In Vitro Fertilization of Horse Follicular Oocytes Matured In Vitro J.J. ZHANG, L.Z. MUZS, AND M.S. BOYLE Thoroughbred Breeders' Association, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk, United Kingdom

ABSTRACT In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: l ) washed and incubated in TALP medium with 3 rng/ml bovine serum albumin (BSA) and 10 pg/ml heparin for 4 h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 pM calcium ionophore A 23187 for 5-1 0 min followed by washing. After a given pretreatment, suspensions were diluted into 82 medium to a concentration of 5 x lo6 sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophoretreated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 1 1 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.

(Cohen, 19861, cows, pigs, sheep (Iritani, 1988), rhesus monkeys (Boatman and Bavister, 1984; Wolf et al., 19891, and several laboratory animal species (Dekel and Shalgi, 1987; Bavister, 1989). Successful IVF of in vivo matured horse oocytes has been reported by Bezard et al. (19891, although there are no reports to date of foals being born following IVF of horse oocytes. During oogenesis, oocytes of most mammalian species, including the horse, remain at the diplotene stage of meiotic prophase from embryonic life until the resumption of meiosis just before ovulation. Resting follicular oocytes can also resume meiosis spontaneously and complete their maturation in vitro when removed from the follicular environment. Zhang et al. (1989b) described a method for recovering horse oocytes from ovaries obtained from an abattoir. After IVM, oocytes were replaced into the oviduct of a mated recipient mare. Fertilization was achieved in vivo and resulted in pregnancy. The present study was conducted to evaluate the ability of stallion spermatozoa to fertilize in vitro matured oocytes after being treated in various ways.

Key Words: Stallion spermatozoa, lonophore, IVF

INTRODUCTION Research on early events of fertilization in the horse has been slow compared with that in other large domestic species. The lack of success when attempting to superovulate mares with exogenous gonadotropin (Squires et al., 1986) remains one major obstacle to the production of large numbers of oocytes and embryos. Another problem is that development of the equine embryo to late morula/early blastocyst stage occurs in the oviduct before entering the uterus between 5 and 6 days after ovulation (Betteridge et al., 1982). Consequently, access to live embryos less than 6 days of age, a requirement both for studies on the earlier events of embryonic development and for micromanipulation to produce multiple monozygotic offspring, is severely limited. For these reasons, the techniques of in vitro maturation (IVM) and in vitro fertilization (IVF) of follicular oocytes offer an attractive alternative solution. IVF has now been used successfully in the human


MATERIALS AND METHODS Oocyte Maturation The procedure used for maturing horse oocytes in vitro was described by Zhang et al. (1989b). In brief, cumulus-oocyte complexes were dissected free a t room temperature from horse ovaries obtained at an abattoir. The cumulus-oocyte complexes were cultured at 37.5"C under 5% C 0 2 in 100% humidified air in maturation medium M199 (Flow Laboratories, Scotland) containing 20% (v/v) foetal calf serum (FCS; Sigma, Poole, England), 2.5 pg/ml follicle-stimulating hormone (FSH) and 2.5 pg/ml LH (Serono, U.K.).After 36-42 h of incubation, maturation of cumulus-oocyte complexes was evaluated under a stereomicroscope,and







Received February 26, 1990; accepted March 9,1990. Address reprint requests to J.J. Zhang, Thoroughbred Breeders' Association, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, U.K.



those complexes with a fully expanded cumulus oophorus and corona radiata and exhibiting even, translucent oocyte cytoplasm were selected for IVF.

