G.R. Marshall 2 ' 3 '4,5 and T.M. Plant 4

Transcription

1 BIOLOGY OF REPRODUCTION 54, (1996) Puberty Occurring Either Spontaneously or Induced Precociously in Rhesus Monkey (Macaca mulatta) Is Associated with a Marked Proliferation of Sertoli Cells 1 G.R. Marshall 2 ' 3 '4,5 and T.M. Plant 4 Department of Pediatrics 3 and Department of Cell Biology and Physiology 4 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Division of Pediatric Endocrinology, 5 Children's Hospital of Pittsburgh Pittsburgh, Pennsylvania ABSTRACT The pubertal ontogeny of Sertoli cell proliferation in primates is controversial. In the study of the rhesus monkey presented here, we investigated this issue during normal sexual maturation and during precocious puberty induced experimentally with pulsatile GnRH treatment. Cell number was calculated by standard stereological methods employing volume fraction of either seminiferous tubule or, whenever possible, cell nuclei. In the first experiment, testes were collected from normal monkeys at three developmental phases: neonates (7-18 days old, n = 5); juveniles (approximately 17 mo old, n = 4); and adults (5 yr and older, n = 4). In the second experiment, tissue was obtained from an additional three untreated juvenile monkeys and from six other juveniles of comparable age that had been subjected to premature activation of the pituitary-testicular axis for 5 or 1 wk with pulsatile GnRH (1 [ig/min for 3 min every 3 h). The number of Sertoli cells in adults was 6-fold greater than that in neonatal and juvenile animals. A similar increase in number of this somatic cell type was also observed during experimentally induced puberty. A less dramatic proliferation of Sertoli cells occurred during the neonataljuvenile transition. Although the increase in Sertoli cell number between the juvenile and adult stages of development was paralleled by a comparable change in Ad and Ap spermatogonia, proliferation of these stem spermatogonia during premature activation of the pituitarytesticular axis was less striking. These results lead to the suggestion that, in primates, the onset of puberty is associated with a rapid and substantial proliferation of Sertoli cells, which is subsequently followed by amplification of stem spermatogonia. They also indicate that while Sertoli cells and stem spermatogonia proliferate during infancy and juvenile development, these earlier changes appear to be more insidious than those at puberty. INTRODUCTION The ontogeny of Sertoli cell proliferation has been described in detail in the rat. In this species, the mitotic activity of this testicular cell is maximal in late fetal life, declines during the first 2-3 wk of life, and is absent in the adult [1-4]. This developmental pattern of Sertoli cell proliferation in the rat appears to be characteristic of nonprimate species, having also been reported in mice and sheep [5, 6]. The foregoing findings have given rise to the notion that in mammalian species in general, proliferation of Sertoli cells is completed before the onset of puberty [7]. Review of the studies of this issue in humans and monkeys, however, suggests that this concept may not be applicable to primates. In this regard, Cortes et al. [8], studying human autopsy tissue, observed a dramatic increase in mitotic activity of Sertoli cells at the time of puberty. Moreover, in juvenile monkeys, administration of testosterone and FSH either alone or in combination resulted in an increase in the number of Accepted Janur 17, Received Septemher 19, 'Prcliminarv reports of this work. which was supported by NIH Grants H)861 and HD13254i (to T.M.P.). and HI)32,73 (to G.R.M.). and by the Division of Pediatric Endocrinology, Dept. of Pediatrics, Children's Hospital of Pittshurgh, were presented at the 24th and 25th Annual Meetings of The Society for the Study of Reproduction held July, 1991, Van couer, Canada, aind July, 1992, Raleigh, NC, respectively. C(orrcspondcnce: I)r. G.R. Marshall, Department of Pediatrics I)ivision of Pediatric Endocrinology, Children's Hospital of Pittsburgh, 375 Fifth Ave., Pittshurgh. PA FAX: ( 1 ) Sertoli cells [9]. On the other hand, Nistal et al. [1, 11], also studying human testes obtained during autopsies, reported a pattern of Sertoli cell proliferation similar to that of the rat. In addition, in vitro studies of thymidine labeling in a limited number of monkey testes have provided evidence that Sertoli cell mitotic activity declines continually throughout the juvenile period [121. In order to further study this controversial issue, testes from neonatal, juvenile, and adult rhesus monkeys were obtained to determine the number of Sertoli cells at these three stages of development. Furthermore, unrelated experiments using the juvenile male rhesus monkey treated with pulsatile GnRH provided an opportunity to examine the effect of precocious activation of the pituitary-testicular axis on Sertoli cell proliferation. During the course of the analysis of Sertoli cells in these paradigms, unexpected concomitant observations on undifferentiated spermatogonia numbers were made. This paper describes the findings on the ontogeny of both Sertoli cells and undifferentiated spermatogonia observed in these studies. Animals MATERIALS AND METHODS Five neonatal (age 7-18 days, BW kg, mean + SD), 13 juvenile (age mo, BW 2.5 ±.8 kg), and 4 adult (age 5-7 yr, kg BW) male rhesus monkeys 1192 on 3 July 218

