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Fertilization of guinea pig eggs in vitro.

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Fertilization of Guinea Pig Eggs IR Vitro
R. YANAGIMACHI
Department of Anatomy and Reproductive Biology, University of
Hawaii School of Medicine, Honolulu, Hawaii 96822
ABSTRACT
Guinea pig eggs can be fertilized in nitro in a chemically defined
medium originally developed by Biggers, Whitten and Whittingham for the cultivation of fertilized mouse eggs. When unfertilized eggs were inseminated in this
medium (abbr. BWW) with fresh epididymal spermatozoa, sperm penetration
occurred not earlier than two hours after insemination. Quick and efficient sperm
penetration occurred when the eggs were inseminated with spermatozoa preincubated in BWW for 11-18 hours. Spermatozoa started to pass through the
zona pellucida as early as 10-15 minutes following insemination and all the eggs
were penetrated by spermatozoa within one hour. The time needed for functional
capacitation of guinea pig spermatozoa is estimated to be no less than two hours
under the experimental conditions used, the optimal time possibly being 8-12
hours or even longer. The possibility of in vitro sperm capacitation and fertilization of eggs in media much simpler than BWW (e.g., Earle's solution with or
without albumin) is suggested.
Prior to 1961, the rabbit was the only
mammalian species for which unequivocal
evidence of in vitro fertilization had been
presented (Auslin, '61a). Since then, in
vitro fertilization has been reported in the
mouse, rat, golden hamster, Chinese
hamster, cow, sheep and human (for review, see Chang, '68; Austin, '70; Brackett,
'70, '71 ).
The guinea pig was used in some of the
pioneer work on in vitro fertilization.
Schenk (1878) inseminated follicular eggs
of guinea pig with epididymal spermatozoa
in the presence of follicular fluid and
uterine mucosa, and claimed that he obtained successful in vitro fertilization after
observing emission of the polar body and
cleavage of the eggs. Unfortunately, emission of the polar body and cleavage of the
eggs may be due to spontaneous activation
or fragmentation of the eggs and cannot
be taken as unequivocal evidence of fertilization (Austin, '61a,b). It is rather surprising that no one since those early experiments has reported further work on the
in vitro fertilization in this species. This
paper reports that in vitro fertilization of
guinea pig eggs can be effected with a
simple technique, using chemically defined
media with relatively simple constituents.
ANAT. REC., 174: 9-20.
MATERIALS AND METHODS
The standard medium used throughout
this study was the one developed by
Biggers, Whitten and Whittingham ('71)
for cultivation of fertilized mouse eggs.
The medium (abbr. BWW) was prepared
without addition of any antibiotics, sterilized by filtration through a millipore filter,
stored in a refrigerator (1"-3" C ) and
used in experiments within one week after
preparation. Spermatozoa from distal caudae epididymides of fertile male guinea
pigs were suspended in warm (30"-38" C )
BWW a t a concentration of approximately
2-3 X lo' spermatozoa per ml. A drop
(0.05-0.1 m l ) of the sperm suspension
was placed under mineral oil in a watchglass and incubated a t 37"-38" C (gas
phase, air). After various times of incubation, the motility and morphology of the
spermatozoa were appraised using a phasecontrast microscope. Unfertilized eggs were
collected from unbred female guinea pigs,
in the morning (9:OO to 12:OO A M ) following the evening of behavioral estrus, by
flushing each of the oviducts with BWW.
The eggs surrounded by cumulus cells
(fig. 1 1 ) were rinsed three times with
BWW by transferring them from one dish
Received March 6, '72. Accepted M a y 12, '72.
