chondria with concentric cristae (large arrows). C: Developing sper-
490 J. PUDNEY
tures of spermatozoa have been reported for several

reptilian species. Thus, for C . picta spermatozoa have a
perforatorium consisting of 2-3 rods which are contig-
uous with nuclear canals, a connecting collar of dense
material that surrounds the base of the nucleus and a
distal centriole that contains central microtubules ex-
tending the entire length of this structure. In L. getulus
and C . constrictus a distinctive nonmitochondrial com-
ponent of the midpiece has been described (Hamilton
and Fawcett, 1968) This region consists of conspicuous
plaques of dense material, interspersed between the
mitochondria, spaced at irregular intervals along the
length of the midpiece. These plaques were not sur-
rounded by a membrane and were thought to be de-
rived from granules that did not participate in the for-
mation of the neck cylinder.
AVES
Birds are amniotes and the organization of the testis
is essentially similar to that of other amniotes. Those
avian species inhabiting temperate climates exhibit
well-defined seasonal cycles of testicular activity. Dur-
ing testicular regression the seminiferous tubules are
composed only of Sertoli cells and spermatogonia. Since
birds are homeotherms they carry out prenuptial sper-
matogenesis. Due to the high metabolic rate of birds,
however, full testicular recrudescence can be accom-
plished in a matter of weeks.
Although nearly 9,000 extant avian species exist,
spermatogenesis has been studied in only a few. Fur-
thermore, these studies have been essentially re-
stricted to domesticated birds, e.g., fowl, quail, or
commercially available song birds, e.g., canary, bud-
gerigar. At the light microscopic level, several reports
have described spermatogenesis in various avian spe-
Fig. 27. Development of mantle of Sertoli cytoplasm (arrowhead) cies, e.g., Cygnus olor (Breuker, 1982); Gallus domes-
that envelopes acrosomal region of spermatid. G . domesticus. (Micro-
graph courtesy of R.L. Sprando and L.D. Russell.) ticus (DeReviers, 1971; Zlotnik, 1947); Branta cana-
densis interior (Mori and George, 1978).
Although a number of electron microscope studies
have been carried out on avian spermatogenesis they
mammalian species. Reptilian spermatozoa (1)retain have been primarily restricted to the stage of spermi-
both centrioles with the proximal centriole orientated ogenesis with few investigations on the complete
at roughly 90" to the distal centriole; (2) develop an breeding cycle, e.g., Passer domesticus (Sotelo and Tru-
additional structure, the neck cylinder, which encom- jillo-Cenoz, 1958); P . montanus saturatus (Yasuzumi,
passes the centriolar region of the flagellum, and for 1956);Gallus domesticus (Cooksey and Rothwell, 1973;
snakes display an unusual concentric arrangement of Gunawardana and Scott, 1977; Maretta, 1977; McIn-
the mitochondrial and fibrous sheaths, and further- tosh and Porter, 1967; Nagano, 1962; Nicander, 1967;
more do not possess a striated connecting piece. The Sprando and Russell, 1988b; Tingari, 1973; Xia et al.,
neck cylinder is a dense structure which is attached to 1986); Streptopelia roseogrisea (Mattei et al., 1972a);
the posterior pole of the nucleus and surrounds the Melopsittacus undulatus (Humphreys, 1975a,b);Gallus
connecting piece and centrioles. The mitochondrial gallus (Okamura and Nishiyama, 1976); Cairina mos-
sheath consists of a number of seperate mitochondria chata (Marchand, 1977); Cygnus olor (Breuker, 1982);
that spiral around the flagellum. For snakes the mito- Lonchura striatu var domestica (Kondo et al., 1988);
chondrial sheath is extensive and extends a consider- Rhea umricans (Phillips and Asa, 1989);and Coturnix
able distance along the length of the flagellum. A sys- coturnixjaponica (Lin and Jones, 1992).In general, the
tem of 9 dense fibers is also present which apparently process of spermatogenesis in birds is very similar to
develops from the proximal centriole and courses down that previously described for the reptiles.
to surround the axoneme. The outer fibrous sheath cov- An excellent study has been reported describing the
ers most of the flagellum. For some reptilian sperma- fine structure of spermatogenesis throughout the
tozoa (particularly turtles) the mitochondria extend breeding season in C . olor (Breuker, 1982). At the be-
