KENJI OHYAMA, MASANORI OHTA, YosHIKo NAKAGOMI, YOSHIE SHIMURA, T0M0AKI SANG, KAZUMASA SATO, SHINPEI NAKAZAWA, AND HIROMICHI ISHIKAWA*

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1 Endocrine Journal 1997, 44 (4), Restoration of Seminiferous Tubular Function after Discontinuation of Long-Term Gonadotropin-Releasing Hormone Agonist Administration in Premature Male Rats KENJI OHYAMA, MASANORI OHTA, YosHIKo NAKAGOMI, YOSHIE SHIMURA, T0M0AKI SANG, KAZUMASA SATO, SHINPEI NAKAZAWA, AND HIROMICHI ISHIKAWA* Department of Pediatrics, Yamanashi Medical University, Yamanashi , and *JCR Pharmaceuticals, Kobe , Japan Abstract. We examined whether seminiferous tubular function develops normally after discontinuation of long-term gonadal suppression in premature male rats. Wistar male rats 4 weeks old were subjected to the injection of gonadotropin-releasing hormone (Gn-RH) agonist or normal saline solution as control for 12 weeks. The rats were sacrificed 0 and 6 weeks after discontinuation of the treatment. Histological examinations of the seminiferous tubules immediately after cessation of the Gn-RH agonist treatment demonstrated a stage-related change in specific germ cells. Seminiferous tubules of Gn-RH agonisttreated rats were narrow and irregular in shape, and contained significantly fewer spermatids and pachytene spermatocytes at stages VII to XIV than those in controls. A complete development of spermatogenesis was histologically observed 6 weeks after cessation of the treatment. Leydig cells became atrophic without any reduction in cell number immediately after the treatment, but Leydig cells grew rapidly and were similar in appearance to those in control rats 6 weeks after cessation of the treatment. Serum testosterone concentrations were noticeably suppressed immediately after cessation of the treatment (P<0.01 vs. control) and reached a similar level to those of controls 6 weeks after the cessation. Testes weights were significantly lower in Gn-RH agonist-treated rats than in control rats and had not fully developed 6 weeks after cessation of the treatment (P<0.01 vs. control). These results suggest that the testicular function develops normally after cessation of the long-term gonadal suppression in premature rats, although the increase in testicular weight may be slightly influenced. Key words: Gonadotropin-releasing hormone agonist, Testis, Spermatogenesis, Sexual development, Leydig cell (Endocrine Journal 44: ,1997) PATIENTS with precocious puberty have been generally treated with long-acting gonadotropinreleasing hormone (Gn-RH) agonistic analog in clinics [1-4]. Jay et. al. [5] reported that gonadal function normally developed after cessation of Gn- RH agonist therapy in patients with central Received: January 23, 1997 Accepted: May 16, 1997 Correspondence to: Dr. Kenji OHYAMA, Department of Pediatrics, Yamanashi Medical University, 1110 Shimogato, Tamaho-cho, Nakakoma-gun, Yamanashi , Japan precocious puberty. These patients were treated during the prepubertal age and discontinued the treatment at the early pubertal age. Recently normal short children were treated with Gn-RH agonist for prolongation of GH therapy [6, 7]. These patients were treated for several years from early to late pubertal age, since they had no sexual precocity. Their pubertal development started at the late pubertal age after cessation of the Gn-RH agonist therapy. It is still not known whether gonadal tissues normally mature after cessation of the therapy in the short normal children. Several researchers reported that the suppression

