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Evaluation of a TEST-Yolk Sperm Capacitation System for Use in Bovine In Vitro Fertilization1
PHYSIOLOGY AND MANAGEMENT Evaluation of a TEST-Yolk Sperm Capacitation System for Use in Bovine In Vitro Fertilization 1 A. IJAZ2 and A. G. HUNTER3 Departments of Animal Science and Large Animal Clinical Sciences University of Minnesota
St Paul 55108 ABSTRACT
to capacitate bull sperm for in vitro fertilization. (Key words: capacitation, bull sperm, in vitro fertilization, TEST-yolk medium)
Bovine sperm acquire the ability to penetrate zona-free hamster oocytes (capacitation) after incubation in TESTyolk buffer. Our objective was to determine whether such spenn could penetrate zona-intact bovine oocytes in vitro. Bovine cumulus enclosed oocytes from 2- to 5-mm follicles were incubated in maturation medium for 24 h at 37°C. Ejaculated bovine semen was diluted 1: 10 in TEST-yolk buffer, cooled to 4°C, and stored for 8 h to induce capacitation. Sperm were then washed thrice in pH 7.6, .15 M NaCI containing .1% bovine serum albumin V (37°C) and diluted to 2 x 1()6 sperrn/rnl in fertilization medium. Droplets of fertilization medium containing capacitated sperm, killed sperm, or no sperm were made under paraffin oil. Oocytes (matured 24 h) were added and cocultured with sperm for 8 h and then transferred to fresh fertilization medium for 40 h. After 24 h, 53% of the oocytes had matured (metaphase 11). The fertilization rate of the metaphase n oocytes (203) with TEST-yolk capacitated sperm was 87%, whereas the parthenogenetic controls were 2 and 0%, respectively. Therefore, TEST-yolk buffer can be used
Abbreviation key: BSA = bovine serum albumin, MI = metaphase I, MIl = metaphase n, PM = premetaphase, SSH = swollen sperm head. INTRODUCTION
Received June 19, 1991. Accepted September 13, 1991. lPublished as Paper Number 19,356 of the Scientific Journal Series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Agricultural Experiment Station Project Number 72 supported by Hatch funds. 2Present address: University of Agriculture, Department of Animal Reproduction, College of Veterinary Sciences, Lahore, Pakistan. 3To whom correspondence should be addressed, Department of Animal Science, 495 Animal Science-Veterinary Medicine, University of Minnesota, 1988 Fitch Avenue, St. Paul, MN 55108. 1992 J Dairy Sci 75:394-398
Progress has been made in developing bovine in vitro fertilization systems using in vitro matured oocytes (3, 6, 9, 10, 13, 19, 20, 24). Such systems have the potential for yielding embryos for embryo transfer and for genetic manipulation. If genes are to be inserted, they must be rnicroinjected into the pronuclei of one-cell embryos (17). Currently, only a small percentage of injected foreign genes actually become incorporated into chromosomes and then expressed (17). Therefore, there is a need to create in vitro large numbers of bovine onecell embryos for genetic manipulation studies. Ijaz and Hunter (7) developed a successful bovine sperm capacitation system that involved the storage of bull sperm in TEST-yolk buffer for 4 to 48 h at 4°C. The capacitating activity was shown to reside in the N-Tris (hydroxymethyl) methyl-2-aminomethane-sulfonic acid and Tris (hydroxylmethyl) amino methane molecules (8). Those studies (7, 8) were performed using zona-free hamster 00cytes to assay for capacitation. The objective of the present study was to determine whether this bovine spermcapacitating technique could be used for the in vitro fertilization of in vitro matured, zonaintact bovine oocytes. MATERIALS AND METHODS Oocyte Maturation
Ovaries were removed immediately after slaughter and transported from slaughter house
394
CAPACITATION OF SPERM
