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Capacitation of Rabbit Spermatozoa in the Uterus*
Vol. 21, No.9, September 1970 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright
© 1970 by The Williams
& Wilkins Co.
CAPACITATION OF RABBIT SPERMATOZOA IN THE UTERUS* BENJAMIN G. BRACKETT, D.V.M., PH.D.,
AND
JOEL B. SERVER, B.S.
Division of Reproductive Biology, Department of Obstetrics and Gynecology, School of Medicine, and Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Since the discovery of a need for rabbit spermatozoa to undergo a conditioning in the female reproductive tract ( capacitation) prior to becoming capable of penetrating an ovum, 1 • 10 several investigations have been carried out in an attempt to understand the hormonal influences on this process. 2 • 3· 11 - 14 • 16· 20 • 21 The ability of spermatozoa to fertilize ova has been tested by in vivo methods in most of these studies. In vitro fertilization has been the method of choice for testing the capacity of rabbit spermatozoa to fertilize ova in our laboratory.3-6· 8 • 9 • 17 • 18 • 22 · 23 By this means it was possible to demonstrate that no direct effect of the oviduct on the ovum prior to fertilization is necessaryY Subsequent experiments revealed that the oviduct is also unnecessary for sperm capacitation and that union of ovum and spermatozoon can occur in vitro in the absence of previous exposure of either gamete to the influence of the oviduct. 18 The purpose of the present investigation is to examine the fertilizing ability of spermatozoa recovered from uteri of ovariectomized and salpingectomized does and to improve the uterus as a sperm-capacitating environment by hormone administration. MATERIALS AND METHODS
Mature New Zealand white rabbits were used. They were individually caged and fed
* Supported by Grants FR 00-340-04 and HD 01810-05 from the National Institutes of Health, United States Public Health Service, and by the Ford Foundation. Presented at the 26th Annual Meeting of the American Fertility Society, Washington, D. C., March 18-21, 1970.
a diet of Purina Rabbit Chow. Seven- to eight-month-old estrus does were used as donors of ova or as donors of uterine spermatozoa ( capacitators). Both ovaries and both oviducts, including the uterotubal junctions, were removed surgically ( ovariectomies and salpingectomies) from most of the does to be used as capacitators. Two does to be used as capacitators in Experiments 23-23 and 23-28 were subjected to ovariectomies and the oviducts were left intact. These surgical procedures were done under aseptic conditions and under pentobarbital sodium anesthesia. Following surgery, the capacitators were returned to individual cages and held for at least 1 month before they were used. They were either untreated or treated by intramuscular injection of cottonseed oil (CSO), by intramuscular injection of 0.5 f'-g. of estradiol cyclopentyl propionate (ECP, Upjohn)/kg. of body wt. for 3 days, by 6 daily intramuscular injections of 0.12, 0.25, 0.50, or 1.00 f'-g. of ECP /kg. of body wt. prior to mating, or by 6 daily intramuscular injections of 0.5 f'-g. of ECP jkg. of body wt. plus a single intramuscular injection of 0.25 mg. of progestin/kg. of body wt. given at the time of mating, which was 6Y2 hr. after the last ECP injection. The progestins used were 17ahydroxyprogesterone (A 4 - pregnen - 17 ;;_ ol - 3, 20 - dione) and 20a - hydroxyproges terone (A 4 - pregnen - 20a - ol - 3 - one) purchased from Sigma Chemical Company. The ECP was diluted with sterile cottonseed oil and the progestins were dissolved in ethanol before use. Comparison of in vitro fertilization results afforded by uterine
687
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BRACKETT AND SERVER
688
spermatozoa from estrogen-treated (0.5 p.g. of ECP /kg. of body wt. for 6 days) capacitators with those afforded by uterine spermatozoa from estrogen- and progestintreated capacitators were made and the data were statistically analyzed by x2 with Yates correction. Ovum donors were superovulated with 150 I.U. of pregnant mare serum gonadotropin (Gestyl, Organon) given intramuscularly, followed by 75 I.U. of human chorionic gonadotropin (HCG) (A.P.L., Ayerst) given intravenously 72-84 hr. later. Ovum recovery was accomplished in some experiments in the manner previously reportedP· 18 These experiments included all of those in which capacitators were given estrogen for 3 days, Experiments 23651 and 24-41, and both experiments in which capacitators were given estrogen and progestin. Briefly this method of ovum recovery involved surgical removal of both fimbriae and a portion of each ampulla 9V2 hr. after HCG injection. The abdominal cavity was closed temporarily and the ani-
I\
I PMSG I
I HCG I
mal was allowed to ovulate while still under pentobarbital sodium anesthesia. The ovaries were removed 11% hr. after HCG injection (in the present work), submerged in sterile 37° C. saline, and carried into a 37° C. culture room, where the ova surrounded by cumulus cells were removed from their ovulation sites on the ovarian surface. In the course of the present study, the surgical procedure (fimbriectomy) prior to ovulation was found to be unnecessary. The experimental procedure adopted in this study is summarized in Fig. 1. Surprisingly, more ova could be recovered from the ovarian surface if the animals were killed 11% hr. after HCG injection with no previous precautions taken to prevent the oviduct from collecting ovulated ova. After removal from the ovum donors, the ovaries were trimmed free of extraneous tissue and held in a 250-ml. beaker of 37° C. sterile saline. One ovary at a time was transferred to a tissue culture dish (with a 40-mm. inside diameter and a depth of 12 mm.), where
I OVARIECTOMY AND
SPERM RECOVERED AND OVA
OVUM RECOVERY
INSEMINATED
OVA TRANSFERRED TO CULTURE MEDIUM
OVA EXAMINED FOR CLEVAGE
-12--o------11314----12 112-----17112--37 HOURS
FIG. 1. Schematic diagram of experimental plan for in vitro fertilization of ovulated ova recovered from the ovarian surfaces used in these experiments.