from the region containing the proximal centriole to ginning of the breeding season spermatogonia are the
the annulus, thus making the identification of a dis- primary germ cells present in the seminiferous epithe-
crete neck region difficult. Unusual morphological fea- lium. Nuclei of the spermatogonia are round or slightly
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 49 1
irregular in shape with a prominent nucleolus. The surround the distal centriole and proximal portion of
cytoplasm contains abundant ribosomes, many mito- the axoneme in the form of a helix (Bakst and
chondria, a well-developed Golgi apparatus, two cen- Howarth, 1975).
trioles orientated at right angles to each other, and an The onset of testicular regression occurs when the
unusual feature comprised of a single long microtubule last generation of spermatids is released with no fur-
associated with a bundle of fine filaments. The primary ther maturation of the remaining germ cells. This re-
spermatocytes possess many mitochondria closely results in a loss of the synchronized stages of spermato-
lated with long cisternae of SER and a chromatoid genesis, degeneration of residual germ cells and
body. A species difference has been described for M. obliteration of the lumen of the seminiferous tubule.
undulatus in which numerous oval mitochondria occur Eventually the germinal epithelium is reduced to a
located at the periphery of spermatocytes (Humphreys, layer of spermatogonia and Sertoli cells. An interesting
1975a). In C . olor, secondary spermatocytes are char- feature of testicular involution was a massive invasion
acterized by the presence of annulate lamellae. With of seminiferous tubules by macrophages which appar-
the onset of spermiogenesis the long cisternae of SER ently assisted the Sertoli cells in the removal of cellu-
present in spermatocytes break down into vesicles in lar debris. These macrophages were prevalent both in
spermatids, and the distal centriole initiates the devel- the lumen as well as the germinal epithelium. During
opment of a flagellum a t the periphery of the cell. The spermiogenesis of several avian species a mantle or
annulus develops around the flagellum where it ex- hood of Sertoli cytoplasm associated with the acroso-
tends from the cell membrane. The proximal centriole ma1 region of elongating spermatids, analagous to that
is orientated roughly 90" with respect to the distal cen- previously described during spermiogenesis of reptiles,
triole. A Golgi apparatus is located in the vicinity of has been described (Fig. 27), e.g., G. domesticus, (Cook-
the centriole and produces an acrosomal vesicle that sey and Rothwell, 1973; Sprando and Russell, 1988b);
apparently lacks an acrosomal granule. Then both cen- 5'. roseogrisea (Mattei et al., 1972a); Lonchura striata
trioles and acrosomal vesicle migrate close to the nu- (Kondo et al., 1988).
cleus. In the flagellar region, five layers of annulate As in P. scripta, the spermatid cytoplasm is pene-
lamellae develop which often appear attached to the trated by Sertoli cell processes in G . domesticus (Sp-
nucleus. Subsequently, the acrosome and centrioles rando and Russell, 1988b). Although these processes
move to their respective anterior and posterior posi- also sequester portions of spermatid cytoplasm, how-
tions on the nucleus. ever, unlike in reptiles there was no evidence to indicate
For S. roseogrisea, however, this alignment of acroso- these packets of spermatid cytoplasm were removed by
ma1 vesicle and flagellum has been reported to occur by the Sertoli cell. Instead, it appeared that excess cyto-
rotation of the nucleus (Mattei et al., 1972a). As the plasm was primarily eliminated by means of a lobe,
spermatid nucleus undergoes elongation, a rod (perfo- developed by spermatid cytoplasm forming the mantle,
ratorium or acrosomal spine) develops beneath the ac- that was pinched off at spermiation to produce a resid-
rosomal vesicle. With further nuclear elongation this ual body (Sprando and Russell, 198813).
rod lengthens to penetrate the nucleus in an endonu- Several reports on avian spermatogenesis have re-
clear canal or for other species in an apical nuclear marked that germ cell stages do not appear to be as
pocket. The perforatorium is not bound by a mem- closely synchronized as in mammals, e.g., Breuker
