Caleb A. Awoniyi, Ph.D.t Woo Kyoon Kim, M.D. Bradley S. Hurst, M.D. William D. Schlaff, M.D.

Transcription

1 FERTILITY A STERILITY Copyright 1992 The American Fertility Society Printed on acid-free paper in U.S.A. Immunoneutralization of gonadotropin-releasing hormone and subsequent treatment with testosterone Silastic implants in rats: an approach toward developing a male contraceptive* Caleb A. Awoniyi, Ph.D.t Woo Kyoon Kim, M.D. Bradley S. Hurst, M.D. William D. Schlaff, M.D. Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado Study Objective: To determine the extent to which increasing doses of exogenous testosterone (T) administered via Silastic implants can restore spermatogenesis and fertility to rats made azoospermic by active immunization against gonadotropin-releasing hormone (GnRH). Design: Male rats were made azoospermic by active immunization against GnRH. Increasing doses of exogenously administered T (via Silastic implants) were administered for 8 weeks, and testicular sperm concentration and ability to impregnate female rats were evaluated. Setting: Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado. Animals: Sexually mature male Sprague Dawley rats (SASCO, Omaha, NE). Results: Suppression of gonadotropins and azoospermia was achieved by actively immunizing rats against GnRH. Testosterone was capable of restoring quantitatively complete spermatogenesis and fertility in GnRH-immunized azoospermic rats. This relationship was dose-dependent, as evidenced by the partial restoration of spermatogenesis and fertility observed in animals replaced with smaller T Silastic implants. Conclusion: Gonadotropin-releasing hormone immunization and T-filled Silastic implants may provide a model to study isolated gonadotropin deficiency and for the development of a reversible male contraceptive. Fertil Steril 1992;58:403-8 Key Words: Azoospermia, fertility, gonadotropin-releasing hormone, Silastic implant Historically, research and development of contraceptive methods have been directed toward the female. As a consequence, females have borne a disproportionate contraceptive burden. Fortunately, in the Western world at least, the notion that contraception is the woman's responsibility is now changing. With this change in attitude and with recent Received February 11, 1992; revised and accepted April 14, * Presented in part at the 39th Annual Society for Gynecologic Investigation, San Antonio, Texas, March 18 to 21, t Reprints requests: Caleb A. Awoniyi, Ph.D., Department of Obstetrics and Gynecology, Campus Box B198, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado progress toward a better understanding of male reproductive physiology, research on development of a reversible male contraceptive has been intensified. It is possible to influence male fertility directly by affecting spermatogenic cells or indirectly by interfering with hypothalamic production of gonadotropin-releasing hormone (GnRH), pituitary production of luteinizing hormone (LH) and folliclestimulating hormone (FSH), and/or Leydig cell production of testosterone (T). Among the studies directed at male contraception in the literature, some have used androgens to suppress LH and T production (1, 2), others have suppressed GnRH with GnRH antibodies (3, 4) or with GnRH analogues (5, 6), and still others have used drugs such as gos- A woniyi et al. Reversible sterility in male rats 403

2 sypol (7, 8) that directly affect spermatogenic cells. Despite these studies, a satisfactory male contraceptive has yet to be developed. The major problems have been inconsistent inhibition of spermatogenesis, lack of spontaneous reversibility, and/or unacceptable side effects. Although many compounds tested as male contraceptive agents possess some antispermatogenic or anti fertilizing capacity, their contraceptive action is invariably overshadowed by their cytotoxic, neurotoxic, or antimetabolic effects, or by their untoward effects on libido, accessory sex glands, and male endocrine system (9). Recent studies indicate that T is capable of restoring quantitatively complete spermatogenesis in rats whose testes were induced to regress by the immunological interdiction of GnRH (3, 10). In contrast, spermatogenesis has not been quantitatively restored by exogenously administered T in chronically hypophysectomized rats (11-13). These results suggest that the use of immunological techniques may provide a promising model to understanding spermatogenic inhibition and restoration and ultimately to develop a male contraceptive. In the present study, we sought to extend our investigation of restoration of spermatogenesis to address the fertility potential of restored animals. By active immunization against GnRH, immunoneutralization of endogenous GnRH by antigen-antibody complexes was produced and resulted in