September 1970
RABBIT SPERM CAPACITATION IN UTERUS
the ova with surrounding cumulus cells were removed from it. This operation was executed most easily under a dissecting microscope by pinching the cumulus close to the ovary with one pair of jeweler's forceps while another pair of jeweler's forceps was used to free the mass from the first forceps. With a Pasteur pipet·· (over-all length of 230 mm.), the ovum in cumulus then was transferred to a smaller (30-mm. diameter and 12 mm. deep) tissue culture dish containing 4.0 ml. of synthetic medium with 20% heated rabbit serum. 5 After all of the ovulated ova were collected, the capacitator was killed and the uterus was flushed with 4.0 ml. of synthetic medium with 20% heated rabbit serum. The resulting suspension, usually containing uterine spermatozoa, was placed into a small tissue culture dish and covered with paraffin oil which had been equilibrated with 5% C0 2 in air. To this fertilization medium were added the ova and with the top of the dish excess oil was forced out, leaving all space above the medium occluded by oil when the top was in place. Then the dish was wrapped in foil to avoid light and incubated under a moist 5% C0 2 -in-air atmosphere. Rapid equilibration to 5% C0 2 tension was ensured by the use of a small chamber (anaerobic culture apparatus, small size, Arthur H. Thomas Company) . Five hours after in vitro insemination the ova were transferred to 10% serum solution8 and incubated under an air atmosphere until the time of examination, approximately 24 hr. postinsemination. All ova were examined at 30X under a dissecting microscope and again at 160X under phase contrast. Normal ovum cleavage with the presence of sperm cells around the ova was taken as evidence of fertilization. Each capacitator, with two exceptions, was mated two to five times to fertile males 17-17Y:! hr. prior to the time of sperm recovery. In each of two experiments (Experiments 24-54 and 24-58) an ejaculate was collected by use of an artificial
689
vagina, washed once by dilution to 5.0 ml. with Krebs-Ringer phosphate solution with 100 U. of penicillin G sodium and 1.0 mg. of streptomycin sulfate added per milliliter, followed by centrifugation at 734 X g for 5 min. The supernatant solution was discarded and the cells were resuspended and counted. The capacitators in these experiments were anesthesized with pentobarbital sodium and their uteri were surgically exposed. Sperm cells (8 to 9 X 106 ) were injected through a 27-gauge needle in 0.2ml. volume into each uterine horn near the cervix 17Y-i hr. before sperm recovery. All solutions, instruments, and glassware were sterile before use and manipulations of ova were carried out aseptically. Tissue was taken from the middle of the uterine horns of untreated capacitators and from those treated with the above mentioned levels of ECP. The uterine tissue was fixed in Bouin's solution, dehydrated, infiltrated with paraffin, and embedded in paraffin. Sections, 5 p. in thickness, were stained with Delafield's hematoxylin and alcoholic eosin Y. RESULTS
Surgical intervention to prevent ovum pickup by the oviductal fimbria was found to be unnecessary. By recovering ovaries from ovum donors after an interval of 11% hr. after the HCG injection, it was found that more ova were clinging to the ovarian surface than was the case when ovaries were recovered at a comparable time but 2 hr. after removal of fimbria. More than twice as many ova were recovered from each donor when surgery was eliminated from the ovum-recovering procedure (Table 1). The mean number of ova recovered from each donor without prior fimbriectomies, 13.3, represented 48.4% of the mean number of ova ovulated by each ovum donor, 27.5, as estimated by counting ovulation points. Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingecto-
690
BRACKETT AND SERVER
TABLE 1. Recovery of Ovulated Ova from the Ovarian Surface Fimbria re-
No. of
Yes No
74
401
58
770
~o:~1f[{~~ d':,';,':,~s
No. of ova Mean no. of ova from each recovered donor (range)
5.4 (0-18.5) 13.3 (6.3-29.0)
TABLE 2. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Experiment
no.
Special treatment of capacitator
Ova ferNo. of sperm Fertilper milliliter Motil- tilized and ity ova of fertilizare- ization tionmedium cover.l'd
-- - 24-23 24-28 24-39 24-45 24-54
24-58
CSO and uterine insemination
32,000
80
12/15 0/11 0/39 0/16 22/46
Uterine insemination
54,000
10
0/24
None None
4,000
50
None found None found None found
cso cso
--
%
%
80
0
0 0 0 47.8
TABLE 3. Fertilizing Ability of Uterine Sperm from Surgically Treated Capacitators given 0.5 1-'Y· of ECP /kg. of Body Wt. for 3 Days No. of Ova fer tilExper- sperm per Surgical treatment iment mlilliliter Motility izedand Fertilof fertiliof capacitator ova re- ization no. zation medium ---
covered
-- -- -% %
Ovariectomized
23-23 23-28
114,000 52,000
30 50
18/25 3/6
72 50
Ovariectomized and salpingectomized
23-28 23-39 23-421 23-431 23-30 23-33
12,000 14,000 18,000 9"8,000 98,000 270,000
80 15 60 65 25 40
0/8 0/8 1/7 25/36 5/7 3/7
0 0 14.3 69.5 71.5 42.8
mized Does (Table 2). Sperm cells were recovered from the uterine horns of only 1 of 4 ovariectomized and salpingectomized capacitators force-mated to fertile males. A high proportion of ova were fertilized by a relatively low concentration of uterine spermatozoa in this instance (Experiment 24-23, Table 2). The usual absence of spermatozoa in the uteri of these capacitators is best explained by failure of the
Vol. 21
cervix to allow entry to the uterine lumen in the absence of any estrogenic influence. Dead sperm cells were found in the vagina of each doe that yielded no uterine spermatozoa. Since sperm capacitation did occur when spermatozoa reached the uterus (Experiment 24-23, Table 2) two experiments were done to see if washed ej aculated spermatozoa could become capacitated following injection directly into the uterine lumen. In one such experiment (Experiment 24-54, Table 2) capacitation did occur. In the other (Experiment 24-58, Table 2) the sperm motility was very poor after recovery from the uterus and the proper conclusion might be that the capacitating system was overloaded by the large number of spermatozoa used. Fertilizing Ability of Uterine Spermatozoa from Surgically Treated Capacitators Given 0.5 p.g. of ECP /kg. of Body W t. for 3 Days (Table 3). In two experiments (Experiments 23-23 and 23-28, Table 3) 21 of 31 ova (67.7%) were fertilized by uterine spermatozoa from ovariectomized capacitators given 0.5 p.g. of ECP /kg. of body wt. for 3 days. A comparable proportion of ova was fertilized, 33 of 50 ova (66%), in three experiments (Experiments 23-431, 23-30, and 23-33, Table 3) by uterine spermatozoa from ovariectomized and salpingectomized capacitators given the same hormone treatment when there were 98,000 or more sperm cells in each milliliter of the fertilization medium. In three experiments (Experiments 23-28, 23-39, and 23-42I, Table 3), fewer uterine spermatozoa were recovered from comparably treated capacitators of the latter type. Only one of 23 ova was fertilized when 12,000 to 18,000 sperm cells were present in each milliliter of the fertilization medium. The estrogen treatment used in these experiments appeared to be insufficient either to ensure consistent passage of large enough numbers of spermatozoa into the uterus or to adequately capacitate the spermatozoa that did reach the uterus.
