Neelakanta Ravindranath, Ph_D_t:\: Venkatarayappa Ramesh, M.Sc. t Heganahalli Narasimha Sastry Krishnamurthy, M.Sc.

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1 FERTILITY AND STERILITY Copyright e 1992 The American Fertility Society Vol. 57, No.3, March 1992 Printed on acid-free paper in U.S.A. Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)* Neelakanta Ravindranath, Ph_D_t:\: Venkatarayappa Ramesh, M.Sc. t Heganahalli Narasimha Sastry Krishnamurthy, M.Sc. Addicam Jagannadha Rao, Ph.D.t Raghuveer N. Moudgal, Ph.D.t 11 Indian Institute of Science, Bangalore, India Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50 p.g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of bus ere lin acetate and T. Fertil Steril1992;57:671-6 Key Words: Male infertility, buserelin, pituitary desensitization, monkey testicular function Gonadotropin-releasing hormone agonists (GnRH-a) administered over a prolonged period of time bring about marked reduction in the secretion of pituitary follicle-stimulating hormone and lu- Received February 20, 1991; revised and accepted October 3, * Supported by grant 85011, Special Programme of Research in Human Reproduction, World Health Organization, Geneva, Switzerland. t Primate Research Laboratory, Center for Reproductive Biology and Molecular Endocrinology. :j: Present address: Department of Obstetrics and Gynecology, Magee Women's Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania. Department of Biochemistry. II Reprint requests: Professor Raghuveer N. Moudgal, Ph.D., Primate Research Laboratory, Department of Biochemistry, Indian Institute of Science, Bangalore , India. teinizing hormone (LH) because of desensitization of pituitary GnRH receptors.! In some cases, such treatment is also known to down regulate the gonadal LH receptor content.2 An end result of such treatment is suppression of testicular function in both animals3-5 and man. 6,7 However, both rhesus monkeys and man have been shown to resist the inhibitory effects of GnRH -a when administered as single or twice daily injections. A constant infusion of GnRH-a through subcutaneous (SC) implants of pellets or via a miniosmotic pump induced better suppression of testicular function in the monkeys,9 and man.lo,n Concomitantly, there was an associated decrease in potency and libido. Because it is also known that testosterone (T) alone can inhibit gonadotropin secretion and spermatogenesis in rats!2 and humans,13 efforts have been made to supplement T along with GnRH -a to achieve total suppression Vol. 57, No.3, March 1992 Ravindranath et al. Testis of pituitary desensitized monkey 671

2 of spermatogenesis but at the same time maintain normal libido. Even though GnRH analogue and T synergistically inhibited gonadotropin secretion, suppression of spermatogenesis has been partial or incomplete in men 14,15 and total only in the monkey.8,9 This has been attributed to the dose of agonist infused and the duration of treatment. Further, even if higher doses of agonist and T have to be administered in men, a long-term study was necessary to determine the toxicity and reversibility of the effects of this combined regimen in a nonhuman primate model. With this in view, a long-term efficacy and safety study was undertaken in the male bonnet monkey, and the results are reported here. MATERIALS AND METHODS Experimental Animals Fifteen adult male bonnet monkeys and 45 adult female bonnet monkeys of proven fertility in the colony were used in this study. They were maintained in individual cages under regulated light:dark conditions (lights on 6 A.M. to 6 P.M.). The standard husbandry practices followed have been reported earlier.16,1? Hormones The GnRH-a, buserelin acetate, was a product of Hoechst AG, Frankfurt, Germany, and the crystalline T of Serva Chemical Co, Heidelberg, Germany. Tritiated T, progesterone (P), and estradiol-17{3 (E 2) were purchased from Steroloids (Wilton, NH). Design of the Study Fifteen adult male bonnet monkeys were divided into two groups: a control group (group I, n = 5) and an experimental group (group II, n = 10). Both groups of monkeys were prescreened for the following parameters: (1) body weight; (2) testicular volume; (3) serum T concentrations during the day (10 A.M.) and night (10 P.M.); (4) total sperm counts; (5) blood chemistry; and (6) pituitary responsiveness to exogenous GnRH -a injection. The monkeys in group I received SC implants of mini-alzet pumps (model: 2002, release rate; ttl/h) containing distilled water mixed with an equal volume of propylene glycol. The group II monkeys received SC implants of mini-alzet pumps containing GnRH-a dissolved in distilled water-propylene glycol mixture (1:1). The concentration of agonist in the