REPRODUCTION

RESEARCH

Stage and season effects on cell cycle and apoptotic activities

of germ cells and Sertoli cells during spermatogenesis in the
spiny dogfish (Squalus acanthias)
L M McClusky
Department of Physiology, Faculty of Health Sciences, University of Pretoria, P.O. Box 2034, Pretoria 0001,
South Africa
Correspondence should be addressed to L M McClusky; Email: [email protected]

Abstract
To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used
standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-
meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression
varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at
the mitosis– meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD
across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12– 13 require 9 –10 months to reach the
mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4– 5-fold less than the num-
ber that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident
with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed.
Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except
during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related.
PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited
to spermatogonia and increased stage-dependently in the PrM region (L-PrM 5 mid-stage PrM @ E-PrM @ GZ), correlating
with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are
stage- and process-specific.
Reproduction (2005) 129 89–102

Introduction indicate that developing Sertoli cells influence the cell-

population dynamics of gonocytes, and undifferentiated
Normal testicular development requires the spatiotem-
and differentiated spermatogonia, and their further matu-
poral coordination of spermatogenic processes and devel-
ration, depending on the age of the Sertoli cell (Boitani
opment to ensure optimal production of spermatozoa. The
et al. 1993, 1995, Zhou et al. 1993, De Rooij & Van
founders of the germ-cell lineage, the spermatogonial Dissel-Emiliani 1997, Schlatt et al. 1999). Our knowledge
stem cells, give rise to progeny that undergo a species- of the inter-relationships between the different stages of
specific number of differentiating divisions (mitosis) and spermatogonia and the various stages of Sertoli cell devel-
apoptosis before they enter meiosis (De Rooij & Van Dis- opment in vivo is incomplete. Although it is known that
sel-Emiliani 1997, De Rooij 2001). Studies primarily in germ cells are organized into clones and cohorts, it is not
adult rodents indicate that, of the numerous regulatory readily apparent whether Sertoli cells themselves are orga-
mechanisms present within the testis, density-dependent nized into clones, cohorts or groups to coordinate their
(De Rooij 2001, Tadokoro et al. 2002) and hormone-con- inhibitory and stimulatory activities during their regulation
trolled (Meachem et al. 1999, Allan et al. 2004) mechan- of spermatogonial numbers in the germinal epithelium.
isms are two of the major physiological regulators that In contrast to that of the adult common laboratory
control these processes in an age-dependent and sperma- rodent, spermatogenesis in the adult spiny dogfish shark,
togonial-stage-dependent manner. In addition, data Squalus acanthias, is different in that spermatogonial
obtained from organ cultures of immature rodent testes proliferation (13 divisions in total) is accompanied by

q 2005 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00177

ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
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via free access
90 L M McClusky

Sertoli-cell proliferation in a 1:1 ratio for the first nine div- Materials and Methods
isions (Holstein 1969). S. acanthias is furthermore an ideal
model in which to study spermatogenic processes stage by Animals
stage because its testicular organization is much simpler Mature male spiny dogfish, S. acanthias, were collected
than that of the conventional laboratory rodent. In Squa- (n ¼ 3 –8/month) at various points during their annual
lus, the spermatogenic wave passes across the entire run along the United States Atlantic coast through the
diameter of the testis (from the germinal ridge, containing facilities of the following institutions: January –March,
the stem cells, to the mature pole), providing a readily vis- North Carolina Division of Marine Fisheries (Cape Hat-
ible zonation in testicular cross-sections of spermatocysts teras, NC, USA; 52 –61 cm snout– vent length); April –
mainly in the premeiotic (PrM; spermatogonia and prelep- May, Massachusetts Division of Marine Fisheries (Cape
totene spermatocytes), meiotic (M; primary and secondary Cod Bay, MA, USA; 46 –57 cm) and the Marine Biologi-
spermatocytes) or postmeiotic (PoM; round, elongating cal Laboratory (MBL; Woods Hole, MA, USA; 43 –
and mature spermatids) stages of development. Moreover, 55 cm); June–September, Mount Desert Island Biological
spermatogenesis in this and other shark species has a ‘cys- Laboratory (MDIBL; Salsbury Cove, ME, USA; 36 –
tic’ mode, in which a single germinal clone and a second 49 cm); October–December, MBL (39 –58 cm). Animals
clonal population of stage-synchronized Sertoli cells form were killed by double-pithing via the olfactory canal or
an anatomically discrete follicle-like unit (spermatocyst). transection of the cervical spinal cord (Cape Hatteras
In addition, male germ cells in Squalus depend primarily samples only).
on Sertoli cells for somatic cell support, since differen-
tiated Leydig cells have not yet evolved at this phyletic
level (Pudney & Callard 1984). Tissue preparation
Follow-up biochemical (Callard et al. 1995) and in situ
Cross-sections (3–5 mm) were taken midway along the
fluorescence analyses (McClusky et al. 1996) to an initial
length of one testis and fixed in 10% (v/v) neutral buffered
report of Simpson & Wardle (1967), which described a
formalin (formaldehyde diluted 1:10 in elasmobranch
zone comprising several layers of degenerating germinal
balanced salt solution, EBSS) at 48 C for 24 h. As reported
tissue that first appears naturally in May in S. acanthias at
previously (Callard et al. 1985), each cross-section
the spermatogonia– spermatocyte transition, confirmed the
includes a full spermatogenic sequence, and cross-sec-
mode of death in this tissue as apoptosis. Since it has
tions are consistent throughout the length of the testis
always been assumed that the month of May also marked
except at the anterior and posterior poles. Fixed tissues
the onset of the spermatogenically active period in Squa-
were washed twice in EBSS (1 h each), stored in cold 70%
lus (Callard et al. 1995, Piferrer & Callard 1995), the
ethanol for no longer than 3 –5 weeks, and embedded in
observation of such a large stage-specific loss of testicular
Paraplast X-TRA (Oxford Labware, St Louis, MO, USA).
cells during presumed normal testicular recrudescence
Two consecutive 6 mm sections (one each for PCNA and
was indeed intriguing.
TUNEL) were floated on to a distilled water bath (478 C),
Proliferating cell nuclear antigen (PCNA) is used widely
collected on slides pre-coated with poly-L -lysine (1 mg/ml,
to provide an immunohistochemical assessment of cell-
Mw 33 000; Sigma) and allowed to air dry. Sections were
cycle activity in normal tissues (Hall & Woods 1990), and
then deparaffinized and rehydrated stepwise through an
its expression is used as a marker of cell proliferation in
ethanol series.
cultured granulosa cells (the female equivalent of Sertoli
cells) following their stimulation with follicle-stimulating
hormone (FSH) and activin (El-Hefnawy & Zeleznik
PCNA immunohistochemistry
2001). PCNA, which is synthesized in early G1 phase of
the cell cycle, is an auxillary protein to d and 1 DNA Immunostaining was carried out using the Unitect Avi-
polymerases during the S phase in cycling cells, and its din-Biotin-Peroxidase Immunohistochemistry Detection
immunoreactivity has a linear relationship with thymidine System Kit (Oncogene Science, Cambridge, MA, USA),
incorporation in a wide range of normal tissues (Hall & which contains the recombinant PCNA mouse mono-
Woods 1990). The terminal deoxynucleotidyl transferase clonal PC10 antibody shown to interact with fish PCNA
(TdT)-mediated dUTP nickend-labelling (TUNEL) assay is antigen (Ortego et al. 1994). The procedure was a modi-
now almost considered a classic apoptosis assay, in which fication of that suggested by the supplier. Endogenous
the free 30 -OH ends of double-stranded DNA fragments peroxidase was quenched by treating sections in dark-
that are formed preferentially in apoptotic cells are ness for 10 min with 3% (v/v) hydrogen peroxide. Fol-
labelled (Gavrieli et al. 1992). Using standard morphome- lowing a rinse in distilled water, the sections were
try, and immunohistochemistry of PCNA and TUNEL, the subjected to an antigen-retrieval procedure (Ortego et al.
aims of this study were (1) to document the seasonal kin- 1994), which involved heating in a microwave oven
etics of spermatogenesis and (2) to elucidate the role of (450 W) in 1% (v/v) anhydrous zinc sulphate for 3 min,
cell division versus cell death in germ cells and Sertoli resting for 1 min and heating a further 3 min. After cool-
cells at each developmental stage and time of year. ing for 15 min, sections were rinsed in distilled water

