Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone and testosterone secretion and testicular histology in male rhesus monkeys*

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1 FERTILITY AND STERILITY Copyright c 1985 The American Fertility Society Printed in U.SA. Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone and testosterone secretion and testicular histology in male rhesus monkeys* David R. Mann, Ph.D.t Ma:tjorie M. Smith, M.D.:j: Kenneth G. Gould, Ph.D. Delwood C. Collins, Ph.D.II Morehouse School of Medicine and Emory University, Atlanta, Georgia Three adult male rhesus monkeys were treated for 20 weeks with a gonadotropinreleasing hormone (GnRH) agonist (Ag; Wy-40972; Wyeth Laboratories, Philadelphia, PA) using osmotic minipumps. Ag administration resulted in a transient increase and then a precipitous decrease in the concentration of luteinizing hormone (LH) and testosterone (T). After 5 weeks, serum levels of LH were undetectable(< 0.2 ILglml), while serum T was always detectable, but continued to fall throughout the period of Ag administration. The serum LH and T response to 50 WJ of GnRH was abolished by 4 weeks of Ag treatment, and this effect persisted through the treatment period. Testicular histology at 20 weeks of Ag treatment exhibited diffuse atrophy of seminiferous tubule$, suppressed germinal cell division, and the absence of spermatids or spermatozoa. There was no evidence of testicular necrosis or calcification of the seminiferous tubules. Following the termination of Ag infusion, serum LH and T concentrations rebounded to levels that exceeded pretreatment values for a 5-week period before falling back to baseline levels. A complete restoration of spermatogenesis and testicular volume occurred by 12 weeks after treatment. These data suggest that continuous Ag administration is an effective method of reversibly disrupting spermatogenesis in the male rhesus monkey. Fertil Steril43:115, 1985 Received August 1, 1984; revised and accepted September 18, *Supported by National Institutes of Health grants RR and RR-00165; Wyeth Laboratories and National Science Foundation grant PCM ; and Veterans Administration project treprint requests: David R. Mann, Ph.D., Department of Physiology, Morehouse School of Medicine, 720 Westview Drive, S.W., Atlanta, Georgia tdepartment of Pathology, Morehouse School of Medicine. Yerkes Regional Primate Research Center, Emory University. IIVeterans Administration Medical Center and Department of Medicine, Emory University School of Medicine. Chronic administration of a gonadotropin-releasing hormone (GnRH) agonist is an effective method of suppressing spermatogenesis in the rat,l- 3 dog, 3 monkey, 4 and human. 5 However, some reports in the rat suggest that agonist treatment causes pathologic changes in testicular histology that may not be reversible. 3 6 Fourteen days of administration of [n-nal(2) 6 ] luteinizing hormone-releasing hormone (LH-RH) induced testicular infarct resulting in a fibrotic calcified lesion in the rat. 6 While spermatogenesis was suppressed, the effects were focal, with euspermic Mann et al. Antifertility effect of a GnRH agonist 115