mixture was adjusted in such a way as to release 50 ttg over a 24-h period. The pumps were changed once in 21 days routinely during the entire 3-year period of study in both the groups. In addition to agonist, each of the monkeys in group II also received an SC silastic implant containing crystalline T (100 mg in lo5-cm length silastic tube of outer diameter and inner diameter 0.132) on day 170 after the initiation of agonist treatment. The T implant was renewed every 60 days until the end of the treatment period. Both the body weight and testicular volume (by using Prader beads according to procedure outlined earlier18) were determined once every 21 days. The serum chemistry was monitored once every 3 months using an auto analyzer (model 705; Hitachi, Tokyo, Japan). The parameters checked included glucose, blood urea, calcium, sodium, potassium, creatinine, cholesterol, triglycerides, total protein, albumin, albumin:globulin ratio, total bilurubin, hemoglobin, packed cell volume, alkaline phosphatase, serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic-pyruvic transaminase (SGPT). Sexual behavior recorded before, during, and after treatment periods was essentially a subjective evaluation of the response of the male to the presentation of a female between days 9 and 14 ofthe cycle. Generally, in the colony a normal male would mount a female in midcycle (between days 9 and 14 of cycle) within 2 to 3 minutes. The occurrence of such a response was recorded as +ve and the absence as -ve. Ability to impregnate during the recovery period was considered as the final proof of restoration of normal sexual behavior. The pituitary responsiveness to bolus injection of buserelin acetate was checked by injecting intravenous [IV] 10 ttg of buserelin acetate at 10 A.M. and analyzing serum (blood) withdrawn at 0, 30, 60, and 120 minutes for T. Responsiveness was checked approximately every 20 days until the end of the study. Semen Analysis The semen was collected by penile electrostimulation twice a month from unanesthetized monkeys by the method of Mastroiani and Manson.19 The procedures followed for detailed analysis of semen is outlined elsewhere.16 Steroid Radioimmunoassays (RIAs) The T, E 2, and P in the serum samples of monkeys were estimated by specific RIAs standardized in the laboratory.16,1? The results obtained were statisti- 672 Ravindranath et al. Testis of pituitary desensitized monkey Fertility and Sterility

3 cally analyzed by Student's t-test and fourfold contingency test. Fertility Evaluation Each male monkey in both the groups were exposed to three females during the midcycle period (day 9 to 14 of cycle). The females were monitored for the establishment of pregnancy based on serum steroid concentrations beyond day 25 of mated cycle, detection of chorionic gonadotropin in the serum, and per rectal examination beyond day 45 of cycle. RESULTS Effect of Constant Infusion of GnRH-a Alone on Testicular Function (Days 1 to 150) Of the 15 monkeys used in both groups I and II, 3 monkeys of group II tended to break the pumps by rubbing their backs on the cage frame. However, after change of pump implantation site to the side of the body in these animals, the pumps remained intact. Apart from this initial problem, the study was carried out with routine changes of pumps as per schedule. In the first phase ofthe study in which a constant infusion of GnRH -a was maintained, the body weights of the animals of group II showed marginal but statistically insignificant reduction. Unlike the control group of monkeys that showed a progressive increase in testicular volume (with onset of prime breeding season July to December), the experimental monkeys continued to show low testicular volumes (Table 1). The difference observed was significant (P < 0.01). Although the serum T during the day (10 A.M.) remained unaltered after buserelin acetate infusion, the nocturnal surge levels of T at 10 P.M. was abolished in all of the experimental monkeys (Table 1, P < 0.05). Despite the presence of basal concentration of T, the sperm counts decreased progressively in these monkeys, azoospermia setting in by day 70 of the start of agonist infusion (Table 1). The animals also lost their ejaculatory reflex beyond day 70. Sexual response (mounting behavior) toward cycling females was absent. Effect of T Supplementation on Testicular Function in Monkeys Under Chronic GnRH-a Infusion (Days 170 to 1,092) In this phase of the study, along with GnRH infusion, the monkeys in group II received supplementation of T through silastic implants. The period of supplementation extended from day 170 after initiation of GnRH-a infusion to day 1,092 when both the implants were removed. The serum T concentration stabilized