Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Theriogenology 38:823-831,

1992

OVULATION AND EMBRYO RECOVERY RATES FOLLOWING IMMUNIZATION OF MARES AGAINST AN INHIBIN ALPHA-SUBUNIT FRAGMENT P.M. McCue 1 N.J. Camey,* J.P. Hughes,l J. Rivier, 31W. Vale3 and B.L. Lasleyl lDepartment of Reproduction *Veterinary Medical Teaching Hospital School of Veterinaq Medicine University of California, Davis Davis, CA 95616 3The Salk Institute The Clayton Foundation Laboratories for Peptide Biology San Diego, CA 92186 Received for publication: March 70, 1992 Accepted: September16, 2992 ABSTRACT Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund’s incomplete adjuvant. Immunized mares ovulated a significantly larger (P
Copyright 0 1992 Butterworth-Heinemann

Theriogenolog

y

INTRODUCTION The physiologic mechanisms responsible for limiting the development and ovulation of multiple follicles during the normal follicular phase in the mare are not yet fully understood. Studies in other species have suggested that the dominant follicle is responsible for the suppression of smaller follicles through the inhibition of follicle stimulating hormone (FSH) secretion (l-3). This decline in FSH that occurs in the mare during late diestrus and early estrus may be the cause of the atresia that occurs in the smaller follicles (4). Maintenance of elevated FSH concentrations during the follicular phase may prevent the regression or atresia of these smaller follicles and thus result in multiple ovulations. Attempts at inducing multiple ovulation in the mare using pregnant mare serum gonadotropin (PMSG; 5-6), equine pituitary extracts (EPE, 7-13) and FSH (4,12) have met with limited success. This is in marked contrast to the multiple ovulations that occur in ruminants following administration of FSH or PMSG (14,15). An alternative strategy to the administration of exogenous gonadotropins for the purpose of superovulation is to increase endogenous FSH by eliminating gonadal factors that inhibit FSH secretion. Active immunization against inhibin, a glycoprotein produced in granulosa cells of the ovary (16) that specifically suppresses FSH, has resulted in an increase in ovulation rates in the ewe (17-20) and gilt (21). The goal of this study was to determine if active immunization of mares with a synthetic fragment of the alpha-subunit of porcine inhibin would result in increased ovulation and embryo recovery rates in the mare. MATERIALS AND METHODS Animals Twenty nonlactating mares between 4 and 12 years of age were used in the study. All mares were maintained in outdoor paddocks under ambient light conditions, and were fed a maintenance ration of alfalfa hay with fresh water and a trace mineral supplement available ad libitum. Immunogen Preparation The immunogen used was a synthetic porcine alpha-subunit fragmenta (pIa(l-26)GY) conjugated to bovine serum albumin (BSA) using 0.2 96 glutaraldehyde (22). In brief, inhibin was incubated with 3 times the weight excess of BSA dissolved in coupling buffer (0.1 M sodium phosphate, pH 7.0) in the presence of 0.2 96glutaraldehyde, for 4 hours at room temperature. The reaction was stopped by dialysis against 6 1 of 0.9 96 NaCl at 4” C with 3 changes over 24 hours, using dialysis tubingb with a molecular weight aThe Salk Institute, La Jolla, CA. bSpectrum Medical Industries, Inc., Los Angeles, CA.

Theriogenology

825

cutoff of 3400. The inhibin:BSA conjugate was diluted to a final concentration of 2 mg protein per ml. Immunizations consisted of 1 ml of the inhibin:BSA conjugate emulsified in 1 ml of Freund’s incomplete adjuvant,c divided into 2 to 4 injection sites. Immunizations Mares were randomly assigned to treatment or control groups. Six mares were immunized 5 times at 3-week intervals beginning on August 20, 1991. Fourteen mares were maintained as nonimmunixed controls. Inhibin Binding The antibody response in immunized and control mares was determined by the ability of serum samples to bind ‘2sI-labeledinhibin or-subunit(22). Serum samples were tested at a final dilution of l:lO,OOO,using normal horse serum as the carrier and high-titer rabbit anti-equine gamma-globulinsd as the second antibody at a dilution of 1: 10. Inhibin binding (46) was determined in samples collected prior to the first immunization and one week after the third and fifth immunizations. Reproductive Evaluation Beginning on the date of the third immunization, the 6 immunized and 6 of the control mares were evaluated daily during estrus and every other day during die&us by palpation per rectum and ultrasonography of the reproductive tract. The diameter of all follicles > 20 mm, the number of ovulations and the interovulatory intervals were recorded. Following the fifth immunization, the 6 immunized mares and the 8 remaining control mares were inseminated with a minimum of 500 million progressively motile spermatoxoa, collected from a stallion of known fertility. Inseminations began on the second day of estrus and continued until ovulation was detected by palpation and ultrasonography. Human chorionic gonadotropine (hCG; 2,500 ID) was administered as an intravenous injection to all mares when the diameter of the largest follicle was 2 35 mm. Embryo recovery was performed on Day 7 postovulation (23). Ten embryo recovery attempts were made from mares in both the immunized and control groups. Additional embryo collection attempts were not possible due to the decline in ovarian activity associated with the winter anestrum.

