Enhanced viability after in vitro fertilization of bovine oocytes matured in vitro with high concentrations of luteinizing hormone*†

Vol. 52, No.2, August 1989

FERTILITY AND STERILITY

Printed in U.S.A.

Copyright" 1989 The American Fertility Society

Enhanced viability after in vitro fertilization of bovine oocytes matured in vitro with high concentrations of luteinizing hormone*t

Benjamin G. Brackett, D.V.M., Ph.D.:j: Abdelmoniem I. Younis, B.V.Sc., M.S.§ RichardA. Fayrer-Hosken, B.V.Sc., Ph.D. II Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia

The purpose of this study was to better understand requirements for oocyte maturation to yield viable embryos after bovine in vitro fertilization (IVF). High proportions (95% to 100%) of cumulus-surrounded oocytes matured in vitro in all treatments, but subsequent development was enhanced after maturation with high concentrations of purified bovine luteinizing hormone (LH). Beneficial effects of undisturbed cumulus cells were demonstrated. Improved IVF followed insemination of cumulus-surrounded oocytes, but not denuded oocytes, after maturation with high LH (100 ~g/ml) versus low LH (10 ~g/ml), implicating cumulus cells in mediating hormonal enhancement. Oocytes matured with high LH resulted in embryos of superior viability, as reflected by cleavage to 4- to 8cell stages. Pregnancy resulting from transcervical embryo transfer further documented embryonic viability. These findings should be useful in further development and implementation of reproductive and genetic technologies. Fertil Steril52:319, 1989

Since initial documentation of bovine in vitro fertilization (IVF) by ultrastructural studies1 and birth of live offspring,2,3 technology has developed rapidly. Variability in quality of in vivo matured oocytes recovered from ovarian follicles or from oviducts near the time of ovulation following

Received November 25, 1988; revised and accepted March 8, 1988. * Presented at the Forty-Fourth Annual Meeting of The American Fertility Society, October 10 to 13, 1988, Atlanta, Georgia. t Supported by The University of Georgia Veterinary Medical Experiment Station. :j: Reprint requests: Benjamin G. Brackett, D.V.M., Ph.D., Department of Physiology and Pharmacology, The University of Georgia, College of Veterinary Medicine, Athens, Georgia 30602. § A fellowship was provided for Dr. Younis by the Faculty of Veterinary Medicine of the University of Khartoum, Khartoum, Sudan. II Present address: Department of Large Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, Georgia. Vol. 52, No.2, August 1989

superovulation protocols has been a deterrent in developing bovine IVF by this approach. Such difficulties led to greater emphasis on in vitro maturation (IVM) of oocytes recovered from unstimulated follicles, and quite recently, several successful experiments, with pregnancy resulting, have been reported. 4- 9 Hormonal conditioning of oocytes is of great importance in achieving cytoplasmic maturation, which is necessary for preparation ofthe female gamete for initiating normal development through fertilization. 7,lo,l1 A prominent role in oocyte maturation is played by gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), and the beneficial influence of 17,B-estradiol (E 2) on normal oocyte development is well recognized. 12- 15 In spite of recent progress, optimal conditioning of oocytes during maturation to enable their continued viability after fertilization has not been adequately described. This article presents data, recently reported in preliminary form,16 on the beneficial influence of Brackett et a1.

LH-enhanced maturation and bovine IVF

319

LH on maturation of bovine oocytes and their capability for continuing development after IVF. Hormonal conditions found in this work have been applied in initial efforts to improve in vitro development of bovine embryos to uterine stages (i.e., at least 8-cell) capable of normal gestational development after transcervical embryo transfer. MATERIALS AND METHODS Gametes

Oocytes with surrounding cumulus complements were aspirated from 2- to 5-mm follicles of ovaries obtained from slaughtered cows. Only those 00cytes with normal morphologic appearance, i.e., compact layers of cumulus cells and homogeneous cytoplasm, were studied. The experimental approach was similar to that recently employed in this laboratory,9.17 with modifications described below. Sperm were used after frozen storage. For this, Holstein semen was processed with egg yolk or milk extender, and frozen in 0.5-ml straws with 107 cells per unit (Atlantic Breeders Cooperative, Lancaster, P A). Three straws were used in each experiment. Oocyte Maturation