at approximately 4 to 6 ng/ml within a few days of T implant; nocturnal elevation in serum T level, however, was not seen (Table 1). The body weights of experimental animals continued to be below that of the control. The testicular volume of monkeys did not return to normal, despite T supplementation (Table 1). However, the males did respond positively to the cycling females presented to them (mounting reflex), indicating an improvement in their sexual responsiveness. Besides, upon penile electrostimulation, ejaculatory reflex was seen, but the volume of semen ejaculated was lower than in controls (data not shown). Although the majority of monkeys exhibited azoospermia until the end of the treatment phase, one to two monkeys showed occasional (20% to 27% of their ejaculates) acute 01- Table 1 Effect of Chronic Buserelin Acetate Infusion Alone and With T Supplementation on Testicular Function in the Male Bonnet Monkeya Before start of the infusion Before T supplementation (day 170) After T supplementation (day 500) Parameter Group Ib Group II' Group I Group II Group I Group II Body weight (kg) Testicular volume (cc) Serum T (ng/ml) 10 A.M. 10 P.M. Sperm counts (X106) 6.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.8 d 3.2 ± ± ± ± 2.1 d 625 ± ± ± ± ± l.4 d 2.5 ± ± ± ± ± ± 15 de a Values are means ± SD. b Group I (n = 5), controls., Group II (n = 10), buserelin acetate infused alone till day 170 and from then on provided with buserelin acetate pump + T implant. d Significantly different from the corresponding control values (P < 0.05). 'Majority of the animals were azoospermic and one or two monkeys showed acute oligospermia occasionally «25 X 10 6 ). Vol. 57, No.3, March 1992 Ravindranath et al. Testis of pituitary desensitized monkey 673

4 Table 2 Reversibility of the Effect on Testicular Function Upon Withdrawal of Buserelin Acetate and T Treatments in the Male Bonnet Monkeya Before withdrawal of the treatment (day 1,092) After withdrawal of treatment (90 to 100 d later) Parameters Group Ib Group II' Group I Group II Body weight (kg) Testicular volume (cc) Serum T (ng/ml) 10 A.M. 10 P.M. Total sperm counts (X10 6 ) 7.9 ± ± ± ± ± ± ± ± ± 2.8 o 8.4 ± ± ± ± ± ± ± 0.8 d 5.8 ± ± 1.2 d 330 ± 35 a Values are means ± SD. b Group I, control (n = 5). 'Group II, experimental (n = 10). d Significantly different from the corresponding values before withdrawal of treatment (P < 0.05). For other experimental details see text or Table 1. igospermia «25 X 106/ejaculate). Because the animals were azoospermic, fertility testing of monkeys was not conducted during this phase. Effect of Withdrawal of GnRH-a Plus T Treatment on Testicular Function (Day 1,093 Onward) The constant infusion of GnRH -a and T supplementation was withdrawn at the end of the 3-year period (day 1,092 after initiation of GnRH-a infusion). Testicular function was assessed at weekly intervals by the same parameters described before. Both the body weight and testicular volume returned to the control range within 90 to 120 days of withdrawal of treatment (Table 2). The sperm counts also increased progressively, reaching control range by day 90. The monkeys started exhibiting nocturnal surges of T within a week of removal of GnRH plus T therapy (Table 2). Testing of Responsiveness of Pituitary Gonadal Axis to Challenge Dose of Buserelin Acetate at Various Phases of Experimentation A bolus injection of 10 Jlg of buserelin acetate given by IV route at 10 A.M. showed an equivalent response in both control and experimental monkeys before start of experimentation. The response was evaluated by monitoring serum T levels at 0, 30, 60, and 120 minutes after a bolus injection (Table 3). Maximal response (threefold to sixfold increment in T levels) occurred at 60 to 120 minutes after injection. After pituitary desensitization, by initiating buserelin acetate infusion, the experimental group of monkeys ceased to show response to GnRH challenge (Table 3); this occurred as early as day 22 of initiation oftreatment and continued until day 1,092 when both the Alzet pump and silastic implant were removed. The response to GnRH challenge returned to normal (threefold to sixfold increment in serum Table 3 Acute Response to Buserelin Acetate Challenge Testa Serum Tb Control Experimental Day o min 30 min 60 min 120 min o min 30 min 60 min 120 min ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.8 1, ± ± ± ± ± ± ± ± ,169' 4.14 ± ± ± ± ± ± ± ± 2.93 ng/ml a Values are means ± SD. b 10 fj,g buserelin acetate given IV at 0 minute and serum collected at different time intervals was analyzed for T., 77 days after Alzet pump and T implant were removed. For other details see text. 674 Ravindranath et al. Testis of pituitary desensitized monkey Fertility and Sterility

