BIOLOGY OF REPRODUCTION 12.

260-274 (1975)

Capacitation of Rabbit Spermatozoa in vitro

BENJAMIN G. BRACKETT AND GENE OLIPHANT’
Section of Clinical Reproduction, Department of Clinical Studies, School of Veterinary Medicine and Division of
Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia.
Pennsylvania 19174

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Accepted September 20, 1974

This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm
to fertilize ova in vitro. During 20 mm exposure of sperm cells to defined media of increasing NaCI
concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic
components was initiated and completed as reflected by an immunological assay at approximately 300
and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or
alteration of these components as determined by the immunological assay during the first 5 mm of
sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration
of some but not all of the antigenic sperm coating seminal plasma components detectable by this means
during 20 mm of exposure.
Sperm within the perivitelline apace, presence of pronuclei. and 2- and 4-cell cleavage stages were
taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm
showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg)
medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged
from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with
isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent
(SI of 69 ova) when results were considered according to individual male sperm donors. No large differ-
ences were found by the immunological assay that could be linked to variability of fertilizing ability
of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested
as a likely reason for differences in fertilizing ability of sperm from different bucks.
In experiments using sperm and ova from the same sources, no differences in fertilization results
were apparent following 305 and 380 mOsm/kg sperm treatments, Initiation, but not completion, of
removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary
prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm pene-
tration into ova occurred in some cases before 7-10 h after insemination (4 of 54 ova penetrated), but
actively motile sperm were found within the perivitelline apace of other ova as late as 25 h after in-
semination.
One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does.
Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos
resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried
successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation
for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and repre-
sents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and
embryo transfer in the rabbit.

Since the initial reports by Austin (1951) penetrate the zona pellucida of the ovum,
and Chang (1951) of a need for sperm of many efforts have been directed toward elimi-
fertile rabbits to reside in the female repro- nating the requirement of the female repro-
ductive tract for some hours before they could ductive tract in this process. Sperm capacita-
tion, the physiological changes that prepare
Present address: Division of Reproductive Biology.
Department of Obstetrics & Gynecology. University of sperm for penetration of
ova, has been re-
Virginia, School of Medicine, Charlottesville, Virginia ported to occur in vitro following vartous
22901. treatments for sperm of the golden hamster
260
Copyright #{174}
1975 by The Society for the Study of Reproduction.
All rights of reproduction in any form reserved.
 CAPACITATION OF RABBIT SPERMATOZOA in vitro 261

(Yanagimachi and Chang, 1963, 1964; Barros removal or alteration of sperm surface semi-
and Austin, 1967a, b, c; Barros, 1968; Gwat- nal plasma components, including decapaci-
kin and Anderson, 1969; Yanagimachi, tation factor, as was shown with the rabbit to
1969a, b; Barros and Garavagno, 1970), occur normally in vivo (Oliphant and Brack-
Chinese hamster (Pickworth and Chang, ett, l973a, b). The purpose of this investiga-
1969), mouse (Iwamatsu and Chang, 1969, tion was to capacitate ejaculated rabbit sperm
1970, 1971; Toyoda, et a!., 197la, b; Miya- in a defined medium and to document this
moto and Chang, 1972a, b; Pavlok and achievement by in vitro fertilization and term

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McLaren, 1972; Mukherjee, 1972; Oliphant development following transfer of resulting
and Brackett, l973b), guinea pig (Yanagima- embryos to recipient does.
chi, 1972), human being (Bavister, et a!.,
1969; Edwards, et a!., 1969; Soupart and MATERIALS AND METHODS
Strong, 1974), gerbil (Noske, 1972), rat (Bre- Animals
gulla, 1969; Toyoda and Chang, 1974a, b)
Sperm used in these experiments were derived primar-
and rabbit (Kirton and Hafs, 1965; Ericsson, ily from 5 rabbits: 4 New Zealand White bucks (B-IS.
1969; Ericsson, et a!., 1971; Brackett, et a!., B-36, B-39, B-45) and one Dutch Belted buck (B-61).
1972; Ogawa, et a!., 1972; Okinaga, et a!., Every buck used in this study was considered to be

