Cleavage Development of Bovine Oocytes Fertilized by Sperm Injection CAROL L. KEEFER, ABDELMONEIM I. YOUNIS, AND BENJAMIN G. BRACKET" Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia

ABSTRACT Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-8. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 FM A23187, sperm nuclear decondensation occurred in the A231 87-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those iniected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the iniected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the iniected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection. Key Words: Microinjection, Bovine ova, lonophore, Activation

INTRODUCTION Microinjection techniques have been used to explore features of fertilization and sperm decondensation that could not be studied using standard in vitro fertilization (IVF) techniques (Uehara and Yanagimachi, 1976; Perreault et al., 1988; Keefer, 1989). The capacity of


heterologous ooplasm to direct decondensation and pronuclear formation has been demonstrated in mice and rat (Thadani, 1979,1980) and in hamsters (Uehara and Yanagimachi, 1976; Perreault et al., 1988). Recent reports have demonstrated the ability of homologous oocytes fertilized by sperm injection to undergo normal development. Mann (1988) obtained offspring following injection of motile spermatozoa into the perivitelline space of mouse oocytes. Offspring resulting from injection of immobilized rabbit spermatozoa into ooplasm of rabbit oocytes have also been reported (Hosoi et al., 1988). These studies indicate the potential of fertilization by sperm injection for domestic species. Sperm injection could be used to overcome problems in capacitation of semen, which affect IVF success rates in domestic species. Injection methods could also be used to extend valuable semen and to allow development of alternate means (e.g., freezedrying) of semen storage, because maintenance of motility is not required. However, species differences have been noted in the ability of oocytes to survive the injection procedure (see Keefer, 1989). Furthermore, sperm treatment prior t o injection can affect the ability of oocytes to activate and direct sperm decondensation and male pronuclear formation (Keefer, 1989). These observations indicate that individualized protocols may need to be established for fertilization by sperm injection in different species. In this series of experiments, whole in vitro capacitated bovine spermatozoa were injected directly into the ooplasm of in vitro matured oocytes. Incubated (capacitated) spermatozoa were used rather than uncapacitated spermatozoa or sperm nuclei since incubated, intact spermatozoa yielded the highest percentage of activated ova with a male pronucleus in studies in the rabbit (Keefer, 1989). It was determined that bovine oocytes survive the process of sperm injection but, unlike hamster and rabbit oocytes, require additional stimulation to activate. Once activated, injected bovine

Received July 20, 1989; accepted October 24, 1989. Address reprint requests to Dr. Carol L. Keefer, W.R. Grace Animal Services, P.O. Box 459, DeForest, WI 53532.



oocytes are capable of directing sperm decondensation, pronuclear formation, and cleavage development.

MATERIALS AND METHODS Media Preparation Follicular oocytes were cultured to maturity in a modified TCM 199 medium (GIBCO 380-2340, Grand Island, NY) containing sodium pyruvate (50 pgiml), sodium bicarbonate (2.6 mgiml), glucose (5.5 mg/ml), gentamicin sulfate (50 ug/ml), 20% (v/v) heat-treated proestrus (Day 20, D20) cow serum, 100 pg/ml luteinizing hormone (LH), and 1 pg/ml estradiol 17-B (Younis, 1988). Injected and/or activated eggs were cultured in a modified Ham’s FlO/basal medium Eagle’s (BME) containing 1.7 mg/ml sodium bicarbonate and 10% heat-treated fetal calf serum (Keefer et al., 1988) or in HEPES-buffered TCM 199 supplemented with 10%fetal calf serum. Sperm were prepared in a modified Tyrode’s balanced salt solution (TALP; Bavister and Yanagimachi, 1977) containing HEPES (2.3 mg/ml), sodium bicarbonate (160 pg/ml), glucose (0.9 mg/ml), sodium pyruvate (112 pg/ml), sodium lactate (110 pgi ml), gentamicin sulfate (50 ug/ml), and bovine serum albumin (6 mg/ml, essentially fatty acid-free, No. A7511; Sigma Chemical Co., St. Louis, MO). The pH of all media was adjusted to 7.4-7.6, and the osmolarity was adjusted to 280-300 mOsm/kg.

Sperm Preparation A modification of the method described by Parrish e t al. (1986) for in vitro sperm capacitation was used (Younis et al., 1989). Bovine semen from a Holstein bull was processed in egg yolk or skim milk extender and frozen in 0.5 ml straws with lo7 sperm cells per straw (Atlantic Breeders Cooperative, Lancaster, PA). Three straws were thawed at 37°C for 10 sec; then, at 39”C, 0.2 ml of thawed semen was layered under 1 ml HEPES-TALP in six small tissue culture tubes (12 x 55 mm) and held at a 45” angle. One hour later, the top 0.8 ml from each tube was removed, pooled in a sterile 15 ml centrifuge tube, and centrifuged (350g) for 10 min. After discarding the supernatant, the sperm pellet was resuspended to 4 ml with HEPES-TALP and centrifuged a s before. The resulting sperm pellet was resuspended to 200 pl with heparin-containing (10 pg/ ml) HEPES-TALP in a microcentrifuge tube (5 x 10 mm) and kept a t 39°C for 30 min to induce capacitation and the acrosome reaction. A portion of the sperm was used for IVF experiments (Younis, 1988); the remainder was stored frozen a t -20°C for subsequent use in sperm injection.