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 Cell cycle and apoptosis in the shark testis 91

and placed in 0.5% (v/v) Tween 20 in PBS (137 mM Microscopy

NaCl, 29 mM NaH2PO4·H2O and 9 mM Na2HPO4, pH
Quantitative measurements were carried out using an
7.4; referred to here as PBS-A) for 5 min. Sections were
Olympus BH2 microscope with a graticule-containing eye-
then incubated with 3.6% (v/v) normal horse serum for
piece and an attached Hamamatsu C2400 CCD camera
30 min at room temperature (RT) to reduce non-specific
serially connected to a Sony monochrome video monitor
binding, and then overnight at 48 C with PCNA antibody
and a Power Macintosh computer equipped with an AG-5
diluted 1:200 (final concentration, 1 mg/ml) in Tween
frame-grabber card (Scion Corporation, Frederick, MD,
20/PBS-A. After three washes in PBS-A, sections were
USA) and NIH Image software (version 1.62). All cysts
incubated with biotinylated horse secondary antibody for
along three lines radiating from the point of origin of cyst
30 min at RT, washed in PBS-A, and then incubated with
development (the germinal ridge) to the proximal border of
the avidin–biotin complex for a further 30 min at RT.
the peripherally located epigonal organ at the mature
Following a 30-s rinse in 1% (v/v) Triton X-100/PBS-A to
testicular pole were counted, classified by stage and
reduce background staining, the antigen was finally
scored for PCNA or TUNEL immunoreactivity (total per
detected by treating the sections, according to the sup-
animal, 240–300 cysts). Since the developmental pro-
plier’s instructions, for 3 min with a diaminobenzidine
gression is accompanied by changes in cyst diameter (see
substrate kit (Vector Laboratories, Burlingame, CA, USA)
Table 1 below), cyst counts were adjusted for the width of
that stained positive cells brown. Sections were counter-
a testicular cross-section at the midpoint of the segment
stained with 0.5% (w/v) Methyl Green in 100 mM
occupied by each stage as follows:
sodium acetate (pH 4), dehydrated in 100% (v/v) buta-
nol, cleared in toluene and mounted with Permount N cyst ; xa ¼ tw=dcyst ·lcount
(Fisher, Fair Lawn, NJ, USA). Negative controls were
generated by substituting the primary antibody with the where Ncyst, xa is the number of cysts/cross-sectional area
kit-supplied trp E antibody (0.5 mg/ml) and by serial per stage, tw is testis width at the midpoint of each cyst
dilution of the primary antibody. PCNA immunoreactivity stage (mm), dcyst is the mean diameter of each cyst stage
was scored as (a) intense brown nuclear staining, (b) (mm) and lcount is the mean number of cysts counted on
weak brown nuclear staining and (c) no staining. Each three radiating lines at each stage.
cyst was scored separately for PCNA-labelled germ cells
or Sertoli cells, and PCNA staining of more than Statistics
approximately 50% of nuclei of a given cell type was
For each time point, data were expressed as the mean
considered positive (not all cells in a clone are synchro-
total number of cysts in the spermatogenic progression
nized precisely).
and in each substage (per cross-section; see Fig. 1 below),
the fractional distribution of different substages as a per-
TUNEL immunohistochemistry centage of the total number of cysts (see Fig. 2 below) and
the mean percentage of cysts with PCNA-labelled germ
Deparaffinized and rehydrated sections were treated with
cells, PCNA-labelled Sertoli cells and TUNEL-labelled
0.1% (v/v) Triton X-100 in 0.1% (w/v) sodium citrate for
germ cells at each substage (see Figs 5 and 7 below). The
3.5 min at RT, and washed twice with PBS-B (50 mM
raw percentage data were arcsine square-root-transformed
sodium phosphate, pH 7.4, and 200 mM NaCl). Endogen-
prior to testing for differences. Data were examined by
ous peroxidase was quenched by treating sections in dark-
one- and two-way ANOVA (see figure legends) using
ness with 3% (v/v) hydrogen peroxide for 7 min, followed
SigmaStat, version 2.0 (Jandel Scientific, San Rafael, CA,
by two washes in PBS-B. Subsequent steps for TUNEL
USA). The Student –Newman –Keuls multiple-comparisons
staining were carried out using the ApopTag-Peroxidase
test was used to determine which values differed signifi-
Kit according to the supplier’s instructions (Oncor,
cantly (P , 0.05).
Gaithersburg, MD, USA), except that the terminal transfer-
ase enzyme (TdT) was diluted 1:16 with sterile water. Fol-
lowing incubation of the sections with the TUNEL Results
reaction mixture in a humidified chamber at 378 C for 1 h,
Staging criteria
anti-digoxigenin–peroxidase complex was added for
30 min at RT. Sections were then treated with diaminoben- To standardize morphometric analyses, the staging criteria
zidine, counterstained and mounted as described for and the nomenclature used for different substages were
PCNA. To generate negative controls, TdT was replaced adopted after initial light-microscopic and immunohisto-
with sterile water, whereas for positive controls chemical observations of the full seasonal series. As sum-
sections were pretreated with DNase I (Sigma; 10 mg/ml in marized in Table 1, staging criteria were based on (a)
distilled water) to generate DNA fragments. For TUNEL position of the cyst in the testis cross-section relative to
scoring, spermatocysts containing more than three the germinal ridge (maturational order), (b) cyst diameter
TUNEL-positive cells were considered apoptotic, a stan- and (c) cyst organization with respect to relative numbers
dard previously applied to rat tubules (Lee et al. 1997). and positions of germ-cell versus Sertoli-cell nuclei, as