brane. Also in the cytoplasm a tubular body appears (1982). This is apparently due to the fact that in birds
that is similar to that produced by reptilian sperma- fewer spermatogonial mitotic divisions are synchro-
tids. nized with the spermatogenic cycle (Lin and Jones,
With further maturation of the spermatid, the annu- 1992). This explains therefore, why only small portions
late lamellae separate from the nucleus to lie free in of the avian germinal epithelium are occupied by dis-
the cytoplasm, and can often be found in contact with tinct germ cell stages and is reminiscent of the primate
the tubular body. Also, annulate lamellae occasionally mosaic pattern of spermatogenesis.
appear to roll up and so form rings which become de- An excellent study has been carried out on the evo-
tached from the tubular body. During spermiogenesis, lution of the RER during spermiogenesis of G. domes-
as the spermatid elongates the manchette develops. ticus (Xia et al., 1986). In early round spermatids the
Initially the microtubules are spirally organized RER is organized into several different systems con-
around the nucleus. With further elongation and consisting of either a loose network of tubular cisternae
densation of the nucleus, however, the microtubules scattered throughout the cytoplasm, or long flattened
become orientated parallel to the long axis of the nu- cisternae occurring singly or in layers of 2-3 running
cleus. This raises the question, therefore, as t o whether parallel to the nucleus, or a collection of tubular cis-
two different sets of microtubules occur or only one that ternae opposite thickened plaques of the plasma mem-
becomes reorientated from a spiral to a longitudinal brane, or as a tight network of anastomosing tubular
pattern. At a later stage of spermiogenesis the micro- cisternae (6-8) associated with spherical vesicles
tubules of the manchette disappear. Late in spermio- which was called an alveolar body. All these elements
genesis, a t the level of the annulus, a fibrous sheath is of the RER appeared contiguous with each other. A
formed which surrounds the axoneme along the length stack of annulate lamellae was usually found in close
of the principal piece. The final step in the formation of proximity to the alveolar body. With elongation of the
the midpiece is the development of a mitochondria1 spermatid nucleus the flattened cisternae of RER dis-
sheath which extends from the base of the nucleus to appear but the tubular cisternae now become ex-
the annulus. In G . domesticus, about 30 mitochondria panded. As the spermatid matured further with con-
492 J. PUDNEY
densation of the nuclear chromatin the network of (Cooksey and Rothwell, 1973; Osman et al., 1980; sp-
tubular cisternae disintegrated into vesicles that be- rando and Russell, 1987a). The structural appearance
came highly distended to give the cytoplasm a vacuo- of the avian ectoplasmic specializations differ from
lated appearance. The alveolar body is apparently those produced by mammalian Sertoli cells. Filaments
maintained just prior to spermiation when it undergoes (6 nm) do not form an organized linear array but ap-
dissolution. All RER remnants are subsequently shed pear as a tangled mass and were often associated with
with the residual body. Although spermatid RER in microtubules. Furthermore, these studies also reported
this avian species undergoes distinct structural change the absence of subsurface cisternae of ER which form
during spermiogenesis the exact physiological rele- part of the mammalian ectoplasmic specializations. Ec-
vance of these interesting transformations is as yet un- toplasmic specializations between elongate spermatids
known. and Sertoli cells were reported not to be present in the
A great deal of interest has centered on the develop- testis of C . olor (Breuker, 1982).
ment of the manchette during avian spermiogenesis, There are several distinctive features of spermato-
and a detailed study of this structure has been carried genesis that distinguish passerines from nonpasserine
out by McIntosh and Porter (1967) in G. domesticus. species. As noted previously, passerines develop only
Initially the highly ordered array of microtubules one set of spiral microtubules during spermiogenesis,
which surround the elongating nucleus was found t o be whereas nonpasserines produce two sets, one spiral fol-