2 460 OHYAMA et a!. of gonadal function by Gn-RH agonist treatment was completely reversible in adult male and female rats [8, 9]. There are, however, insufficient data on the sexual development after cessation of Gn-RH agonist treatment when the gonadal function was suppressed before the natural onset of puberty by the treatment. It is therefore necessary to demonstrate whether the gonadal function suppressed by long-term Gn-RH agonist administration at the prepubertal stage normally develops before clinical trials can be started. In this study, we examine changes in testicular function in premature male rats, especially in spermatogenesis after long-term Gn-RH agonist treatment. of right testes were measured. Testes were then fixed by immersion in Bouin's solution, dehydrated, and embedded in historesin and stained with periodic acid-schiff's reagent and haematoxylin. Germ cells per seminiferous tubule were counted at stages 1-VI, VII-VIII, IX-XI and XII-XIV [10]. Fifteen tubules in each stage per testis were counted. The cell counts were corrected with the Abercrombie formula [11]. The intra- and interassay coefficients of variation in testosterone radioimmunoassay were 2.6% and 8.2%, respectively. The minimum limit of sensitivity was 50 pg/m1. Data were analyzed with one-way analysis of variance and Tukey's test to determine significant differences at the 5% level. Methods Wistar male rats aged four weeks were housed at 22 C on a 12-h light, 12-h dark cycle. All animals were fed a pelleted diet ad libitum and given free access to water. The animals were divided into two Groups of 12 rats and were subjected to the injection of Gn-RH agonist (D-Leu6-[des-Glylo- NH2]-Gn-RH ethylamide acetate; sustained release leuprorelin acetate, Takeda Pharmaceutical) or normal saline solution as control for the following periods. Six hundred,ug/body weight (kg) of Gn- RH agonist were subcutaneously administered every two weeks for 12 weeks (from 4 weeks old to 16 weeks old). Six rats were sacrificed at 0 or 6 weeks after cessation of Gn-RH agonist treatment. Serum testosterone concentrations and wet weights Results In 4-week-old rats (before treatment), serum testosterone concentrations were below 10 ng/dl, and wet weights of testes were 0.37 ± 0.02 g (mean ± SEM, n=6). The serum testosterone concentrations and testes weights after Gn-RH agonist treatment are shown in Table 1. In Gn-RH agonist-treated rats, serum testosterone concentrations were niticeably suppressed immediately after cessation of the treatment (P<0.01 vs. control), but they reached a similar level to those of control rats at 6 weeks after cessation of the treatment. Testes weights were significantly lower in Gn-RH agonist-treated rats than in control rats and had not fully developed 6 weeks after cessation Table 1. Testis weight and serum testosterone concentration in rats treated with Gn-RH agonist for 12-week

3 SPERMATOGENESIS Fig. 1. IN Gn-RH AGONIST-TREATED RATS 461 Light micrographs of a representative tubular section at stage VII in a rat treated with Gn-RH agonist for 12 weeks. a) control rat 16 weeks old b) a tubule immediately after 12 weeks treatment with Gn-RH agonist (16-week-old rat), showing irregularity in tubular shape and a decreased number of spermatids, especially mature spermatids. c) control rat 22 weeks old d) a tubule at 6 weeks after cessation of the treatment (22-week-old rat), showing a similar appearance to the age-matched control. of the Gn-RH agonist treatment (P<0.01 vs. control). Histological examinations of the seminiferous tubules after the Gn-RH agonist treatment demonstrated stage-related changes in specific germ cells. Figure 1 shows light micrographs of seminiferous tubules at stage VII. The seminiferous tubules of Gn-RH agonist-treated rats were narrow and irregular in shape compared to non-treated rats (Fig.1 a, b). In Gn-RH agonist-treated rats, there were noticeably fewer spermatids, especially elongated spermatids, compared to those in controls. Six weeks after cessation of the treatment, the shape and size of the seminiferous tubules were similar to the controls, and complete development of spermatogenesis was observed (Fig. 1 c, d). The quantitative analyses of spermatogenesis are shown in Table 2. Numbers of spermatogonia (type A, intermediate and type B) in each stage were similar in Gn-RH agonist-treated and control rats. In seminiferous tubules immediately after cessation of Gn-RH agonist treatment, the number of elongated spermatids (step 8-19) was reduced in comparison to the controls (26-42% of control), and numbers of round spermatids (step 1-7: 60-70% of controls) and pachytene spermatocytes at stage VII to XIV (73-79% of controls) were also significantly decreased (P<0.01). Nevertheless, in rats 6 weeks after cessation of the treatment, the numbers of all types of germ cells in seminiferous tubules were quite similar to the control rats. There were no significant difference between Gn-RH agonisttreated and untreated rats in the numbers of Sertoli cells at each stage. As shown in Fig. 2, immediately after cessation