Within 3 hours after slaughter, oocytes were removed from follicular fluid aspirates, washed (five times) with TCM 199 (Sigma Chemical Co., St. Louis, MO), and cultured in droplets (30 to 40 00cytes per 200-p,1 droplet) of TCM 199 containing sodium pyruvate (50 p,l/ml) , sodium bicarbonate (2.6 mg/ml), glucose (5.5 mg/ml), and gentamicin sulfate (50 p,g/ml) supplemented with heat-treated proestrus (day 20) bovine serum (20% vol/vol). Experimental treatments included the addition of E2 (1 p,g/ml), and varying concentrations of LH (USDA-bLH-B-5), i.e., 1, 10, 100, or 1,000 p,g/ml. In the last series of experiments, HEPES (N-2hydroxyethyl piperazine-N' -2-ethanesulfonic acid)-buffered TCM 199 (Gibco, Grand Island, NY) modified by addition of pyruvate and gentamicin, as above, and supplemented with proestrus serum (20% vol/vol), was employed for oocyte maturation in an effort to compare influences of alterations in conditions for in vitro maturation (IVM) and zygote culture on early development in ~itro. Incubations of oocytes were carried out at 39°C under 5% CO2, 5% O2,90% N2 in a humid atmo320

Brackett et al.

LH-enhanced maturation and bovine IVF

sphere. After 24 to 26 hours, the oocytes were examined for cumulus expansion denoting maturation and readiness for exposure to sperm. Sperm Preparation

For sperm treatment, a modified Tyrode's balanced salt solution (T ALP) was prepared18 with HEPES (2.3 mg/ml), sodium bicarbonate (160 p,g/ ml), glucose (0.9 mg/ml), sodium pyruvate (112 p,g/ ml), sodium lactate (2.90 p,g/ml), gentamicin sulfate (50 p,g/ml), and bovine serum albumin (6 mg/ ml, essentially fatty acid-free, Sigma). From pooled semen, 0.2-ml aliquots were layered under 1.0 mlofHEPES-TALP in six small (12 X 55 mm) tissue culture tubes, each held at a 45-degree angle for 1 hour. The top 0.8 ml from each tube was then pooled into a sterile centrifuge tube and centrifuged (350 X g for 10 minutes), and the sperm pellet (after discarding supernatant) was resuspended to 200 p,l with HEPES-T ALP containing heparin (10 p,g/ml).18 Sperm concentration and motility were assessed and 6- to 10-p,1 aliquots were added to 50-p,1 droplets of fertilization medium to give a final sperm concentration of 106/ml. In Vitro Insemination

For insemination, IVF-TALP medium 18 containing sodium bicarbonate (2.1 mg/ml), sodium pyruvate (20 p,g/ml), sodium lactate (1.35 p,g/ml), glucose (0.9 mg/ml), hypotaurine (1 p,g/ml), and bovine serum albumin (6.0 mg/ml) was used. Following the maturation interval, oocyte-cumulus complexes were examined for cumulus expansion (40X). Some of these were exposed to hyaluronidase (1 mg/ml) in HEPES-TALP for 2 to 3 minutes. Surrounding cumulus cells were removed by fine bore pipetting for visualization of first polar bodies and homogeneity of ooplasm. Treated 00cytes were washed (three times) in HEPES-TALP and transferred to droplets (5 oocytes per 50-p,1 droplet) of IVF-TALP to which sperm cells were added. Incubation conditions for fertilization and subsequent culture were as for maturation. The insemination interval was 16 to 18 hours. Zygote Culture and Assessment

Oocytes were transferred from the sperm suspension into a modified Ham's F-10 medium (Sigma Chemical Co., St. Louis, MO)/Basal Eagle's medium (BME, Sigma) with 1.7 mg/ml sodium bicarbonate added and prepared with 10% Fertility and Sterility