CGibco Laboratories, Inc., Grand Island, NY. dAntibodies, Inc., Davis, CA. eLyphoMed, Inc., Rosemont, IL.

Theriogenology

826 Statistical Analysis

Comparisons of the number of ovulations per estrous cycle, embryo recovery rates and interovulatory intervals between immunized and control groups were made by the Mann-Whitney test (24). The correlation coefficient between inhibin antibody titer and ovulation rate was calculated for the cycle following the fifth immunization by the least squares method (24). All values are expressed as the mean f SEM. RESULTS Immune Response A significant antibody response (3 to 15 % iBI-labeled porcine inhibin binding) was present in 4 of the treated mares following the third immunization, while high titers (> 20 % binding) were present in 5 of 6 mares following the fifth immunization (Figure 1). In contrast, binding of labeled inhibin remained undetectable in the control mares throughout the experiment.

40

g/26/91 a6524

m8547

t

10/7/91 t t t t Date of Sample Collection a9609

1148504

m8593

11/11/91 m8507

Figure 1. Antibody response in mares following immunization against a synthetic alphasubunit inhibin fragment as estimated by binding of ‘2sI-labeled porcine inhibin. Arrows indicate approximate dates of immunization.

827

Theriogenology

Ovulation Rate The 6 immunized mares averaged 1.3 f 0.2, 2.3 f 0.6 and 2.8 f 1.1 ovulations per cycle following the third, fourth and fifth immunizations, respectively (Table 1). The ovulation rate for nonimmunized control mares (n = 14) was 1.1 f 0.1 ovulations per cycle. The ovulation rate for mares immunized against inhibin following the fifth immunization was significantly higher (P0.05). The interovulatory interval for immunized mares (22.6 f 1.6 days) was not significantly different (P>O.O5) from that of control mares (23.1 f 0.6 days).

Table 1.

Ovulation rates in 6 mares following the third, fourth and fifth immunizations against inhibin.

Mare

3

6524 8504 8507

1 1 2

8547 8593 9609

1 2 1

Mean

1.3 f 0.2

Immunization # 4

5

3

2

1 1 5 2 2

1 1 8 2 3

2.3 f 0.6

2.8 f 1.1

Embryo Recovery Rate The Day-7 embryo recovery rate for immunized mares (1.6 f 0.5 embryos per flush; range 0 to 4; n= 10) tended to be greater (P< 0.1) than the recovery rate for the control mares (0.7 f 0.2 embryos per flush; range 0 to 2; n=lO). The ratio of embryos recovered per ovulation was not significantly different (P > 0.05) between the immunized and control groups (0.58 and 0.57, respectively). Four healthy blastocysts and 1 unfertihzed oocyte were recovered on 2 occasions from a mare that ovulated 8 and 5 follicles per cycle, respectively.