2 tubules existing with aspermic tubules in histological sections. After 4 weeks of treatment of male rats with n-ala 6 -LH-RH-ethylamide (100 ng, twice weekly) tubular degeneration was very advanced, 25% of the tubules exhibiting an almost complete absence of germ cells, only Bertoli cells persisting. 6 After treatment with a larger dose of the same agonist, some of the tubules also appeared to lack Bertoli cells as well. In contrast, the Leydig cells retained their normal appearance. The testicular infarcts associated with agonist administration in the rat may be restricted to this species. Although the dog is quite sensitive to the inhibitory effects of agonists on spermatogenesis, fibrotic lesions of the tubules have not been reported. 3 Moreover, ultrasound examination of the testes of children treated for precocious puberty with GnRH agonists have not indicated any testicular calcification. 7 The rhesus monkey has been the animal model of choice of investigators wishing to draw parallels with the human situation. While initial studies indicated a resistance of the male rhesus monkey to the inhibitory effects of GnRH analogs,8-10 recent studies have shown that continuous infusion of these analogs is an effective method of suppressing reproductive processes in this species.4 11 The effects of long-term administration of GnRH agonists on testicular histology in the rhesus monkey have not been examined. GnRH agonists are currently being tested in clinical trials as a treatment for precocious puberty and as a male contraceptive. Therefore, it is important that the long-term effects ofthese compounds on testicular function and the reversibility of these effects be examined. We examined the effects of chronic treatment with a GnRH agonist on LH and testosterone (T) secretion, on the sensitivity to native GnRH, and on testicular histology in adult male rhesus monkeys. MATERIALS AND METHODS Three adult male rhesus monkeys were treated continuously for 20 weeks with a GnRH agonist (Ag; Wy:4Q972; Wyeth Laboratories, Philadelphia, PA; n-trp 6 -N-Me-Leu 7 -des-gly10-pro 9 - NHEt-GnRH; 25 JJ.g/day) with a subcutaneously implanted Alzet (2 ml-4) minipump (Alza Corporation, Palo Alto, CA). Minipumps were replaced at 4-week intervals throughout the study. Pumps had a mean fill volume of 2070 JJ.l and a flow rate 116 Mann et al. Antifertility effect of a GnRH agonist of 2.41 JJ.llhour. The animals were maintained in a light-controlled environment (12 hours light/12 hours dark) throughout the study. Blood samples (10:00 A.M. to 12:00 noon) were taken from the saphenous vein during the pretreatment period (two or three samples from each animal taken at weekly intervals), at 2, 8, and 14 days of treatment, and at biweekly intervals thereafter. At 4-week intervals, each animal was immobilized with 10 mg/kg of ketamine and given an intravenous bolus of 50 JJ.g GnRH. Blood samples were collected at 15 minutes before administration, at the time of administration, and 15, 30, 60, and 120 minutes after GnRH injection. Serum was stored at - 20 C until assayed for LH and T. Concentrations of LH in the serum were determined using the mouse interstitial cell-t bioassay.12 The National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases rhesus monkey pituitary gonadotropin standard (LER ) was used as the reference preparation. The within- and between-assay coefficients of variation were 8.6% and 15.5%, respectively; and the minimal level of detection was 0.2 JJ.g/ml. Serum Twas determined by radioimmunoassay as described previously.13 The within- and betweenassay coefficients of variation for the T assay were 4.9% and 6.8%, respectively. Testicular tissue was recovered by needle hiopsy before Ag treatment, after 20 weeks of Ag administration, and at 4 and 12 weeks after treatment. For this procedure, the animal was anesthetized with ketamine, and the scrotal skin was shaved and prepared by washing and coating with a betadine solution. A 15-gauge cannula with trochar was introduced into the body of the testis by direct puncture of the skin. Care was taken to avoid contact with the epididymis. After insertion, the trochar was removed, and a split biopsy needle was introduced. The needle was advanced through the cannula until the split tip protruded approximately 0.5 em. After the needle was rotated through 180 degrees it was withdrawn, and the trapped tissue was fixed in buffered formalin or Bouin's solution. The puncture in the skin was not sutured. This method of biopsy can result in more tissue disruption than that associated with open biopsy methods, but it was preferred because multiple biopsies can be obtained more readily. Biopsy specimens were routinely processed for paraffin embedding, and serial sections were cut Fertility and Sterility

3 E -4 :I:...I ~ 2 :r I WY p,g/dayi 40 eao -Ia c20 10 WEEKS Figure 1 Mean serum LH and T concentrations ± SEM before, during, and after Ag treatment. The 0 week LH and T values represent the mean values of two or three serum samples taken at weekly intervals from each animal during the pretreatment period. Serum LH values were undetectable ( < 0.2 ng/ml) from 5 through 20 weeks of Ag administration. n = 3. at 4 f.l and stained with hematoxylin and eosin (H & E). In addition, biopsies taken at 20 weeks of Ag treatment and 4 weeks after treatment were stained with Masson's trichrome for connective tissue. The number of seminiferous tubules collected from biopsies varied from 13 to 51, with a mean ± standard error of the mean (SEM) of 31.6 ± 3.7. Testicular measurements were made with Lange calipers with a compression pressure of 15 gm. The long and short axes were measured and corrected for skin thickness. Testicular volume was calculated from the formula for the volume of a prolate spheroid (V = 4/3 1r x long axis x short axis 2 ). The statistical significance of hormonal data, testicular volume, and tubular diameters was tested initially with analysis of variance. Multiple comparisons were then made with the least significant difference test. 14 RESULTS Basal levels of serum LH increased more than.14-fold (P < 0.001) over the first 2 days of Ag treatment but then fell precipitously to values that were undetectable ( < 0.2 JLg/ml) by 5 weeks of Ag administration (Fig. 1). Serum T concentrations increased (P < 0.001) from a mean pretreatment value ± SEM of 4.30 ± 2.15 ng/ml to ± 6.90 ng/ml on the second day of Ag administration and remained elevated (P < 0.05; ± 4.38 ng/ml) above pretreatment values on day 8 of Ag treatment.. Levels of serum T then fell rapidly below pretreatment values by 2 weeks of Ag treatment (1.42 ± 0.51 ng/ml) and continued to decline during the remainder of Ag administration, reaching 0.34 ± 0.15 ng/ml by 20 weeks. During the pretreatment period, animals exhibited normal LH and T responses to an intravenous bolus of 50 JLg GnRH (Fig. 2). Serum LH levels had increased more than 15-fold over basal levels by 30 minutes after GnRH administration, and serum concentrations of T had risen more than 10-fold by 120 minutes. The LH and T response to GnRH was abolished by 4 weeks of Ag treatment. The animals exhibited no significant E 15 -:I:...I C) 10 :::$.. E 'c,20 c 10 f_ f} WEEKS.. Figure 2 Mean serum LH and T responses ± SEM to an intravenous bolus of 50 J.Lg GnRH before, during, and after continuous Ag treatment (25 J.Lg/day). The response was tested before (0), at 4-week intervals during treatment, and at 2 and 4 weeks after Ag treatment was terminated (indicated by arrow). Serum samples were taken at 15 minutes before administration, at the time of administration, and 15, 30, 60, and 120 minutes after GnRH administration. n = 3. Mann et al. Antifertility effect of a GnRH agonist 117