Injection Procedure Whole in vitro capacitated spermatozoa were thawed Oocyte Isolation and Maturation and diluted (approximately 1:2) with 10% polyviBovine ovaries were obtained a t a n abattoir 10-15 nylpyrrolidone (PVP; Sigma) in 0.9% saline solution min postslaughter, rinsed with normal saline solution (Perreault and Zirkin, 1982). Spermatozoa were non(“normal saline, 0.9%, USP’; Travenol Laboratories, motile due to frozen storage without extender. A small Inc., Deerfield, IL) supplemented with 4,000 U penicil- drop of sperm-PVP mixture was placed on a depression lirdml and stored in normal saline in a thermos bottle slide near a larger drop of culture medium, and the (approximately 35-37°C). Small antral follicles (2-5 drops were covered with lightweight paraffin oil. Apmm in diameter) were aspirated within 1 h of slaughter proximately six partially denuded ova were placed in with a 10 cc syringe fitted with a n 18 gauge needle. The the drop of culture medium, and the slide was placed on follicular fluid aspirates were pooled in a sterile 50 ml a Leitz fixed-stage microscope equipped with Leitz miErlenmeyer flask and held for a n additional 1h during cromanipulators. The injection pipette and holding transit to the laboratory. Subsequent handling of ga- pipette were prepared a s described by Perreault and metes was done in a controlled environmental room at Zirkin (1982). Injection needles were pulled on a 39°C. The follicular fluid was transferred to sterile 15 Brown-Flaming micropipette puller (model P-80; Sutml conical glass centrifuge tubes. Cumulus-oocyte ter Instruments, San Rafael, CAI. The injection needle complexes, sedimented by gravity, were recovered was thinned by dipping the tip into 25% hydrofluoric within 10-15 min under low-power (16x ) magnifica- acid while air was expelled. The needle was washed by tion. Oocytes were washed and cultured in maturation dipping the tip first in distilled water and then in acmedium (50 oocytes in 0.2 ml drops) under sterile par- etone. The size of the tip could be judged by the size of affin oil at 39°C in humidified 5% CO,, 5% O,, and 90% the air bubbles observed during the thinning procedure. The resulting inner diameter of the tip was Nz.. After a maturation culture interval of 24 h, oocyte- slightly larger than the width of the sperm. A small cumulus complexes were treated with hyaluronidase (1 (1-2 mm) bolus of mercury was placed in the back end mg/ml) in HEPES-TALP for 2-3 min. With a finely of the injection needle and the needle was connected to drawn Pasteur pipette, cumulus cells surrounding each the syringe system, which was filled with silicone oil oocyte were removed to allow visualization of ooplasm (Uehara and Yanagimachi, 1976). An individual sperm and polar bodies. Oocytes exhibiting a polar body were cell was picked up from the sperm drop, and the needle washed in HEPES-TCM 199 and transferred to drops of was moved to the ova-containing drop. The sperm cell either Ham’s FlO/BME- or HEPES-buffered TCM 199 was injected into the ooplasm of a n ovum, which was medium under paraffin oil. held steady by a suction-controlled holding pipette. Af-



TABLE 1. Classification of Bovine Ova After 3.5 h of Culture Decondensed Condensed sperm sperm Other” Number (%) (%I (%) injected Injected A23187 + 0 9 (47) 10 (53) 19 + + 11 (37)* l(3) 18 (60) 30 ““Other”includes those ova that had no identifiable sperm in the cytoplasm or had degenerated. *P < .05.

ter injection, the ova were returned to the incubator in drops of embryo culture medium under paraffin oil.

Oocyte Activation and Zygote Culture Oocytes were activated by the method of Ware et al. (1989) using the calcium ionophore A23187. Injected or noninjected control oocytes were washed twice through Dulbecco’s phosphate-buffered saline solution (PBS) and then incubated in 1 FM A23187 (Sigma) in PBS for 5 min a t 39°C. An equal volume of PBS containing bovine serum albumin (6 mg/ml) was added to stop the activation process. The oocytes were washed twice through PBS and once through culture medium. The activated oocytes were cultured for a n additional 48 h in Ham’s FlO/BME or HEPES-TCM 199 supplemented with serum. Unactivated controls (injected or noninjected) were treated in the same manner except that they were incubated for 5 min in PBS containing 0.06% dimethyl sulfoxide (DMSO), the solvent for A23187. Because bovine oocytes had a dense cytoplasm, which prevented visualization of cytoplasmic sperm or pronuclei, some injected eggs were fixed in acetic alcohol (1:3, glacial acetic acid:ethanol) and stained with 1% aceto-orcein after 3.5 h and examined by interference contrast microscopy for the occurrence of sperm decondensation. For a n ovum to be considered successfully fertilized, the egg must have initiated the activation process (i.e., completion of meiosis, extrusion of the second polar body, and female pronucleus formation), and the injected sperm must have decondensed and be forming a male pronucleus. After 48 h, injected eggs and controls were scored for cleavage development (two- and four-cell stages) and then either were transferred to fresh drops of culture medium for additional incubation or were fixed for observation. Statistical Analysis Data were appropriately partitioned into 2 x N contingency tables and analyzed using the X2 test. Data were classified by 1) sperm decondensation (presence or absence) and by A23187 treatment (treated or not treated) and 2) by cleavage development (presence or absence) and by A23187 treatment. Differences below the 0.05 probability level (P < 0.05) were considered statistically significant. Fisher’s exact test was used when the numbers of ova were too small for the X2 test.

Fig. 1. Decondensed bovine sperm nucleus inside activated bovine ovum 3.5 h after injection. x 544.

RESULTS In the first series of injection experiments, in vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm of in vitro matured bovine oocytes. The injected oocytes were cultured for a n additional 48 h . Noninjected control oocytes remained in metaphase I1 (67%,16/24), cleaved parthenogenetically (8%,2/24>, or degenerated (25%).Sham-injected oocytes remained in metaphase I1 (31%, 13/42), cleaved parthenogenetically (5%,2/42), or degenerated (64%).Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm but generally failed to activate; only 2% (2401) cleaved. I n 30% of the injected oocytes, the sperm was found on the ooplasmic membrane. To enhance activation, injected oocytes were exposed briefly to the calcium ionophore A23187 or 0.06% DMSO (control). After 3.5 h of culture, decondensed sperm (Fig. 1) were observed only in those injected oocytes t h a t had been exposed to the calcium ionophore (Table 1). Spermatozoa observed in the ooplasm of the injected control oocytes remained condensed. In a third series of experiments, 107 ova were divided into four treatments; 1) noninjected and nonactivated, 2) injected and nonactivated, 3) noninjected and activated, and 4) injected and activated (Table 2). Injected and activated oocytes had the highest rate of cleavage development (28%), although 18% of the noninjected and activated oocytes also cleaved parthenogenetically to the two- to four-cell stage. Both injected and noninjected oocytes that were not activated by A23187 had low rates of cleavage division (9% and 4%, respectively).


C.L. KEEFER ET AL. TABLE 2. Classification of Bovine Ova After 48 h of Culture

Injected -

+ +

Pronuclear stage" 0 3 6 6

A23187 -

+ +



Total activated


2 (9) 4 (15) 13 (34) 12 (57)

19 (90) 23 (85) 25 (66) 9 (43)


2 (9)" 1 (4Y 7 (18Tsd 6 (28Id


Total ova 21 27 38 21

aFemale pronucleus and male pronucleus (or swollen head) plus tail remnant observed in injected ova and female pronucleus (-ei) observed in noninjected ova. b"Degenerated ova" includes those that died following injection and those that failed to activate. Due to the difficulty in scoring degenerated ova, the presence or absence of spermatozoa was not determined. C.dNumbersof cleaved embryos with different letters differ significantly by Fisher's exact test P < 0.05).

TABLE 3. Classification of Bovine Ova After 48 h of Culture: Effect of HEPES-Buffered Medium Injected

+ + + +


+ -


A23187 -

+ +

Pronuclear stagea 3 7 6 19



1(4Y 1 (8)",d 6 (28Id." 19 (38)"

Total activated (%) 4 (15) 8 (67) 12 (57) 38 (76)



23 (85) 4 (33) 9 (43) 12 (24)

Number injected 27 12 21 50

aFemale pronucleus and male pronucleus (or swollen head) plus tail fragment observed in all injected ova except for two in row 2 and two in row 4, in which two pronuclei but no tail fragments were identified. bDegenerated includes those ova that died immediately following injection and those that failed to activate. Due t o the difficulty in scoring degenerated bovine ova, the presence or absence of sperm was not determined. c,d,eNumbersof cleaved embryos with different letters differ significantly by Fisher's exact test (P< .05).