September 1970
RABBIT SPERM CAPACITATION IN UTERUS
Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingectomized Does Given ECP for 6 days (Table 4). Uterine spermatozoa from ovariectomized and salpingectomized capacitators treated for 6 days with levels of ECP ranging from 0.25-1.00 p.g.jkg. of body wt. fertilized a high proportion of ova (Experiments 24-35, 23-651, 24-37, 24-41, and 24-56, Table 4). The 0.12 p.g.j kg. of body wt. level of ECP was not enough to provide access of the spermatozoa to the uterus (Experiment 24-48, Table 4). Dead sperm cells were found in the vagina of the capacitator in this experiment. The optimal level of ECP appeared to be 0.5 p.g./kg. of body wt. given each day for 6 days. Uterine spermatozoa from ovariectomized and salpingectomized capacitators given this treatment fertilized 85 of 112 ova TABLE 4. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Given ECP for 6 Days Treatment
Ova Experi- No. of sperm fer tilment per milliliter Motility izedand Fertilof fertilizano. ova re- ization tion medium covered
--
,.g. ECP/kg. of body wt.
%
0.12
24-48
None found
0.25
24-35
200,000
0.50
23-65! 24-37 24-41
1.00
24-56
--
% 0/34
0
75
15/25
60
414,000 152,000 8,000
80 85 20
24/24 52/72 9/16
100 72 56
132,000
50
25/49
51
I
691
(75.9%) in three experiments (Experiments 23-651, 24-37, and 24-41, Table 4). Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingectomized Does Given 0.5 p.g. of ECP /kg. of Body Wt. for 6 Days and 0.25 mg. of Progestin/kg. of Body Wt. at Time of Mating (Table 5). When 0.25 mg. of either 17a-hydroxyprogesterone (A. 4 -pregnen-17aol-3, 20-dione) or 20a- hydroxyprogesterone (A. 4 -pregnen-20a-ol-3-one) was given at the time of mating to estrogen-treated (0.5 p.g. of ECP/kg. of body wt. for 6 days) capacitators without ovaries and oviducts, the resulting uterine spermatozoa were capable of fertilizing a high proportion of ova. Of 53 ova, 48 (90.6%) were fertilized in vitro in these two experiments (23-571 and 23-531, Table 5). This represents a statistically significant (p < 0.05) increase over the proportion of ova that were fertilized by spermatozoa capacitated in uteri of animals treated with estrogen only. This proportion of in vitro fertilization exactly equals that (90.6%) reported previously by the same procedure in which intact estrus capacitators were used. 18 Histologic Observations. Sections of the uterine horns of capacitators that were untreated were compared with those of capacitators given the various levels of estrogen described above. Figure 2 depicts a typical unstimulated endometrium from a capacitator given no estrogen. This section was taken from the capacitator of Experiment 24-23, Table 2, and the results
TABLE 5. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Given 0.5 p.g. of ECP/kg. of Body Wt.for 6 Days and 0.25 mg./kg. of Body Wt. of Progestin at Time of Mating Experiment no.
Treatment
No. of sperm per milliliter of fertilization medium
Motility
Ova fertilized and ova recovered
Fertilization
23-57!
17a-OH-progesterone (4. 4 -pregnen -17a-ol -3, 20-dione)
200, ()()()
% 60
28/31
% 90
23-531
20a-OH-progesterone (4. 4 -pregnen -20a-ol -3-one)
218,000
65
20/22
91
692
BRACKETT AND SERVER
Vol. 21
FIG. 2. Unstimulated endometrium from the capacitator of Experiment 24-23 (Table 2). Note the absence of well-developed glands and capillaries.
or estrogen but that it could not be achieved in the progesterone-dominated uterus.n He obtained similar in vivo fertilization result::: with uterine spermatozoa from untreated and estrogen-treated ovariectomized doe:::. Chang injected a small volume of diluted semen into the uterine horns as was done with washed spermatozoa on two occasions (Experiments 24-54 and 24-58, Table 2) in the present work. The earliest time following uterine insemination of epididymal spermatozoa that ova could be penetrated when placed into the uterus was found to be 17Y2 hr. by Bedford. 2 Although consistent results were not obtained in the present study, the impression was gained that under ideal conditions, especially DISCUSSION regarding sperm numbers 'and length of Twelve years ago, Chang reported that uterine incubation, a high degree of capacicapacitation was achieved in the uterus of tating efficiency can be attained in the immature and ovariectomized rabbits with unstimulated uterus. Bedford has recently or without treatment with gonadotrophin reached a similar conclusion regarding the
of this experiment clearly show that capacitation of spermatozoa occurred in this uterine environment, although only approximately 16,000 sperm cells were recovered from it. Figure 3 depicts a stimulated endometrium from the capacitator of Experiment 24-37, Table 4, which was typical of those observed following six daily injections of 0.5 p.g. of ECP /kg. of body wt. Sperm capacitation also occurred in this uterine environment and in this case approximately 608,000 sperm cells were recovered. It appears that the estrogen stimulation may enable the uterine environment to capacitate a greater number of spermatozoa.