1974). Capacitation of sperm of the golden normal and sired at least one litter. New Zealand White
does were used exclusively as ovum donors in these
hamster (Bavister, 1969, 1973), guinea pig
experiments. New Zealand White and Dutch Belted does
(Yanagimachi, 1972), rat (Toyoda and were used as recipients for the embryo transfers. All
Chang, l974a, b), rabbit (Ogawa, eta!., 1972) females were isolated for three weeks prior to use to
and mouse (Toyoda, et a!., 1971a, b; Miya- avoid pseudopregnancy. The rabbits were fed Purina
rabbit chow and water ad libitum. The bucks were
moto and Chang, 1972b) has been observed to
exposed to 8 h of light and 16 h of darkness, while the
occur in defined media, as evidenced by does were exposed to 14 h of light and 10 h of darkness
subsequent fertilization of ova in vitro. Al- each day.
though evidence for fertilization in these
experiments is, in general, acceptable, only in Sperm Treatments
the mouse and rat has the potential for Semen was collected from bucks by use of an artificial
continuation of development of the embryos vagina (Walton, 1958) and a teaser doe. Only the liquid
portion of semen was used and any jelly present in the
resulting from fertilization by sperm capaci-
ejaculate was discarded. The sperm cells were washed to
tated in vitro been demonstrated (mouse: remove excess seminal plasma by suspending them in 3-5
Miyamoto and Chang, 1972b, Mukherjee, ml of defined medium and centrifugation at 734 x g for 5
1972; rat: Toyoda and Chang, l974a). Epi- mm, discarding the suspernatant fluid, and resuspending
didymal sperm was the starting material for the cells in fresh medium. The defined medium used in
these experiments was that previously used for fertiliza-
both the mouse and rat experiments.
tion of rabbit ova in vitro (Brackett, 1970a) supple-
Although much speculation regarding the mented with 1.25 mM pyruvate (Table 1). The osmolality
mechanism of capacitation of sperm of vari- of this medium (305 mOsm/kg) was increased by the
ous mammalian species has been written, addition of NaCI. Osmolalities reported here were the
only recently has it been shown by immuno- actual or measured osmolalities. For this purpose, an
osmette precision osmometer (Precision Systems) was
logical methods that the process involves
employed. The usual sperm treatment consisted of
removal or alteration of seminal plasma washing the sperm cells immediately after their recovery
components that normally coat the sperm from the buck, then resuspending the cells to a volume of
surface (Oliphant and Brackett, 1973a, b; 2.0 ml with defined medium of the desired osmolality in
Aonuma, et a!., 1973). In addition, recent which the cells were incubated for IS mm, followed by
another washing procedure and final resuspension of cells
experiments have shown that mouse sperm
in 2.0 ml of isotonic (305 mOsm/kg) defined medium.
can be capacitated more rapidly in media of The sperm cells were, therefore, exposed to experimental
elevated ionic strength, and the mechanism of media for 20 mm including the 5 mm centrifugation. The
capacitation induced in this way involves the centrifugations were carried out at room temperature,
 262 BRACKETT AND OLIPHANT

TABLE I tion and the ovaries were immediately removed and

MEDIUM FOR in vitro FERTILIZATION AND EARLY placed in a 250 ml beaker containing warm (approxi-
DEVELOPMENT OF RABBIT OVA mately 38#{176}C)sterile saline and carried into the adjacent
warm (approximately 38#{176}C)tissue culture room. One
Component g/l mM ovary at a time was transferred to a large tissue culture
dish (40 mm inside diameter and 12 mm deep) where the
NaCI 6.550 112.00 ova with surrounding cumulus cells were removed from
KCI 0.300 4.02 the ovarian surface and pipetted into a small tissue
CaCI,.2H,O 0.330 2.25 culture dish (30 mm diameter and 12 mm deep) contain-

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NaH,PO4.H,O 0.113 0.83 ing 4.0 ml of defined medium (305 mOsm/kg). If the
MgCI,.6H,O 0.106 0.52 sperm were not ready for inseminating at this time, the
NaHCO, 3.104 37.00 medium containing ova was overlaid with paraffin oil
Glucose 2.500 13.90 (Saybolt Viscosity 125/135, Fisher Scientific Co.) and
Pyruvic acidt 0.110#{176} 1.25 held under a moist 5 percent CO, in air atmosphere at
Crystalline bovine albumin 3.000 38#{176}Cuntil insemination. This interval was generally no
Penicillin, sodium salt 0.031 longer than 30 mm. Prior to use, the medium and
Distilled water to 1000 ml paraffin oil were equilibrated with 5 percent CO2 in air at
38#{176}C.Insemination was carried out by adding approxi-
t
When pyruvic acid is used, the pH is adjusted by mately 10’ sperm cells in 0.1 ml or less to a dish
dropwise addition of 1.5 N NaOH to yield final pH of 7.8 containing freshly ovulated ova in 4.0 ml volume of
under 5% CO2 atmosphere at 38#{176}C. medium. After insemination, the gamete-containing
* 0.1375 g/l anhydrous sodium pyruvate can be used. dishes were incubated under a moist 5 percent CO2 in air
in which case the pH adjustment with NaOH becomes atmosphere at 38#{176}Cin a small chamber (Anaerobic
unnecessary. Culture Apparatus, small size, Arthur, H. Thomas Co.).
Osmolality of approximately 305 mOsm/kg. Pyruvate was added to the medium as the sodium salt
which made possible the elimination of the tedious
25#{176}C;
and at other times, the sperm cells were held in a adjustment of pH with NaOH when pyruvic acid was