to laboratory within 4 h. Tissue temperature during transit was maintained at 33 to 3S"C, and laboratory dissections were carried out at 22 to 26"C. Zona-enclosed oocytes were harvested as described previously (9) and placed in a 3S-mm petri dish containing 4 to S mI of pH 7.4 TC-199 with Earle's salts (Sigma Chemical Co., St. Louis, MO), 2.2 gIL of NaHC03, and 100 mg/L of L-glutamine. 00eytes were washed thrice in the medium and distributed to SOO-fJ.I droplets of maturation medium (10 oocytes per droplet) under paraffin oil. Maturation medium consisted of the TC-I99 supplemented with 10 fJ.g/ml of porcine follicle-stimulating hormone (BumsBiotech, Omaha, NE), 1.0 IU/mI of human chorionic gonadotropin (Sigma), and 20% heat-inactivated fetal calf serum (Sigma) with 1.5 fJ.g/ml of estradiol-17~, 75 fJ.g/ml of streptomycin, and 100 IU/mI of penicillin G. The pH was adjusted to 7.4 with sodium bicarbonate. Medium contained .2% phenol red as a pH indicator. Medium was sterilized by filtering through a .2-fJ. filter. The oocytes were then incubated at 37"C in an atmosphere of S% COz in air for 24 h. After culture, oocytes were transferred to fertilization medium (fC-I99; medium supplemented with 20% fetal calf serum, 75 fJ.g/ml of streptomycin, and 100 IU/ml of penicillin G). semen Collection and Sperm Capacitation
Semen was collected by artificial vagina from two fertile, normal Holstein bulls. Semen was processed and capacitated in TEST-yolk: buffer at 4"C as described previously (7). At 8 h, 1 mI of extended semen was pipetted into a 15-ml plastic conical centrifuge tube at 37"C and washed in S mI of pH 7.6, .15 M NaCI containing .1% bovine serum albumin (BSA), Fraction V (Sigma). The extended semen was centrifuged for 5 min at 1000 x g, the supernatant was discarded, and washing was repeated twice in 5 mI of BSA-saline. Final sperm resuspension was made to 2 x loti sperm/ml in fertilization medium at 37"C. Sperm motility was visually estimated using a phase microscope. Sperm Oocyte Coculture
Sperm droplets (SOO fll) were made under paraffin oil. In addition, droplets of dead
39S
sperm, killed by repeated freezing and thawing, were made for a control (control 1). An additional control (control 2) for parthenogenesis consisted of fertilization medium droplets without any sperm. Oocyte complexes (matured for 24 h) were then transferred to these droplets and cocultured with sperm or spermfree medium for 8 h. The oocyte complexes were then transferred to sperm-free droplets of fertilization medium. Cultures were maintained at 37"C in an atmosphere of 5% C02 and 100% relative humidity for another 40 h. Oocyte Staining and Evaluation
Oocytes were evaluated for stage of nuclear maturation and for fertilization as described by Ijaz and Hunter (9). An oocyte was classified as penetrated when a swollen, decondensing sperm head or two or more pronuclei were present in the ooplasm. Male pronuclei were judged to have formed when two or more pronuclei were present. An oocyte with a single pronucleus without accompanying sperm was considered parthenogenetically activated. RESULTS AND DISCUSSION Meiotic Nuclear Maturation of Oocytes
The nuclear maturation data of the in vitro matured bovine oocytes prior to sperm addition are presented in Table 1. At 0 h of culture, 96.8% oocytes were in the germinal vesicle stage, and none was mature (metaphase n with a polar body, MIl). At the end of 24 h of culture in maturation medium, only 3.7% of the oocytes remained in the germinal vesicle stage. In 9.2% of the cultured oocytes, nuclear maturation began and proceeded to an early premetaphase (PM) stage. In 27.5% of the cultured oocytes, nuclear maturation stopped at metaphase I (MI). Thus, 41.3% of the oocytes began maturation but failed to mature to MIl. However, 53.2% of the initial oocytes matured to MIl, the meiotic stage at ovulation for the cow. Meiotic Nuclear Maturation of Oocytes During the In Vitro Fertilization
Nuclear maturation of the 24 h in vitro matured oocytes did not continue during the following 48 h in fertilization medium in the Joumal of Dairy Science Vol. 75, No.2, 1992
396
DAZ AND HUNTER.
TABLE 1. Maturation at 24 and 72 h of culture fO£ bovine oocytes isolated from 2- to 4-mm follicles. OocyteslculturedfO£ 72 ~ without sperm
Maturation stage (n)
Germinal vesicle Premetaphase Metapbase I Anaphase I Metaphase D Degenerated Tolll1
4
10 30 1 58 6 109
(%)
3.7 9.2 27.5 .9 53.2 5.5 100
(%)
(n)
(%)
o
o
5 22
6.0 26.5
o
1 2 11 1
46 10 83
55.4 12.0 100
47 5 67
1.5 3.0 16.41.5 70.17.5 100
(n)
o
IThree replicates per group. 2Cultured in maturation medinm for 24 h.