september 1970
RABBIT SPERM CAPACITATION IN UTERUS
1.0 p.g. of estradiol benzoate/kg. of body wt., given for 14 days after ovariectomy provided maximal capacitating efficiency, as evidenced by a fertilization rate of 70%. Hamner and Sojka treated ovariectomized does with 0.5 p.g. of ECP /kg. of body wt. for 6 days and obtained a fertilization rate of 86%. 14 In the present report the latter level of ECP gave better results than did other dosages tried. Hilliard, Archibald, and Sawyer reported the synthesis and release of 20a-hydroxyprogesterone and progesterone to be acti-
vated by gonadotrophic stimulation in the preovulatory period in the rabbitP The 20a-hydroxy steroid was produced in quantities approximately 10-fold greater than progesterone. These progestins were isolated and identified in ovarian venous blood. 19 Progesterone is converted to 17a- hydroxyprogesterone by cumulus clots recovered from rabbit oviducts soon after ovulation. 7 It was postulated by Soupart that 20ahydroxyprogesterone might participate in the capacitation process. 20 Recently, however, he reported an absence of synergy when increasing levels of 20a-hydroxyprogesterone were given to estrogen-stimulated ovariectomized does. 21 A statistically significant (p < 0.05) enhancement of capacitating efficiency was observed in the present investigation when a low dose of progestin was given to estrogen-primed ovariectomized and salpingectomized capacitators. The experimental conditions
694
Vol. 21
BRACKETT AND SERVER
differed from those of Soupart.20 • 21 The manner of hormone administration might prove to be a significant factor in the discrepancy of results. In this study the capacitators were operated on at least 1 month before they were used, whereas Soupart began injections of estradiol benzoate the day after operation. Other striking differences include the longer uterine incubation of spermatozoa following its ejaculation into the capacitator's vagina and the in vitro fertilization method for assaying for capacitation in this work. Soupart injected epididymal spermatozoa directly into the uterine lumen and tested capacitation efficiency by tubal insemination 13 hr. after an ovulating injection of HCG and duplicated his findings by using a different method for in vitro fertilization.21 In comparing the capacitating efficiency of the estrogen-treated capacitator's uterus with that of the estrogen- and progestintreated capacitator's uterus, certain assumptions were necessarily made. Technically, all conditions were comparable; however, whether animal variations, difference in number of uterine spermatozoa recovered, or other -less obvious factors were responsible for the observed results are all questions to be answered through further experimentation. SUMMARY
Capacitation of rabbit spermatozoa in the uterus of ovariectomized and salpingectomized does has been investigated. Spermatozoa were recovered from uteri 17-17Y2 hr. after coitus or uterine insemination. The capacitating efficiency was assayed by in vitro fertilization of ovulated ova recovered from the ovarian surface. It was found that surgical intervention was unnecessary in preventing pickup of ova by the fimbria prior to ovum recovery in this procedure. When fimbriectomies were not performed, an average of 13.3 ova were
recovered from each superovulated ovum donor. Sperm capacitation could be accomplished in the uterus of untreated does with no ovaries or oviducts, provided that the sperm cells gained access to the uterus. Capacitation occurred in the uterus of ovariectomized does and also ovariectomized and salpingectomized does after 3 days of estrogen treatment (0.5 p.g. of ECP /kg. of body wt.). More consistent results were obtained with ovariectomized and salpingectomized does when they were given the same treatment but for 6 days. This estrogen treatment (0.5 p.g. of ECPI kg. of body wt. for 6 days) led to a fertilization rate of 75.9%, which represented better capacitating efficiency than was observed with other levels of estrogen tried. A significant (p < 0.05) increase in the fertilization rate, to 90.6%, was observed when 0.25 mg. of either 17a-hydroxyprogesterone or 20a-hydroxyprogesterone was given to the estrogen-treated capacitator at the time of mating. The fertilization rate resulting from the latter treatment was exactly the same as that previously found to be afforded by uterine spermatozoa from intact estrus capacitators.18 Acknowledgments. We are grateful to Miss Mary Peace, Mrs. Joan Trammell, Mr. Don Killen, Mrs. Karen Wright, and Mr. John Klaus for their excellent technical assistance during the course of this research. We also thank Mr. William Harper for care of the animals and Mrs. Harriette Turner for typing the manuscript. REFERENCES 1. AusTIN, C. R. Observations of the penetration
of the sperm into the mammalian egg. Aust J &iRes B4:581, 1951. 2. BEDFORD, J. M. The influence of oestrogen and progesterone on sperm capacitation in the reproductive tract of the female rabbit. J Endocr 46:191, 1970. 3. BRACKETT, B. G. Respiration of spermatozoa after in utero incubation in estrus and pseudo-
September 1970
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
RABBIT SPERM CAPACITATION IN UTERUS
pregnant rabbits. VI Int Congr Anim Reprod (Paris) 1 :43, 1968. BRACKETT, B. G. Effects of washing the gametes on fertilization in vitro. Fertil Steril 20:127,1969. BRACKETT, B. G. "In .Vitro Fertilization of Mammalian Ova." In Advances in the Biosciences, Schering Symposium on Mechanisms Involved in Conception, Berlin, 1969 (Vol. 4), Raspe, G., Ed. Pergamon and Vieweg, New York,1969. BRACKETT, B. G. In vitro fertilization of rabbit ova: time sequence of events. Fertil Steril 21 :169, 1970. BRACKETT, B. G., AND FLICKINGER, G. L. Unpublished data. BRACKETT, B. G., AND WILLIAMS, W. L. In vitro fertilization of rabbit ova. J Exp Zool 160:271, 1965. BRACKETT, B. G., AND WILLIAMS, W. L. Fertilization of rabbit ova in a defined medium. Fertil Steril19 :144, 1968. CHANG, M. C. Fertilizing capacity of spermatozoa deposited in the fallopian tubes. Natture (London) 168 :697, 1951. CHANG, M. C. Capacitation of rabbit spermatozoa in the uterus with special reference to the reproductive phases of the female. Endocrinology 63 :619, 1958. CHANG, M. C. "Hormonal Regulation of Sperm Capacitation." In Advances in the Biosciences, Schering Symposium on M echanisms Involved in Conception, Berlin, 1969 (Vol. 4), Raspe, G., Ed. Pergamon and Vieweg, New York, 1969. HAMNER, C. E., JONES, J. P., AND SOJKA, N. J. Influences of the hormonal state of the female on the fertilizing capacity of rabbit spermatozoa. Fertil SteriI19:137, 1968. HAMNER, C. E., AND SOJKA, N. J. Requirements
15.
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23.
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for capacitation of rabbit sperm. Nature (London) 220: 1042, 1968. HILLIARD, J., ARCHIBALD, D., AND SAWYER' C. H. Gonadotropic activation of prevulatory synthesis and release of progestin in the rabbit. Endocrinology 72 :59, 1963. NOYES, R. W., WALTON, A., AND ADAMS, C. E. Capacitation of rabbit spermatozoa. J Endocr 17 :374, 1958. SEITZ, H. M., JR., BRACKETT, B. G., AND MASTROIANNI, L., JR. In vitro fertilization of ovulated rabbit ova recovered from the ovarian surface. Bioi Reprod 2 :262, 1970. SEITZ, H. M., JR., ROCHA, G., BRACKETT, B. G., AND MASTROIANNI, L., JR. Influence of the oviduct on sperm capacitation in the rabbit. Fertil Steril21 :325, 1970. SIMMER, H. H., HILLIARD, J., AND ARCHIBALD, D. Isolation and identification of progesterone and 20 a-hydroxy-pregn-4-en-3-one in ovarian venous blood of rabbits. Endocrinology 72: 67, 1963. SOUPART, P. Studies on the hormonal control of rabbit sperm capacitation. J Reprod Fertil (Suppl. 2) :49, 1967. SOUPART, P. "The Effect of 20a-Hydroxyprogesterone on Sperm Capacitation in the Rabbit Uterus." In Society jor the Study oj Reproduction: Abstracts oj Papers, 2nd Annual Meeting, University of California, Davis, Calif., 1969. STAMBAUGH, R., BRACKETT, B. G., AND MASTROIANNI, L., JR. Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors. Bioi Reprod 1 :223, 1969. WILLIAMS, W. L., HAMNER, C. E., WEINMAN, D. E., AND BRACKETT, B. G. Capacitation of rabbit spermatozoa and initial experiments on in vitro fertilization. V Int Congr Anim Reprod (Trento) 7 :228, 1964.