9
10
R. YANAGIMACHI
of BWW to another. The eggs were kept in
0.1-0.2 ml of fresh BWW (37"-38" C )
under mineral oil in a watchglass for no
longer than 30 minutes before insemination. Insemination was performed by introducing 0.01-0.02 ml of the sperm suspension into 0.1-0.2 ml of BWW containing
the eggs. After the eggs and spermatozoa were thoroughly mixed with a needle,
the preparation was incubated at 37"38" C (gas phase, air). At various times
after insemination, the eggs were examined with a high power dissecting microscope for dispersion of the cumulus cells
and attachment of spermatozoa to the
zona pellucida. The eggs were then
mounted between a slide and coverslip,
compressed moderately, and examined
with a phase-contrast microscope for the
evidence of sperm penetration. For karyological details of the eggs, the mounted
eggs were fixed overnight with a mixture
of glacial acetic acid and ethanol ( I : 3 ) ,
stained with 0.25% lacmoid in 45%
acetic acid, cleared with 45% acetic acid,
and examined with a phase-contrast microscope.
RESULTS
Prominent features of guinea pig spermatozoa are the extraordinarily large size
of their acrosomes and a tendency to stack
by their heads (acrosomal area to acrosomal area). Even within the epididymis,
many spermatozoa show the head to head
stacking (fig. 1 and Fawcett and Hollenberg, '63). A mucus-like substance over
the sperm plasma membrane of the acrosomal region (fig. 2 ) may be responsible
for such stacking. When fresh epididymal
spermatozoa were suspended in BWW,
these stacked spermatozoa as well as free
spermatozoa showed active motility, and
within ten minutes many large masses of
agglutinated spermatozoa appeared (compare figs. 7 and 8 with fig. 3). These large
masses of agglutinated spermatozoa could
be broken up by pipetting, but the spermatozoa soon reagglutinated into large
masses. At 30 minutes after suspension in
BWW, a l l or the vast majority of motile
spermatozoa, either agglutinated or nonagglutinated, had apparently intact acrosomal caps (figs. 4-6). When examined
between two and five hours after suspension, a few spermatozoa (usually 0.050 . 5 % , sometime 2-5%) had lost their
acrosomal caps and showed free, active
movement. The number of such freely
swimming spermatozoa lacking acrosomal
caps (figs. 9, 10) increased sharply after
incubation for about ten hours (sometimes
7-8 hours) or longer. Many of these spermatozoa showed very vigorous serpentine
movement which was somewhat similar to
the movement of capacitated hamster spermatozoa (Yanagimachi, '70). On many OCcasions, a large proportion of the spermatozoa lacking acrosomal caps showed only
slow movements after a long (e.g., 12-24
hours) incubation. However, on dilution
of these sperm suspensions with fresh
BWW (37"-38" C ) , many of these spermatozoa resumed vigorous movement.
I n order to determine how quickly the
spermatozoa begin to enter the eggs, unfertilized eggs were inseminated with
fresh epididymal spermatozoa (no preincubation of sperm). Within 30 minutes
after insemination, most of the cumulus
cells surrounding the eggs had dispersed,
and a few spermatozoa without acrosomal
caps had attached to the surface of the
zona pellucida (fig. 14). The spermatozoa
with intact acrosomal caps, either agglutinated or non-agglutinated, came into
contact with the zona surfaces, but none
of them made permanent attachment. The
number of the eggs penetrated by spermatozoa (with swelling sperm heads or male
pronuclei within the ooplasm) was 0 out
of 3, 0 out of 6, 1 out of 6 (fig. 15) and
2 out of 5 at 1, 2, 4 and 6 hours after
insemination, respectively.
Quick and very efficient sperm penetration occurred when the eggs were inseminated with spermatozoa preincubated in
BWW for 11-18 hours. The cumulus cells
surrounding the eggs were completely dispersed within ten minutes after insemination, and many active spermatozoa without acrosomal caps firmly attached to the
zona pellucida (figs. 16, 17). Rotation of
the eggs by these spermatozoa was commonly observed. Sperm heads lacking
acrosomal caps could be seen within the
zona pellucida as early as 10-15 minutes
after insemination (fig. 18). By 20-30
minutes after insemination, sperm heads
I N VITRO FERTILIZATION
had already reached the ooplasm surface
(fig. 19) or were within the ooplasm
(fig. 20). Cortical granules seen in the
unfertilized eggs (fig. 12) had disappeared
completely or almost completely by this
time. A total of 32 eggs examined between
one and four hours after insemination all
showed definitive signs of sperm penetration (figs. 21-27). The number of spermatozoa that entered the ooplasm of a n egg
varied from one to five. In a series of experiments, seven eggs were inseminated
with spermatozoa preincubated in BWW
for 11-13 hours. About 30 minutes after
insemination, the eggs were repeatedly
washed by pipetting to remove excess
spermatozoa attached to the zona pellucida.