3Cultured in matwation medinm for 24 h and fertilization medium for 48 h. Sperm and oocytes were cocultured only during Ihe first 8 h in fertilization medium.
-P < .05 compared with 24 h of maturation or 72 h of maturation with dead sperm.
absence of sperm (53.2% Mll at 24 h vs. 55.4% MIl at 72 h; Table 1). However, nuclear matmation of the 24-h in vitro matured bovine oocytes continued (P < .05) during the following 48 h in fertilization medium with either live or dead sperm [53.2% MIl at 24 h vs. 70.1 % MIl with dead sperm (Table 1) and 71.1 % MIl with live sperm (Table 2; sum of unfertilized MIl oocytes and fertilized oocytes)] at 72 h. This increase was associated with a decrease in the number of oocytes blocked at MI. Baker (1) hypothesized that MI-blocked oocytes may come from the be smaller follicles and may not "programmed" by the follicle for full meiotic development. Programming errors could be either in the ability to synthesize sufficient RNA (16) or in the cyclic AMP regulatory system governing phosphorylation or dephosphorylation of proteins that control meiosis (14). Whatever the mechanism, our resuhs strongly suggest that the presence of sperm around the oocytes somehow helps to overcome the MI block. In Vitro Ferullzatlon
Exposure of the 24-h in vitro-matured 00cytes to sperm, incubated in the TEST-yolk buffer for 8 h and then washed with BSAsaline, resulted in 62.1% (Table 2) of the oocytes becoming penetrated by the spenn. The parthenogenetic controls were negative Journal of Dairy Science Vol. 75, No.2, 1992
(Table 2). Therefore, the TEST-yolk buffer system resulted in sperm capacitation, and the capacitated sperm were able to penetrate the bovine oocytes and initiate development leading to cleavage in some cases. Table 3 shows the in vitro fertilization rate based on only oocytes that had matured to MIl (the normal in vivo fertilization stage). Using this stage as a standard for measuring sperm penetration, 87.2% of the oocytes that had matured to Mll were penetrated by the TESTyolk capacitated sperm. The penetrated oocytes included 5.4% Mll with swollen sperm heads (SSII), 22.7% anaphase II or telophase II with SSH, 16.8% female pronucleus with SSH, 27.6% male and female pronuclei, and 14.8% two-cell or four-cell embryos. Therefore, the TEST-yolk buffer system was an efficient capacitating system for bovine ejaculated sperm and functioned well in a bovine in vitro fertilization system as was previously shown using the zona-free hamster oocyte penetration assay (7). Our data also document that the zona-free hamster oocyte penetration assay for determining capacitation status is a very conservative assay. Under identical capacitation conditions in our lab, the TEST-yolk capacitating system yielded a 29.2% penetration rate with zonafree hamster oocytes (1). However, the penetration rate for unselected, matured, cumulus enclosed, zona-intact bovine oocytes was 62.1% (Table 2) and was 87.2% (fable 3) for
397
CAPAClTAll0N OF SPERM
TABLE 2. Effect of capacitation in TEST-yolk buffer on the ability of bovine sperm to in vitro fertilize in vitro matured bovine oocytes.l Parthenogenesis Control, no sperm2
Oocyte condition
Control, dead sperm2
(no./total no.) (%)
Germinal vesicle Prem.etaphase Metaphase I Anaphase I Metaphase II Degenerate Metaphase II with swollen sperm Anaphase II or telophase II with swollen sperm Female pronucleus with swollen sperm Female and male pronucleus Two-cell
0/63 2/63 17/63 1/63 35/63 8/63
Fertilization live sperm2
(oo./total no.) (%)
0 3.2 27.0 1.6 55.6 12.7
1/67 2/67 11/67 1/67 46/67 5/67 1/67
1.5 3.0 16.4 1.5 68.7 7.5 1.5
(no./total no.) (%) 4/285 5/285 38/285 2/285 26/285 33/285 11/285
1.4 1.8 13.3 .7 9.1* 11.6 3.9
46/285 34/285 56/285 30/285
16.1* 11.9* 19.6* 10.5*
lCu1tured in maturation medium for 24 h and fertilization medium. for 48 h. Sperm and oocytes were cocultured only during the first 8 h in fertilization medium. 2At least 30 oocytes per replicate. All tests replicated twice except fertilization with live sperm was replicated thrice. *p < .05 compared with parthenogenetic controls.