4 462 OHYAMA et al. Table 2. Effects of Gn-RH agonist on numbers of germ cells and Sertoli cells in s permatogenic cycle in rats of the Gn-RH agonist treatment, Leydig cells became atrophic without any reduction in cell number. Six weeks after cessation of the treatment, Leydig cells exhibited rapid growth and were similar in appearance to those of the control rats. Discussion Agonistic analogs of Gn-RH down-regulate the secretion of pituitary gonadotropins and circulating hormones fall to prepubertal levels [12-15]. Recent indications for the possible use of Gn-RH agonist for short normal children [6, 7,16] as well as for the treatment of precocious puberty [17-19] have led to reinvestigation of sexual maturation after discontinuation of long-term suppression of gonadal function from the prepubertal stage. Previous studies on adult male rats indicated qualitative and quantitative recovery of spermatogenesis after cessation of Gn-RH antagonists treatment [8, 9]. Bokser et al. [8] reported that 2 to 3 months after cessation of Gn- RH antagonist (SB-75) treatment, no differences between adult treated and untreated rats were observed in gonadal weights, sex hormone levels and histological findings. Hikim and Swerdlof f [9] studied the time-course of recovery of spermatogenesis in the 6 weeks after the cessation of Gn-RH antagonist treatment in adult rats. They presented evidence indicating complete reversal in spermatogenesis and the progression of various germ cells during the recovery period following the normal time-schedule of germ cell

5 SPERMATOGENESIS Fig. 2. IN Gn-RH AGONIST-TREATED RATS 463 Light micrographs of representative Leydig cells. a) control rat 16 weeks old b) Leydig cells immediately after 12 weeks treatment with Gn-RH agonist (16-week-old rat), showing atrophic shape. c) control rat 22 weeks old d) Leydig cells at 6 weeks after cessation of the treatment (22-week-old rat), showing a similar appearance to the age-matched control. development. Our present study demonstrated that germ cells and Leydig cells fully matured and the number of germ cells in seminiferous tubules reached normal levels in the 6 weeks after the discontinuation of the long-term suppression of gonadal function. These results indicate that spermatogenic function develops normally even though the onset of puberty is extremely delayed in premature rats. Morphological responses to the long-term gonadotropin withdrawal in premature rats were characteristic stage-related changes in seminiferous epithelium. Immediately after cessation of the GnRH agonist treatment, pachytene spermatocytes at stage VII to XIV were significantly reduced and furthermore the acrosome-phase spermatids (steps 8-14) and the maturation-phase spermatids (steps 15-19) were greatly reduced in number. Similar stage-specific degeneration of germ cells can be found in adult rats after hypophysectomy [20, 21] or with Gn-RH antagonist treatment [22]. These observations in adult rats with gonadotropin deprivation presented no further progression of spermatogenesis beyond step 7 of spermiogenesis. In contrast, spermatogonia did not decrease in number during this treatment. These results indicate that stages VII-VIII in the cycle of the seminiferous epithelium are the most sensitive to withdrawal of gonadotropins, pubertal maturation of germ cells is also suppressed at stages VII-VIII by Gn-RH agonist treatment, and that the proliferation of spermatogonia responds to minimum secretion of gonadotropins or may be independent of gonadotropins. The effect of Gn-RH agonist administration was manifested by a marked suppression of testis