FERTILITY AND STERILITY
Copyright
© 1970 by The Williams
& Wilkins Co.
CAPACITATION OF RABBIT SPERMATOZOA IN THE UTERUS* BENJAMIN G. BRACKETT, D.V.M., PH.D.,
AND
JOEL B. SERVER, B.S.
Division of Reproductive Biology, Department of Obstetrics and Gynecology, School of Medicine, and Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Since the discovery of a need for rabbit spermatozoa to undergo a conditioning in the female reproductive tract ( capacitation) prior to becoming capable of penetrating an ovum, 1 • 10 several investigations have been carried out in an attempt to understand the hormonal influences on this process. 2 • 3· 11 - 14 • 16· 20 • 21 The ability of spermatozoa to fertilize ova has been tested by in vivo methods in most of these studies. In vitro fertilization has been the method of choice for testing the capacity of rabbit spermatozoa to fertilize ova in our laboratory.3-6· 8 • 9 • 17 • 18 • 22 · 23 By this means it was possible to demonstrate that no direct effect of the oviduct on the ovum prior to fertilization is necessaryY Subsequent experiments revealed that the oviduct is also unnecessary for sperm capacitation and that union of ovum and spermatozoon can occur in vitro in the absence of previous exposure of either gamete to the influence of the oviduct. 18 The purpose of the present investigation is to examine the fertilizing ability of spermatozoa recovered from uteri of ovariectomized and salpingectomized does and to improve the uterus as a sperm-capacitating environment by hormone administration. MATERIALS AND METHODS
Mature New Zealand white rabbits were used. They were individually caged and fed
* Supported by Grants FR 00-340-04 and HD 01810-05 from the National Institutes of Health, United States Public Health Service, and by the Ford Foundation. Presented at the 26th Annual Meeting of the American Fertility Society, Washington, D. C., March 18-21, 1970.
a diet of Purina Rabbit Chow. Seven- to eight-month-old estrus does were used as donors of ova or as donors of uterine spermatozoa ( capacitators). Both ovaries and both oviducts, including the uterotubal junctions, were removed surgically ( ovariectomies and salpingectomies) from most of the does to be used as capacitators. Two does to be used as capacitators in Experiments 23-23 and 23-28 were subjected to ovariectomies and the oviducts were left intact. These surgical procedures were done under aseptic conditions and under pentobarbital sodium anesthesia. Following surgery, the capacitators were returned to individual cages and held for at least 1 month before they were used. They were either untreated or treated by intramuscular injection of cottonseed oil (CSO), by intramuscular injection of 0.5 f'-g. of estradiol cyclopentyl propionate (ECP, Upjohn)/kg. of body wt. for 3 days, by 6 daily intramuscular injections of 0.12, 0.25, 0.50, or 1.00 f'-g. of ECP /kg. of body wt. prior to mating, or by 6 daily intramuscular injections of 0.5 f'-g. of ECP jkg. of body wt. plus a single intramuscular injection of 0.25 mg. of progestin/kg. of body wt. given at the time of mating, which was 6Y2 hr. after the last ECP injection. The progestins used were 17ahydroxyprogesterone (A 4 - pregnen - 17 ;;_ ol - 3, 20 - dione) and 20a - hydroxyproges terone (A 4 - pregnen - 20a - ol - 3 - one) purchased from Sigma Chemical Company. The ECP was diluted with sterile cottonseed oil and the progestins were dissolved in ethanol before use. Comparison of in vitro fertilization results afforded by uterine
687
Vol. 21
BRACKETT AND SERVER
688
spermatozoa from estrogen-treated (0.5 p.g. of ECP /kg. of body wt. for 6 days) capacitators with those afforded by uterine spermatozoa from estrogen- and progestintreated capacitators were made and the data were statistically analyzed by x2 with Yates correction. Ovum donors were superovulated with 150 I.U. of pregnant mare serum gonadotropin (Gestyl, Organon) given intramuscularly, followed by 75 I.U. of human chorionic gonadotropin (HCG) (A.P.L., Ayerst) given intravenously 72-84 hr. later. Ovum recovery was accomplished in some experiments in the manner previously reportedP· 18 These experiments included all of those in which capacitators were given estrogen for 3 days, Experiments 23651 and 24-41, and both experiments in which capacitators were given estrogen and progestin. Briefly this method of ovum recovery involved surgical removal of both fimbriae and a portion of each ampulla 9V2 hr. after HCG injection. The abdominal cavity was closed temporarily and the ani-
I\
I PMSG I
I HCG I
mal was allowed to ovulate while still under pentobarbital sodium anesthesia. The ovaries were removed 11% hr. after HCG injection (in the present work), submerged in sterile 37° C. saline, and carried into a 37° C. culture room, where the ova surrounded by cumulus cells were removed from their ovulation sites on the ovarian surface. In the course of the present study, the surgical procedure (fimbriectomy) prior to ovulation was found to be unnecessary. The experimental procedure adopted in this study is summarized in Fig. 1. Surprisingly, more ova could be recovered from the ovarian surface if the animals were killed 11% hr. after HCG injection with no previous precautions taken to prevent the oviduct from collecting ovulated ova. After removal from the ovum donors, the ovaries were trimmed free of extraneous tissue and held in a 250-ml. beaker of 37° C. sterile saline. One ovary at a time was transferred to a tissue culture dish (with a 40-mm. inside diameter and a depth of 12 mm.), where
I OVARIECTOMY AND
SPERM RECOVERED AND OVA
OVUM RECOVERY
INSEMINATED
OVA TRANSFERRED TO CULTURE MEDIUM
OVA EXAMINED FOR CLEVAGE
-12--o------11314----12 112-----17112--37 HOURS
FIG. 1. Schematic diagram of experimental plan for in vitro fertilization of ovulated ova recovered from the ovarian surfaces used in these experiments.