hypogonadotropic hypogonadism and azoospermia. We then examined the degree to which graded doses of exogenous T, administered via Silastic implants, could restore not only spermatogenesis but fertility as well. MATERIALS A METHODS Animals and Experimental Design Adult male Sprague Dawley rats (300 to 350 g) were housed under standard controlled temperature (22 C) and lighting (14 hours of light, 10 hours of darkness) with food and water ad libidum. Male rats were first proven to be successful breeders. Briefly, each rat was placed with two normal cycling sexually mature female rats for 5 days. Twenty days after mating, the female rats were euthanized, and the number of embryos was determined. A male rat was considered fertile if one or both of the females became pregnant and infertile if neither of the two females became pregnant. Twenty-five male rats that successfully passed this fertility test were actively immunized against GnRH as previously de- scribed (3). In brief, 100 jlg of GnRH conjugated to human serum globulin was dissolved in 0.2 ml sterile saline and emulsified with an equal volume (0.2 ml) of Freund's adjuvant (GIBCO BRL, Grand Island, NY). After emulsification, the total dose of immunogen (0.4 ml/rat) was injected intradermally at multiple sites (12 to 15 sites). Primary immunization was given in Freund's complete adjuvant (GIBCO BRL), and boosters were given in Freund's incomplete adjuvant at weeks 4, 6, and 8 thereafter. Five control rats received injections of saline and adjuvant throughout the trial. Two weeks after the second booster injections (i.e., week 8), GnRH-immunized rats received subdermal implantation of T filled Silastic implants of 4, 8, 12, or 24 cm, or empty implants (n = 5/group). The five control rats received empty implants. Two months after implantation, each male rat was placed with 3 female rats for 5 days. After 20 days, female rats were euthanized. The ovaries were removed, and the number of corpora lutea (CL), embryos, and implantation sites were determined. All GnRH-immunized and control rats were euthanized after the fertility trial. Serum was collected for measurement of T, LH, and FSH by radioimmunoassay (RIA). Testis, ventral prostate, and seminal vesicle weights were determined. One testis from each rat was used for the determination of the number of advanced spermatids by the hemacytometric counting of testicular homogenate under phase-contrast microscopy (14). Preparation of Silastic Implants Testosterone implants were prepared by filling Silastic tubings (Dow Corning, Midland, MI) with T according to a previously described method (15) and then sealing both ends with medical adhesive A (Dow Corning). The inner diameter of the tubing was 1.98 mm, with an outer diameter of 3.18 mm. From previous studies, T release rate was known to be approximately 30 jlg/cm per day (16). Hormone Measurements Serum T concentrations were determined in duplicate samples by a previously described RIA procedure (17). The sensitivity ofthe assay was 10 pg/ tube with intra-assay and interassay coefficients of variation (CVs) of 10.7 and 9.5, respectively. Concentrations of LH and FSH were measured by a specific homologous RIA as previously described (18), using reagents graciously supplied by the National Hormone and Pituitary Program (NIDDK). The minimal amounts of hormone detected per assay 404 A woniyi et al. Reversible sterility in male rats Fertility and Sterility

3 tube were ng rat LH RP-2 and 0.25 ng rat FSH RP-2. All samples were measured in a single assay. The intra-assay CV was <10% for both LH and FSH. Statistical Analysis All data were analyzed by one-way ANOV A. Duncan's multiple range test was used to identify differences between the treatment groups (19). Values were considered significant at P < RESULTS Effects of T Treatment on GnRH-Immunized Azoospermic Rats The effects of 8 weeks of T treatment to GnRHimmunized azoospermic rats on serum T, LH, and FSH, and on seminal vesicle and ventral prostate weights are presented in Table 1. Serum levels of T, LH, and FSH in control rats were 2.1 ± 0.4, 1.3 ± 0.3, and 7.4 ± 1 ng/ml, respectively. Testosteronefilled implants of 4, 8, 12, and 24 cm resulted in a graded increase in serum levels of T. Serum levels of LH and FSH were non detectable in all GnRHimmunized rats. There was approximately 95% reduction in both seminal vesicle and ventral prostate weights in GnRH-immunized rats that received empty Silastic implants when compared with controls. When T-filled implants of 4 to 24 cm were administered to GnRH-immunized rats, seminal vesicles and ventral prostate weights increased approximately twofold when compared with controls. However, increasing doses of T did not cause a significant graded increase in the weights of seminal vesicle and ventral prostate. Figure 1 shows the effects of active immunization against GnRH and subsequent T treatment on the testis weight and testicular sperm contents. A sig- l X CJ iii 1 ~ en i= en w I- '" - 2~ ' c o A,,,,,,",/,,,...,'.. "",",, ',"",, " ','"..."",... '... '.. '",,,,, " '...'" 