the selected MIl oocytes. Although using only MIl oocytes gave a high penetration rate, 31.6% of the penetrated bovine oocytes had two pronuclei, and 16.9% of the penetrated bovine oocytes had undergone cleavage. Thus, in approximately half the penetrated bovine oocytes, male pronuclei formation failed to occur. Other studies (2, 5, 9, 10, 11, 18) with different systems have reported that approximately one-third or more of in vitro matured oocytes fail to develop male pronuclei following penetration. We hypothesize that this was due to either improper in vitro oocyte maturation or embryo culture conditions and was not due to the sperm capacitation conditions, because 87.2% of the MIl oocytes were penetrat-
ed. Sperm must become capacitated before they can penetrate oocytes (2). The 87.2% penetration rate documents that capacitation had occurred. The inability of an oocyte to transform a penetrated sperm into a male pronucleus may be due to the absence of a cytoplasmic male pronucleus growth factor (15,21), because immature oocytes commonly do not form a male pronucleus after penetration (22). The penetration results with our in vitro system (fable 3) were comparable with other successful different systems (2, 4, 9, 10, 12, 18, 23). In conclusion, the TEST-yolk sperm capacitating technique is an effective methodology for use in bovine in vitro fertilization. Its
TABLE 3. In vitro fertilization rate of only bovine oocytes that had matured to metaphase II. Fertilization stage
Number/total no. Percentage
lMII
= Metaphase II;
AIl
MIl with SSH
AIl with SSH
Female pronucleus with SSH
11/203 5.4
46/203 22.7
34/203 16.8
= anaphase II;
SSH
~
pronuclei
Two-cell
Total fertilized
56/203 27.6
30/203 14.8
177/203 87.2
= swollen sperm head. Joumal of Daily Science Vol. 75, No.2, 1992
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UAZ AND HUNTER
major advantage is that the sperm can be stored at 4°C for 4 to 48 h and still retain motility and penetrability (7). REFERENCES 1 Baker, T. G. 1982. Oogenesis and ovulation. Page 17 in Germ cells and fertilization. C. R. Austin and R. V. Short, ed. Cambridge Univ. Press, New York, NY. 2 Ball, G. D., M. L. Leibfried, R. W. Leoz, R. L. Ax, B. D. Bavister, and N. L. First 1983. Factors affecting successful in vitro fertilization of bovine follicular oocytes. BioI. Reprod. 28:717. 3 Brackett, B. J., D. Bousquet, M. L. Boice, W. J. Bonawick, J. F. Bvans, and M. A. Dressel. 1982. Normal development following in vitro fertilization in the cow. BioI. Reprod. 27:147. 4 Fukui, Y., K. Imai, N. F. Alfonso, and H. Ono. 1987. Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro. J. Anim. Sci. 64: 935. 5 Fulka, J., A Pavlok., and J. Fu1ka. 1982. In-vitro fertilization of zona-free bovine oocytes matured in culture. I. Reprod. Ferfil. 64:495. 6 Goto, K., Y. Kajihara, S. Kosaka, M. Kolla, Y. Nakanishi, and K. Ogawa. 1988. Pregnancies after coculture of cumulus cells with bovine embryos desived from in vitro fertilization of in-vitro matured follicular oocytes. 1. Reprod. Fertil. 83:753. 7 Ijaz, A., and A. G. HIIlIter. 1989. Induction of bovine sperm capacitation by TEST-yolk semen extender. J. Dairy Sci. 72:2683. 8 Ijaz, A, and A. G. HIIlIter. 1989. Identification of the capacitating agent for bovine sperm in egg yolk-TEST semen extender. J. Dairy Sci. 72:2700. 9Ijaz, A, and A G. HIIlIter. 1989. Bvaluation of calcimn-free Tyrode's sperm capacitation medimn for use in bovine in vitro fertilization. J. Dairy Sci. 72: 3280. 10Leibfried-Rutledge, M. L., B. S. Critser, W. H. Byestone, D. L. Northey, and N. L. First. 1987. Development potential of bovine oocytes matured in vitro and in vivo. Biol. Reprod. 36:376. 11 Leibfried-Rutledge, M. L., E. S. Critser, and N. L. First 1986. Effects of fetal calf sermn and bovine senun albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes. Biol. ~od. 35:850. 12 Ln, K. H., and I. Gordon. 1988. Effect of heparin on the capacitation of frozen-thawed bovine spermatozoa used in the in vitro fertilization (IVF) of oocytes matured in vitro. 11th Int Congr. Anim. Reprod.