September 1970
RABBIT SPERM CAPACITATION IN UTERUS
the ova with surrounding cumulus cells were removed from it. This operation was executed most easily under a dissecting microscope by pinching the cumulus close to the ovary with one pair of jeweler's forceps while another pair of jeweler's forceps was used to free the mass from the first forceps. With a Pasteur pipet·· (over-all length of 230 mm.), the ovum in cumulus then was transferred to a smaller (30-mm. diameter and 12 mm. deep) tissue culture dish containing 4.0 ml. of synthetic medium with 20% heated rabbit serum. 5 After all of the ovulated ova were collected, the capacitator was killed and the uterus was flushed with 4.0 ml. of synthetic medium with 20% heated rabbit serum. The resulting suspension, usually containing uterine spermatozoa, was placed into a small tissue culture dish and covered with paraffin oil which had been equilibrated with 5% C0 2 in air. To this fertilization medium were added the ova and with the top of the dish excess oil was forced out, leaving all space above the medium occluded by oil when the top was in place. Then the dish was wrapped in foil to avoid light and incubated under a moist 5% C0 2 -in-air atmosphere. Rapid equilibration to 5% C0 2 tension was ensured by the use of a small chamber (anaerobic culture apparatus, small size, Arthur H. Thomas Company) . Five hours after in vitro insemination the ova were transferred to 10% serum solution8 and incubated under an air atmosphere until the time of examination, approximately 24 hr. postinsemination. All ova were examined at 30X under a dissecting microscope and again at 160X under phase contrast. Normal ovum cleavage with the presence of sperm cells around the ova was taken as evidence of fertilization. Each capacitator, with two exceptions, was mated two to five times to fertile males 17-17Y:! hr. prior to the time of sperm recovery. In each of two experiments (Experiments 24-54 and 24-58) an ejaculate was collected by use of an artificial
689
vagina, washed once by dilution to 5.0 ml. with Krebs-Ringer phosphate solution with 100 U. of penicillin G sodium and 1.0 mg. of streptomycin sulfate added per milliliter, followed by centrifugation at 734 X g for 5 min. The supernatant solution was discarded and the cells were resuspended and counted. The capacitators in these experiments were anesthesized with pentobarbital sodium and their uteri were surgically exposed. Sperm cells (8 to 9 X 106 ) were injected through a 27-gauge needle in 0.2ml. volume into each uterine horn near the cervix 17Y-i hr. before sperm recovery. All solutions, instruments, and glassware were sterile before use and manipulations of ova were carried out aseptically. Tissue was taken from the middle of the uterine horns of untreated capacitators and from those treated with the above mentioned levels of ECP. The uterine tissue was fixed in Bouin's solution, dehydrated, infiltrated with paraffin, and embedded in paraffin. Sections, 5 p. in thickness, were stained with Delafield's hematoxylin and alcoholic eosin Y. RESULTS
Surgical intervention to prevent ovum pickup by the oviductal fimbria was found to be unnecessary. By recovering ovaries from ovum donors after an interval of 11% hr. after the HCG injection, it was found that more ova were clinging to the ovarian surface than was the case when ovaries were recovered at a comparable time but 2 hr. after removal of fimbria. More than twice as many ova were recovered from each donor when surgery was eliminated from the ovum-recovering procedure (Table 1). The mean number of ova recovered from each donor without prior fimbriectomies, 13.3, represented 48.4% of the mean number of ova ovulated by each ovum donor, 27.5, as estimated by counting ovulation points. Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingecto-
690
BRACKETT AND SERVER
TABLE 1. Recovery of Ovulated Ova from the Ovarian Surface Fimbria re-
No. of
Yes No
74
401
58
770
~o:~1f[{~~ d':,';,':,~s
No. of ova Mean no. of ova from each recovered donor (range)
5.4 (0-18.5) 13.3 (6.3-29.0)
TABLE 2. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Experiment
no.
Special treatment of capacitator
Ova ferNo. of sperm Fertilper milliliter Motil- tilized and ity ova of fertilizare- ization tionmedium cover.l'd
-- - 24-23 24-28 24-39 24-45 24-54
24-58
CSO and uterine insemination
32,000
80
12/15 0/11 0/39 0/16 22/46
Uterine insemination
54,000
10
0/24
None None
4,000
50
None found None found None found
cso cso
--
%
%
80
0
0 0 0 47.8
TABLE 3. Fertilizing Ability of Uterine Sperm from Surgically Treated Capacitators given 0.5 1-'Y· of ECP /kg. of Body Wt. for 3 Days No. of Ova fer tilExper- sperm per Surgical treatment iment mlilliliter Motility izedand Fertilof fertiliof capacitator ova re- ization no. zation medium ---
covered
-- -- -% %
Ovariectomized
23-23 23-28
114,000 52,000
30 50
18/25 3/6
72 50
Ovariectomized and salpingectomized
23-28 23-39 23-421 23-431 23-30 23-33
12,000 14,000 18,000 9"8,000 98,000 270,000
80 15 60 65 25 40
0/8 0/8 1/7 25/36 5/7 3/7
0 0 14.3 69.5 71.5 42.8
mized Does (Table 2). Sperm cells were recovered from the uterine horns of only 1 of 4 ovariectomized and salpingectomized capacitators force-mated to fertile males. A high proportion of ova were fertilized by a relatively low concentration of uterine spermatozoa in this instance (Experiment 24-23, Table 2). The usual absence of spermatozoa in the uteri of these capacitators is best explained by failure of the
Vol. 21
cervix to allow entry to the uterine lumen in the absence of any estrogenic influence. Dead sperm cells were found in the vagina of each doe that yielded no uterine spermatozoa. Since sperm capacitation did occur when spermatozoa reached the uterus (Experiment 24-23, Table 2) two experiments were done to see if washed ej aculated spermatozoa could become capacitated following injection directly into the uterine lumen. In one such experiment (Experiment 24-54, Table 2) capacitation did occur. In the other (Experiment 24-58, Table 2) the sperm motility was very poor after recovery from the uterus and the proper conclusion might be that the capacitating system was overloaded by the large number of spermatozoa used. Fertilizing Ability of Uterine Spermatozoa from Surgically Treated Capacitators Given 0.5 p.g. of ECP /kg. of Body W t. for 3 Days (Table 3). In two experiments (Experiments 23-23 and 23-28, Table 3) 21 of 31 ova (67.7%) were fertilized by uterine spermatozoa from ovariectomized capacitators given 0.5 p.g. of ECP /kg. of body wt. for 3 days. A comparable proportion of ova was fertilized, 33 of 50 ova (66%), in three experiments (Experiments 23-431, 23-30, and 23-33, Table 3) by uterine spermatozoa from ovariectomized and salpingectomized capacitators given the same hormone treatment when there were 98,000 or more sperm cells in each milliliter of the fertilization medium. In three experiments (Experiments 23-28, 23-39, and 23-42I, Table 3), fewer uterine spermatozoa were recovered from comparably treated capacitators of the latter type. Only one of 23 ova was fertilized when 12,000 to 18,000 sperm cells were present in each milliliter of the fertilization medium. The estrogen treatment used in these experiments appeared to be insufficient either to ensure consistent passage of large enough numbers of spermatozoa into the uterus or to adequately capacitate the spermatozoa that did reach the uterus.