4 Table 1. Effect of Continuous Treatment with Ag on Mean Testicular Volumes ± SEM (in Milliliters) in Male Rhesus Monkeys Pretreatment Week of treatment Weeks after treatment Mean a 6.3a 7.oa 4.1a 17.9a 35.0 SEM ± 14.0 ± 10.2 ± 2.7 ± 1.1 ± 1.9 ± 1.4 ± 3.9 ± 5.1 asignificantly different from pretreatment value at p < 0.05 or better. LH and T response to GnRH throughout the course of Ag administration. Ag administration for 20 weeks reduced testicular volume from a mean pretreatment value ± SEM of 43.3 ± 14.0 ml to 4.1 ± 1.4 ml (Table 1). Pretreatment biopsies revealed prominent cellularity of the seminiferous tubules with active spermatogenesis (Figs. 3A and 4A). The lamina propria of tubules was thin and inconspicuous. Twenty weeks of Ag treatment induced a complete arrest of spermatogenesis in one of the animals (Fig. 3A to D). After 20 weeks of Ag administration, histologic study revealed diffuse tubular atrophy, with a marked reduction in tubular diameter (pretreatment mean ± SEM = 0.27 ± 0.03 mm versus 20 weeks Ag = 0.14 ± 0.02 mm; P < 0.01), along with an almost total absence of spermatogenic activity (Fig. 3B). Most tubules were lined by a single layer of epithelium, and none contained spermatids. Because of the sparse cellularity of the seminiferous tubules, the lamina propria and interstitial tissue appeared more prominent with H & E staining. However, Masson's trichrome stain did not demonstrate significant thickening or hyalinization of the lamina propria. In the other two animals, the changes were somewhat less dramatic (biopsies from one of Figure 3 Testicular histology in a male rhesus monkey. (A), Before Ag treatment ( x 100); (B), after 20 weeks of Ag treatment ( x 100); (C), at 4 weeks after treatment ( x 200); and (D), at 12 weeks after treatment ( x 100). 118 Mann et al. Antifertility effect of a GnRH agonist Fertility and Sterility

5 Figure 4 Testicular histology in a male rhesus monkey. (A), Before Ag treatment ( x 100); (B), after 20 weeks of Ag treatment ( x 200); (C), at 4 weeks after treatment ( x 200); and (D), at 12 weeks after treatment ( x 100). these animals are shown in Fig. 4A to D). Although tubular diameters were decreased (P < 0.001) and there was less spermatogenic activity, some tubules were moderately cellular and contained spermatocytes, but no spermatids were evident. None of the biopsies exhibited a focal block of spermatogenesis (euspermatic tubules present with aspermatic tubules) or foci of calcification or necrosis. Within 2 weeks following the termination of Ag treatment, basal concentrations of serum LH (P < 0.01) and T (P < 0.001) had risen to values that exceeded pretreatment levels (Fig. 1). The serum values of both hormones slowly returned to pretreatment levels by 7 weeks after treatment. These changes in basal secretion of LH and T reflected exaggerated LH and T responses to GnRH shortly after (2 weeks after treatment) the termination of Ag administration, but by 4 weeks after treatment these responses had returned to pretreatment levels (Fig. 2). A complete restora- tion of testicular volume occurred by 8 weeks after treatment (Table 1). Four weeks following the cessation of Ag treatment, testicular biopsies revealed increased cellularity, with spermatogenesis recovering in most tubules, although the tubular diameter had not fully recovered to the pretreatment state (Figs. 3C and 4C). Spermatocytes were evident at this time in the tubules but few if any spermatids. Some of the tubules showed a striking vacuolization of the epithelial cells. Twelve weeks after treatment the histologic features of biopsy specimens were indistinguishable from those of pretreatment biopsy specimens (Figs. 3D and 4D). DISCUSSION The results of the present study confirmed our earlier work 11 and that of others, 4 showing that continuous infusion of a GnRH Ag is an effective Mann et al. Antifertility effect of a GnRH agonist 119