5 T levels) within 15 days of pump and implant removal (Table 3). Assessment of the Fertility Status Upon Return of Normal Testicular Function The monkeys recruited to this study were proven fertile males. None of the monkeys were tested for their fertility during the treatment phase because the majority of experimental monkeys (8 of 10) showed consistent azoospermia. With the return of testicular function to normal after treatment withdrawal, all monkeys (both experimental and control) were assessed for their fertility by undertaking mating studies with proven fertile females. The results showed that 7 of 10 experimental monkeys had regained fertility, whereas in the control group 4 of 5 male monkeys impregnated the females, the overall fertility of the two groups being 70% and 80%. This regaining of fertility could be achieved within 90 to 120 days of pump removal. DISCUSSION Though a combined regimen of GnRH -a and T has been shown to suppress testicular function of both animals and man,8,14,20 studies carried out hitherto in men15,20,21 have shown that consistent azoospermia is difficult to achieve. It is not clear if this is because of the method of drug delivery, dose, or choice of the agonist. Besides, the critical effects of long-term treatment with high doses of GnRH-a and T have thus far not been established. Whether T alone can initiate spermatogenesis22 and bring about attenuation of the antifertility effect of GnRH _a23,24 is also not clear. Further, the effects of long-term desensitization of pituitary to GnRH -a and T substitution on the general physiology of the test animal has hitherto not been documented. In our present study, GnRH-a infusion in bonnet monkeys brought about only a marginal reduction in the body weights of animals (statistically not significant from the controls), but the testicular volumes of experimental animals were decreased significantly. Other than this specific reduction in testicular weight, the animals remained healthy, periodic analysis of sera for a variety of biochemical constituents (glucose, blood urea, calcium, sodium, potassium, creatinine, cholesterol triglycerides, total protein, albumin:globulin ratio, bilirubin, hemoglobin, packed cell volume, alkaline phosphatase, SGOT and SGPT) not showing any deviation from normal. Though the nocturnal surge of T characteristic of the male bonnet monkey25 was abolished in experimental animals, basal concentrations of serum T (day levels) remained unaltered. The pituitary response to challenge dose of GnRH -a, as tested by gonadal steroid hormone release, was absent in the experimental group of monkeys. The semen analysis carried out at every 15-day intervals revealed a progressive decline in sperm counts. By day 70 of infusion, 7 of 10 animals showed azoospermia, the remaining 3 showing acute oligospermia. Beyond day 70, electroejaculation was not possible in these 7 animals. At approximately 100 days of treatment, ejaculatory reflex was also totally absent. The above results are in confirmation with the earlier reported work in rhesus monkeys.8 Within 2 weeks of T supplementation, however, the ejaculatory reflex returned, and seminal ejaculates were obtained, but the ejaculates were azoospermic. This study clearly demonstrates that maintenance of basal levels of T is inadequate to sustain sperm production. All the animals continued to show azoospermia till discontinuation of treatments (3 years). Apart from marked improvement in sexual behavior (positive mating response on presentation of a cycling female), the T supplement did not bring about any change in testicular volume. With the withdrawal of treatment at the end of 3 years, a progressive return of all functional parameters of testes was observed within 60 to 90 days. Similar results have earlier been reported by Akhtar et al.8 and Sundaram and Tahu. 9 However, in their studies, the duration of treatment was short, and fertility studies were not undertaken. The final confirmation of total recovery came from the fact that 7 of 10 monkeys were able to impregnate females within 90 to 180 days after discontinuation of treatment. Four of 5 control monkeys implanted with minipumps containing vehicle were able to impregnate when tested at the same time as experimentals. The reversibility of the effect induced by GnRH-a and T indicate that there may not be permanent damage to the testes after long-term infusion of buserelin acetate. This would be an important determinant if this combination therapy were ever considered for development as a male contraceptive. Acknowledgments. Thanks are due to Mr. Shanmugavelu and Mrs. Srilatha for their technical assistance. REFERENCES 1. Nett TM, Crowder ME, Moss GE, Duello TM: GnRH-receptor interaction. V. Down regulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous hormone. Bioi Reprod 24:1145, 1981 Vol. 57, No.3, March 1992 Ravindranath et al. Testis of pituitary desensitized monkey 675