After these eggs had been transferred to
fresh BWW under mineral oil and cultured
for 24 hours under a n atmosphere of 5%
COZ in air, all but one of them were at the
two-cell stage (fig. 28).
In another series of experiments, 12
eggs were treated with 0.2% hyaluronidase
(bovine testicular, 300 USP units/mg, Nutritional Biochem.) and 0.2% pronase
(from Streptomyces griseus, B grade, 450
proteolytic units/mg, Calbiochem.) in
BWW. As soon as the cumulus cells had
dispersed and the zonae had dissolved
completely ( i n about 10 minutes at room
temperature), the eggs were thoroughly
rinsed with BWW free of enzymes. When
seven of these “naked” eggs were inseminated with fresh epididymal spermatozoa
(no preincubation of sperm), many spermatozoa with intact acrosomal caps firmly
attached to the eggs (figs. 29, 30). In spite
of such immediate sperm attachment, none
of these eggs showed signs of sperm penetration (presence of swelling sperm heads
within ooplasm) earlier than two hours
after insemination. I t appeared from repeated observations that the spermatozoa
with intact acrosomal caps could not penetrate into the ooplasm; only those that had
lost their acrosomal caps while retaining
high motility could do so. When the five
remaining “ n a k e d eggs were inseminated
with spermatozoa that had been preincubated in BWW for 12-18 hours, many
active spermatozoa lacking acrosomal caps
immediately attached to the egg surfaces.
By one hour after insemination, all of
11
these eggs were heavily penetrated by
spermatozoa.
Finally, mention should be made of the
possibility of in vitro fertilization of guinea
pig eggs in chemically defined media much
simpler than BWW. After suspension ‘of
Earle’s (’43) balanced salt solution and
incubation under mineral oil for 10-18
hours (gas phase, either air or 5% CO1
in air), a few guinea pig epididymal spermatozoa (usually 0.05-0.5% , sometimes
2-5% ) lost their acrosomal caps and
showed vigorous movement. Addition of
bovine or human serum albumin (2-10
mg/ml) to Earle’s solution significantly
increased the incidence of such spermatozoa (sometimes 50-60% ). A similar
phenomenon was found with Tyrode’ solution, although this solution was considerably less effective than Earle’s solution in
its ability to induce acrosome reaction.
When spermatozoa were preincubated in
Earle’s solution containing albumin ( 4
mg/ml) for 14-16 hours a t 37”-38” C
(gas phase, either air or 5% CO, in air)
and used for in vitro insemination of unfertilized eggs with both cumulus cells and
zona pellucida, 100% (12 out of 12) of
the eggs were penetrated by spermatozoa
within one hour following insemination.
Even with spermatozoa preincubated for
15-16 hours in pure Earle’s solution (no
albumin at all), 100% ( 5 out of 5 ) of the
eggs in Earle’s solution were penetrated by
spermatozoa within one hour after insemination.
DISCUSSION
The data presented above clearly indicate that epididymal spermatozoa of the
guinea pig can fertilize the eggs in vitro
in chemically defined media with relatively
simple constituents. This does not necessarily imply that the spermatozoa of this
species do not require “capacitation” to
fertilize the eggs. It seems more feasible
to consider that the spermatozoa were
capacitated in these media and then
effected fertilization .