6 464 OHYAMA et a1. growth and testosterone secretion. In adult rats, the testicular weight, which was 14 to 27% of the control at the end of treatment [8, 9], had fully recovered by 6 to 8 weeks posttreatment. Nevertheless, in our study, when the gonadal function was suppressed before the natural onset of puberty, the testicular weights were significantly reduced even 6 weeks after cessation of the treatment, although the diameter of the seminif erous tubules in the treated-rats appeared to be similar to that in the control. These results suggest that the length of the seminiferous tubules in the developing testes of the premature rats may be influenced negatively by the long-term suppression of gonadal function. In summary, we have presented evidence which indicates that 1) premature testis is seen to fully develop in number of various types of germ cells in morphological examination and normally secretes testosterone after discharge from the longterm gonadotropins deprivation, 2) the maturation of germ cells in developing testis is suppressed at stages VII-VIII by Gn-RH agonist treatment, and 3) the testicular volume does not reach normal size even 6 weeks after cessation of the treatment. References 1. Comite F, Cutler GB, Rivier J, Vale WW, Loriaux DL, Crowley WF (1981) Short-term treatment of idiopathic precocious puberty with a long-acting analogue of luteinizing hormone-releasing hormone. N Engl J Med 305: Lee PA, Page JG, Leuprolide Study Group (1989) Effects of leuprolide in the treatment of central precocious puberty. J Pediatr 114: Neely EK, Hintz RL, Parker B, Bachrach LK, Cohen P, Olney R, Wilson DM (1992) Two-year results of treatment with depot leuprolide acetate for central precocious puberty. J Pediatr 121: Cook JC, Doty KL, Conn PM, Hansen JR (1992) Assessment of depot leuprolide acetate doseadequacy for central precocious puberty. J Clin Endocrinol Metab 74: Jay N, Mansfield MJ, Blizzard RM, Crowley Jr WF, Schoenfeld D, Rhubin L, Boeple PA (1992) Ovulation and menstrual function of adolescent girls with central precocious puberty after therapy with gonadotropin-releasing hormone agonists. J Clin Endocrinol Metab 75: Job JC, Toublanc JE, LandierF (1994) Growth of short normal children in puberty treated for 3 years with growth hormone alone or in association with gonadotropin- releasing hormone agonist. Horm Res 41: Saggese G, Cesaretti G, Bersanti S, Rossi A (1994) Combined therapy with GnRH agonists and GH in short subjects without a GH deficiency and with a normal onset of puberty [Abstract 161]. Horm Res 41: Bokser L, Srkalovic G, Szepeshazi K, Schally SA (1991) Recovery of pituitary-gonadal function in male and female rats after prolonged administration of a potent antagonist of luteinizing hormonereleasing hormone (SB-75). Neuroendocrinology 54: Shinha Hikim AP, Swerdloff RS (1994) Time course of recovery of spermatogenesis and leydig cell function after cessation of gonadotropin-releasing hormone antagonist treatment in the adult rat. Endocrinology 134: Leblond CP, Clermont Y (1952) Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci 55: Abercrombie, M (1946) Estimation of nuclear population from microtome sections. Anatomical Record 94: Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E (1978) Hypophyseal response to continuous and intermittent delivery of hypothalamic gonadotropinreleasing hormone. Science 202: Comite F, Pescovitz OH, Rieth K, Rieth KG, Dwyer AJ, Hench K, McNemar A, Loriaux DL, Cutler Jr GB (1984) Luteinizing hormone releasing hormone (LHRH) analogue treatment of true precocious puberty secondary to a hypothalamic hamartoma in males. J Clin Endocrinol Metab 59: Kappy M, Stuart T, Perelman A, Clemons R (1989) Suppression of gonadotropin secretion by a longacting gonadotropin-releasing hormone analog (leuprolide acetate, Lupron depot) in children with precocious puberty. J Clin Endocrinol Metab 69: Parker KL, Lee PA (1991) Depot leuprolide for treatment of precocious puberty. J Clin Endocrinol Metab 69: Bulducci R, Toscano V, Mangiantim A, Municchi G, Vaccaro F, Picone S, Rito AD, Boscherini B (1995) Adult height in short normal adolescent girls treated with gonadotropin-releasing hormone analog and growth hormone. J Clin Endocrinol Metab 80:

7 SPERMATOGENESIS IN Gn-RH AGONIST-TREATED RATS Cara JF, Kreiter ML, Rosenfield RL (1992) Height prognosis of children with true precocious puberty and growth hormone deficiency: Effect of combination therapy with gonadotropin releasing hormone agonist and growth hormone. J Pediatr 120: Attie KM, Ramirez NR, Conte FA, Kaplan SL, Grumbach MM (1990) The pubertal growth spurt in eight patient with precocious puberty and growth hormone deficiency: Evidence for a direct role of sex steroids. J Clin Endocrinol Metab, 71: DiMartino J, Wu R, Varner R, Wong WLT, Saenger P (1994) The effect of luteinizing hormone-releasing hormone analog for central precocious puberty on growth hormone (GH) and GH-binding protein. J Clin Endocrinol Metab 78: Russel LD, Clermont Y (1977) Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. Anat Rec 187: Ghosh S, Sinha Hikim AP, Russel LD (1991) Further observations of stage-specific effects seen after short-term hypophysectomy in the rat. Tissue Cell 23: Sinha Hikim AP, Swerdloff RS (1993) Temporal and stage-specific changes in spermatogenesis of rat after gonadotropin deprivation by a potent gonadotropin-releasing hormone antagonist treatment. Endocrinol 133: