Maintenance of Sexual Function with Testosterone in the Gonadotropin-Releasing Hormone-Immunized Hypogonadotropic Infertile Male Rat

BIOLOGY OF REPRODUCTION 49, 1170-1176 (1993) Maintenance of Sexual Function with Testosterone in the Gonadotropin-Releasing Hormone-Immunized Hypogonadotropic Infertile Male Rat CALEB A. AWONYI,1'2 MELANIE S. REECE,2 BRADLEY S. HURST, 2 KENNETH A. FABER, 2 VARADARAJ CHANDRASHEKAR, 3 and WILLIAM D. SCHLAFF2 Division of Reproductive Endocrinology,2 Department of Obstetrics and Gynecology University of ColoradoHealth Sciences Center, Denver, Colorado 80262 Department of Physiology,3 Southern Illinois University, Carbondale, Illinois 62901 We have previously shown that active immunization against GnRH in the mature male rat can predictably produce hypogonadotropic hypogonadism and azoospermia and, further, that normospermia and normal fertility can be restored by testosterone (T) administration alone. The objective of this study was to explore the hypothesis that GnRH-immunized azoospermic rats could be supplemented with T doses sufficient to restore sexual behavior, but insufficient to support adequate spermatogenesis or to allow restoration of fertility. Adult male rats of proven fertility were immunized against GnRH and supplemented with 2-, 4-, or 8-cm T implants or with empty implants. Eight weeks later, fertility was evaluated; concentrations of serum T, LH, FSH, growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) were determined; sperm number was obtained from the testis; and weights of androgen-dependent organs were measured. GnRH immunization and T supplementation resulted in restoration of organ weights and of fertility in a dose-dependent manner. GnRH immunization with or without T supplementation resulted in the absence of circulating gonadotropins, but had no effect on serum GH, PRL, or TSH levels. Whereas all control animals were fertile, rats that received either empty or 2-cm T implants were completely infertile. Rats that received 4-cm or 8-cm T implants were fertile in 60% and 100% of cases, respectively. Sexual behavior of rats with empty and with 2-cm T implants was compared at 10-18 wk after immunization with GnRH. GnRH-immunized rats given empty implants displayed negligible sexual activity, but those with 2-cm T implants displayed sexual activity equivalent to that of untreated controls despite complete infertility. These data indicate that 1) GnRH immunization selectively eliminates gonadotropins without affecting other pituitary hormones (GH, PRL, and TSH) and 2) supplementation with exogenous T at a low dose (2-cm implant) maintains sexual behavior without restoring fertility in GnRH-immunized azoospermic rats. INTRODUCTION The objective in devising a nonsurgical male contraceptive is to prevent the release of functional spermatozoa at the time of ejaculation. There are three possible sites of action for a male contraceptive: 1) the testis during spermatogenesis, 2) the caput epididymidis during sperm maturation, and 3) the cauda epididymidis during sperm storage. Of the three, suppression of spermatogenesis has been studied most extensively. It is possible to inhibit sperm production indirectly by suppressing the secretion of testosterone (T), which is required for spermatogenesis. This has been achieved variously by using exogenous androgens [1-5] to inhibit the release of LH, and by blocking the action of GnRH [6-12]. However, drugs such as gossypol [13-15], tolnidamine [16], and sulfasalazine [1721] can also directly inhibit spermatogenesis. While these methods suppress sperm production, they also cause decreased libido, irreversible sterility, and other unacceptable side effects. Recently, we reported that active immunization against Accepted August 6, 1993. Received June 16, 1993. Correspondence: Dr. Caleb A. Awoniyi, Department of Ob-Gyn, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. FAX: (303) 270-4804. GnRH in male rats induces complete azoospermia and that T supplementation to immunized rats restores both sperm production and sperm function in a dose-dependent manner [22]. Specifically, subsequent treatment of GnRH-immunized azoospermic rats with T implants 8-24 cm in length restored normal spermatogenesis and fertility, whereas 4-cm T implants restored spermatogenesis and fertility only partially. These results suggest an experimental paradigm with potential application for male contraception. If T implants of 4 cm or less release T at a rate that is insufficient to restore normal spermatogenesis in GnRHimmunized rats but high enough to maintain sexual behavior, and if these implants do not suppress other pituitary hormones, then the combination of GnRH immunization and low-level T replacement is a viable model for male contraception. In the present studies, we examined 1) the effect of GnRH immunization on pituitary secretion of LH, FSH, growth hormone (GH), prolactin (PRL), and thyroid stimulating hormone (TSH); and 2) fertility and sexual behavior in GnRHimmunized rats receiving low levels of exogenous T. The results indicate that GnRH immunization in rats selectively eliminates gonadotropin without affecting other pituitary hormones, and that low-dose T implants of 2 cm are capable of maintaining normal sexual behavior without restoring fertility. 1170 ABSTRACT TESTOSTERONE AND SEXUAL FUNCTION IN MALE RATS MATERIALS AND METHODS Animals and Fertility Screening Test Sexually mature male Sprague-Dawley rats (350-400 g) were housed in an approved vivarium under climate-controlled conditions, with a constant 14L:10D cycle. Rats were provided with rat chow and water ad libitum. Rats used in these studies were first proven to be fertile by their ability to impregnate females during a five-day period. The purpose of experiment 1 was to determine a) the effects of GnRH immunization on serum levels of gonadotropins, GH, PRL, TSH, and T; and b) the threshold dose of T below which GnRH-immunized rats remain infertile. Twenty-five rats were divided into five groups of 5 rats each. Rats in four groups were actively immunized against GnRH as previously described [1, 7, 23]. The control group received saline and adjuvant injections. Two booster injections were given at Weeks 4 and 6. At Week 8, T implants of 2, 4, or 8 cm were administered s.c. to three groups of GnRH-immunized rats with the remaining two groups (1 control and 1 GnRH-immunized) receiving empty silastic implants of 2 cm. Two months after implantation, each male rat was placed with 3 female rats for 5 days. After 20 days, female rats were killed. The ovaries were removed; and the number of corpora lutea, embryos, and implantation sites were determined. All GnRH-immunized and control rats were killed after the fertility trial. Serum was collected for measurement of T, LH, FSH, GH, PRL, and TSH by RIA. Testis, epididymis, prostate, and seminal vesicle weights were determined. One testis from each rat was used for determining the number of advanced spermatids by the hemacytometric counting of testicular homogen (...truncated)