a left-handed double helix with cross-bridges connect- lowed by a longitudinal array. Furthermore, in contrast
ing each helix. With further maturation the helical set to nonpasserines, passerine spermatozoa do not possess
of microtubules is replaced by a system of straight mi- a perforatorium. Also during spermiogenesis, nonpas-
crotubules running parallel to the long axis of the nu- serine spermatids are arranged as individual cells em-
cleus. It is uncertain from the literature whether two bedded in Sertoli cytoplasm similar to other amniotes,
different transitory sets of microtubules appear during whereas in passerines the spermatids are collected into
spermiogenesis, e.g., McIntosh and Porter (1967) and bundles reminiscent of spermatogenesis in the anam-
Nicander (1967), or whether only one system of micro- niotes (Denehy, 1976; Humphreys, 1972).
tubules occurs which simply changes orientation, e.g., Several studies have reported on the fine structure of
Okamura and Nishiyama (1976); Breuker (1982); the avian spermatozoon, e.g., G. domesticus (Bakst and
Mattei et al, (1972a). This development of two sets of Howarth, 1975; Thurston and Hess, 1987; Tingari,
differently orientated microtubules is characteristic 1973);Eudromia elegens elegens (Asa et al., 1986);Grus
of nonpasserine spermiogenesis, e.g., G. domesticus vipio (Phillips et al., 1987);Numida meleagrir and Me-
(McIntosh and Porter, 1967; Nicander, 1967); C . olor leagris gallopavo (Thurston and Hew, 1987); Rhea
(Breuker, 1982).R. americans (Phillips and Asa, 1989). americans (Phillips and Asa, 1989).In general, nonpas-
By contrast, in passerine species only one set of micro- serines produce what has been termed sauropsid sper-
tubules appears which forms a twisted bundle that matozoa due to their resembling reptilian spermatozoa.
winds around the short nucleus and proximal portion of These sauropsid spermatozoa are characterized by a
the flagellum during formation of the corkscrew- small acrosome, a perforatorium, a well-defined mid-
shaped spermatozoa that is characteristic of these birds piece containing two centrioles, and they move by a
(Kondo et al., 1988; Nicander, 1967). lashing motion of the flagellum; by contrast passerines
At a later stage of spermiogenesis, the manchette produce spiral spermatozoa that do not have a perfora-
disappears. There is some controversy as to the func- torium and progress by helical forward movement (Bac-
tion of the manchette. Several studies have produced cetti et al., 1980; Humphreys, 1972; Yasuzumi, 1974).
evidence to suggest microtubules act as a scaffold to Species differences obviously occur in the morphology of
mold the shape of the nucleus as it elongates in the avian spermatozoa. Thus, Serinus canaria and P. do-
developing spermatid, e.g. Kondo et al. (1988); McIn- mesticus produce spermatozoa which possess an undu-
tosh and Porter (1967). Other reports indicate that the lating membrane that runs from the tip of the acrosome
manchette is not involved in nuclear shaping that is to the end of the flagellum (Humphreys, 1972; Nagano,
controlled by intranuclear mechanisms such as conden- 1962). In G. vipio it has been reported that chromatin
sation of the chromatin (Fawcett et al., 1971).It may be condensation is not complete in mature spermatozoa
that species vary in the involvement of the manchette which was assumed to be responsible for the large vari-
microtubules in the molding of the nucleus. Certainly ation in the shape of the sperm head in this species
in passerine species there appears good evidence that (Phillips et al., 1987).Spermatozoa of the primitive bird
the helical microtubules play a role in the formation of E. elegens elegens develop a sheath of cytoplasm con-
the spiral nucleus characteristic of these birds. taining glycogen that surrounds the flagellum and a
During spermatogenesis a variety of Sertoli cell/ long axial canal that extends through the length of the
germ cell junctions has been described in the avian nucleus (Asa et al., 1986).
testis. Contact between germ cells (spermatocytes
and/or spermatids) and Sertoli cells via desmosomes, CONCLUSION
desmosome-like or presumed gap junctions has been As can be appreciated from this review, a large body
commonly detected (Breuker, 1982; Cooksey and Roth- of literature exists describing spermatogenesis in non-
well, 1973; Osman, et al., 1980; Scheib, 1972; Sprando mammalian vertebrates. This is, however, limited com-