In the last series of experiments, HEPES-buffered medium was used to stabilize the pH during the injection process (Table 3). This treatment slightly enhanced the cleavage rate of injected and activated oocytes; 38% cleaved in HEPES-buffered medium vs. 28% in bicarbonate-buffered medium (not significant). Of the 19 cleaved ova in HEPES-TCM 199 (table 3), seven (36.8%)developed to the eight-cell stage (Fig. 2) after a total incubation period of 72 h postinjection.

DISCUSSION Studies have shown that following microinjection into the ooplasm, hamster (Uehara and Yanagimachi, 1976; Perreault and Zirkin, 1982), mouse (Thadani, 1979,1980), human (Lanzendorf et al., 19881, and rabbit (Keefer, 1989) spermatozoa can undergo decondensation and pronuclear formation. Mouse and rabbit ova fertilized by injection can undergo cleavage development (Markert, 1983; Hosoi et al., 1988; Keefer, 1989) and live offspring following embryo transfer have been obtained in the rabbit (Hosoi et al., 1988). Our goal was to determine whether bovine oocytes could be fertilized by sperm injection into the ooplasm. A limiting factor was that bovine oocytes were less sensitive to the injection process than were hamster or rabbit oocytes in that only a low percentage (2%)of bovine oocytes activated following injection. The calcium ionophore A23187 can be used to stimulate egg activation and pronuclear development of in vitro matured bovine oocytes (Ware et al., 1989). In our

Fig. 2. Eight-cell bovine embryo resulting from sperm injection and A23187 activation. x 300.

studies, A23187 activated both injected and noninjected oocytes, and activated injected oocytes directed normal sperm decondensation and polar body formation. Cleavage-staged embryos resulting from injection and activation contained normal-appearing nuclei within the blastomeres; however, chromosomal analysis was not performed to demonstrate paternal chromo-

SPERM INJECTION somes definitively. Although some of the injected oocytes may have developed parthenogenetically without participation of the spermatozoa, the high incidence of injected oocytes containing a decondensed sperm a t 3.5 h and male pronucleus at 48 h suggests that at least some of the embryos developed from ova containing a n injected sperm and were diploid. These results demonstrate that in vitro matured bovine oocytes can be fertilized by sperm injection and that the resulting zygotes may be capable of development to a t least the eight-cell stage. Improvements in culture conditions, such a s the use of oviductal cell coculture, with subsequent embryo transfer should permit continued growth and development of bovine embryos obtained by sperm injection.

ACKNOWLEDGMENTS This work was supported by NIH New Investigator Research Award grant HD20128 and UGA VMES 87019 to C.L.K. The authors thank Dr. Sally Perreault for her valuable comments. REFERENCES Bavister BD, Yanagimachi R (1977):The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol Reprod 16228-237. Hosoi Y, Miyake M, Utsumi K, Iritani A (1988): Development of rabbit oocytes after microinjection of spermatozoa. Proc 11th Int Cong Anim Reprod Artif Insemination, Abstract 331. Keefer CL (1989): Fertilization by sperm injection in the rabbit. Gamete Res 22:59-69.


Keefer CL, Fayrer-Hosken RA, Brown LM, Brackett BG (1988): Culture of in vitro fertilized rabbit ova. Gamete Res 20:431-436. Lanzendorf SE, Maloney MK, Veeck LL, Slusser J , Hodgen GD, Rosenwaks Z (1988): A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil Steril 49:835-842. Mann J (1988): Full-term development of mouse eggs fertilized by a spermatozoon microinjected under the zona pellucida. Biol Reprod 381077-1083. Markert CL (1983): Fertilization of mammalian eggs by sperm injection. J Exp Zoo1 228:195-201. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL (1986): Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25:591. Perreault SD, Barbee RR, Elstein KH, Zucker RM, Keefer CL (1988): Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm injection and in vitro by flow cytometry. Biol Reprod 39:157-167. Perreault SD, Zirkin BR (1982): Sperm nuclear decondensation in mammals: Role of sperm-associated proetinase in vivo. J Exp Zoo1 224:252-257. Thandani VM (1979): Injection of sperm heads into immature rat oocytes. J Exp Zoo1 210:161-168. Thadani VM (1980): A study of heterospecific sperm-egg interactions in the rat, mouse and deer mouse using in vitro fertilization and sperm injection. J Exp Zoo1 212:435-453. Uehara T, Yanagimachi R (1976): Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 15467-470. Ware CB, Barnes FL, Maki-Laurila M, First NL (1989): Age dependence of bovine activation. Gamete Res 22265-275. Younis A1 (1988): In vitro maturation and fertilization of bovine oocytes. Master’s thesis, University of Georgia, Athens, Georgia. Younis AI, Brackett BG, Fayrer-Hosken RA (1989): Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 23:189-201.

Cleavage development of bovine oocytes fertilized by sperm injection.

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine wh...
830KB Sizes 0 Downloads 0 Views