6 2. Labrie F, Auclair C, Cusan L, Kelly PA, Pelletier G, Ferland F: Inhibitory effects of LHRH and its agonists on testicular gonadotropin receptors and spermatogenesis in the rat. Int J Androl (suppl 2):303, Pelletier G, Cusan L, Auclair C, Kelly PA, Desy L, Labrie F: Inhibition of spermatogenesis in the rat by treatment with n-ala6desglylo-lhrh EA. Endocrinology 103:641, Vickery BH, McRae GI: Effect of continuous treatment of male baboons with superagonists of LHRH. Int J Fertil 25: 179, Wickings EJ, Zaidi P, Nieschlag E: Effect of chronic high dose LHRH agonist treatment on pituitary and testicular functions in rhesus monkeys. J Androl 2:72, Linde R, Doelle GC, Alexander N, Kirchner F, Vale W, Rivier J, Rabin D: Reversible inhibition of testicular steroidogenesis and spermatogenesis by a potent gonadotropin-releasing hormone agonist in normal men. N Engl J Med 305:663, Faure N, Labrie F, Lemay A, Belanger A, Gourdeau Y, Laroche B, Robert G: Inhibition of serum androgen levels by chronic intranasal and subcutaneous administration of a potent luteinizing hormone-releasing hormone (LH -RH) agonist in adult men. Fertil Steril 37:416, Akhtar FB, Marshall GR, Wickings EJ, Nieschlag E: Reversible induction of azoospermia in rhesus monkeys by constant infusion of a GnRH agonist using osmotic minipumps. J Clin Endocrinol Metab 56:534, Sundaram K, Tahu B: Reversal of testicular function after prolonged suppression with a GnRH agonist in rhesus monkeys. J Androl 8:103, Schurmeyer TH, Knuth UA, Freischmen CW, Sandow J, Akhtar FB, Nieschlag E: Suppression of pituitary and testicular function in normal men by constant GnRH agonist infusion. J Clin Endocrinol Metab 59:19, Bhasin S, Steiner BS, Swerdloff RS: Does constant infusion of gonadotropin releasing hormone agonist lead to greater suppression of gonadal function in man than its intermittent administration? Fertil Steril 44:96, Ewing LL, Desjardins C, Irby DC, Robaire B: Synergistic interaction of testosterone and oestradiol inhibits spermatogenesis in rats. Nature 269:409, Swerdloff RS, Campfield LA, Palacios A, McClure RD: Suppression of human spermatogenesis by depot androgen: potential for male contraception. J Steroid Biochem 11:663, Bhasin S, Heber D, Steiner BS, Swerdloff RS: Hormonal effects of GnRH agonist in the human male. III. Effects of long term combined treatment with GnRH agonist and androgen. J Clin Endocrinol Metab 60:998, Bhasin S, Yuan QX, Steiner BS, Swerdloff RS: Hormonal effects of gonadotropin releasing hormone (GnRH) agonist in men: effects of long term treatment with GnRH agonist infusion and androgen. J Clin Endocrinol Metab 65:568, Murty GSRC, Sheela Rani CS, Moudgal NR, Prasad MRN: Effect of Passive immunization with specific antiserum to FSH on the spermatogenic process and fertility of adult male bonnet monkeys (Macaca radiata). J Reprod Fertil 26(Suppl): 147, Ravindranath N, Moudgal NR: Use of Tamoxifen, an antioestrogen in establishing a need for oestrogen in early pregnancy in the bonnet monkey (Macaca radiata). J Reprod Fertil 81:327, Aravindan GR, Ravindranath N, Gopalakrishnan K, Moudgal NR: DNA flow cytometric analysis of testicular germ cell populations of the bonnet monkey (Macaca radiata). J Reprod Fertil 89:397, Mastroiani L, Manson W A: Collection of monkey semen electroejaculation. Proc Soc Exp Bioi Med 112:1025, Doelle GC, Alexander AN, Evans RM, Linde R, Rivier J, Vale W, Rabin D: Combined treatment with a LHRH agonist and testosterone in man: reversible oligospermia without impotence. J Androl 4:298, Pavlou SN, Interlandi JW, Wakefield G, Rivier J, Vale W, Rabin D: Heterogeneity of sperm density profiles following 16-week therapy with continuous infusion of high dose LHRH analogue plus testosterone. J Androl 7:228, Marshall GR, Wickings EJ, Ludecke DK, Nieschlag E: Stimulation of spermatogenesis in stalk sectioned rhesus monkeys by testosterone alone. J Clin Endocrinol Metab 57:152, Akhtar FB, Marshall GR, Nieschlag E: Testosterone supplementation attenuates the antifertility effects of an LHRH agonist in male monkeys. Int J Androl 7:228, Michel E, Bents H, Akhtar FB, Honigl W, Sandow J, Nieschlag E: Failure of high dose sustained release GnRH agonist plus testosterone to suppress male fertility. (Abstr.) J Androl 6(Suppl):37, Mukku VR, Prahalada S, Moudgal NR: Effect of constant light on nycthemeral variations in serum testosterone in male Macaca radiata. Nature 260:778, Ravindranath et al. Testis of pituitary desensitized monkey Fertility and Sterility