and Russell, 198713). Ectoplasmic specializations are pared to the mass of information that is available on
developed by Sertoli cytoplasm associated with the ac- spermatogenesis in mammals. Furthermore, several
rosomal region of elongate spermatids in G. domesticus caveats hold concerning the quality and quantity of
SPERMATOGENESIS I N NONMAMMALIAN VERTEBRATES 493
reports which can be obtained on mammalian versus amphibia, at least, the advantages of in vitro experi-
nonmammalian spermatogenesis. mental techniques can be used to further understand
First, relatively few comprehensive studies have processes involved in maturation of germ cells. The
been carried out describing spermatogenesis through- ability to culture isolated amphibian germ cells, and
out the reproductive cycle for nonmammalian species. possibly germ cells from other anamniote species, is
Moreover, in many cases cytological analysis of germ probably related to the fact that during spermatogen-
cell development and maturation has been limited to esis in vivo, the early germ cells mature in association
only a few representative species of a particular non- with a morphologically poorly differentiated Sertoli
mammalian class or order. Due to the extreme diver- cell. Since usually structure reflects function it is pos-
sity in reproductive strategies which can be displayed sible that the Sertoli cells, therefore, are not highly
by nonmammalian species, it is often difficult, there- active and so play a minor role in maintaining and
fore, to generalize on the cytology of spermatogenesis regulating germ cell development. This would suggest
in many classes or even orders of these vertebrates. that in many species of anamniotes the early stages of
Second, this problem is compounded by the fact that spermatogenesis progress mostly independent of the
spermatogenesis in few nonmammalian vertebrates Sertoli cell.
has been analyzed using modern microscopic tech- In conclusion, as previously discussed (Pudney et al.,
niques such as scanning electron microscopy, freeze- 19851, to approach and elucidate basic problems in re-
fracture analysis, immunocytochemistry, morphome- productive biology more attention should be paid to
try. This has resulted, unfortunately, in placing choosing the correct and appropriate animal model. A
reliance on older reports for cytological descriptions of wide range of species displaying different reproductive
spermatogenesis in many nonmammalian vertebrates. strategies is available to investigators of male repro-
These studies were often carried out using, by contem- ductive biology. No one species per se is capable of pro-
porary standards, suboptimal conditions for preserva- viding all the answers. By judicious selection, however,
tion of tissue and microscopic analysis. This is espe- a particular species can be chosen because, due to some
cially true for many seminal papers on the fine novel morphological parameter, it is more suited for
structure of germ cell maturation of various nonmam- studying one particular aspect of male reproduction.
malian species. This illustrates and defines the importance of studying
Due to the great variety in the organization of the so-called unconventional animal models to understand
testis and pattern of spermatogenesis that can be found more completely phenomena associated with male re-
in nonmammalian vertebrates, certain species provide production.
excellent models for analyzing mechanisms that regu-
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MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995)

Spermatogenesis in Nonmammalian Vertebrates

KEY WORDS Anamniotes, Spermatocysts, Amniotes

ABSTRACT Spermatogenesis appears to be a fairly conserved process throughout the verte-

INTRODUCTION spermatogenesis depend primarily on several parame-

0 1995 WILEY-LISS, INC.

cold-blooded (poikilotherm) or warm-blooded (homeo-

THE ORGANIZATION OF THE TESTIS AND

EXTERNAL VERSUS trioles orientated roughly a t 90" to each other, one of

the nucleus and into the flagellum. This endonuclear

Fig. 16. Schematic diagrams illustrating spermatogenesis in the

with transfer of spermatophores to the efferent ducts.

ier and Spink, 1971); R. pipiens (Poirier and Spink,

tures of spermatozoa have been reported for several

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