Jberiogenology

828 DISCUSSION

Ovulation rate following the immunization series was 2.8 f 1.1 ovulations per cycle, with 1 mare ovulating as many as 8 follicles during 1 estrous period. Four out of the 6 mares exhibited multiple ovulations during successive estrous cycles following active immunization. It is possible that the ovulation rate would have been increased further had immunizations been continued over a longer period of time, performed earlier in the breeding season, or had a higher dosage of immunogen been used. The physiologic mechanism responsible for the increased ovulation rate in immunized mares is presumed to be the neutralization of endogenous inhibin secreted by granulosa cells of the developing follicles ( 17-19). Reduction in bioactive inhibin would allow FSH concentrations to remain elevated during follicular recruitment, development and maturation, culminating in an increased ovulation rate. Concentrations of FSH are normally decreasing during folliculogenesis, limiting growth of all but the most mature follicles (l-4). No significant correlation was found between ‘2sI-labelled inhibin binding and ovulation rate in immunized mares. This was due, in part, to the low sample size and the fact that one mare (# 8504) had a high antibody titer (Figure l), yet consistently ovulated one follicle (Table 1). A similar lack of correlation was reported for ewes immunized with a recombinant inhibin subunit (13). Previous attempts to induce multiple ovulations in the mare with PMSG have been unsuccessful (5,6), possibly due to the low binding of PMSG exhibited by equine ovarian FSH receptors (25). Ovulation rates of 1.7 to 4.6 ovulations per cycle were reported following administration of equine pituitary extract to cyclic mares (7-13), while 1.6 to 1.7 ovulations per cycle were reported following twice-daily administration of porcine FSH (4,12). Induction of multiple ovulations with pituitary extract or FSH requires daily or twice daily treatment, respectively, from the end of die&us until ovulation. Collection and extraction of gonadotropins from equine pituitaries is labor intensive (26), and treatment with FSH is both less effective and prohibitively expensive (12). In contrast, immunization against inhibin may be done once every third week and be effective over several subsequent estrous cycles. One mare in the present study ovulated an average of 5.7 f 1.2 follicles per cycle for 3 consecutive cycles following the fifth immunization. The number of embryos recovered per induction in immunized mares (1.6 + 0.5) was approximately double that recovered from the nonimmunized control mares (0.7 f 0.2), and yet the percentage of embryos recovered per ovulation was similar (58 and 5796, respectively). This suggests that immunization against inhibin and induction of multiple ovulations do not significantly affect embryo viability, and immunization may be useful in providing multiple embryos for transfer. The percentage of embryos recovered per ovulation following treatment with equine pituitary extract has ranged from 35 to 60% (9,11,13), while embryo recovery rates reported from normal nonstimulated mares range from 43 to 75% (23,27).

829

Theriogenology

Currently, most embryo collection attempts in the mare follow the ovulation of a single follicle. Induction of multiple ovulations and collection of multiple embryos would improve the efficiency of equine embryo transfer. The viability of embryos produced following multiple ovulations induced by inhibin immunoneutralization, however, must still be addressed. While some studies suggest that embryos collected from multipleovulating mares are less viable than embryos collected from single ovulating mares (11,28,29), Squires et al. (13) report that the viability of embryos collected from spontaneous and induced multiple-ovulating mares was equal to that of embryos from single-ovulating mares. In summary, immunization of mares against a synthetic porcine inhibin alpha-subunit fragment resulted in an increase in ovulation and embryo recovery rates and did not affect the length of the estrous cycle. This technique may be beneficial in providing multiple embryos for collection and transfer in the mare and, possibly, in stimulating development of multiple follicles prior to collecting ova for in vitro fertilization.

REFFXENCES

1.

Baird, D.T. Factors regulating the growth of the preovulatory follicle in the sheep and human. J. Reprod. Fertil. @: 343-352 (1983).

2.

Driancourt, MA. Follicular dynamics in sheep and cattle. Theriogenology s: 55-79 (1991).

3.

Ireland, J.J. Control of follicular growth and development. Fertil. 3 (Suppl.): 39-54 (1987).

4.

Irvine, C.H.G. Endocrinology of the estrous cycle of the mare: applications to embryo transfer. Theriogenology fi: 85-104 (1981).

5.

Day, F.T. Ovulation and the descent of the ovum in the fallopian tube of the mare after treatment with gonadotrophic hormones. J. Agri. Sci. 2:459-469 (1939).

6.

Day, F.T. Clinical and experimental observations on reproduction in the mare. J. Agri. Sci. 3: 244-261 (1940).

7.

Douglas, R.H., Nuti L. and Ginther, O.J. Induction of ovulation and multiple ovulation in seasonally-anovulatory mares with equine pituitary fractions. Theriogenology 2: 133-142 (1974).

8.

Lapin, D.R. and Ginther, O.J. Induction of ovulation and multiple ovulations in seasonally anovulatory and ovulatory mares with an equine pituitary extract. J. Anim. Sci. M:834-842 (1977).

J. Reprod.

830

Theriogenolog y

9.

Douglas, R.H. Review of superovulation and embryo transfer in the equine. Theriogenology j&33-46 (1979).

10.

Woods, G.L. and G&her, O.J. Induction of multiple ovulations during the ovulatory season in mares. Theriogenology 2Q: 347-355 (1983).

11.

Woods, G.L. and G&her, O.J. Collection and transfer of multiple embryos in the mare. Theriogenology 21: 461-469 (1984).

12.