300~ , c I- Z ::I o 0100 ~ II: W Q. en o 24 c o Testosterone Implant Length (em) Figure 1 The effect of implanting GnRH azoospermic rats with T-filled Silastic implant of 4 to 24 cm for 2 months on testis weight (A) and sperm numbers per testis (B). For each graph, bars with asterisks (*) are significantly different (P < 0.05) from controls. nificant reduction in the testis weights (Fig. la) was observed in GnRH-immunized rats that received empty Silastic implants when compared with controls. Implantations of T-filled Silastic implants of 4 to 24 cm resulted in a graded increase in the testis weights. With the longer implant (24 cm), there was no significant difference between testis weights when compared with controls. In control rats, the testicular sperm content was 207 ± 18 X In contrast, there were no detectable spermatozoa in the testis of GnRH-immunized rats that received empty im- B Table 1 Effects of Treatment of GnRH-Immunized Azoospermic Rats With T Implants on the Serum T, LH, and FSH Concentrations, and on Seminal Vesicle and Prostate Weights* Treatment group T LH FSH Seminal vesicle Prostate Control Anti-GnRH + ot Anti-GnRH + 4 Anti-GnRH + 8 Anti-GnRH + 12 Anti-GnRH ± 0.4 :j: 4.2 ± ± ± ± 1.6 ng/ml 1.3 ± ± ± ± ± ± ± ± ± ± 0.1O 3.5 ± ± ± ± 0.23 g * Values are means ± SEM; n = 5 rats/group. t T implant length in cm. :j:, nondetectable. Significantly different from controls (P < 0.05). Awoniyi et al. Reversible sterility in male rats 405

4 plants. With T-filled implants of 4 cm in GnRHimmunized rats, the testicular sperm number was 8.8 ± 1 X 10 6, which is approximately 4% of controls. There was a further increase in testicular sperm numbers when GnRH-immunized rats were treated with T -filled implants of 8 and 12 cm. With T -filled implants of 24 cm, the testicular sperm number, 213 ± 13 X 10 6, was not significantly different from controls (Fig. 1B). Fertility Effects of T Treatment in GnRH Immunized Rats The effects of T-filled implants in GnRH-immunized azoospermic rats on restoration of fertility and on the numbers of embryos and CL when exposed to females are presented in Table 2. None of the GnRH-immunized rats that received empty Silastic implants was able to impregnate the female rats when cohabitated for 5 days. There was a slight improvement in the ability of GnRH-immunized rats to impregnate females when given T-filled implants of 4 cm (2 of 5 males impregnated female rats). In GnRH-immunized rats that received T filled Silastic implants of 8 to 24 cm, fertility of these male rats was similar to that of control rats (5 of 5 in each group). The mean number of embryos and CL present in female rats 22 days after cohabitation with control male rats were 14.0 ± 1 and 16.2 ± 1.3, respectively. There were no embryos in any of the female rats that were mated with GnRH-immunized rats that received empty implants. However, the number of CL was not significantly different from controls, confirming that an equal number of oocytes was available for fertilization by the male. In female rats that were cohabitated with GnRH-immunized male rats that received T-filled implant of 4 cm, the number of embryos was reduced significantly to 5.0 ± 3.1, and again, the number of CL was similar to controls. When GnRH-immunized rats were treated with T-filled implants of 8 to 24 cm, there were no significant differences in the number of embryos or CL when compared with controls (Table 2). The ratio of implantation sites to embryos was one for all treatment groups in which fertilization occurred, thus suggesting that there were no defects in fertilization capability of spermatozoa remaining in the ejaculate of animals restored partially or fully. DISCUSSION In our previous study (3), we reported that exogenous administration of T -filled Silastic implants measuring 24 cm, similar to one of the implants used in this study, restored quantitatively complete spermatogenesis to rats in which spermatogenesis was suppressed by active immunization against GnRH. Although this was an important finding, we did not know whether the spermatozoa produced in these restored animals were able to function normally, nor did we know how much T would be required to produce normal fertility. Therefore, in the present study, we evaluated the extent to which T could restore not only sperm production but fertility as well. Similar to our previous study (3), active immunization against GnRH in the present study resulted in precipitous suppression of gonadotropin levels that persisted uniformly in all immunized animals. Testicular, seminal vesicle, and