2 PUBERTY AND SERTOLI CELL PROLIFERATION 1193 (Macaca mulatta) were used in this study. In this species, the initiation of puberty is observed at approximately 3 yr of age [13]. The neonatal animals were housed with their mothers while the juvenile and adults were housed in single cages; all animals were maintained under a controlled photoperiod (lights-on 6-18) as previously described [14] in accordance with NIH Guidelines for the Care and Use of Experimental Animals. The animals were used in two studies as described below. All experimental procedures were approved by the Institutional Animal Committee on Use and Care. Experimental Design Study 1. To determine the number of Sertoli cells in the testes of neonatal, juvenile, and adult rhesus monkeys. For this study, testes were removed by aseptic procedures from 5 neonatal, 4 juvenile, and 4 adult male monkeys after anesthesia. For this purpose, the neonates received i.m. injections (1 mg/kg BW) of ketamine hydrochloride (Ketaset; Fort Dodge Laboratories, Inc., Fort Dodge, IA), whereas the juveniles and adults were first sedated with ketamine hydrochloride (5 mg i.m.) and then anesthetized with sodium pentobarbital (3 mg/kg BW i.v., Nembutal; Abbott Laboratories, North Chicago, IL). After the testes were removed, they were weighed, and specific gravity was determined according to standard procedures in order to calculate absolute testicular volume [15]. The gonads were cut into pieces and fixed overnight in Bouin's solution. Each piece of the fixed testicular tissue was individually embedded in paraffin in preparation for morphometric analysis as described below. Study 2. To determine Sertoli cell numberfollowing precocious activation of the pituitary-testicular axis. This study was made possible because of the need, in unrelated experiments [14, 16], to orchidectomize juvenile rhesus monkeys (age mo old, n = 6) that had received a continuous intermittent i.v. infusion of GnRH (1 Lpg/min for 3 min every 3 h) in order to elicit an adult pattern of endocrine activity in the pituitary-testicular axis. This experimental paradigm, which requires the implantation of indwelling venous catheters, has been described in detail previously [17]. For the present study, the testes that had been removed after either 5 or 1 wk of GnRH stimulation were employed. The hormonal profiles typically induced by pulsatile GnRH treatment in the juvenile monkey resemble those typically observed in the adult [14]. The morphological data from the precociously activated testes were compared to those obtained from testes of noncatheterized, non-gnrh-stimulated juvenile rhesus monkeys (age mo, n = 3). The procedures for orchidectomy and the preparation of testicular tissue for morphometric analysis were similar to those described above for study 1. The GnRH-treated animals were sedated briefly with ketamine hydrochloride (1 mg/kg BW; Ketaset) at weekly intervals in order to monitor the condition of the animals. Histological Evaluation After overnight fixation in Bouin's solution, testicular tissue was dehydrated and embedded in paraffin. The number of blocks prepared from each testis were 1 per neonate, 2 per untreated juvenile, 2-4 per treated juvenile, and 1 or more per adult. Ten histological sections (4 $m thick) were cut from each paraffin block and stained with periodic acid Schiff-hematoxylin. After a number was assigned to each section, those for analysis were selected by use of a random number generator. The volume fractions (Vv) of the interstitium, seminiferous compartment (cord or tubule), and Sertoli cell and undifferentiated spermatogonial nuclei were determined by the point-counting method [15, 18]. Briefly, a grid of intersecting lines of known area was superimposed over a section. The number of intersections on the grid (test points) overlying the tissue component of interest (Pn) was counted, and the ratio of these points to total number (PT) was the volume fraction of that component (equation 1). Thus, VV = P + PT Eq. 1 A total of 4 test points were examined on a randomly selected histological section. The diameter (Ds) of twenty-five cross sections of seminiferous cords in the neonatal and unstimulated juvenile testes was measured with a calibrated ocular micrometer. In testes from adults and GnRH-stimulated juveniles, a similar number of seminiferous tubules was analyzed. The numbers of Sertoli cells and of Ad and Ap spermatogonia were determined in randomly chosen cross sections of seminiferous cords (25-5 per testis) or tubules (1-2 per testis), defined by circular profiles and, where appropriate, by centrally located circular lumens. All cell counts were corrected by the method of Abercrombie [19], and the results were expressed as cell number per cross section of seminiferous tubule (Ns). The total number of Sertoli cells and Ad and Ap spermatogonia per testis was calculated in two different ways: First, the total numbers of Sertoli cells and undifferentiated spermatogonia per testis were estimated from the product of total length of seminiferous compartment (L) and Ns of the particular cells (equation 2): Total No. Cell = N X (L (Section Thickness)) Eq. 2 The seminiferous cords or tubules were assumed to be cylindrical, and their lengths were estimated from absolute volume (Vs) and Ds as indicated in equations 3 and 4 [2]: L = V s + (r X (Ds - 2) 2 ) Eq. 3 on 3 July 218