The necessity of the acrosome reaction
(the loss of the acrosomal caps from living
spermatozoa, not the loss of degeneration
of acrosomes in moribund or dead spermatozoa) for successful sperm penetration
through the zona pellucida has been well
12
R. YANAGIMACHI
documented in several mammalian species tions of the medium could be responsible
(Austin and Bishop, '58; Austin, '61b; Bed- for the induction of capacitation. Under in
ford, '70a,b,c) and has been confirmed in vitro conditions, the spermatozoa of these
the present study. The exact relationship species may not require the presence of
between the acrosome reaction and capaci- female genital tract fluid, but this should
tation is not known a t the present time, not be taken as an indication that female
but it appears reasonable to assume that genital tract fluid or its Components have
the acrosome reaction occurs either in the nothing to do with capacitation of spermafinal stage of capacitation (Austin, '64; tozoa under natural (in vivo) conditions.
Yanagimachi, '69) or as a sequential event We should consider that the chemically defollowing the completion of capacitation fined media have some characteristics in
(Bedford, '70a). The time necessary for common with genital tract fluid and simply
sperm capacitation seems to vary from mimic its action.
species to species and also to depend on
ACKNOWLEDGMENTS
the physiological conditions of the animals
as well as the experimental systems used
This study was supported by grants from
(Austin, '70; Bedford, '70a,b; Chang, '70). NIH-USPHS (HD-03402 and HD-02066),
No one has ever reported or suggested the the Population Council and the Ford
time needed for the capacitation of guinea Foundation. The author expresses his appig spermatozoa, although there have been preciation to Dr. Y. D. Noda for preparaseveral papers dealing with fertilization in
this species (Squier, '32; Austin and tion of a n electron micrograph (fig. 2)
Bishop, '58; Hunt and Chang, '64; Hunter, used i n this paper, and to Mrs. C. A. Mahi,
Hunt and Chang, '69). The present experi- Dr. J. M. Cummins and Dr. B. F. Lino for
ments were not designed to determine the their assistance in preparation of the
capacitation time of guinea pig sperma- manuscript.
tozoa, but the data obtained seem to sugLITERATURE CITED
gest that it is no less than two hours under Austin, C. R. 1961a Fertilization eggs in vitro.
the experimental (in vitro) conditions
Int. Rev. Cytol., 12: 337-359.
1961b The Mammalian Egg. Charles
used. The optimal time could be 8-12
C Thomas, Springfield.
hours or even longer.
1964 Behaviour of spermatozoa in the
According to Bavister ('69), golden
female genital tract and i n fertilization. Proc.
hamster eggs can be fertilized in vitro by
Vth Int. Congr. Anim. Reprod., 3: 7-22.
epididymal spermatozoa in the presence
1970 Sperm capacitation-Biological significance i n various species. In: Advances i n
of a modified Tyrode's solution containing
Biosciences, 4-Schering Symposium on Mechaalbumin and pyruvate (with a possible
nisms Involved i n Conception. G. Rasp-4, ed.
trace of oviductal fluid). He inferred that
Pergamon Press, Vieweg, pp. 5-11.
capacitation of hamster spermatozoa could Austin, C. R., and M. W. H. Bishop 1958 Role
of the rodent acrosome and perforatorium i n
be spontaneous with the fluid in the ovifertilization. Proc. Roy. SOC., Ser. B, 149:
duct (combination of oviductal and follicu24 1-248.
lar fluids) merely creating a favorable Bavister,
B. D. 1969 Environmental factors
environment of capacitation. A similar
important for in vitro fertilization in the
hamster. J. Reprod. Fertil., 1 8 : 544-545.
inference was made by Toyoda, Yokoyama
and Hosi ('71a,b) who obtained in vitro Bedford, J. M. 1970a Sperm capacitation and
fertilization i n mammals. Biol. Reprod. Suppl.,
fertilization of mouse eggs following in2 128-158.
semination of the eggs with epididymal
1970b The saga of mammalian sperm
spermatozoa in a modified Krebs-Ringer
from ejaculation t o syngamy. In: Mammalian
Reproduction. H. Gibian and E. J. Poltz, eds.
solution containing albumin and pyruvate.
Springer-Verlag, New York, pp. 124-182.