water bath at 38#{176}C. used, and a moist 5 percent CO,. 8 percent 02 and
balance N1 atmosphere was used for gassing the paraffin
Immunological Assay oil and the incubation chamber in the last 10 of the 55 in
vitro insemination experiments reported here. The inclu-
Guinea pigs were injected for preparation of anti-
sion of pyruvate in the present fertilization medium
serum to whole rabbit seminal plasma from which sperm
eliminated the necessity of transferring ova into a more
were removed by centrifugation. The y-globulin fraction
complete medium for cleavage to occur following sperm
of the resulting antiserum was labeled with [“C] formal-
penetration.
dehyde by reductive alkylation (Oliphant and Brackett,
Ova were usually examined on the day following
1972). Radioimmunological evaluation of changes in the
insemination for evidence of fertilization. Morphological
sperm bound seminal plasma antigens was made as
examinations were carried out in the fresh state under
described by Oliphant and Brackett (1973a). Results are
phase contrast and interference contrast microscopy; and
expressed as an uptake ratio of [“C-antibody uptake by
some ova were fixed with 2% glutaraldehyde in 0.1 M
treated sperm minus nonspecific adsorption] divided by
Cacodylate buffer, pH 7.4, post fixed with 1.0% osmium
[“C-antibody uptake by control washed, non-incubated,
tetroxide, and embedded in Epon 812 prior to serial
ejaculated sperm minus nonspecific adsorption]. Expres-
sectioning at 10’ m and staining with toluidine blue for
sion of data in this manner has been previously discussed
more detailed light microscopic study. Pronuclear devel-
(Oliphant and Brackett, 1973a). The measured osmolal-
opment and ovum cleavage are events not seen under
ity and the time of sperm incubation in the various media
these conditions in the absence of sperm cells. The
are indicated in the results of each experiment.
presence of two well-developed pronuclei was taken as
evidence that the fertilization process was initiated, and
In Vitro Fertilization symmetrically cleaved ova resembling those recovered
The ability of treated sperm to penetrate and fertilize from oviducts of mated rabbits (normal cleavage) were
ova was tested in vitro. The procedure for in vitro taken as evidence that fertilization had occurred. Addi-
fertilization was similar to that previously reported tional evidence for fertilization included the presence of
(Brackett and Server, 1970). Briefly, this involved su- sperm cells around and stuck on the zona pellucida. and
perovulation of two ovum donors with ISO lU. of the presence of sperm inside the perivitelline space,
Pregnant Mare’s Serum Gonadotropmn (“Gestyl”, Orga- demonstration of chromatin in each blastomere of
non) given intramuscularly, followed by 75 lU. of cleaved ova following lacmoid staining (Chang. 1952).
Human Chorionic Gonadotropin (HCG) (“APL”, and continued in vivo development following transfer to
Ayerst) given intravenously 72-84 h later. Ovum donors recipient does.
were killed 12 h after HCG injection by cervical disloca- Embryo transfers were carried out as previously
 CAPACITATION OF RABBIT SPERMATOZOA fl vitro 263

described (Brackett, e a!., 1972; Mills, et a!., 1973). high as twice nontreated sperm. This was
Recipient does were injected with 75 lU. of HCG
attributed to a sperm membrane change
approximately 24 h prior to the transfer of 2- and 4-cell
which nonspecifically allowed penetration of
stagc embryos into their oviducts. Laparotomy was
performed on each recipient doe approximately two the ‘4C labeled antiserum into the sperm. The
weeks after the transfer, and the ability of transferred labeled antiserum bound in this way could not
embryos to develop further in vivo was assessed at that be completely removed during washing. Con-
time. Implantations, sites of resorption and complete
ditions which resulted in varying degrees of
failures to implant were noted. A few animals with
dissociation or alteration of sperm surface

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normally developing gestation were retained for final
assessment of fetal development at term. antigenic components of seminal plasma,
prior to apparent membrane damage, were
RESULTS
used for in vitro capacitation of sperm.
Immunological Evaluation of Ionic Strength When sperm pooled from several bucks
Effect were incubated in a medium of 380 mOsm/kg
Using the radioimmunological assay to and samples removed at varying times of
quantitate the amount of sperm bound semi- incubation, the sperm bound seminal plasma
nal plasma antigens, the effect of treatment of antigens were readily removed or altered
sperm for 20 mm with solutions of varying (Fig. 2). The results of the ‘4C-antibody assay
ionic strength rapidly results in a decrease of suggested that the ionic strength mediated
‘4C-antibody binding to the sperm (Fig. 1). dissociation or alteration of the seminal
The sperm cells were from pooled ejaculates plasma components was initiated within two
of several bucks. These results indicate a minutes and virtually completed within five
rapid ionic strength mediated dissociation or minutes of the onset of incubation.
alteration of the sperm bound seminal plasma Removal or alteration of these bound semi-
antigens. This appears as progressive loss or nal plasma antigens from the sperm cells of
alteration of bound seminal plasma antigens individual bucks used in these experiments
beginning at around 300 mOsm/kg and con- are compared in Fig. 3. The ‘4C-antibody
tinuing up to approximately 380 mOsm/kg. uptake of each individual’s sperm after treat-
However, beyond 380 mOsm/kg, levels of ment was compared to that for fresh washed,
t4C taken up by the sperm were observed as non-incubated ejaculated sperm. Sperm treat-
ments included incubation for 20 mm in
BINDING OF TO SPERM TATED IN HIGH SALT MEDIA isotonic defined medium (305 mOsm/kg) and
incubation for 20 mm in high ionic strength
medium (380 mOsm/kg). Results (Fig. 3)
0 1.0 indicated that that there was little, if any,
difference in removal or alteration of sperm
E
bound seminal plasma antigens from buck to
0.15