Sperm Treatment and IVF Procedure Semen samples were collected from two fertile pony stallions using an artificial vagina. After conventional analysis (volume, motility, and concentration), the samples were assigned to various treatment groups. Immediately before use, sperm motility was again evaluated visually under phase-contrast illumination at about 37°C. Group 1 This procedure is modified from the method used for bovine IVF. Sperm were washed twice in TALP medium (Bavister, 1989)with 10 mM Hepes and 3 mg/ml BSA at 320g for 4 min and resuspended in TALP with 10 pg/ml heparin to give a concentration of 50 x lo6 sperm/ml. After 4 h of incubation in a test tube placed in a water bath at 37.5”Cin air, 50 pl of sperm suspension was added to 450 pl B2 medium (Menezo, 1976; API Laboratory Products Ltd.) enriched with 15% (v/v) foetal calf serum (FCS). Group 2 Spermatozoa were washed as above and incubated for 3 h at 395°C.Stock solutions of dibutyryl cyclic adenosine monophosphate (dbcAMP) and caffeine were then diluted into the sperm suspension to final concentrations of 5 and 1 mM respectively. After a further 1 h of incubation in a water bath at 37.5“C,50 ~1 of sperm suspension was diluted into 450 ~1 B2 medium containing 15% (v/v) FCS.This sperm treatment procedure has been successfully used in the monkey (Boatman and Bavister, 1984;Wolf et al., 1989). Group 3 After being washed as above, spermatozoa were resuspended in TALP medium in which the pH had been adjusted to 7.9-8.2 with 1 N NaOH. The concentration was adjusted to give 4 x 10’ s p e d m l , and the suspension was incubated for 2 h a t 37.5”Cin a water bath. Five microliters of the sperm suspension was then diluted into 495 p1 B2 medium containing 15% (v/v)FCS. Group 4 Fresh, gel-free semen was diluted into TALP medium containing 10 mg/ml BSA t o a concentration of 25 x lo6 sperdml. The sample was incubated in a water bath a t 375°C for 5 min before a stock solution of calcium ionophore A 23187 (E7250;Sigma) in dimethyl sulfoxide (DMSO) was added to give a concentration of 7.14 pM. After a further 5-10 min of incubation, the sperm suspension was washed once a t 320g for 4 min before being resuspended in B2 medium to a final concentration of 5 x lo6 sperm/ml. Sperm suspensions, treated in the same way but without calcium ionophore, were used as a control. After partly removing the cumulus cells by gentle pipetting, the selected oocytes were washed three times in M199 containing 10% (v/v) FCS. The oocytes were

TABLE 1. Changes in Sperm Motility Under Various Pre-Incubation Conditions

Initial Final Premotility motility incubation” (%) (%) groups 4 h in TALP with 10 pglml heparin (group 1) 75 65 3hinTALP + l h with caffeine and dbcAmp (group 2) 80 50 2-4 h in TALP at PH 7.9-8.2 (group 3) 80 50 10 min in TALP with 1%BSA and 7.14 pM A 23187 (group 4) 89 75

Acquisition of hyperactivated Ahb motility









“For details, see Materials and Methods. bAmplitude of lateral sperm head displacement.

then transferred into tubes containing sperm suspensions treated as above and incubated for periods of 12 or 24-48 h under 5% COPin air at 385°C.With longterm incubations, the medium was changed at 24 h. One two-cell stage zygote resulting from the above procedure was transferred surgically to the oviduct of an unmated, synchronized recipient pony mare.

RESULTS Table 1 shows the changes in sperm motility following various treatments. With all treatments, sperm velocity decreased with increasing incubation time. The amplitude of lateral displacement of the sperm head (Ah) increased dramatically, and a pattern of sperm movement resembling “hyperactivity” was noticed in samples treated with ionophore, whereas spermatozoa in the group treated with dbcAMP and caffeine showed only a minor degree of Ah. When the sperm suspensions were finally diluted in B2 medium to a concentration of 5 x lo6 sperdml, all motile spermatozoa agglutinated within 30 min in groups 1-3, whereas in samples treated with A 23187 in group 4 agglutination was delayed for 2 h and was limited to only 30-40% of the motile sperm population. Fertilization did not occur when oocytes were co-incubated with spermatozoa treated with dbcAMP caffeine, heparin, or high pH. Spermatozoa that were bound to the zona pellucida of oocytes in these groups were removed easily with a pipette. On the other hand, pretreatment of spermatozoa with 7.14 FM A 23187 for 5-10 min had a significant effect. As is shown in Table 2, 18 of 54 (33%) oocytes inseminated in vitro were seen to have been fertilized (Fig. 1)when they were fixed at 12 h; a swollen sperm head or two pronuclei with an associated tail or midpiece was regarded as evidence of fertilization. When