Journal of Dairy Science Vol. 75, No. 2, 1992
Artif. Insem. 3:339. 13 Ln, K. H., I. Gordon, M. Gallagher, and H. McGovern. 1987. Pregnancy established in cattle by ttansfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet Rec. 121:259. 14 Maller, J. L., and E. G. Krebs. 1977. Progesteronestimulated meiotic cell division in Xenopus oocytes. I. BioI. Chem. 252:1712. 15 Mattioli, M., G. Galeati, M. L. Bacci, and E. Sereno 1988. Male pronucleus formation depends on soluble factors produced by follicle somatic cells during pig oocyte maturation. 11th Int Congr. Anim. Reprod. Artif. Insem. 3:343. 16 Moor, R. M., and G. M. Warnes. 1979. Meiosis in mammalian oocytes. Br. Med. Bull. 35:99. 17 Palmiter, R. D., R. L. Brinster, R. B. Hammer, M. B. Trumbaure, M. G. Rosenfled, N. C. Brinberg, and R. M. Bvans. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothioneingrowth hormone fusion genes. Nature (Lond.) 300: 611. 18 Parrish, J. J., J. L. Susko-Parrish, M. L. LeibfriedRutledge, E. S. Crister, W. H. Eyestone, and N. L. First 1986. Bovine in vitro fertilization with frozenthawed semen. Theriogenology 25:591. 19 Sirard, M. A.• R. D. Lambert, D. P. Menard, and M. Bedoya. 1985. Pregnancies after in vitro fertilization of cow follicular oocytes, their incubation in rabbit oviducts and their transfer to the cow uterus. J. Reprod. Fertil. 75:551. 20 Sirard, M. A., 1. 1. Parrish, C. B. Ware, M. L. Leibfried-Rutledge, and N. L. First 1988. The culture of bovine oocytes to obtain developmentally competent embryos. BioI. Reprod. 39:546. 21Thibaull, C., M. Gerard, and Y. Mcnezo. 1975. In vitro acquired ability of rabbit and cow oocyte to ensure sperm nucIens decondensation during fertilization (MPGF). Ann. Biol. Anim. Biochim. Biophys. 15: 705. 22 Usui, N., and R. Yanagimachi. 1976. Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. The appearance of factors involved in sperm chromatin decondensation in egg cytoplasm. J. Ulttastruct Res. 57:276. 23 Xu, K. P., and T. Greve. 1988. A detailed analysis of early events during in vitro fertilization of bovine follicular oocytes. J. Reprod. Fertil. 82:127. 24 Xu, K. P., T. Greve, H. CaIlesen, and P. Hyttel. 1988. Birth of a calf following in vitro fertilization of in vitro matured oocytes. J. Reprod. Fertil. (Abstr. Ser.) 1:14.
St Paul 55108 ABSTRACT
to capacitate bull sperm for in vitro fertilization. (Key words: capacitation, bull sperm, in vitro fertilization, TEST-yolk medium)
Bovine sperm acquire the ability to penetrate zona-free hamster oocytes (capacitation) after incubation in TESTyolk buffer. Our objective was to determine whether such spenn could penetrate zona-intact bovine oocytes in vitro. Bovine cumulus enclosed oocytes from 2- to 5-mm follicles were incubated in maturation medium for 24 h at 37°C. Ejaculated bovine semen was diluted 1: 10 in TEST-yolk buffer, cooled to 4°C, and stored for 8 h to induce capacitation. Sperm were then washed thrice in pH 7.6, .15 M NaCI containing .1% bovine serum albumin V (37°C) and diluted to 2 x 1()6 sperrn/rnl in fertilization medium. Droplets of fertilization medium containing capacitated sperm, killed sperm, or no sperm were made under paraffin oil. Oocytes (matured 24 h) were added and cocultured with sperm for 8 h and then transferred to fresh fertilization medium for 40 h. After 24 h, 53% of the oocytes had matured (metaphase 11). The fertilization rate of the metaphase n oocytes (203) with TEST-yolk capacitated sperm was 87%, whereas the parthenogenetic controls were 2 and 0%, respectively. Therefore, TEST-yolk buffer can be used
Abbreviation key: BSA = bovine serum albumin, MI = metaphase I, MIl = metaphase n, PM = premetaphase, SSH = swollen sperm head. INTRODUCTION
Received June 19, 1991. Accepted September 13, 1991. lPublished as Paper Number 19,356 of the Scientific Journal Series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Agricultural Experiment Station Project Number 72 supported by Hatch funds. 2Present address: University of Agriculture, Department of Animal Reproduction, College of Veterinary Sciences, Lahore, Pakistan. 3To whom correspondence should be addressed, Department of Animal Science, 495 Animal Science-Veterinary Medicine, University of Minnesota, 1988 Fitch Avenue, St. Paul, MN 55108. 1992 J Dairy Sci 75:394-398