I buck.
of removal
Also, these data
or alteration
demonstrate a degree
of seminal plasma
0.50 antigenic components by incubation in de-
fined medium with an osmolality of 305
mOsm/kg that is less than that effected by
025
200 300 400 sperm treatment with the high-salt medium.
Osmoloilty (m0sn/kg)
in Vitro Fertilization
FIG. I. Binding of “C-antibodies to sperm treated in
media of varying osmolalities (ionic strength). The Initial efforts to capacitate sperm in vitro
immunological assay was carried out on a sample of l0
were under conditions which resulted in maxi-
sperm taken from a pool of sperm previously treated with
medium of specified osmolality, washed by centrifuga-
mal dissociation or alteration of sperm sur-
tion, resuspended and sperm concentrations determined. face coating antigenic components of seminal
Each point is the average of duplicate determinations. plasma, as detected by the radioimmunoassay
 0
0

a
4-
a

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U

TI ME(min)
FIG. 2. Time course of removal or alteration of sperm bound seminal plasma antigens by the high ionic
strength medium. Sperm were assayed by the “C-antibody technique for sperm bound seminal plasma
antigens after sperm had been incubated for various lengths of time in the high ionic strength medium (380
mOsm/kg). Incubation in the hypertonic medium was terminated by dilution of 0.1 ml sperm in 3.0 ml of
physiological saline. Sperm were then recovered by centrifugation and a portion used for the assay.

B 15 B36 B39 B45 B 57 861

W DS WOS WDS WDS W OS WDS
I.00

0
4-
a 0.75
0

2
a
D
C-)
F
0.50

FIG. 3. Quantitation of removal or alteration of sperm bound seminal plasma antigens for the sperm of
individual bucks. Ejaculated sperm of each buck was assayed with the ‘4C-antibody assay for sperm-bound
seminal plasma antigens immediately after one wash in defined medium (W), after 20 mm incubation in
isotonic (305 mOsm/kg) medium (D), and after 20 mm incubation in high ionic strength (380 mOsm/kg)
medium (S). Results indicated are of two determinations on each sample with the experimental spread
indicated.
264
 CAPACITATION OF RABBIT SPERMATOZOA !fl vitro 265

(Figs. I & 2); namely, sperm incubation in vitro in 13 of 16 experiments, and this data is
380 mOsm/kg defined medium. A summary shown in Table 3. Sperm from B-IS failed to
of the achievement of the capacity to fertilize fertilize any of 19 ova in 2 experiments and
ova in vitro by treatment of ejaculated sperm that from B-45 failed when 3 ova were
with 380 mOsm/kg defined medium is shown inseminated in another trial.
in Table 2. In this series, capacitation of In 5 experiments, sperm washed once in
rabbit sperm occurred in 23 of 31 experi- defined medium (305 mOsm/kg), but not
ments. Sperm from B-IS failed to fertilize incubated, were tested for the ability to

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any of 19 ova in 2 experiments, sperm from fertilize ova in vitro. None of 34 ova provided
B-39 failed to fertilize any of 124 ova in two evidence of fertilization, but one fragmented
experiments and sperm from B-45 failed to ovum was seen 27 h after insemination.
fertilize any of 36 ova in 4 experiments. Gametes used in these “negative control”
Sperm from B-36 and B-61 afforded some experiments were from the same pools di-
success in every trial. vided for the tests of fertilizing ability after
In 15 of the 31 experiments listed in Table incubating the sperm cells in hypertonic
2, ova and spermatozoa recovered from the (Table 2) and isotonic (Table 3) media. These
same donor animals were divided and used in experiments with ejaculated sperm from
efforts to achieve capacitation in vitro by males B-IS, B-45, B-61 and B-39 reinforce the
sperm treatment with 305 mOsm/kg defined contention that once-washed rabbit sperm are
medium. Results of the latter efforts provided unable to fertilize ova in vitro and thereby
evidence that capacitation had occurred in emphasize the significance of positive results

TABLE 2
In vitro FERTILIZATION WITH EJACULATED SPERM FROM 5 BucKs FoLLowING SPERM TREATMENT WITH 380
mOsm/kg DEFINED MEDIUM

No.o f ova showing signs of fertilization’

No. of ova
Buck no. inseminated Total Pronuclear 2-Cell 4-Cell

B-IS 152 63 (41.4%) 22 22 19

B-36 72 52 (72.2%) 2 25t 25
B-39 148 12 (8.1%) 5 5 2t
B-45 134 36(26.8%) 13 1St 8
B-61 114 71(62.3%) 6 47 18t

* Ova were examined approximately 27 h after insemination.

t These figures each include 2 degenerating ova.