IN VITRO FERTILIZATION IN THE HORSE TABLE 2. Fertilizing Ability of Spermatozoa Treated With Ionophore A23187 No. oocytes

Sperm treatment 9.14 KM A23187 for 5-10 min

Replicates 1 2 3 4 5 6

Total (%) Control"


2 3 4 5 6

fertilizedl No. insem. (12h) 6/16 1/6 418


3/10 4114 18/54(33) 018 015




No. oocytes


No. insem. (24-48 h)

215 2/10 218 2/13 319 11/45(24)

014 016 018

015 015 0128

Total 0117 aSpermatozoa were incubated in T A W with 10 mglml BSA and without ionophore A23187.

Fig. 1. Twelve hours postinsemination, oocyte shows a swollen sperm head and an associated midpiece (arrow). x 250. Fig. 2. Twenty-six hours postinsemination,two-cell embryo. x 150. Fig. 3. Forty-eight hours postinsemination,stained embryo shows six nuclei (arrow). x 150.

the other 45 oocytes were cultured for 24-48 h, 11 (24%) showed cleavage, of which 10 had developed to the two-cell stage and one to the six-cell stage (Figs. 2,3).One two-cell-stage egg from this group was transferred surgically into the fimbria of an unmated, synchronized recipient pony mare, but no pregnancy resulted.

DISCUSSION In contrast to the situation in many other domestic species, development of IVF has been slow in equids.


This has been due, partly, to the limited availability of equine oocytes but also to the difficulty in capacitating stallion spermatozoa outside the mare's reproductive tract (Blue et al., 1989;Zhang et al., 1989b).The shortage of experimental material has been partly overcome by the use of slaughterhouse ovaries, which, on average, each yield five to ten follicular oocytes. However, about half of these oocytes frequently show signs of degeneration. With regard to capacitation, several methods which are used successfully in other species do not appear to capacitate stallion spermatozoa in vitro. For example, preincubation with caffeine and dbcAMP is crucial for capacitation of rhesus monkey spermatozoa in vitro (Boatman and Bavister, 1984;Wolf et al., 19891, and, in bovine IVF, heparin has been widely used to achieve capacitation (Lu et al., 1988;Xu et al., 1987). Recently, Parrish et al. (1989)suggested that heparin, as a sulphate glycoconjugate, may prepare spermatozoa for fertilization across a wide range of species. In our system, however, spermatozoa treated with either dbcAMP + caffeine or heparin did not fertilize horse oocytes. The question of whether stallion spermatozoa are exceptional in their requirements for these agents remains open. In a similar way, the oviducal environment of pseudopregnant rabbits, which have been shown to support xenogenous fertilization in several species (Hirst et al., 1981;Rao et al., 19841,did not prove suitable for fertilization of horse oocytes matured in vivo (McKinnon et al., 1988) or in vitro (J. Zhang, L. Muzs, and C. Galli, unpublished data). It is noteworthy, in the present study, that treatment with calcium ionophore appeared to promote the fertilizing ability of stallion spermatozoa; ionophore-treated spermatozoa penetrated 33% of one batch of oocytes fixed after 12 h culture, and 24% of another batch of oocytes cultured for 24-48 h developed into two- to six-cell embryos. From the results of our previous study (Zhang et al., 1989b) and the present studies, spontaneous parthenogenic division of unfertilized horse oocytes appears to be very rare either in vitro or in vivo. IVF of in vivo matured horse oocytes was reported by Bezard et al. (19891,who used ionophore-treated spermatozoa. In their study, all oocytes were co-cultured with ionophore-treated spermatozoa for at least 14 h, and six of 20 eggs were cleaved. Pre-incubation of stallion spermatozoa in TALP at pH 7.9-8.2 facilitates penetration of zona-free hamster eggs (Zhang et al., 1989a1, as does treatment with lysophosphatidylcholin and ionophore A 23187 (Blue et al., 1989). In both these studies, similarly treated spermatozoa failed to penetrate zona intact horse oocytes. In the present experiment, stallion spermatozoa treated with ionophore for 5-10 min were able to fertilize zona-intact horse eggs. Also, from immunofluorescent staining with a monoclonal antibody raised against sperm acrosomes (Zhang et al., 19901,it appeared that a large proportion of spermatozoa still had an intact acrosome after 10 min of ionophore treat-