Although there seems to be no doubt that
1970c Morphological aspects of sperm
the spermatozoa of these two species as
capacitation i n mammals. In: Advances in
well as the guinea pig (the present study)
Biosciences, 4-Schering Symposium on Mechanisms Involved in Conception. G. Rasp&, ed.
can be capacitated in simple chemically
Pergamon Press, Vieweg, pp. 35-50.
defined media, we have to be cautious Biggers,
J. D., W. K. Whitten and D. G. Whittingabout the use of the word "spontaneous,"
ham 1971 The culture of mouse embryos
since some specific components or condiin uitro. In: Methods of Mammalian Embryol-
IN VITRO FERTILIZATION
ogy. J. C. Daniel, ed. Freeman and Co., San
Francisco, pp. 86-116, Table 6-5.
Brackett, B. G. 1970 In vitro fertilization of
mammalian ova. In: Advances in Biosciences,
4-Schering Symposium on Mechanisms Involved
in Conception. G . Raspe, ed. Pergamon Press,
Vieweg, pp. 73-94.
1971 Recent progress in investigations
of fertilization in vitro. In: The Biology of the
Blastocyst. R. J. Blandau, ed. Univ. Chicago
Press, Chicago, pp. 329-348.
Chang, M. C. 1968 Im vitro fertilization of
mammalian eggs. J. Anim. Sci., 27 (Suppl. 1):
15-22.
1970 Hormonal regulation of sperm
capacitation. In: Advances in Biosciences,
4-Schering Symposium on Mechanisms Involved
in Conception. G. RaspC, ed. Pergamon Press,
Vieweg, pp. 13-33.
Earle, W. R. 1943 Production of malignancy
in vitro. IV. The mouse fibroblast cultures and
changes seen i n the living cells. J. Nat. Cancer
Inst., 8: 165-212.
Fawcett, D. W., and R. D. Hollenberg 1963
Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis.
2. Zellforsch., 60: 276-292.
Hunt, D. M., and M. C. Chang 1964 Fertilization and cleavage of guinea pig ova. Anat. Rec.,
148: 378. Abstr.
13
Hunter, R. H. F., D. M. Hunt and M. C. Chang
1969 Temporal and cytological aspects of
fertilization and early development in the
guinea pig, Cavia poreellus. Anat. Rec., 165:
411-430.
Schenk, S. L. 1878 Das Saugethierei kunstlich
befruchtet ausserhalb des Mutterthieres. Mitt.
Embryol. Inst. K. K. Univ. Wien, I: 107-118.
Squier, R. R. 1932 The living egg and early
stages of its development in the guinea-pig.
Contr. Embryol. Carnegie Inst., 32: 223-254.
Toyoda, Y., M. Yokoyama and T. Hosi 1971a
Studies on the fertilization of mouse eggs in
vitro. I. In vitro fertilization of eggs by fresh
epididymal sperm. Japan. J. Anim. Reprod.,
16: 147-151.
1971b Studies on fertilization of mouse
eggs in vitro. 11. Effects of in vitro pre-incubation of spermatozoa on time of sperm penetration of mouse eggs in vitro. Japan. J. Anim.
Reprod., 16: 152-157.
Yanagimachi, R. 1969 In vitro capacitation of
hamster spermatozoa by follicular fluid. J. Reprod. Fertil., 18: 275-286.
1970 The movement of golden hamster
spermatozoa before and after capacitation.
J. Reprod. Fertil., 23: 193-196.
PLATE 1
EXPLANATION O F FIGURES
14
1
Spermatozoa in the distal cauda epididymis, showing head-to-head
stacking. Phase-contrast. x 900.
2
Sagittal sections of the heads of stacked spermatozoa, shortly
after being suspended in BWW. Note the presence of mucus-like
substance ( m ) over the acrosomal area of the spermatozoa. Fixed
i n 2.5% glutaraldehyde, postfixed with 1% O s 0 4 and embedded
i n Epon. The section was stained with lead citrate and uranyl
acetate. x 10,000.
3-6
Spermatozoa immediately after being suspended in BWW. Figure 3,
dark field, x 90; figure 4, stacked spermatozoa, phase-contrast,
X 900; surface (fig. 5 ) and side (fig. 6) views of free spermatozoa, phase-contrast, x 900.