TABLE 3
In vitro FERTILIZATION WITH EJACULATED SPERSI FROM 5 BUCKS FOLLOWING SPERM TREATMENT WITH 305
mOsm/kg DEFINED MEDIUM

No. of Ova Showing Signs of Fertilization’

No. of Ova
Buck No. Inseminated Total Pronuclear 2-Cell 4-Cell

B-IS 19 0 (0.0%) 0 0 0
B-36 69 51 (73.9%) 5 27 19
B-39 8 I (12.5%) I 0 0
B.45 16 8 (50.0%) 4 2 2
B-61 56 28 t(50.O%) 7 12 7

* Ova were examined approximately 27 h after insemination.

t Two of these had motile sperm within the perivitelline space.
266 BRACKETT AND OLIPHANT

reported in Tables 2 and 3. This result suggests more rapid completion of

Data in Tables 2 and 3 were recorded capacitation, at least most of the time, by
approximately 27 h (with a few exceptions in sperm from B-36 than was achieved by the
the range of 25-28 h) after in vitro insemina- same treatment of sperm from B-39.
tion, a time at which development of 4- to In 3 experiments, ova were examined for
8-cell stage embryos would normally be ex- sperm penetration through the zona pellucida
pected. In these experiments, however, vari- 7-10 h after insemination with treated sperm
ous stages of fertilization and cleavage were from B-36 and B-39. One of 23 and 3 of 31

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seen. These included the presence of motile ova exposed to sperm treated with high ionic
sperm cells within the perivitelline space, strength (380 mOsm/kg) and isotonic (305
pronuclear stage, and 2- to 4-cell stages. The mOsm/kg) media, respectively, were pene-
overall impression was that sperm penetra- trated. Support for the idea of great variabil-
tion and/or the fertilization process was ity of the required time interval following
retarded. It can also be seen that there is insemination for sperm penetration through
much variability in development following the zona was also afforded by the finding of
exposure of ova to sperm of various bucks. actively motile sperm cells within the perivi-
From Table 2, for example, 25 (48.1%) of 52 telline space (Fig. 4) of 2 ova, still lacking
fertilized ova developed to the 4-cell stage well-developed pronuclei 25 h after insemina-
when sperm from B-36 were used while only 2 tion with B-61’s sperm treated with isotonic
(16.7%) of 12 fertilized ova developed to the medium. Late pronuclear stage ova were
4-cell stage when sperm from B-39 were used. found in the same incubation dish (Fig. 5),

FIG. 4. Rabbit ovum penetrated by sperm capacitated in vitro. Sperm from 8-61 was treated with 305
mOsm/kg defined medium prior to in vitro insemination approximately 25 h before the ovum was
photographed. Note sperm cells, which were still motile, within the perivitellmne space at II o’clock and 3
o’clock. Photographed under interference contrast.
 CAPACITATION OF RABBIT SPERMATOZOA ifl vitro 267

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FIG. 5. Rabbit ovum in late pronuclear stage approximately 25 h after in vitro insemination with sperm
from B-6l treated with 305 mOsm/kg defined medium. Photographed under interference contrast.

while ova from the same ovum pool were in In general, lower sperm motility correlated
the 2-cell stage 25 h after insemination with with lower fertilization, but subjective esti-
high-salt treated sperm. mates of the percentage of motile sperm cells
Similar proportions of ova showed signs of is not a completely reliable criterion for
fertilization following in vitro insemination prediction of success with in vitro fertiliza-
with sperm treated with isotonic defined tion. The quality of sperm motility seems
medium (Table 3) as those following insemi- more important than the actual proportion of
nation with sperm treated with hypertonic a sample which is motile. Addition of high
defined medium (Table 2). Also, similar salt medium to sperm cells was frequently
proportions of ova were found in each devel- followed by alteration of the quality of motil-
opmental stage, regardless of the sperm treat- ity, which was manifested by an increased
ment. These results taken with those obtained flagellation resulting in a more forceful, pro-
by the use of the radioimmunoassay indicate gressive type movement similar to that previ-
that complete removal or alteration of the ously described for hamster spermatozoa fol-
sperm surface seminal plasma components lowing in vitro capacitation (Yanagimachi,
detected by the labeled antibodies prior to 1969a, 1970). A distinct impression developed
incubation of sperm with ova is not an that the rate of progressive movement of
essential prerequisite for the achievement of sperm cells from males B-36 and B-61 was
fertilization under these conditions. However, considerably greater than that of other males
the initiation of removal or alteration of these used.
sperm surface antigenic components seems to Individual ejaculates gave varying re-
be required prior to insemination for success- sponses to treatments in this work, For
ful achievement of fertilization. example, sperm from bucks B-36 and B-61
268 BRACKETT AND OLIPHANT

were capable of fertilizing ova in every experi- Zealand White buck, B-57, was subjected to
ment, whereas sperm from bucks B-15, B-39 the same treatment and retained good motil-
and B-45 gave variable results. In addition, ity, i.e., greater than 50 percent; and cleavage
sperm motility from these latter three bucks of 4 of 45 ova inseminated resulted (3 in 2-cell
was absent or severely depressed following and one in 4-cell stages).
treatments in some experiments which In addition to observations of ova in the
yielded capacitated sperm in other experi- fresh state for evidence of fertilization with in
ments. Therefore, the impression was that vitro capacitated sperm, in many cases nor-