6 method of suppressing basal LH and T secretion and desensitizing the pituitary to native GnRH. Serum LH levels rose initially after the beginning of Ag treatment and then fell to undetectable values by 5 weeks of treatment. Serum T values showed a similar pattern, rising initially, but then falling as LH concentrations decreased. The fall in basal LH secretion was related to a desensitization of the pituitary to native GnRH. Ag infusion totally abolished the serum LH and T response to an intravenous bolus of GnRH by 4 weeks of Ag treatment. This effect persisted for the duration of Ag administration (20 weeks). Following the termination of Ag infusion, serum LH and T concentrations recovered quickly. In fact, within 2 weeks of the cessation of Ag treatment, basal LH and T levels exceeded pretreatment values. These elevated levels of serum LH and T persisted for about 5 weeks after treatment before falling back toward pretreatment levels. This rebound phenomenon of LH and gonadal steroid secretion may be of clinical concern in the use of GnRH Ag for the treatment of endometriosis or breast cancer. During the recovery period, one might expect an exacerbation of the condition unless efforts are made to neutralize increased gonadal activity during this period. The present study is the first to examine the long-term effects of Ag administration on testicular histology in a primate. Seminiferous tubules in all animals exhibited diffuse atrophy. In the monkey that exhibited the most dramatic changes, there was a marked decrease in tubular diameter, germinal cell division was absent, and tubules consisted of a single layer of epithelium. The other two animals exhibited diffuse atrophy of the seminiferous tubules and suppressed germinal cell division, but some tubules were moderately cellular and contained some spermatocytes. Spermatids were absent from the tubules of all three animals. Most importantly, there was no evidence of tubular calcification or necrosis or a focal block of spermatogenesis. It must be emphasized that while needle biopsies were preferred in this study because multiple samples could be obtained more readily, the limited number of tubules obtained per biopsy (13 to 51) may not necessarily be characteristic of all tubules in the testes of these animals. The effects of 20 weeks of Ag treatment on testicular histology appeared fully reversible. Within 4 weeks of the terminati_on of Ag infusion, seminiferous tubules in all animals exhibited re- 120 Mann et al. Antifertility effect of a GnRH agonist newed spermatogenic activity. By 12 weeks after treatment, testicular biopsies from all animals were indistinguishable from pretreatment specimens. The present study extends the work of Akhtar et al. 4 Using a similar experimental protocol, it was found that Ag infusion eliminated spontaneous and electroejaculatory activity and induced azoospermia in rhesus monkeys. T therapy reestablished ejaculatory behavior, but there was a persistence of azoospermia. Testicular histology was not evaluated. One of the unanswered questions concerning the use ofgnrh analogs as a male contraceptive is the use of androgen therapy to maintain libido and the possible effect on Ag-induced azoospermia. As mentioned above, T therapy, when initiated after the onset of azoospermia, did not reinitiate spermatogenesis in Ag-infused male monkeys. 4 However, when T supplementation was begun simultaneously with Ag infusion in monkeys, azoospermia was not achieved. 15 More importantly, men treated by continuous infusion of a GnRH Ag and given androgen therapy when they complained of loss of libido failed to become azoospermic although sperm counts were suppressed. 5 Obviously, more work needs to be done relative to the effect of androgen therapy on GnRH analog-induced azoospermia in primates. In summary, we have found that Ag infusion effectively blocks spermatogenesis in male rhesus monkeys. Needle biopsies collected after 20 weeks of Ag treatment exhibited diffuse seminiferous tubular atrophy, suppressed to absent germinal cell division, and a lack of spermatids in all animals. The effects of Ag treatment on testicular histology appeared totally reversible. Within 12 weeks after treatment, testicular tubular architecture was comparable to that before treatment. There was no evidence of testicular tubule necrosis or calcification, as has been reported in rats. However, longer term studies will have to be performed in order to fully determine whether the effects of Ag treatment on testicular function are fully reversible. Acknowledgments. We thank Drs. Frederick Bex and Alan Corbin of Wyeth Laboratories, Philadelphia, PA, for providing Wy-40972, and the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases for providing the rhesus monkey gonadotropin reference preparation (LER ). Fertility and Sterility

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