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 Reproduction (2005) 129 89–102

92
L M McClusky
Table 1 Criteria used for classification of spermatocysts during the spermatogenic progression and stage-related patterns of PCNA and TUNEL immunohistochemistry during annual cycle in
S. acanthias.

Germinal Germinal Early (e-) Mid (m-) Late (l-) Early (e-) Mid (m-) Late (l-)
Substage (zone) ridge (GR) zone (GZ) premeiotic premeiotic premeiotic Meiotic postmeiotic postmeiotic postmeiotic

Cyst diameter (mm) 10 –15 15 –60 60 –150 150–300 300–380 380–400 400 –420 340–360 250–290
Stage of germ cell development Gonocytes & 18 s’gonia 28 s’gonia 28 s’gonia 28 s’gonia 18 & 28 s’cytes Round s’tids Elongating s’tids Mature s’tids
18 s’gonia
a
Spermatogonial generation 1 –2 2 –4 5 –9 12 –1310 –11 NA NA NA NA
Lumen No Yes Yes Yes Yes Yes Yes Yes Yes
Location: Sertoli-cell nuclei Random Random Adluminal Adluminal
Migrating Peripheral Peripheral Peripheral Peripheral
peripherally
Number of germ cell layers NA NA 1 –3 4 –8 8 –9 8–9 9– 11 NA NA
a
Germ-cell:Sertoli-cell ratio per cyst 1:1 1:1 1:1 2–8:1 8 –16:1 16 –64:1 64:1 64:1 64:1
a
Number of Sertoli cells per cyst 1 –8 8 –16 32 –512 512 512 512 512 512 512
d
% Cysts with PCNA-positive germ cells Low – high, Low – high, cLow – high, High, year High, year bLow – high, None None None
seasonal seasonal seasonal round round seasonal &
stage-related
d
% Cysts with PCNA-positive Sertoli cells Low – high, Low – high, cLow – high, None None None None None None
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seasonal seasonal seasonal

d
% Cysts with TUNEL-positive germ cells None None Low Low – high, Low – high, None None None None
seasonal seasonal
d
% Cysts with TUNEL-positive Sertoli cells None None None None None None None None None
a
Estimates based on Holstein (1969).
b
Cell cycle stage-dependent: intense (leptotene ! early pachytene); weak or negative (mid ! late pachytene).
c
Labelling of germ cell and Sertoli cells alternates.
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d
This report.
NA, not applicable; s’gonia, spermatogonia; s’cyte, spermatocyte; s’tid, spermatotid.
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 Cell cycle and apoptosis in the shark testis 93