Progress has been made in developing bovine in vitro fertilization systems using in vitro matured oocytes (3, 6, 9, 10, 13, 19, 20, 24). Such systems have the potential for yielding embryos for embryo transfer and for genetic manipulation. If genes are to be inserted, they must be rnicroinjected into the pronuclei of one-cell embryos (17). Currently, only a small percentage of injected foreign genes actually become incorporated into chromosomes and then expressed (17). Therefore, there is a need to create in vitro large numbers of bovine onecell embryos for genetic manipulation studies. Ijaz and Hunter (7) developed a successful bovine sperm capacitation system that involved the storage of bull sperm in TEST-yolk buffer for 4 to 48 h at 4°C. The capacitating activity was shown to reside in the N-Tris (hydroxymethyl) methyl-2-aminomethane-sulfonic acid and Tris (hydroxylmethyl) amino methane molecules (8). Those studies (7, 8) were performed using zona-free hamster 00cytes to assay for capacitation. The objective of the present study was to determine whether this bovine spermcapacitating technique could be used for the in vitro fertilization of in vitro matured, zonaintact bovine oocytes. MATERIALS AND METHODS Oocyte Maturation
Ovaries were removed immediately after slaughter and transported from slaughter house
394
CAPACITATION OF SPERM
to laboratory within 4 h. Tissue temperature during transit was maintained at 33 to 3S"C, and laboratory dissections were carried out at 22 to 26"C. Zona-enclosed oocytes were harvested as described previously (9) and placed in a 3S-mm petri dish containing 4 to S mI of pH 7.4 TC-199 with Earle's salts (Sigma Chemical Co., St. Louis, MO), 2.2 gIL of NaHC03, and 100 mg/L of L-glutamine. 00eytes were washed thrice in the medium and distributed to SOO-fJ.I droplets of maturation medium (10 oocytes per droplet) under paraffin oil. Maturation medium consisted of the TC-I99 supplemented with 10 fJ.g/ml of porcine follicle-stimulating hormone (BumsBiotech, Omaha, NE), 1.0 IU/mI of human chorionic gonadotropin (Sigma), and 20% heat-inactivated fetal calf serum (Sigma) with 1.5 fJ.g/ml of estradiol-17~, 75 fJ.g/ml of streptomycin, and 100 IU/mI of penicillin G. The pH was adjusted to 7.4 with sodium bicarbonate. Medium contained .2% phenol red as a pH indicator. Medium was sterilized by filtering through a .2-fJ. filter. The oocytes were then incubated at 37"C in an atmosphere of S% COz in air for 24 h. After culture, oocytes were transferred to fertilization medium (fC-I99; medium supplemented with 20% fetal calf serum, 75 fJ.g/ml of streptomycin, and 100 IU/ml of penicillin G). semen Collection and Sperm Capacitation
Semen was collected by artificial vagina from two fertile, normal Holstein bulls. Semen was processed and capacitated in TEST-yolk: buffer at 4"C as described previously (7). At 8 h, 1 mI of extended semen was pipetted into a 15-ml plastic conical centrifuge tube at 37"C and washed in S mI of pH 7.6, .15 M NaCI containing .1% bovine serum albumin (BSA), Fraction V (Sigma). The extended semen was centrifuged for 5 min at 1000 x g, the supernatant was discarded, and washing was repeated twice in 5 mI of BSA-saline. Final sperm resuspension was made to 2 x loti sperm/ml in fertilization medium at 37"C. Sperm motility was visually estimated using a phase microscope. Sperm Oocyte Coculture
Sperm droplets (SOO fll) were made under paraffin oil. In addition, droplets of dead
39S
sperm, killed by repeated freezing and thawing, were made for a control (control 1). An additional control (control 2) for parthenogenesis consisted of fertilization medium droplets without any sperm. Oocyte complexes (matured for 24 h) were then transferred to these droplets and cocultured with sperm or spermfree medium for 8 h. The oocyte complexes were then transferred to sperm-free droplets of fertilization medium. Cultures were maintained at 37"C in an atmosphere of 5% C02 and 100% relative humidity for another 40 h. Oocyte Staining and Evaluation