September 1970
RABBIT SPERM CAPACITATION IN UTERUS
Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingectomized Does Given ECP for 6 days (Table 4). Uterine spermatozoa from ovariectomized and salpingectomized capacitators treated for 6 days with levels of ECP ranging from 0.25-1.00 p.g.jkg. of body wt. fertilized a high proportion of ova (Experiments 24-35, 23-651, 24-37, 24-41, and 24-56, Table 4). The 0.12 p.g.j kg. of body wt. level of ECP was not enough to provide access of the spermatozoa to the uterus (Experiment 24-48, Table 4). Dead sperm cells were found in the vagina of the capacitator in this experiment. The optimal level of ECP appeared to be 0.5 p.g./kg. of body wt. given each day for 6 days. Uterine spermatozoa from ovariectomized and salpingectomized capacitators given this treatment fertilized 85 of 112 ova TABLE 4. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Given ECP for 6 Days Treatment
Ova Experi- No. of sperm fer tilment per milliliter Motility izedand Fertilof fertilizano. ova re- ization tion medium covered
--
,.g. ECP/kg. of body wt.
%
0.12
24-48
None found
0.25
24-35
200,000
0.50
23-65! 24-37 24-41
1.00
24-56
--
% 0/34
0
75
15/25
60
414,000 152,000 8,000
80 85 20
24/24 52/72 9/16
100 72 56
132,000
50
25/49
51
I
691
(75.9%) in three experiments (Experiments 23-651, 24-37, and 24-41, Table 4). Fertilizing Ability of Uterine Spermatozoa from Ovariectomized and Salpingectomized Does Given 0.5 p.g. of ECP /kg. of Body Wt. for 6 Days and 0.25 mg. of Progestin/kg. of Body Wt. at Time of Mating (Table 5). When 0.25 mg. of either 17a-hydroxyprogesterone (A. 4 -pregnen-17aol-3, 20-dione) or 20a- hydroxyprogesterone (A. 4 -pregnen-20a-ol-3-one) was given at the time of mating to estrogen-treated (0.5 p.g. of ECP/kg. of body wt. for 6 days) capacitators without ovaries and oviducts, the resulting uterine spermatozoa were capable of fertilizing a high proportion of ova. Of 53 ova, 48 (90.6%) were fertilized in vitro in these two experiments (23-571 and 23-531, Table 5). This represents a statistically significant (p < 0.05) increase over the proportion of ova that were fertilized by spermatozoa capacitated in uteri of animals treated with estrogen only. This proportion of in vitro fertilization exactly equals that (90.6%) reported previously by the same procedure in which intact estrus capacitators were used. 18 Histologic Observations. Sections of the uterine horns of capacitators that were untreated were compared with those of capacitators given the various levels of estrogen described above. Figure 2 depicts a typical unstimulated endometrium from a capacitator given no estrogen. This section was taken from the capacitator of Experiment 24-23, Table 2, and the results
TABLE 5. Fertilizing Ability of Uterine Sperm from Ovariectomized and Salpingectomized Does Given 0.5 p.g. of ECP/kg. of Body Wt.for 6 Days and 0.25 mg./kg. of Body Wt. of Progestin at Time of Mating Experiment no.
Treatment
No. of sperm per milliliter of fertilization medium
Motility
Ova fertilized and ova recovered
Fertilization
23-57!
17a-OH-progesterone (4. 4 -pregnen -17a-ol -3, 20-dione)
200, ()()()
% 60
28/31
% 90
23-531
20a-OH-progesterone (4. 4 -pregnen -20a-ol -3-one)
218,000
65
20/22
91
692
BRACKETT AND SERVER
Vol. 21
FIG. 2. Unstimulated endometrium from the capacitator of Experiment 24-23 (Table 2). Note the absence of well-developed glands and capillaries.
or estrogen but that it could not be achieved in the progesterone-dominated uterus.n He obtained similar in vivo fertilization result::: with uterine spermatozoa from untreated and estrogen-treated ovariectomized doe:::. Chang injected a small volume of diluted semen into the uterine horns as was done with washed spermatozoa on two occasions (Experiments 24-54 and 24-58, Table 2) in the present work. The earliest time following uterine insemination of epididymal spermatozoa that ova could be penetrated when placed into the uterus was found to be 17Y2 hr. by Bedford. 2 Although consistent results were not obtained in the present study, the impression was gained that under ideal conditions, especially DISCUSSION regarding sperm numbers 'and length of Twelve years ago, Chang reported that uterine incubation, a high degree of capacicapacitation was achieved in the uterus of tating efficiency can be attained in the immature and ovariectomized rabbits with unstimulated uterus. Bedford has recently or without treatment with gonadotrophin reached a similar conclusion regarding the
of this experiment clearly show that capacitation of spermatozoa occurred in this uterine environment, although only approximately 16,000 sperm cells were recovered from it. Figure 3 depicts a stimulated endometrium from the capacitator of Experiment 24-37, Table 4, which was typical of those observed following six daily injections of 0.5 p.g. of ECP /kg. of body wt. Sperm capacitation also occurred in this uterine environment and in this case approximately 608,000 sperm cells were recovered. It appears that the estrogen stimulation may enable the uterine environment to capacitate a greater number of spermatozoa.