7-8
Spermatozoa after one hour of suspension i n BWW, showing large
masses of head-to-head stacking of spermatozoa. Figure 7, dark
field, x 90; figure 8, somewhat strongly compressed under the
coverslip, phase-contrast, x 900.
9-10
Surface (fig. 9) and side (fig. 10) views of spermatozoa lackin?
acrosomal caps (compare with figs. 5, 6 ) , after 14 hours incubation in BWW. These spermatozoa were extremely active before
being photographed. Phase-contrast, x 900.
IN VITRO FERTILIZATION
R. Yanagimachi
PLATE 1
15
PLATE 2
EXPLANATION OF FIGURES
11
An unfertilized egg surrounded by cumulus cells. Phase-contrast, X 400.
12 Cortical granules ( c g ) of an unfertilized egg. The cortical granules located
over large cytoplasmic granules are obscured by refraction effects. Phasecontrast, x 900.
13
The second meiotic division metaphase chromosomes of a n unfertilized egg.
Fixed with acetic alcohol and stained with aceto-lacmoid. Phase-contrast,
x 900.
14 A n egg (unfertilized) one hour after insemination with fresh epididymal
spermatozoa. The egg was slightly compressed under the coverslip. Phasecontrast, x 400.
15
A swelling sperm head (arrow) in a n egg four hours after insemination with
fresh epididymal spermatozoa. The nucleus of this egg was at the second
meiotic division telophase. Fixed with acetic alcohol and stained with acetolacmoid. Phase-contrast, X 900.
16-17
Eggs 20 minutes after insemination with spermatozoa preincubated for 14
hours in BWW, showing many spermatozoa attached to the zona pellucida.
The eggs were slightly compressed under the coverslip. Figure 16, x 400;
figure 17, note the absence of acrosomal caps from the spermatozoa, x 900.
Phase-contrast.
18 Egg with a sperm head (arrow) in the zona pellucida ( z ) 15 minutes after
insemination with preincubated spermatozoa. The tail of this spermatozoon
was vigorously beating when photographed. Phase-contrast, x 900.
19 Egg with a sperm head (arrow) which has reached the surface of the
ooplasm 20 minutes after insemination with preincubated spermatozoa. The
posterior portion of the sperm head may have been lifted because of rolling
the egg under coverslip. Ps, perivitelline space; z, zona pellucida. Phasecontrast, x 900.
16
IN VITRO FERTILIZATION
R. Yanagimachi
PLATE 2
17
PLATE 3
EXPLANATION O F FIGURES
20-21
Eggs 30 minutes (fig. 2 0 ) and 60 minutes (fig. 21) after insemination with
preincubated spermatozoa. Arrows indicate sperm tails i n the ooplasm.
Phase-contrast, x 900.
22-23
Sperm head (fig. 22) and egg nucleus at the second meiotic division anaphase
(fig. 23), one hour after insemination with preincubated spermatozoa. A n
arrow (fig. 22) indicates the sperm tail. Fixed with acetic alcohol and stained
with aceto-lacmoid. Phase-contrast, x 900.
24-25
Sperm head (fig. 24) and egg nucleus a t the second meiotic division telophase (fig. 25), between one and one-half and two hours after insemination
with preincubated spermatozoa. A n arrow (fig. 24) indicates sperm tail. Fixed
with acetic alcohol and stained with aceto-lacmoid. Phase-contrast, x 900.
26-27
Male (fig. 2 6 ) and female (fig. 2 7 ) pronuclei in a n egg, four hours after
insemination with preincubated Spermatozoa. Fixed with acetic alcohol and
stained with aceto-lacmoid. Phase-contrast, X 900.
28
29-30
18
A n egg a t the two-cell stage, fertilized and cultured in nitro. Bright field, X 400.
Zona-free eggs two hours after insemination with fresh epididymal spermatozoa, showing attachment to ooplasm surface of many spermatozoa with intact
acrosomal caps. The egg was strongly compressed under the coverslip. Phasecontrast. Figure 29, X 400; figure 30, X 900.
IN VlTRO FERTILIZATION
R. Yanagimachi
PLATE 3
19
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