ment. These findings lead to the hypothesis that only capacitated, but not acrosome-reacted, spermatozoa can bind to and penetrate the zona pellucida. This is similar to the situation shown in the mouse by Bleil et al. (1988). The changes in sperm motility in the present experiment were assessed visually. Although this method is far from accurate, some distinct changes could, nevertheless, be seen. Stallion spermatozoa always showed the highest linear velocity, with the smallest amplitude of lateral head displacement (Ah) afier washing or dilution. Percentage motility and velocity decreased with increasing incubation time. “Hyperactivated” spermatozoa (with the greatest Ah and lowest linear velocity) were observed only in the ionophore-treated samples, in which a large proportion of spermatozoa also showed greater Ah. The motility changes induced by ionophore may represent an increased Ca2+ level in the cytoplasm, which is necessary for spermatozoa to be able to fertilize oocytes. Other workers have found a good correlation between changes in movement characteristics, including the onset of hyperactive motility and fertilizing ability, in the monkey (Boatman and Bavister, 1984)and the rabbit (Johnson et al., 1981;for review, see Katz et al., 1989). Compared with results in other species, the fertilization rate achieved in the present study is low. Several reasons may account for this. First, the cellular investment of in vitro matured oocytes may differ from those matured in vivo, thus restricting sperm access to the oocytes (Saling, 1989). Second, the zona pellucida may undergo hardening during maturation under some in vitro culture conditions, preventing sperm attachment and penetration. This has been reported to occur in the mouse (Downs et al., 1986). Third, the concentration of ionophore used in the present in vitro system may not have been optimal. Stallion spermatozoa appear to be particularly sensitive to this agent, and high concentrations of ionophore, or incubation in lower concentration for a prolonged period, will increase intracellular Ca2+ concentration to toxic levels. On the other hand, suboptimal levels of A 23187 may not be able to evoke the changes in intracellular Ca2 which allow spermatozoa to achieve fertilization. We found that considerable variation exists between individual stallions in the response of their spermatozoa to A 23187 (unpublished observation). This makes it necessary to work out the most effective ionophore concentration for each stallion. In conclusion, whereas commonly used in vitro procedures for sperm capacitation are less efficient in capacitating stallion spermatozoa, exposure to the calcium ionophore A 23187 does induce the ability to fertilize in vitro matured horse oocytes. +

ACKNOWLEDGMENTS This study was financed by the Thoroughbred Breeders’ Association and the Horserace Betting Levy Board (project no. 396).L.Z.M. was supported by a Carlsberg-

Wellcome Scholarship. We thank Drs. R.A.P. Harrison and R.M. Moor of the Institute of Animal Physiology and Genetics Research, Cambridge, for critical reading of this manuscript.