Oocytes were evaluated for stage of nuclear maturation and for fertilization as described by Ijaz and Hunter (9). An oocyte was classified as penetrated when a swollen, decondensing sperm head or two or more pronuclei were present in the ooplasm. Male pronuclei were judged to have formed when two or more pronuclei were present. An oocyte with a single pronucleus without accompanying sperm was considered parthenogenetically activated. RESULTS AND DISCUSSION Meiotic Nuclear Maturation of Oocytes
The nuclear maturation data of the in vitro matured bovine oocytes prior to sperm addition are presented in Table 1. At 0 h of culture, 96.8% oocytes were in the germinal vesicle stage, and none was mature (metaphase n with a polar body, MIl). At the end of 24 h of culture in maturation medium, only 3.7% of the oocytes remained in the germinal vesicle stage. In 9.2% of the cultured oocytes, nuclear maturation began and proceeded to an early premetaphase (PM) stage. In 27.5% of the cultured oocytes, nuclear maturation stopped at metaphase I (MI). Thus, 41.3% of the oocytes began maturation but failed to mature to MIl. However, 53.2% of the initial oocytes matured to MIl, the meiotic stage at ovulation for the cow. Meiotic Nuclear Maturation of Oocytes During the In Vitro Fertilization
Nuclear maturation of the 24 h in vitro matured oocytes did not continue during the following 48 h in fertilization medium in the Joumal of Dairy Science Vol. 75, No.2, 1992
396
DAZ AND HUNTER.
TABLE 1. Maturation at 24 and 72 h of culture fO£ bovine oocytes isolated from 2- to 4-mm follicles. OocyteslculturedfO£ 72 ~ without sperm
Maturation stage (n)
Germinal vesicle Premetaphase Metapbase I Anaphase I Metaphase D Degenerated Tolll1
4
10 30 1 58 6 109
(%)
3.7 9.2 27.5 .9 53.2 5.5 100
(%)
(n)
(%)
o
o
5 22
6.0 26.5
o
1 2 11 1
46 10 83
55.4 12.0 100
47 5 67
1.5 3.0 16.41.5 70.17.5 100
(n)
o
IThree replicates per group. 2Cultured in maturation medinm for 24 h.
3Cultured in matwation medinm for 24 h and fertilization medium for 48 h. Sperm and oocytes were cocultured only during Ihe first 8 h in fertilization medium.
-P < .05 compared with 24 h of maturation or 72 h of maturation with dead sperm.
absence of sperm (53.2% Mll at 24 h vs. 55.4% MIl at 72 h; Table 1). However, nuclear matmation of the 24-h in vitro matured bovine oocytes continued (P < .05) during the following 48 h in fertilization medium with either live or dead sperm [53.2% MIl at 24 h vs. 70.1 % MIl with dead sperm (Table 1) and 71.1 % MIl with live sperm (Table 2; sum of unfertilized MIl oocytes and fertilized oocytes)] at 72 h. This increase was associated with a decrease in the number of oocytes blocked at MI. Baker (1) hypothesized that MI-blocked oocytes may come from the be smaller follicles and may not "programmed" by the follicle for full meiotic development. Programming errors could be either in the ability to synthesize sufficient RNA (16) or in the cyclic AMP regulatory system governing phosphorylation or dephosphorylation of proteins that control meiosis (14). Whatever the mechanism, our resuhs strongly suggest that the presence of sperm around the oocytes somehow helps to overcome the MI block. In Vitro Ferullzatlon
Exposure of the 24-h in vitro-matured 00cytes to sperm, incubated in the TEST-yolk buffer for 8 h and then washed with BSAsaline, resulted in 62.1% (Table 2) of the oocytes becoming penetrated by the spenn. The parthenogenetic controls were negative Journal of Dairy Science Vol. 75, No.2, 1992