(vol/vol) fetal calf serum (FCS)19 for culture. Approximately 32 hours later (48 hours after insemination), eggs were examined for cleavage (to 2- and 4-cell stages). For subsequent culture, the embryos were transferred into fresh droplets for an additional24-hour interval, at which time observations were made (for 6- to 8-cell stages). Oocytes handled similarly but not inseminated were examined for evidence of parthenogenetic cleavage in each experiment. In the last experiments, comparisons were carried out involving zygote culture in HEPES-TCM 199 supplemented with pyruvate (50 JLg/ml) and gentamicin sulfate (50 JLg/ml), and 10% FCS with bicarcarbonate-TCM 199 prepared as indicated above for routine oocyte maturation, and with the standard zygote culture conditions involving use of Ham's F-10:BME. In these experiments, cumulus cells freed initially during washing of oocyte-cumulus complexes were cultured separately and included in the media for zygote and embryo culture. Chi-square analyses were performed for assessment of statistical differences in data acquired. Cleavage to 2- to 4-cell stages by 48 hours after insemination provided the endpoint for fertilization and continued development to 6- to 8-cell stages during the subsequent interval was evidence for continued viability. Embryo Transfer

Following one experiment, three 8-cell stage embryos were loaded into the tip of a tom cat catheter connected by tubing to a 1-ml syringe. The catheter with attached tubing was threaded through an insemination pipette for transcervical delivery of embryos into the uterine horn ipsilateral to the corpus luteum of the recipient cow. The cow was selected to be within 12 hours of synchrony with the developing embryos. The recipient was examined subsequently for return to estrus and by rectal palpation to assess pregnancy at 45 and 90 days after transfer. RESULTS

Oocytes were obtained from approximately 150 ovaries taken from slaughtered cows, predominantly Holstein but with some Angus and other beef breeds represented. An average of 6 good oocyte-cumulus complexes were isolated from each ovary for study. In all experiments, consistently high proportions (95% to 100%) of selected immaVol. 52, No.2, August 1989

Table 1 Influences of High LH and Cumulus Oophorous on Bovine IVM, IVF, and Early Development

Hormones forIVM E2

LH

IVF without (-) or with (+) cumulus a

Proportions (%) of inseminated oocytes reaching: 2-cell b

4- to 8-cell'

"g(ml

1

10

+ 1

100

+

21/65 45/95 29/66 60/80

(32.3) (47.4) (43.9) (75.0)

3/21 (14.3) 16/45 (35.6) 9/29 (31.0) 30/60 (50.0)

a In all cases, presence of cumulus cells was associated with superior results (P < 0.05). b A higher proportion of oocytes inseminated with cumulus cells left intact cleaved after maturation with the higher LH concentration (P < 0.05). C Separate comparisons of development of oocytes without cumulus and with cumulus at insemination demonstrated that higher proportions of embryos resulting from IVF of oocytes matured with the higher concentration of LH continued to develop in vitro (P < 0.05).

ture bovine oocytes matured in vitro as evidenced by characteristic cumulus expansion and presence of first polar bodies (apparent on cumulus cell removal). No evidence of parthenogenetic cleavage was observed in 120 control oocytes (approximately 10/treatment) handled similarly to those in experimental treatments but without insemination. Higher proportions of oocytes cleaved after in vitro insemination (P < 0.05), and higher proportions of embryos reached 4- to 8-cell stages in vitro (P < 0.05) when cumulus cells were not removed after IVM (Table 1). Following IVM in the presence of added E2 (1 JLg/ml) , when cumulus cells were left intact at insemination, higher proportions of oocytes were fertilized (P < 0.05) when high (100 JLg/ml) than when low (10 JLg/ml) concentrations of LH were present during IVM. When cumulus cells were removed before insemination, the improvement of IVF after maturation with high LH concentration over IVF after maturation with low LH, was not statistically significant (Table 1). This finding suggested that the favorable influence of high LH concentrations during IVM was carried over and mediated by cumulus cells as reflected in improved IVF. That the high concentration of LH improved (P < 0.05) cytoplasmic maturation was indicated by improved in vitro development of embryos resulting from IVF even when cumulus cells were removed before insemination (Table 1). Subsequently, it was found that E2 supplementation ofthe serum-containing medium for IVM was Brackett et al.