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individual ejaculates from the same male vary mal cleavage was documented by demonstra-
in their ability to undergo capacitation by the tion of chromatin in each blastomere follow-
in vitro means reported here. In one experi- ing staining with lacmoid. Also, stained sec-
ment, sperm from male B-45 was capacitated tions of representative ova were carefully
in vitro by exactly the same hypertonic treat- examined by light microscopy for morpholog-
ment and with the same solutions which ical details, including the presence of supple-
resulted in the apparent death of male B-IS’s mentary sperm cells within the perivitelline
sperm. In an additional experiment, treated space (Fig. 6). Good evidence of fertilization
sperm from male B-IS was judged to have was provided in many experiments by devel-
motility too poor to justify its use, i.e., less opment to the apparently normal 4-cell stage
than 5 percent. Sperm from another New (Fig. 7). For further documentation of au-
thentic fertilization, an effort was made to

Fic. 6. Toluidine blue stained section of a 2-cell stage

rabbit ovum fertilized in vitro by B-IS’s sperm capaci- FIG. 7. Four-cell stage rabbit embryo fertilized in
tated in vitro following treatment with 380 mOsm/kg vitro by sperm from B-6l treated with 380 mOsm/kg
defined medium. Note supplementary sperm within defined medium prior to in vitro insemination. Photo-
perivitelline space at I o’clock. graphed 28 h after insemination under phase contrast.
 CAPACITATION OF RABBIT SPERMATOZOA lfl vitro 269

assess subsequent in vivo development of 2- resorbed (or were aborted early and missed).
and 4-cell stage ova resulting from insemina- Fifty-two 2-cell stage and 22 4-cell stage ova,
tion with in vitro capacitated sperm. Forty- resulting from in vitro capacitation and ferti-
three 2-cell and 38 4-cell stage ova fertilized lization experiments in which sperm of B-61
in vitro by in vitro capacitated sperm from was used, were transferred into 5 recipient
male B-36 were surgically transferred into does. Three of the animals became pregnant.
oviducts of 5 recipients. Only two of the Three normal-sized implantation sites and 8
recipients became pregnant. Ten embryos smaller-than-normal implantation sites were

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implanted but only two of the implantation observed. The 3 embryos that developed
sites appeared to be of normal size, i.e., at normally through implantation were carried
least 3.5 cm in diameter at laparotomy 14 successfully to the completion of gestation in
days after the transfer. Further development 2 recipient does; one of which was New
was not observed. Five 2-cell and 12 4-cell Zealand White and the other, Dutch Belted.
stage ova resulting from in vitro insemination Two of the offspring, from the New Zealand
with in vitro capacitated sperm of male B-45 White recipient, were male (Fig. 8).
were transferred into oviducts of 3 recipients,
DISCUSSION
but implantation failed to occur. In an experi-
ment with sperm from male B-57, three 2-cell The immunological approach has provided
ova and one 4-cell stage ovum were trans- a means for assessing changes on the sperm
ferred; and 3 of the 4 cleaved ova implanted. surface; and, by combining this approach
Two of the implantation sites appeared nor- with in vitro assessment of fertilizing ability,
mal at 2 wks after transfer but were later a better understanding of capacitation is

FIG. 8. Male offspring resulting from embryo transfer of cleaved ova fertilized in vitro by in vitro
capacitated sperm. Ejaculated sperm of Dutch Belted buck, 8-61, was washed, incubated in defined
medium, and re-washed prior to insemination of New Zealand White ova.
 270 BRACKETT AND OLIPHANT

possible. Differential binding of antibodies The absence of striking evidence for more
against rabbit seminal plasma to the sperm rapid penetration of ova by the sperm treated
surface reflects removal or alteration of sur- with the 380 mOsm/kg medium lends support
face antigens. Removal of antigens from the to this interpretation.
sperm surface is the most likely explanation Since individual bucks, specifically B-36
of the immunological results presented, but and B-61, yielded sperm that provided better
the data might also be interpreted as modifi- evidence for fertilization following these
cation of surface antigens such as changes in treatments, it might be expected that, for