REFERENCES Bavister BD (1989): A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 23:139-158. Betteridge KJ, Eaglesome MD, Mitchell D, Flood PF, Beriault R (1982): Development of horse embryos up to 22 days after ovulation: Observations on fresh specimens. J Anat 135:191-209. Bezard J, Magistrini M, Duchamp G, Palmer E (1989): Chronology of equine fertilization and embryonic development in vivo and in vitro. Equine Vet J Suppl8105-110. Bleil JD, Gereve JM, Wassermann PM (1988):Identification of a secondary sperm receptor in the mouse egg zona pellucida: Role in the maintenance of binding of acrosome-reacted sperm to eggs. Dev Biol 128~376-385. Blue BJ, McKinnon AO, Squires EL, Seidel GE Jr, Muscari KT (1989): Capacitation of stallion spermatozoa and fertilization of equine oocytes in vitro. Equine Vet J Suppl8:lll-116. Boatman DE, Bavister BD (1984): Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J Reprod Fertil 71~357-366. Cohen J (1986): Pregnancy, abortion and birth after in vitro fertilization. In S Fishel, and EM Symmonds, (eds): “In Vitro Fertilization, Past, Present and Future.” Oxford: IRL Press, pp 136-261. Dekel N, Shalgi R (1987): Fertilization in vitro of rat oocytes undergoing maturation in response to a GnRH analogue. J Reprod Fertil 80531-535. Downs SM, Schroeder AC, Eppig JJ (1986): Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida. Gamete Res 15:115-122. Hirst PJ, DeMayo FJA, Dukelow WR (1981):Xenogenousfertilization of laboratory and domestic animals in the oviduct of the pseudopregnant rabbit. Theriogenology 15:67-75. Iritani A (1988): Current status of biotechnological studies in mammalian reproduction. Fertil Steril 50:543-551. Johnson LI, Katz DF, Overstreet JW (1981): The movement characteristics of rabbit spermatozoa before and after activation. Gamete Res 4275-282. Katz DF, Drobnis EZ, Overstreet JW (1989):Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res 22:443-469. Lu KH, Gordon I, McGovern H (1988):Development in vitro of bovine morulae derived from in vitro fertilization of follicular oocytes matured in vitro. In: 11th International Congress on Animal Reproduction & Artificial Insemination, Dublin, Ireland, June 2 6 J u l y 3, 1988, Vol. 3. Brief Communications, University College, Dublin. Abstract, Paper No. 342. McKinnon AO, Squires EL, Carnevale EM, Voss JL, Seidel GE J r (1988): Heterogenous and exogenous fertilization of equine oocytes. (abstract.) Theriogenology 29:278. Menezo Y (1976):Milieu synthetique pour la survie et la maturation des gametes et pour la culture de l’oeuf feconde. CR Acad Sci 282: 1967-1970. Parrish JJ, Susko-Parrish JL, Handrow RR, Ax RL, First NF (1989): Effect of sulfated glycoconjugateson capacitation and the acrosome reaction of bovine and hamster spermatozoa. Gamete Res 24403413. Rao VH, Sarmah BC, Bhattacharyya NK (1984): Xenogenous fertilization of goat ova in the rabbit oviduct. J Reprod Fertil71:377-379. Saling PM (1989): Mammalian sperm interaction with extracellular matrices of the egg. In SR Milligan (ed): “Oxford Review of Reproductive Biology, Vol. 11.”Oxford: Oxford University Press, pp 339368. Squires EL, Garcia EH, Ginther OJ,Voss JR, Seidel GE Jr (1986): Comparison of equine pituitary extract and FSH-Pfor superovulating mares. Theriogenology 26:661-670.

IN VITRO FERTILIZATION IN THE HORSE Wolf DP, Vandevoort CA, Meyer-Haas GR, Zelinski-Wooten MB, Hess DL, Baughamn WL, Stouffer RL (1989): In vitro fertilization and embryo transfer in the rhesus monkey. Biol Reprod 41: 335-346.

Xu KP, Greve, T, Hyttel P (1987): Pregnancy resulting from in vitro fertilization of bovine oocytes matured in vitro. J Reprod Fertil 81:50 1-504. Zhang JJ, Boyle MS, Allen WR (1989a): Studies on in vitro culture of


horse spermatozoa and oocytes. Abstract from the Symposium on Fertilization in Mammals, Boston, August, 1989. Zhang JJ, Boyle MS, Allen WR Galli C (1989b): Recent studies on in vivo fertilization of in vitro matured horse oocytes. Equine Vet J SUppl8101-104. Zhang JJ, Boyle MS, Hartman H, Moore HMD (1990): The acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. Mol Reprod Dev (in press).

In vitro fertilization of horse follicular oocytes matured in vitro.

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermato...
579KB Sizes 0 Downloads 0 Views