LH-enhanced maturation and bovine IVF

321

Table 2 Cumulus-Mediated LH Effect During Maturation on Bovine IVF and Early Development Hormones added" E2

Proportions (%) of inseminated oocytes reaching:

LH

2- to 4-cell

6- to 8-cell b

0 1 10 10 100

17/40 (42.5) 7/30 (23.3) 21/40 (52.5) 24/40 (60.0) 30/40 (75.0)

3/17 (17.6) 1/7 (14.3) 7/21 (33.3) 7/24 (29.2)

PI1/ml

0 0 0 1 0

20/30 (66.7)C

" Control and other maturation media contained 20% proestrous cow serum. b Ten-fold increments in LH concentration during oocyte maturation were reflected in significant improvements in viability of embryos (P < 0.05). C Pregnancy resulted after uterine transfer of three 8-cell embryos from this group via cervix.

unnecessary (Table 2). Incremental LH supplementation of the medium for IVM (by 10-fold steps) was reflected in increased proportions of 00cytes undergoing IVF and in increased proportions (P < 0.05, for each step) of fertilized ova that developed in vitro to 6- to 8-cell stages (Table 2). Pregnancy following nonsurgical transfer of three 8-cell embryos provided additional documentation of continued viability. Another experiment was carried out to extend the comparison of beneficial influences of high LH concentrations, i.e., 100 versus 1,000 JLg LH/ml supplementation of media for IVM. When IVM occurred in the presence of media supplemented with LH 100 JLg/ml, 41 (65.1 %) of63 oocytes were fertilized, 10 (24.4%) reached 4- to 6-cell and 9 (22.0%) reached at least 6- to 8-cell stages. Comparable data for oocytes matured with LH 1,000 JLg/ml included IVF of 33 (77.7%) of 46 oocytes, and development of 10 (30.3%) to 4- to 6-cell and of 11 (33.3%) to at least 6- to 8-cell stages in vitro. The improvements afforded at each stage by the increased LH in this experiment were not statistically significant. Results of experiments designed to study influences of alterations in conditions for IVM and zygote culture on early development in vitro are presented in Table 3. In these experiments, HEPES-TCM 199 and standard conditions, i.e., bicarbonate-TCM 199 for maturation and Ham's F-10: BME for culture, provided superior IVF and in vitro embryo culture results when compared with the use of bicarbonate-TCM 199 for maturation and zygote/embryo culture. 322

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LH-enhanced maturation and bovine IVF

DISCUSSION

That oocytes must be supported by cumulus and underlying granulosa, with some direct cell-oocyte contact, for full maturation and normal subsequent embryonic development, has been shown in sheep.20 Addition of LH to culture media did not affect the completion of maturation, but increased the fertilizability and normal development of extrafollicular oocytes (sheep12, rat 13 ). The undisturbed cumulus contributed to enhanced IVF and subsequent viability in the present work (Table 1). Improved incidences of sperm penetration followed bovine oocyte maturation in presence of FSH or cyclic adenosine monophosphate when compared with untreated controls.21 Addition of FSH + LH + E2 to the medium for bovine oocyte maturation improved IVF results in other experiments. 15 Recent work in our laboratory further defined beneficial addition of gonadotropins and 17,B-estradiol, confirmed necessity for selection of intact oocytecumulus complexes with homogeneous cytoplasm, and extended previous efforts of others22 to identify optimal sera for inclusion in media for oocyte maturation. These efforts revealed inclusion of proestrus (day 20) bovine serum to be optimal, and of sera tested from all phases of the estrous cycle, it had the highest concentration of LH. 9 Steroids, particularly estrogens, have been implicated in cytoplasmic maturation, including synthesis of the presumed male pronucleus growth factor necessary for normal fertilization. 1o,11 Proestrous bovine serum adequately satisfied these requirements (Table 2). Table 3 Influences of Alterations in Conditions for IVM" and Zygote Culture on Early Development In Vitro Proportions (%) of inseminated oocytes reaching: Media for IVM and culture