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protein tertiary structure or regrouping of ejaculates from these bucks, sperm surface
antigens brought about by the treatments. seminal plasma components would be more
Although the results of in vitro fertilization readily removed or altered by the gentler,
(Tables 2 & 3), taken together with the isotonic treatment, or that the seminal
radioimmunoassay data (Figs. 1, 2 & 3), plasma components might be removed or
indicate complete removal or alteration of altered to a greater degree by the high ionic
seminal plasma components detected by the strength medium. However, the data (Fig. 3)
labeled antibodies is not a necessary prerequi- revealed no large discrepancies in this param-
site to in vitro insemination for successful eter between 380 mOsm/kg treated sperm of
accomplishment of fertilization, optimal in individual males suggesting complete removal
vitro fertilization results have been shown or alteration of the surface components de-
previously to follow use of in vivo capacitated tected by the antibodies and only small
sperm with seminal plasma components not differences between isotonically treated
detectable by the antibodies in an agglutina- sperm of bucks in this regard. Refinements of
tion test (Oliphant and Brackett, l973a). the techniques might conceivably provide
Under the latter conditions, sperm penetra- meaningful insight regarding the fertilizing
tion through the zona pellucida occurs soon ability of an individual ejaculate, but this
after mixing the gametes, i.e., within 3 h remains for further research, In light of
(Brackett, l970b). The present work demon- present results, the variability afforded by
strates that an immunologically detectable sperm of individual bucks might be explained
initiation of change(s) in seminal plasma by differences in metabolic characteristics of
components is necessary for fertilization to the cells which enable certain ones to better
occur in vitro, since use of once-washed sperm survive the rigors of surface antigen removal
cells, presumably with inhibitory seminal or alteration, sequential enzyme release and
plasma components still coating the sperm the acrosome reaction, and to maintain ade-
surface, led to no evidence of fertilization. quate propulsive motility for penetration of
Complete loss of recognition of surface anti- the ovum.
gens by the antibodies occurred in much less In evaluating the fertilizing ability of sperm
time in the 380 mOsm/kg medium than the by in vitro fertilization, one point that must
time interval used for incubation in these be remembered is the possibility of variation
experiments (Fig. 2); and, since 305 in fertilizing ability of the ova. Individual
mOsm/kg medium provided similar in vitro variation in quality of ova remains a possible
fertilization results, the progressive changes explanation for fertilization failure. An addi-
initiated presumably continued following in- tional point for academic consideration is the
semination in 305 mOsm/kg medium. This possibility of variation of fertilizing ability of
might have enhanced the fertilizing ability of individual sperm cells within the sperm popu-
the 305 mOsm/kg treated sperm; and, on the lation under study. The progress of the fertili-
other hand, it might have been detrimental to zation process resulting from insemination
the high-salt medium treated sperm, since with in vitro capacitated sperm showed great
harsher treatment with high-salt medium variability in these experiments. The variabil-
seems to lead to sperm membrane damage. ity seemed to reflect variation in the time at
 CAPACITATION OF RABBIT SPERMATOZOA in vitro 271

which sperm penetration could occur. This adenosine 3’,5’-monophosphate (cAMP), as

might be due, in part, to differences in time suggested in recent experiments with the rat
required for the acrosome reaction of individ- (Toyoda and Chang, l974b) and in experi-
ual fertilizing sperm cells to take place under ments with bull sperm in which a marked
the influence of products of ovulation or to stimulation of fructolysis was demonstrated
the variability of barriers presented by the (Hoskins, 1973). Toyoda and Chang (1974b)
ova to the sperm. The most reasonable expla- found an increased K7Na ratio, along with
nation is that the acrosome reaction was the addition of cAMP, to contribute to their

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delayed due to dilution of factors such as best conditions for in vitro capacitation of rat
follicular fluid that are normally present in sperm. Sperm from individual males might be
higher concentrations in association with re- variably benefited by these and other treat-
cently ovulated ova. The question of whether ments that influence metabolic activity.
rabbit sperm capacitated in vitro, as de- Earlier reports of in vitro capacitation of
scribed here, can fertilize ova following exper- rabbit sperm include those of Kirton and
imental removal of follicular cells must be Hafs (1965) and Ericsson (1969) in which
explored. Recent data demonstrate that rab- fertilizing ability of treated sperm was as-
bit sperm undergo an acrosome reaction in sessed following tubal insemination of test
response to products of ovulation, especially does. Since this approach was inadequate to
follicular fluid (Oliphant, 1974). Support satisfactorily discriminate between in vitro
along this line is also provided by experiments and in vivo effects, these efforts have been
reported by Bavister (1973) in which he interpreted more recently as the partial ac-
obtained capacitation of hamster sperm dur- complishment of capacitation in vitro. More
ing incubation in culture medium and subse- recently, Ericsson, et a!. (1971), reported
quently observed the acrosome reaction under evidence for in vitro fertilization by rabbit
the influence of products of ovulation in vitro. epididymal sperm pre-incubated with Sendai
The importance of the quality of motility of virus. The authors speculated that the sperm
in vivo capacitated sperm for successful ferti- membrane was altered to effect a combined
lization of rabbit ova in vitro has long been capacitation-acrosome reaction, enabling the
recognized (Bedford and Chang, 1962). The sperm cells to penetrate and activate the ova.
impression that a change in character of Ogawa, et a!. (1972), reported that epididy-
sperm motility occurs soon after exposure of mal sperm from males treated with HCG
the cells to the high ionic strength medium fits could fertilize ova recovered near the end of
with previously described transitions of sperm their fertilizable life and that the ova could be
motility during the capacitation process of cultured to the blastocyst stage. Several com-
the hamster (Yanagimachi, 1970), mouse plex tissue culture media were used in combi-
(Iwamatsu and Chang, 1969) and guinea pig nation, and the observed effect might well
(Yanagimachi, 1972). This change reflects an have been due to the increased osmolality of
increased metabolic rate which is most likely the medium (measured in our laboratory to
related to the dramatic increase in oxygen be 326 mOsm/kg) rather than to supplying
uptake known to occur in sperm undergoing the gametes with needed nutritional factors or
capacitation in vivo (Hamner and Williams, to any effect of HCG treatment on epididy-
1963; Brackett, 1968). This increased meta- mal sperm. A recent report by Okinaga, eta!.
bolic activity also seems to involve increased (1974), claimed accomplishment of in vitro
levels of adenyl cyclase activity, as reported fertilization with diluted ejaculated rabbit
for hamster sperm undergoing capacitation sperm. In this work, the best cleavage rate
(Morton and Albagli, 1973; Rogers and Mor- was obtained when tubal ova recovered 18 h
ton, 1973). Enhancement of sperm capacita- after HCG injection were exposed to trypsin
tion in the present experiment might be and inseminated in a complex medium with
possible by elevating levels of sperm cyclic added serum, estradiol, estriol, and HCG.
272 BRACKETF AND OLIPHANT