Squires, E.L., Garcia, R.H., Ginther, O.J., Voss, J.L. and Seidel, G.E. Comparison of equine pituitary extract and follicle stimulating hormone for superovulating mares. Theriogenology 26: 661-670 (1986).

13.

Squires, E.L., MCKiMOn, A.O., Camevale, E.M., Morris, R. and Nett, T.M. Reproductive characteristics of spontaneous single and double ovulating mares and superovulated mares. J. Reprod. Fertil. % (Suppl.): 399403 (1987).

14.

Maplecroft, R.J. Bovine embryo transfer. I&: Morrow, D.A. (ed), Current Therapy in Theriogenology. W.B. Saunders Co., Philadelphia, 1986, pp. 5458.

15.

Bondurant, R.H. Embryo Transfer in sheep and goats. In: Morrow, D.A., (ed) Current Therapy in Theriogenology, 2nd ed. W.B. Saunders Co., Philadelphia, 1986, pp. 63-66.

16.

Cuevas, P., Ying, S., Ling, N., Ueno, N., Esch, F. and Guillemin, R. Immunohistochemical detection of inhibin in the gonad. B&hem. Biophys. Res. Comm. .IQ: 23-30 (1987).

17.

Forage, R.G., Brown, R.W., Oliver, K.J., Attache, B.T., Devine, P.L., Hudson, G.C., Goss, N.H., Bertram, K.C., Tolstoshev, P., Robertson, D.M., de Krester, D.M., Doughton, B., Burger, H.G. and Findlay, J.K. Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. J. Endocrinol. u:Rl-R4 (1987).

18.

Mizumachi, M., Voglmayr, J.K., Washington, D.W., Chen, C.L.C. and Bardin, C.W. Superovulation of ewes immunized against the human recombinant inhibin a-subunit associated with increased pre- and postovulatory follicle stimulating hormone levels. Endocrinology 626: 1058-1063 (1990).

19.

Wrathall, J.H.M., McLeod, B.J., Glencross, R.G., Beard, A.J. and Knight, P.G. Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin alpha subunit: effects on gonadotropin concentrations and ovulation rate in sheep. J. Endocrinol. 124: 167-176 (1990).

Theriogenology

831

20.

Schanbacher, B.D., Schemm, S.R. and Rhind, S.M. Gonadotrophin concentrations and ovulation rates in Suffolk ewes actively or passively immunized against inhibin alpha. J. Reprod. Fertil. es: 133-139 (1991).

21.

Brown, R.W., Hungerford, J.W., Greenwood, P.E., Bloor, R.J., Evans, D.F., Tsonis, C.G. and Forage, R.G. Immunixation against recombinant bovine inhibin alpha subunit causes increased ovulation rates in gilts. J. Reprod. Fertil. $jQ:199-205 (1990).

22.

Vaughan, J.M., Rivier, J., Conican, A.Z., McClintock, R., Campen, CA., Jolley, D., Voglmayr, J.F., Bardin, C.W., Rivier, C. and Vale, W. Detection and purification of inhibin using antisera generated against synthetic peptide fragments. In: COM, P.M. (ed), Methods in Enzymology, Vol. 168 Academic Press, Inc., Orlando, FL, 1989, , pp. 588-617.

23.

Imel, K.J., Squires, E.L., Elsden, R.P. and Shideler, R.K. Collection and transfer of equine embryos. J. Am. Vet. Med. Assoc. m:987-991 (1981).

24.

Daniel, W.W. Biostatistics: a foundation for analysis in the health sciences. 5th ed., John Wiley & Sons, New York, 1991, pp. 366-630.

25.

Stewart, F. and Allen, W.R. The binding of FSH, LH and PMSG to equine gonadal tissues. J. Reprod. Fertil. z (Suppl.): 431-440 (1979).

26.

Braselton, W.E. and McShan, W.H. Purification and properties of folliclestimulating and l&nixing hormones from horse pituitary glands. Arch. B&hem. Biophys. .Q9: 45-48 (1970).

27.

Vogelsang, S.G. and Vogelsang, M.M. Influence of donor parity and age on the success of commercial equine embryo transfer. Eq. Vet. J. 8 (Suppl.): 71-72 (1989).

28.

Woods, G.L. and Ginther, O.J. Ovarian response, pregnancy rate and incidence of multiple fetuses in mares treated with an equine pituitary extract. J. Reprod. Fertil. z (Suppl.): 415-421 (1982).

29.

Squires, E.L., Garcia, R.H. and Gin&r, O.J. Factors affecting success of equine embryo transfer. Eq. Vet. J. 9 (Suppl.): 82-95 (1985).