september 1970
RABBIT SPERM CAPACITATION IN UTERUS
1.0 p.g. of estradiol benzoate/kg. of body wt., given for 14 days after ovariectomy provided maximal capacitating efficiency, as evidenced by a fertilization rate of 70%. Hamner and Sojka treated ovariectomized does with 0.5 p.g. of ECP /kg. of body wt. for 6 days and obtained a fertilization rate of 86%. 14 In the present report the latter level of ECP gave better results than did other dosages tried. Hilliard, Archibald, and Sawyer reported the synthesis and release of 20a-hydroxyprogesterone and progesterone to be acti-
vated by gonadotrophic stimulation in the preovulatory period in the rabbitP The 20a-hydroxy steroid was produced in quantities approximately 10-fold greater than progesterone. These progestins were isolated and identified in ovarian venous blood. 19 Progesterone is converted to 17a- hydroxyprogesterone by cumulus clots recovered from rabbit oviducts soon after ovulation. 7 It was postulated by Soupart that 20ahydroxyprogesterone might participate in the capacitation process. 20 Recently, however, he reported an absence of synergy when increasing levels of 20a-hydroxyprogesterone were given to estrogen-stimulated ovariectomized does. 21 A statistically significant (p < 0.05) enhancement of capacitating efficiency was observed in the present investigation when a low dose of progestin was given to estrogen-primed ovariectomized and salpingectomized capacitators. The experimental conditions
694
Vol. 21
BRACKETT AND SERVER
differed from those of Soupart.20 • 21 The manner of hormone administration might prove to be a significant factor in the discrepancy of results. In this study the capacitators were operated on at least 1 month before they were used, whereas Soupart began injections of estradiol benzoate the day after operation. Other striking differences include the longer uterine incubation of spermatozoa following its ejaculation into the capacitator's vagina and the in vitro fertilization method for assaying for capacitation in this work. Soupart injected epididymal spermatozoa directly into the uterine lumen and tested capacitation efficiency by tubal insemination 13 hr. after an ovulating injection of HCG and duplicated his findings by using a different method for in vitro fertilization.21 In comparing the capacitating efficiency of the estrogen-treated capacitator's uterus with that of the estrogen- and progestintreated capacitator's uterus, certain assumptions were necessarily made. Technically, all conditions were comparable; however, whether animal variations, difference in number of uterine spermatozoa recovered, or other -less obvious factors were responsible for the observed results are all questions to be answered through further experimentation. SUMMARY
Capacitation of rabbit spermatozoa in the uterus of ovariectomized and salpingectomized does has been investigated. Spermatozoa were recovered from uteri 17-17Y2 hr. after coitus or uterine insemination. The capacitating efficiency was assayed by in vitro fertilization of ovulated ova recovered from the ovarian surface. It was found that surgical intervention was unnecessary in preventing pickup of ova by the fimbria prior to ovum recovery in this procedure. When fimbriectomies were not performed, an average of 13.3 ova were
recovered from each superovulated ovum donor. Sperm capacitation could be accomplished in the uterus of untreated does with no ovaries or oviducts, provided that the sperm cells gained access to the uterus. Capacitation occurred in the uterus of ovariectomized does and also ovariectomized and salpingectomized does after 3 days of estrogen treatment (0.5 p.g. of ECP /kg. of body wt.). More consistent results were obtained with ovariectomized and salpingectomized does when they were given the same treatment but for 6 days. This estrogen treatment (0.5 p.g. of ECPI kg. of body wt. for 6 days) led to a fertilization rate of 75.9%, which represented better capacitating efficiency than was observed with other levels of estrogen tried. A significant (p < 0.05) increase in the fertilization rate, to 90.6%, was observed when 0.25 mg. of either 17a-hydroxyprogesterone or 20a-hydroxyprogesterone was given to the estrogen-treated capacitator at the time of mating. The fertilization rate resulting from the latter treatment was exactly the same as that previously found to be afforded by uterine spermatozoa from intact estrus capacitators.18 Acknowledgments. We are grateful to Miss Mary Peace, Mrs. Joan Trammell, Mr. Don Killen, Mrs. Karen Wright, and Mr. John Klaus for their excellent technical assistance during the course of this research. We also thank Mr. William Harper for care of the animals and Mrs. Harriette Turner for typing the manuscript. REFERENCES 1. AusTIN, C. R. Observations of the penetration
of the sperm into the mammalian egg. Aust J &iRes B4:581, 1951. 2. BEDFORD, J. M. The influence of oestrogen and progesterone on sperm capacitation in the reproductive tract of the female rabbit. J Endocr 46:191, 1970. 3. BRACKETT, B. G. Respiration of spermatozoa after in utero incubation in estrus and pseudo-
September 1970
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
RABBIT SPERM CAPACITATION IN UTERUS
pregnant rabbits. VI Int Congr Anim Reprod (Paris) 1 :43, 1968. BRACKETT, B. G. Effects of washing the gametes on fertilization in vitro. Fertil Steril 20:127,1969. BRACKETT, B. G. "In .Vitro Fertilization of Mammalian Ova." In Advances in the Biosciences, Schering Symposium on Mechanisms Involved in Conception, Berlin, 1969 (Vol. 4), Raspe, G., Ed. Pergamon and Vieweg, New York,1969. BRACKETT, B. G. In vitro fertilization of rabbit ova: time sequence of events. Fertil Steril 21 :169, 1970. BRACKETT, B. G., AND FLICKINGER, G. L. Unpublished data. BRACKETT, B. G., AND WILLIAMS, W. L. In vitro fertilization of rabbit ova. J Exp Zool 160:271, 1965. BRACKETT, B. G., AND WILLIAMS, W. L. Fertilization of rabbit ova in a defined medium. Fertil Steril19 :144, 1968. CHANG, M. C. Fertilizing capacity of spermatozoa deposited in the fallopian tubes. Natture (London) 168 :697, 1951. CHANG, M. C. Capacitation of rabbit spermatozoa in the uterus with special reference to the reproductive phases of the female. Endocrinology 63 :619, 1958. CHANG, M. C. "Hormonal Regulation of Sperm Capacitation." In Advances in the Biosciences, Schering Symposium on M echanisms Involved in Conception, Berlin, 1969 (Vol. 4), Raspe, G., Ed. Pergamon and Vieweg, New York, 1969. HAMNER, C. E., JONES, J. P., AND SOJKA, N. J. Influences of the hormonal state of the female on the fertilizing capacity of rabbit spermatozoa. Fertil SteriI19:137, 1968. HAMNER, C. E., AND SOJKA, N. J. Requirements
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for capacitation of rabbit sperm. Nature (London) 220: 1042, 1968. HILLIARD, J., ARCHIBALD, D., AND SAWYER' C. H. Gonadotropic activation of prevulatory synthesis and release of progestin in the rabbit. Endocrinology 72 :59, 1963. NOYES, R. W., WALTON, A., AND ADAMS, C. E. Capacitation of rabbit spermatozoa. J Endocr 17 :374, 1958. SEITZ, H. M., JR., BRACKETT, B. G., AND MASTROIANNI, L., JR. In vitro fertilization of ovulated rabbit ova recovered from the ovarian surface. Bioi Reprod 2 :262, 1970. SEITZ, H. M., JR., ROCHA, G., BRACKETT, B. G., AND MASTROIANNI, L., JR. Influence of the oviduct on sperm capacitation in the rabbit. Fertil Steril21 :325, 1970. SIMMER, H. H., HILLIARD, J., AND ARCHIBALD, D. Isolation and identification of progesterone and 20 a-hydroxy-pregn-4-en-3-one in ovarian venous blood of rabbits. Endocrinology 72: 67, 1963. SOUPART, P. Studies on the hormonal control of rabbit sperm capacitation. J Reprod Fertil (Suppl. 2) :49, 1967. SOUPART, P. "The Effect of 20a-Hydroxyprogesterone on Sperm Capacitation in the Rabbit Uterus." In Society jor the Study oj Reproduction: Abstracts oj Papers, 2nd Annual Meeting, University of California, Davis, Calif., 1969. STAMBAUGH, R., BRACKETT, B. G., AND MASTROIANNI, L., JR. Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors. Bioi Reprod 1 :223, 1969. WILLIAMS, W. L., HAMNER, C. E., WEINMAN, D. E., AND BRACKETT, B. G. Capacitation of rabbit spermatozoa and initial experiments on in vitro fertilization. V Int Congr Anim Reprod (Trento) 7 :228, 1964.