described in earlier studies of this and other dogfish Sertoli-cell nuclei was used to demarcate early- and mid-
species (Stanley 1966, Simpson & Wardle 1967, Holstein stage PrM cysts. In addition to the substages defined in
1969). For example, based on actual cell counts per sper- Table 1 and quantified in Fig. 1, several layers of degener-
matocyst, Sertoli cells have been reported to undergo ating cysts comprising a zone of degeneration (ZD) were
nine, and germ cells an additional four, mitotic divisions located at different positions relative to the germinal ridge
in Squalus (Holstein 1969). However, the cyst size associ- and other stages (Fig. 2 and see Fig. 6f, below). Also,
ated with the cessation of Sertoli-cell proliferation, an large, empty cysts with a few loose spermatids and promi-
important landmark in the PrM region, cannot be nent peripheral Sertoli-cell nuclei were located beneath
discerned by routine histological methods. Thus, in this the capsule at the mature surface of the testis at some
analysis, the transition from PCNA-positive to -negative times of the year and designated evacuated cysts (Fig. 2,
and see Fig. 4b, below).
Seasonal variations in the number, fraction and spatial
distribution of staged spermatocysts
To examine seasonal effects on spermatogenic pro-
gression, the number (Fig. 1) and fraction (Fig. 2) of devel-
oping cysts at defined substages were quantified monthly
over a full annual cycle. In order to account for changes
in cyst diameter during developmental progression, the
formula used for the calculation of cyst counts incorpor-
ated the parameters of testis width and mean cyst diam-
eter of each stage. Except for immature spermatogonial
cysts (germinal zone (GZ) and early-stage PrM (E-PrM)
cysts; cyst diameter, 15 –150 mm; Fig. 1b), the number of
cysts in all subsequent stages (Fig. 1c –1h) and the total
number of cysts (Fig. 1a) varied with time of year
(P , 0.001). Regardless of time of year, approximately
50 –70% of germinal clones were in PrM stages of devel-
opment, 25 –40% in PoM stages and , 10% in meiosis
(Fig. 2). Although spermatogonial cysts (GZ, E-PrM and
mid-stage PrM (M-PrM) cysts) were the most abundant
cyst stages (approximately 1200 cysts compared with a
total of approximately 750 cysts for the rest of the stages)
in the spermatogenic progression (Fig. 1) in May/June–
August, testis width was nevertheless at its yearly mini-
mum, because it completely lacked late-stage PrM (L-PrM)
cysts (Figs. 1d and 2) and M-stage cysts (spermatocytes;
Figs. 1e and 2; P , 0.05), the two cyst stages which
together have the largest cyst diameters. Conversely,
during the fall/early winter months, testis width reached
R
Figure 1 Seasonal changes in the number of spermatocysts at defined
spermatogenic substages in S. acanthias. Values represent the mean
number of spermatocysts^ S.E.M. per testicular cross-section (see the
Materials and methods section). Note scale differences on the y-axis
of each panel. Untransformed values were analyzed by one-way
ANOVA. Seasonal variations were significant in all stages (P , 0.001)
except (b). Within each stage, data points with different letters
differed significantly (P , 0.05) from each other. Numbers of animals
collected monthly for this and all subsequent analyses are shown in
parentheses at the bottom of the figure. Letters at the bottom indicate
months of the year starting at the left with May (M). Criteria used in
substaging are described in Table 1. GZ, germinal zone; E-, M-,
L-PrM, early-, mid-, late-stage premeiotic cysts; E-, M-, L-PoM, early-,
mid- and late-stage postmeiotic cysts. Dashed lines indicate the tem-
poral progression of spermatogenesis by extrapolating back from
peak production of L-PoM cysts to the previous season’s peak of
M-stage cysts, and forward from the current M-stage cysts to next
season’s cysts with mature spermatids (L-PoM).

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94 L M McClusky

Figure 2 Seasonal changes in the fractional compo-

sition and spatial arrangement of staged spermato-
cysts in the developmental progression (vertical
arrow) in S. acanthias. Values represent the mean
percentages of cysts at a given stage relative to the
total number per cross-section at each time point
(see Fig. 1 for actual values ^ S.E.M. ). Percentage
values were arcsine square-root-transformed and
analyzed by one-way ANOVA. Except for GZ and
E-PrM cysts, all stages varied significantly by time
of year (P , 0.004). Numbers of animals collected
and abbreviations are shown in Fig. 1. Ev, evacu-
ated cysts.

its maximum, due to the resumption of mature spermato- developmental progression from newly formed spermato-
gonial development (L-PrM; Figs 1d and 2), leading to cysts in the germinal ridge to cysts with fully mature
transition into meiosis (M stage; Figs 1e and 2), and sub- spermatids requires two annual cycles, interrupted by a
sequently the formation of round and elongating sperma- summer period of developmental arrest at the spermatogo-
tids (Figs 1f, 1g and 2; P , 0.05). If the temporal nium –spermatocyte and the elongating-spermatid–
progression of spermatogenesis is traced by extrapolating mature-spermatid transitions. This interpretation is consist-
back from the peak accumulation of L-PoM cysts (mature ent with a 2-year cycle of folliculogenesis, ovulation,
spermatids), or by extrapolating forward from the peak mating, gestation and parturition in females of the same
accumulation of M-stage cysts (spermatocytes; Fig. 1, species (Simpson & Wardle 1967, Jones & Geen 1977).
dotted lines), it is evident that germinal clones in the final
stages of spermiogenesis entered meiosis approximately
PCNA immunoreactivity
9–11 months earlier. Although the peak number of cysts
was 4–5-fold greater at the beginning than at the end of To determine how seasonal variations in the formation of
spermatogenesis (approximately 1200 versus 300; com- new cysts, and the expansion of germinal- and Sertoli-cell
pare Fig. 1b and 1h), this estimated net loss of developing clones in developing cysts, contribute to the spermato-
clones, though conservative, may be even greater as the genic progression, PCNA immunohistochemistry was used
actual net loss cannot be computed from these data with- to observe and quantify cell-cycle activity of germ cells
out knowing the duration of intervening substages. and Sertoli cells in the testis (Figs. 3 –5). As shown in
The spatial and temporal appearance of the ZD in April Fig. 3a–3c, the longitudinal germinal ridge was tongue-
(year 2), interposed between M-PrM and L-PrM stages, like in shape and collapsed when prespermatocyst stem
preceded a deficit of L-PrM cysts initially and M-stage cells (gonocytes and Sertoli-cell precursors) lacked PCNA
cysts later between May and September (Fig. 2; immunoreactivity (September– March), but had a rigid
P , 0.004). The temporal troughs in all cyst stages sub- triangular shape when clusters of PCNA-positive germ
sequent to the M-PrM stage (Fig. 1d –1g; P , 0.05) corre- cells were present (April– August). Although the number of
spond exactly with the position of the ZD in time and cells in the germinal ridge was not counted, this spring/
space in the spermatogenic progression (Fig. 2; summer period of stem-cell proliferation (Fig. 5, horizon-
P , 0.004). Once formed, the number of cysts in ZD did tal bars) was coincident with the seasonal appearance of
not change (187 ^ 12.88 (mean ^ S.E.M. )), suggesting that the ZD (Fig. 2). Once formed, each subsequent spermato-
degenerate cysts were neither added nor entirely reab- cyst stage had a unique PCNA-labelling pattern with
sorbed as the season progressed. The spatiotemporal dis- regards to both germ and Sertoli cells (Fig. 4). In contrast
placement of the ZD occurs as a result of the resumption to mature spermatogonial cysts (M-PrM and L-PrM cysts),
of premeiotic activities and the mitosis–meiosis transition PCNA labelling in immature spermatogonial cysts was
behind the ZD band in August (Fig. 2; P , 0.004). The characterized by season-dependent alternating peaks in
rate of advance of ZD observed in this and an earlier germ-cell and Sertoli-cell labelling, which was already
study (Simpson & Wardle 1967) indicates that spermato- evident in GZ cysts and was more pronounced in E-PrM
gonial generations 12 –13 (L-PrM cysts) require 9 –10 cysts (GZ, Fig. 5a, germ-cell index, P , 0.001; Sertoli-cell
months to reach the mature-spermatid stage. The first index, P , 0.03; E-PrM cysts, Fig. 5b, germ-cell index,
appearance of evacuated cysts in January agrees with the P , 0.05; Sertoli-cell index, P , 0.002). Thus, in these
reported time of spermiation and mating in the Atlantic two stages, when germ cells were labelled, Sertoli cells
stocks of this species (Simpson & Wardle 1967). Consist- were not, and vice versa, and all cells of each type in a
ent with the data in Fig. 1, these results suggest that the given cyst were synchronized in or out of the cell cycle