2- to 4-cell d

6- to 8-cell e

HEPES-TCM 199 b Bicarbonate-TCM 199 b Standard c

44/60 (73.3) 15/34 (44.1) 14/25 (56.0)

22/44 (50.0) 4/15 (26.7) 8/14 (57.1)

" All contained 100 p.g LH per milliliter. b Zygote culture with cumulus cells. C Bicarbonate-TCM 199 for IVM with zygote culture in Ham's F-I0:BME. d Higher proportions reached 2- to 4-cell stages when HEPES-TCM 199 ami standard conditions were used than following use ofbicarbonate-TCM 199 (P < 0.05). e Higher proportions of embryos continued in vitro development to 6- to 8-cell stages after IVF under HEPES-TCM 199 and standard conditions than under bicarbonate-TCM 199 conditions (P < 0.05).

Fertility and Sterility

Previous investigators have supplemented oocyte maturation media with up to 1,000-fold physiologic levels of gonadotropins (referred to in this work as low LH concentrations, e.g., 10 p.g/ml). Enhancement of results followed an additionall0-fold amplification of LH (described here as high LH) to which oocyte-cumulus complexes were exposed. This is the first report in which highly purified LH of bovine origin (USDA-bLH-B-5) was employed, thereby amplifying LH activity in the bovine system to an even greater extent by comparison with other LH preparations. Culture conditions, including presence of cumulus cells, enabling bovine uterine-stage embryos to be reached, were similar to those employed for culture of IVF rabbit embryos19,23 and to those used recently to achieve normal bovine pregnancies following oocyte maturation, IVF, and culture before nonsurgical transfer. 6 These advances obviate the necessity for in vivo culture of bovine IVF embryos in oviducts of rabbits,24 sheep,4,7,25 or heifers5before transfer to recipient cows. The present report further documents, in cattle, feasibility for complete in vitro development from immature oocytes to transferable uterine-stage embryos. Recent laparoscopic improvements, including the facility to transfer oocytes and/or early embryos directly into the oviduct,17 provide the means for clinical application ofIVF for extending fertility of valuable breeding cattle. Pregnancy obtained in the present report further advances the potential applicability of IVF in cattle. Additionally, the economic production of bovine embryos by IVF with culture to stages that can be most efficiently frozen presents a means for upgrading cattle herds around the world. The potential for further development of reproductive and genetic technologies employing IVF can be looked to for improvements in nutrition and many other avenues influencing the quality of human life in the future. Improvement of oocyte quality by an enhancing influence of homologous LH during in vitro maturation resulted in improved IVF and embryonic viability in this work. One might speculate that greater control of human oocyte quality can be afforded, as is the case in cattle, by in vitro oocyte maturation in contrast to in vivo maturation resulting from hormonal treatment of patients. Obvious difficulties with this approach include availability of purified human LH (or LH with this activity) and retrieval of oocytes from unstimulated ovarian follicles. Nevertheless, these data suggest this approach as a potential alternative in certain Vol. 52, No.2, August 1989

patients who do not respond optimally to hormonal treatments but from whom normal immature 00cytes might be obtained. Such an approach might facilitate more appropriate conditions, within the treatment cycle, for synchronous embryo transfer apart from excessive exogenous hormonal influences. Alternatively, one would expect more viable embryos to better withstand the rigors of frozen storage with development following appropriately scheduled transfer during a subsequent menstrual cycle.

Acknowledgments. We wish to thank Atlantic Breeders Cooperative, Lancaster, Pennsylvania for semen; USDA Animal Hormone Program and National Hormone and Pituitary Program, for LH; Shapiro Packing Co., Augusta, Georgia, for ovaries; Dan C. Cabaniss, D.V.M., Cabaniss Dairy Farm, Madison, Georgia for assistance with the recipient cow; Mr. Ken Smith and Mr. Andrew Wooten, for technical assistance; and Ms. Joanne Foster, for typing.

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