(Table 2). Therefore, the TEST-yolk buffer system resulted in sperm capacitation, and the capacitated sperm were able to penetrate the bovine oocytes and initiate development leading to cleavage in some cases. Table 3 shows the in vitro fertilization rate based on only oocytes that had matured to MIl (the normal in vivo fertilization stage). Using this stage as a standard for measuring sperm penetration, 87.2% of the oocytes that had matured to Mll were penetrated by the TESTyolk capacitated sperm. The penetrated oocytes included 5.4% Mll with swollen sperm heads (SSII), 22.7% anaphase II or telophase II with SSH, 16.8% female pronucleus with SSH, 27.6% male and female pronuclei, and 14.8% two-cell or four-cell embryos. Therefore, the TEST-yolk buffer system was an efficient capacitating system for bovine ejaculated sperm and functioned well in a bovine in vitro fertilization system as was previously shown using the zona-free hamster oocyte penetration assay (7). Our data also document that the zona-free hamster oocyte penetration assay for determining capacitation status is a very conservative assay. Under identical capacitation conditions in our lab, the TEST-yolk capacitating system yielded a 29.2% penetration rate with zonafree hamster oocytes (1). However, the penetration rate for unselected, matured, cumulus enclosed, zona-intact bovine oocytes was 62.1% (Table 2) and was 87.2% (fable 3) for
397
CAPAClTAll0N OF SPERM
TABLE 2. Effect of capacitation in TEST-yolk buffer on the ability of bovine sperm to in vitro fertilize in vitro matured bovine oocytes.l Parthenogenesis Control, no sperm2
Oocyte condition
Control, dead sperm2
(no./total no.) (%)
Germinal vesicle Prem.etaphase Metaphase I Anaphase I Metaphase II Degenerate Metaphase II with swollen sperm Anaphase II or telophase II with swollen sperm Female pronucleus with swollen sperm Female and male pronucleus Two-cell
0/63 2/63 17/63 1/63 35/63 8/63
Fertilization live sperm2
(oo./total no.) (%)
0 3.2 27.0 1.6 55.6 12.7
1/67 2/67 11/67 1/67 46/67 5/67 1/67
1.5 3.0 16.4 1.5 68.7 7.5 1.5
(no./total no.) (%) 4/285 5/285 38/285 2/285 26/285 33/285 11/285
1.4 1.8 13.3 .7 9.1* 11.6 3.9
46/285 34/285 56/285 30/285
16.1* 11.9* 19.6* 10.5*
lCu1tured in maturation medium for 24 h and fertilization medium. for 48 h. Sperm and oocytes were cocultured only during the first 8 h in fertilization medium. 2At least 30 oocytes per replicate. All tests replicated twice except fertilization with live sperm was replicated thrice. *p < .05 compared with parthenogenetic controls.
the selected MIl oocytes. Although using only MIl oocytes gave a high penetration rate, 31.6% of the penetrated bovine oocytes had two pronuclei, and 16.9% of the penetrated bovine oocytes had undergone cleavage. Thus, in approximately half the penetrated bovine oocytes, male pronuclei formation failed to occur. Other studies (2, 5, 9, 10, 11, 18) with different systems have reported that approximately one-third or more of in vitro matured oocytes fail to develop male pronuclei following penetration. We hypothesize that this was due to either improper in vitro oocyte maturation or embryo culture conditions and was not due to the sperm capacitation conditions, because 87.2% of the MIl oocytes were penetrat-
ed. Sperm must become capacitated before they can penetrate oocytes (2). The 87.2% penetration rate documents that capacitation had occurred. The inability of an oocyte to transform a penetrated sperm into a male pronucleus may be due to the absence of a cytoplasmic male pronucleus growth factor (15,21), because immature oocytes commonly do not form a male pronucleus after penetration (22). The penetration results with our in vitro system (fable 3) were comparable with other successful different systems (2, 4, 9, 10, 12, 18, 23). In conclusion, the TEST-yolk sperm capacitating technique is an effective methodology for use in bovine in vitro fertilization. Its
TABLE 3. In vitro fertilization rate of only bovine oocytes that had matured to metaphase II. Fertilization stage
Number/total no. Percentage
lMII
= Metaphase II;
AIl
MIl with SSH
AIl with SSH
Female pronucleus with SSH
11/203 5.4
46/203 22.7
34/203 16.8
= anaphase II;
SSH
~
pronuclei
Two-cell
Total fertilized
56/203 27.6
30/203 14.8
177/203 87.2
= swollen sperm head. Joumal of Daily Science Vol. 75, No.2, 1992
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UAZ AND HUNTER
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