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 Cell cycle and apoptosis in the shark testis 95

Figure 3 Photomicrographs showing PCNA immunolabelling of PrM spermatocysts at different substages and times of year (Methyl Green coun-
terstained) in S. acanthias. Germinal ridge showing stem cell nests when gonocytogenesis is (a) inactive in December and (b) active in June.
For orientation, the position of the GZ (the next stage in maturation) is shown (arrowhead). (c) High magnification of boxed area in (b) showing
PCNA labelling of gonocytes but not somatic (pre-Sertoli) elements (arrows). (d and e) GZ region showing earliest stages of spermatocyst for-
mation. In (d), both spermatogonial (white arrows) and Sertoli-cell nuclei (black arrows) are PCNA-positive (August). In (e) spermatogonia
(white arrows) are PCNA-negative, but Sertoli nuclei (black arrows) are PCNA labelled (November). Note labelling of nuclei of collecting duc-
tules (cd), which become patent at the end of spermatogenesis. (f and g) E-PrM-stage spermatocysts showing alternating pattern of PCNA label-
ling: (f) labelled spermatogonia (white arrows) and unlabelled adluminal Sertoli nuclei (black arrows; August), and (g) labelled Sertoli nuclei
(black arrows) and unlabelled peripheral spermatogonia (white arrows; November). Inset in (f) shows negative control (no primary antibody).
Scale bars: (a, b), 100 mm; (c), 20 mm; (d–g), 25 mm.

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96 L M McClusky

Figure 4 Photomicrographs showing PCNA immunolabelling of M- and PoM-stage spermatocysts (Methyl Green counterstained) in S. acanthias.
(a) In a single spermatocyst, intensity of PCNA labelling of spermatocytes decreases from intense (white arrows) to weak (concave-headed
arrows) as meiotic division proceeds. Area containing nuclei with Methyl Green-stained metaphase chromosomes is indicated by*. Note the
absence of labelling of Sertoli cells in spermatocyte cysts (straight-headed arrows). (b) Spermatocysts with mature spermatid bundles are PCNA-
negative, as are Sertoli nuclei (straight-headed arrows), but bundled spermatid heads are strongly Methyl Green-stained. Note strongly PCNA-
positive Sertoli nuclei in evacuated cyst (concave-headed arrows). Scale bars: 20 mm (a) and 25 mm (b).

(Fig. 3d– 3g). The alternating cell-type-dependent PCNA TUNEL labelling inside individual cysts took on several
labelling in the immature spermatogonial cysts was also forms, indicative of the progression from early to late
stage-dependent; i.e. the Sertoli-cell PCNA index was stages of the degenerative process: (a) scattered, single,
GZ @ E-PrM (P , 0.001) and the germ-cell PCNA index small TUNEL spots, consistent with the labelling of frag-
was GZ ! E-PrM (P , 0.001). Sertoli cells of M-PrM and mented DNA in individual spermatogonial nuclei, in
L- PrM cysts (when these cysts were present) were consist- otherwise normal cysts (Fig. 6b); and (b) very large TUNEL
ently PCNA-negative, while M-PrM-cyst spermatogonia spots, consistent with labelling of fragmented DNA in
averaged a PCNA-labelling index of 50% or more during multinucleate germ cells (Fig. 6c and 6e).
the year (Fig. 5c). In addition, the germ-cell PCNA indices As stated above, a cyst was considered TUNEL-positive
of L-PrM and M-stage cysts (when they were present) var- if it had three or more TUNEL-positive germ cells.
ied seasonally (Fig. 5d, L-PrM, P , 0.001; Fig. 5e, M Temporally, the total TUNEL index for the entire PrM
stage, P , 0.03), with both cyst stages lacking PCNA region was at its lowest (2 –8%) during the mid-winter
immunoreactivity in summer. PCNA immunostaining in period (December –February), after which, starting in April
M-stage cysts was also dependent on the stage of meiosis and through to September, it reached maximum levels in a
(Fig. 4a). Secondary spermatocyte, round, elongating (all distinct stage-dependent manner (P , 0.001). The most
not shown) and mature-spermatid cysts (Fig. 4b) were con- frequently labelled germinal clones were M-PrM cysts
sistently PCNA-negative for all cell types. By contrast, Ser- (TUNEL index increased to 20 –55% between April and
toli cells in evacuated cysts were strongly PCNA- September, P , 0.001), indicating that further development
immunoreactive (Fig. 4b), which is unlikely to be an arti- of these cysts in early summer would cease as they would
fact because Sertoli cells in adjacent mature-spermatid- form the current season’s ZD (Fig. 7). Although L-PrM cysts
containing cysts in the same section and negative controls (spermatogonial generations 12/13) were absent from the
(not shown) were not PCNA-labelled. spermatogenic progression between May and July (year 1),
the result of M-PrM cysts aborting and forming the ZD,
TUNEL labelling their TUNEL index was high (20 –45%) when a few cysts in
To examine the cellular basis of ZD formation, and the this stage were seen at the trailing edge of ZD in August
role of apoptosis in the seasonal onset, offset and pro- and September (Fig. 7; P , 0.001). The TUNEL index of
gression of spermatogenesis, TUNEL immunohistochemis- E-PrM cysts never exceeded 10% of the total cyst popu-
try was used to identify apoptotic cells. TUNEL labelling lation (Figs. 6a, 6b and 7; P , 0.001), regardless of the
had distinct spatial (stage-dependent; Fig. 6) and temporal time of year. A complete apoptotic E-PrM cyst, with Sertoli
(seasonal; Fig. 7) patterns. TUNEL-positive staining was cells of normal appearance and positioning, was observed
limited to spermatogonia, never observed in GZ cysts (not only once during the summer in the full seasonal series
shown), occasionally seen in E-PrM cysts (Fig. 6a and 6b) (Fig. 2B in Callard et al. 1998), but their rare observation
and observed frequently in M-PrM (Fig. 6c), L-PrM (Fig. 6e) could perhaps be explained by either their rapid removal
and ZD cysts (Fig. 6f) in a season-dependent manner. and/or their low susceptibility to apoptosis. However, all

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 Cell cycle and apoptosis in the shark testis 97

cell types and stages were labelled after DNase I treatment negative-control reaction, which lacked any background
of tissue sections, evidence that the observed negative labelling (Fig. 6d). Moreover, a regular observation in this
labelling was actually due to the absence of fragmented study and those of others (Simpson & Wardle 1967), was
DNA rather than a lack of access of TUNEL reagents in situ that of ZD-like-shaped cysts (oval-shaped) at the trailing
(Fig. 6h). Specific staining was further verified in the edge of the ZD in sharks caught in August – October, and
which apparently recovered and a few months later devel-
oped to contain some maturing spermatids.

Discussion
This study is the first to quantitatively correlate seasonal
variations in the number and proportions of various sper-
matocyst stages with cell-proliferative and cell-death
activities at each stage during the spermatogenic cycle in
a squalomorph elasmobranch (S. acanthias). Although all
spermatocyst stages appear to be present all year round,
the protracted spermatogenic cycle of 2 years causes tem-
poral overlapping in the developmental peaks of early and
late spermatocyst stages in consecutive years. Thus, even
though the peak in the numbers of mature spermatogonial
cysts coincides in time and space with the peak in the
numbers of mature spermatid cysts in one annual cycle,
the latter really represents the completed development of
germ-cell clones that entered the M-PrM-cyst stage
approximately 1 year earlier. A major factor contributing
to the protracted spermatogenic cycle in Squalus is that
spermatogenic development is characterized by substan-
tial clonal deletion. Specifically, the number of germinal
clones completing spermatogenesis (i.e. mature spermatid
cysts) is approximately 4 –5-fold less than the number of
germinal clones that entered the differentiative pathway
(i.e. GZ and E-PrM cysts) 2 years earlier. Analysis reveals
that the most earliest spermatogenic stage in the pro-
gression, showing a 4 –5-fold seasonal reduction in its
numbers, is the M-PrM stage (the first spermatogonial cyst
stage with postmitotic Sertoli cells). Consequently, the
temporal peaks and troughs in M-PrM cyst numbers are
also reflected in subsequent spermatogenic stages over
the ensuing months. Results show that the 4 –5-fold loss
of M-PrM cysts (the aborted cysts become the ZD) from
the spermatogenic progression at the spring–summer

R
Figure 5 Seasonal and stage-related changes in the percentage of
spermatocysts with PCNA immunolabelling of germ cells (†) and
Sertoli cells (W) in S. acanthias. Percentage values were arcsine
square-root-transformed and analyzed by two-way ANOVA (a and b)
and one-way ANOVA (c–e), followed by the Student–Newman–
Keuls multiple-comparison test. The significance of the monthly vari-
ations at each stage was: GZ (germ cells, t–w, P , 0.001; Sertoli
cells, a–b, P , 0.03); E-PrM (germ cells, t –w, P , 0.05; Sertoli cells,
a– b, P , 0.002); M-PrM (germ cells, non-significant); L-PrM (germ
cells, P , 0.001); M (germ cells, P , 0.03). For GZ (a) and E-PrM (b),
the interaction between cell type and time of year was significant
(P , 0.001). In (d) and (e), cysts in these categories were absent at
certain times of year (see Figs. 1 and 2). Sertoli cells were PCNA-
negative after E-PrM stages, and germ cells after M-stages (see Fig. 3).
For reference, horizontal bars above (a) show period of stem-cell
proliferation in the germinal ridge (cells not quantified).

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98 L M McClusky

Figure 6 Photomicrographs of spermatocysts after TUNEL staining (May/June). GZ (not shown) and (a) E-PrM cysts are usually unlabelled, but
(b) a few E-PrM cysts have a small number of TUNEL-positive germ cells (black star) or very occasionally are degenerate (white star). (c) TUNEL-
positive germ cells are frequently observed in M-PrM cysts of S. acanthias. (d) Negative control of PrM cyst (no TdT). (e) L-PrM spermatocyst
earmarked for degeneration with numerous TUNEL-labelled nuclei. Multinucleated giant cells may be TUNEL-negative (concave-headed arrows)
or, more often, TUNEL-positive (straight-headed arrows). Inset: Sertoli-cell nucleus of a ZD cyst closely apposed to a phagocytosed germ-cell
corpse. (f) ZD region with spermatocysts in advanced state of degeneration occupy the predicted position of L-PrM cysts in this specimen (see
Figs. 1 and 2). Cysts are atrophic and have Sertoli cells only with/without TUNEL-positive germ-cell remnants. (g) M-stage cysts (bottom) and
round spermatid stage cysts (upper left) are consistently TUNEL-negative. (h) Positive control of M-stage and round spermatid-stage spermato-
cysts after DNase-I treatment showing that access of the TdT enzyme is not impeded. Scale bars, 25 mm.

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 Cell cycle and apoptosis in the shark testis 99

Figure 7 Seasonal and stage-related changes in TUNEL labelling of spermatocysts in S. acanthias. Values represent the mean percentage^ S.E.M.
of TUNEL-positive spermatocysts at E-, M- and L-PrM stages of development. GZ cysts were rarely labelled and not scored. Percentage values
were arcsine square-root-transformed and analyzed by two-way ANOVA. Month-by-month differences within each stage (P , 0.001), between
stages (P , 0.001) and their interaction (P , 0.001) were significant. The Student-Newman–Keuls multiple-comparison test was used to deter-
mine which months differed significantly within a given stage (P , 0.05) (E-PrM, r– s; M-PrM, a–e; L-PrM, x–y). Differences between stages in a
given month are indicated with*. Where no bar is shown for a given stage, TUNEL staining was absent. L-PrM cysts were absent from the pro-
gression between May and July of year 1.

transition is due to spermatogonial apoptosis, suggesting other assays, i.e. biochemical analysis of DNA fragmenta-
that the advance from E-PrM to M-PrM stages is a season- tion (Callard et al. 1995), Acridine Orange vital staining of
ally regulated control point that determines the cyst num- living testis slices (McClusky et al. 1996) and transmission
bers of subsequent stages. Thus apoptosis is a major electron microscopy (L.M. McClusky, unpublished obser-
mechanism controlling spermatogenesis, and determines vations). Our findings, however, differ from those reported
the number of cysts that progress to the final spermatogo- in the spotted ray, Torpedo marmorata, which show no
nial division (L-PrM stage), advance into meiosis and, stage-dependency and cell-type specificity of testicular
ultimately, reach the mature spermatid stage. apoptosis (Prisco et al. 2003). Since a ZD has also been
Within the aborted M-PrM cysts, spermatogonia noted in each testicular lobe of the Atlantic stingray,
coalesce to form multinucleate giant cells, a phenomenon Dasyatis sabina (Maruska et al. 1996), and a ZD and
probably facilitated by the opening or dissolution of the stage-dependent premeiotic apoptosis seen in the little
intercellular bridges between germ cells (Pudney 1995). skate, Raja erinacea (L.M. McClusky, unpublished obser-
The multinucleate giant cells are eventually phagocytosed vations), the different mode of testicular apoptosis in
by the Sertoli cells over a period of 10 months after which Torpedo is difficult to explain other than to suggest the
the ZD cysts are resorbed in the epigonal organ, a lym- tempering effects of environmental factors peculiar to
phomyeloid organ associated with the mature surface of the Mediterranean sea where this species was studied.
the testis. This protracted nature of apoptosis is rather unu- The seasonal appearance of a ZD implicates the brain–
sual considering the rapid clearance of apoptotic cells hypothalamo –pituitary–testicular axis in the control of
(a few hours at most) in other tissues (Manfredi et al. germ-cell apoptosis specifically at the spermatogonia –
2002). Immature spermatogonial cysts rarely undergo total spermatocyte transition in the spiny dogfish shark testis
degeneration (see Fig. 2B in Callard et al. 1998), but often in vivo (Simpson & Wardle 1967), but it is likely that
contain a scattering of single apoptotic spermatogonia environmental factors are also involved. This conclusion
throughout the cyst. The criterion chosen to designate a is based on findings in the European spotted dogfish
cyst as TUNEL-positive was actually conservative (a sper- (Scyliorhinus canicula), in which a ZD with a fixed
matocyst was designated as TUNEL-positive if it contained number of cyst layers, more or less in the same position to
three or more TUNEL-positive cells, a standard previously that occurring seasonally in Squalus, can be induced by
applied to rat seminiferous tubules (Lee et al. 1997)), hypophysectomy (provided sea-water temperature is near
which further validates the data of the temporal nature summer-season values), while replacement with shark
and strict stage-dependency of apoptosis reported here. hypophysial extracts, but not mammalian gonadotrophins,
These descriptions of germ-cell apoptosis in Squalus, as can prevent the appearance of the ZD (Dobson & Dodd
assessed by the TUNEL assay, have been verified by three 1977a). Although the identity of the shark gonadotrophin

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100 L M McClusky

is not yet known, these findings are in broad agreement circulating androgen (Dobson & Dodd 1977a), overall
with the demonstrated role of one of the pituitary gonado- reconciliation of all these observations in dogfish sharks
trophins (FSH) as an indispensable survival factor for the suggests that the developmental advance of mature sper-
last spermatogonial cyst stage in another lower vertebrate, matogonial cysts in sharks is dependent not only on de
the newt (Yazawa et al. 2002). These data in lower ver- novo testosterone and/or oestrogen synthesis in PrM cysts,
tebrates, together with data in rodents which demonstrate but also on those synthesized in mature-spermatid cyst
the acute dependency of differentiating spermatogonia stages, when they are present in the spermatogenic
and preleptotene spermatocytes on FSH for their survival progression. These conclusions are consistent with the
(Boitani et al. 1995, Meachem et al. 1999, Allan et al. coincident increased M-PrM-cyst apoptosis in April –May
2004), probably suggest an evolutionarily conserved and a concurrent deficiency of mature spermatids in the
feature of gonadotrophin action on differentiating sperma- spermatogenic progression.
togonia. Since the population of differentiating spermato- Other novel observations in the immature PrM region
gonia in rodents is also subject to strict density-dependent were the apparent coordination of spermatogonial and Ser-
regulation via apoptosis (De Rooij 2001), it might be that toli-cell proliferative activities, Sertoli-cell dominance of
the latter is phylogenetically a relatively late-derived cyst-proliferative activities during the year, except for a
feature in the regulation of spermatogonial cell kinetics. brief period in December and during the spring–summer
In all vertebrates, the spatiotemporal synchronization of months, and the relative resistance of immature cysts to
steroidogenic and spermatogenic developments ensures seasonal apoptosis during the first nine divisions. The
timeous and optimal production of spermatozoa. Results steady increase in cyst diameter during the spermatogonial
presented here show that the resumption of the develop- stages is solely due to the proliferation of spermatogonia
mental progression of mature spermatogonial cysts in the and Sertoli cells, at least for the first nine divisions. Careful
fall coincides in time and space with a corresponding analysis of DNA synthesis data in organ-culture exper-
increase in the number of mature spermatid cysts, findings iments of immature rat testes similarly reveal an inverse
which are also noted in other elasmobranchs (Garnier relationship between germ-cell and Sertoli-cell prolifer-
et al. 1999, Tricas et al. 2000). These findings in Squalus, ation, such that germ-cell DNA synthesis is low when
to some extent, complement those of previous in vitro Sertoli-cell DNA synthesis is elevated and vice versa
experiments on isolated spermatocysts. For example, (Schlatt et al. 1999). Furthermore, FSH regulates both
when a mixed population of PrM cysts, isolated from Sertoli-cell and differentiating spermatogonial DNA
Squalus that lack mature spermatid cysts and have a natu- synthesis (Boitani et al. 1995, Schlatt et al. 1999). By
ral ZD at the spermatogonia –spermatocyte transition, are extension then, one explanation could be that seasonal
placed in basal medium, these cysts gradually, spon- alternating patterns in PCNA immunoreactivity in Sertoli
taneously and stage-dependently (large PrM cysts @ small and germ cells in sharks reflect variations in gonadotrophin
PrM cysts) enter apoptosis in vitro (Callard et al. 1995, secretion and/or changes in cell-type-specific responses to
McClusky et al. 1996), results which are consistent with gonadotrophin secretion in these immature cysts. In this
the absence of survival factors. However, addition of a regard, it is worth noting that both mRNA and protein
gonadotrophin-mimicking agent (3-isobutyl-1-methylxan- PCNA expression are known to be under direct gonado-
thine (IBMX), 1 mM), which is known to raise testosterone trophin (FSH) control in another gonadal cell system, rat
and oestradiol secretion in such PrM cysts to some extent granulosa cells (the female equivalent of Sertoli cells;
(Cuevas & Callard 1992a), causes a significant decrease in El-Hefnawy & Zeleznik 2001), and Sertoli-cell proliferation
both DNA synthesis and DNA fragmentation in PrM cysts in foetal and neonatal rodent testis is FSH-stimulated
(Callard et al. 1995). Similarly, added oestradiol and (Sharpe et al. 2003, Allan et al. 2004). Nevertheless, the
androgens significantly reduce the percentage of apoptotic data presented here show that although Sertoli-cell pro-
cysts with no effect on [3H]thymidine incorporation liferation dominates immature spermatogonial cyst cell
in spermatogonial cysts in vitro (Betka & Callard 1998; kinetics for most of the year, the decreases in Sertoli-cell
L.M. McClusky, unpublished observations). By extension PCNA immunoreactivity correspond temporally exactly
and given that (1) mature spermatid cysts, which are with the time of M-PrM-cyst abortions culminating in ZD
absent in April –May, are major sites for the synthesis of formation, suggesting that April –May is the beginning of a
androgen-binding protein and for the secretion of testos- 4-month period of gonadotrophin deficit, as was also
terone and its precursors (Simpson & Wardle 1967, Mak suggested by Simpson & Wardle (1967). Secondly, the
& Callard 1989, Sourdaine et al. 1990, Cuevas & Callard developmental progression of immature shark spermatogo-
1992a), (2) classical nuclear oestrogen and androgen nia (GZ and E-PrM cysts) is most likely directly gonado-
receptors are concentrated in the PrM region of the shark trophin-independent (Dobson & Dodd 1977b) and/or may
testis (Callard et al. 1985, Cuevas & Callard 1992b), (3) be Sertoli cell-mediated, e.g. via seasonally regulated
the vascular pathway in Squalus is countercurrent to the diffusable factors (Loir 1994) and contact-dependent
spermatogenic progression, i.e. PoM cysts to GZ cysts mechanisms (Mather et al. 1990, Zhou et al. 1993, Loir
(Cuevas et al. 1992) and (4) hypophysectomy of male 1999). Indeed, unidentified growth-stimulatory and -inhibi-
dogfish (Scyliorhinus) leads only to a small decrease in tory bioactivities present in spent media of cultured

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 Cell cycle and apoptosis in the shark testis 101

Squalus Sertoli cells regulate spermatogonial DNA syn- waning of testicular activities of other elasmobranchs
thesis in vitro, depending on the time of year (DuBois & (Garnier et al. 1999, Tricas et al. 2000), further studies are
Callard 1993). With regards to this last point, it should be required to establish whether environmental cues also
noted that when a mixed population of PrM cysts, isolated have a role in cell-proliferative and cell-death activities
from Squalus caught in late April (these animals have no during spermatogenesis in the spiny dogfish shark.
ZD yet), are placed in culture in basal medium, none of
these cysts enter apoptosis spontaneously, and are comple- Acknowledgements
tely unresponsive to gonadotrophin-mimicking agents, ster-
oids or growth factors (L.M. McClusky, unpublished This research was carried out in part at the Mount Desert
observations), despite the fact that Sertoli cells in immature Island Biological Laboratory, Salisbury Cove, ME, USA and
Boston University, and supported in part by a fellowship from
spermatogonial cysts are proliferatively active (this study).
the National Research Foundation of South Africa. The assist-
These observations, together with data of the observed
ance of Andy Sexton (Marine Biological Laboratory, Woods
complete lack of PCNA immunoreactivity in immature Hole, MA, USA), Janet Fields and Vic Nordahl (National Mar-
spermatogonia in winter, are further evidence that imma- ine Fisheries Service, Woods Hole, MA, USA), Jim Frances-
ture spermatogonia are in a state of developmental arrest coni (North Carolina Division of Marine Fisheries, Morehead
during winter, an effect that is probably Sertoli-cell- City, NC, USA) and Arnold Howe (Massachusetts Division of
mediated. Marine Fisheries, Cape Cod Bay, MA, USA) in the collection
An interesting observation was the pronounced gonocy- of shark specimens is greatly appreciated. A special word of
togenesis in the germinal ridge coincident with the thanks is extended to Dr Gloria Callard (Boston University)
M-PrM-cyst abortions culminating in ZD formation in for helpful discussions, and to Dr Jeffrey Pudney (Brigham
April/May–September, and vice versa in winter. These and Women’s Hospital and Harvard Medical School) for criti-
cally reading the manuscript.
germinal stem cells are not found in a continuous line
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