Again, the possibility that increased osmolal- tated) by incubation in hypertonic medium.
ity of the medium might have contributed to Although many ova fertilized by in vitro
this reported success should be explored. capacitated sperm in these experiments were
In previous experiments from our labora- able to undergo implantation in uteri of
tory, rabbit spermatozoa were found capable recipients, only a few completed gestational
of inducing cleavage of freshly ovulated ova development to term, Fraser and Dandekar
in vitro following conditioning of the sperm (1973) reported fertilization of ova held under
cells with uterine fluid recovered from gona- similar conditions for 12 h before insemina-

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dotropin-treated does near the time of ovula- tion in vitro. However, in their experiments in
tion (Brackett, el a!., 1972). Such uterine which 2- to 4-cell stage embryos were trans-
fluid was also shown to effect removal or ferred to recipients, they found ova recovered
alteration of sperm coating antigens, as de- within approximately 3 h after ovulation and
tected by decreased binding of labeled anti- fertilized in vitro had low implantation rates;
bodies against seminal plasma. Restoration and none of the implants developed to full-
of antibody binding was observed following term fetuses, in contrast to approximately
incubation in seminal plasma or with deca- 21% development of ova recovered within
pacitation factor (Oliphant and Brackett, approximately 2 h of the onset of ovulation.
l973a). The observed change in seminal This result suggests a striking decrease in
plasma components on the sperm surface embryonic viability due to even a brief inter-
under physiological conditions is probably val of aging of ova prior to sperm penetra-
brought about enzymatically rather than, or tion. The embryonic wastage in the present
in addition to, being mediated by elevated experiments might be explained by inade-
ionic strength. The osmolality of rabbit semi- quate preparation of sperm cells prior to
nal plasma is 296-3 17 mOsm/kg (Mann, fertilization. But, most likely, the explanation
1964) and that of rabbit oviductal fluid is lies in the fact that the ova fertilized were
approximately 310 mOsm/kg, which is quite old at the time penetration occurred;
higher than that of blood plasma, approxi- and this aging decreased the viability of
mately 293 mOsm/kg (Brunton and Brusi- resulting embryos. Polyspermy represents an
low, 1972). In the present experiments, incu- additional possible explanation for the fetal
bating the sperm cells in defined media be- wastage, but the ova observed in the pronu-
tween two washing procedures was found to clear stage uniformly contained only 2 pronu-
alter seminal plasma components coating the clei. The fact that male offspring resulted
sperm surface, which presumably prevent from transfer of embryos fertilized in vitro by
fertilization from taking place. These find- in vitro capacitated sperm documents the
ings, combined with those from our mouse authenticity of achievement of sperm capaci-
experiments (Oliphant and Brackett, l973b), tation and fertilization in vitro.
suggest that the capacitation process takes
place by a similar mechanism in various ACKNOWLEDGM ENTS

mammalian species. Optimal osmolalities for The authors gratefully acknowledge the encourage-
ment of Dr. M. C. Chang. The authors thank Mr.
penetration of mouse and hamster ova by
Michael J. DeMarco. Mr. George G. Jeitles, Jr., Dr. Y.
epididymal sperm under in vitro conditions K. Oh, Mrs. Joan Trammell, and Mr. David Zimrin for
reported by Miyamoto and Chang (1973) expert assistance with various facets of this work.
were 308 to 372 mOsm/kg and 292 to 392 This work was supported by National Institutes of
mOsm/kg, respectively, findings compatible Health Grants HDO 6274 and HDO 0130, Career
Development Award HD 15861. and by a grant from the
with the presently reported data. A similar
Ford Foundation.
process may even be common to vertebrates
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