ventral prostate weights underwent a drastic reduction, and complete suppression of sperm production was achieved in GnRH-immunized rats. The administration of T filled implants of 4 cm to GnRH-immunized azoospermic rats for 8 weeks restored only 4 % of testicular sperm found in the controls. Furthermore, it should be noted that only two of the five rats in this group were able to impregnate all female rats. In comparison, T implants of 8 cm and longer restored Table 2 Effects of Treatment of GnRH -Immunized Azoospermic Rats With T Implants on Restoration of Fertility and on the Numbers of Embryos and CL When Exposed to Females Treatment group No. of (n = 5) fertile male Control 5 of 5 Anti-GnRH + ot o of 5 Anti-GnRH of 5 Anti-GnRH of 5 Anti-GnRH of 5 Anti-GnRH of 5 * Values are means ± SEM; n = 15 rats/group. t T implant length in cm. Implantation Embryos sites/embryo CL* 14.0 ± ± 1.3 0:1: 14.5 ± ± 3.1:1: ± ± ± ± ± ± ± 1.6 :I: Significantly different from controls (P < 0.05). 406 Awoniyi et al. Reversible sterility in male rats Fertility and Sterility

5 more spermatozoa to the testis, and these rats were able to impregnate all female rats with which they were housed. The result obtained with T-filled implants of 4 cm is particularly significant because it will now be possible to use this immunological method in combination with lower doses of T implant «4 cm) to evaluate libido and possible untoward effects that may result from chronic T administration for contraceptive purposes. Pathological changes of liver function have been reported in men treated with high doses of various androgen preparations (20); however, there are no data to suggest that modest elevation of serum T levels would necessarily produce the same undesirable effects. In fact, in our previous study, we showed that T -filled implant of 2.5 cm to hypophysectomized rats was able to maintain normal serum T levels without restoring spermatogenesis (11), but whether this is enough to maintain normal sexual function is not known. The effect of smaller T-filled implant (1 to 3 cm) given to GnRH-immunized rats requires further study and is presently being investigated in our laboratory. One of the issues in male contraceptive development is whether drug-induced azoospermia is a prerequisite or whether severe oligospermia is adequate. However, unless azoospermia is attained, the clinical efficacy of a male contraceptive will always be in question. Several reports in the literature (5, 21-23) have used GnRH analogues together with subsequent T replacement to accomplish both suppression of sperm production and support sexual function in laboratory animals as well as in humans. In some of these studies, reversible oligospermia but not azoospermia was achieved (21, 23). In the experiment described herein, when azoospermia was achieved, the rats were unable to impregnate female rats. However, with severe oligospermia, 40% ofthe male rats were still fertile. Active immunization against GnRH, like GnRH analogue administration, is a promising male contraceptive because of its ability to induce azoospermia and because it is reversible (3, 11, 24). In addition to these qualities, once the antibodies are generated actively, the animals usually remain suppressed for a longer period of time and require only periodic booster injections. In contrast, the use of a GnRH analogue will require frequent dosing to keep spermatogenesis consistently suppressed. Gonadotropin-releasing hormone is a weak antigen because of its low molecular weight and because it is not species specific. Therefore, to make it antigenic, one must conjugate it to a high molecular weight carrier. For the development of a GnRH vaccine, it will also have to be administered with adjuvant. Freund's adjuvant is one ofthe most powerful immunopotentiators known and has been used extensively in most animal studies as well as the present study. Unfortunately, Freund's adjuvant cannot be used in humans because it contains mineral oil that is not degraded, thus persisting at the injection site and eliciting granuloma formation. Two other adjuvants, liposomes and N-acetylmuramyl-Lalanyl-D-isoglutamine (muramyl dipeptide) show the most promise for use with human contraceptive vaccine (25). The potential use of these two adjuvants with GnRH immunization and the determination of the minimal amount of T required to maintain libido without restoring fertility are presently being investigated in our laboratory. In summary, the results presented herein clearly demonstrate that immunoneutralization of endogenous GnRH can induce complete azoospermia and infertility in male rats and that subsequent T administration can cause partial or complete restoration of sperm production and fertility in a dosedependent manner. 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