3 1194 MARSHALL AND PLANT FIG. 1. Photomicrographs of testes from representative male rhesus monkeys: A) neonatal, B) juvenile, and C) adult. Ad and Ap stem spermatogonia (arrowheads and arrows, respectively) and Sertoli cells were observed at all 3 stages of development, while differentiated spermatogonia, spermatocytes, and spermatids were seen only in adults. In the latter, all 12 cellular associations of cycle of seminiferous epithelium were present. S, Sertoli cells; D, differentiated spermatogonia; Sy, spermatocytes; Sd, spermatids. Solid bar = 5 Am. Vs = (Vv of ST) X ((testicular weight) - (specific gravity of testis)) Eq. 4 The number of the particular cell per cross section was then multiplied by total cord or tubule length to yield the number of this cell type per testis. Second, the total number of cells per testis was estimated by dividing the absolute volume (Vc) of all nuclei of the particular cell type under consideration by the mean nuclear volume (V,,) of this cell (equation 5): Total No. Cell = V(, Vn Eq. 5 The absolute volume (Vc) of Sertoli cell nuclei was estimated by determining the product of V, for these nuclei and the absolute testicular volume (equations 6 and 7): Vc = (V, of Cell nuclei) x ((testicular weight). (specific gravity of testis)) Eq. 6 V, = (D,, 3 ) + 6 Eq. 7 In neonates and juveniles, Sertoli cell nuclei were spherical, and their volume was derived from the mean diameter (Dc) of 25 such nuclei per testis. Because the nuclei of adult Sertoli cells were not spherical, this method of estimating total Sertoli cell number was not performed at this stage of development. The nuclei of the stem spermatogonia were spherical to ovoid, and V,, of each was calculated by the formula for the volume of a sphere using the mean diameter (D(.) of 25 nuclei of each cell type. Statistical Analysis After determining graphically that the results of both studies were normally distributed, we used Bartlett's test to assess the homogeneity of variances among the groups of each study. It was found that the variances of the three groups in study 1 were heterogeneous, and these data were TABLE 1. Testicular parameters (mean ± SD) of neonatal, juvenile, and adult rhesus monkeys. Total Seminiferous V, seminiferous seminiferous No. Sertoli No. Ad/ No. Ap/ tubule tubule or cord tubule or cord cells/cross cross cross Developmental stage (n) Weight (g) Specific gravity diameter (am) (%) length (m)* section section section Neonate ± Juvenile ± ± 1..5 ±.2.3 ±.2 Adult ± ± *Derived values. on 3 July 218

4 -- ~ PUBERTY AND SERTOLI CELL PROLIFERATION 1195 (El o ICC fl)c,, 3 2 e4) Co (a 4) 1 t Neonate Juvenile Adult FIG. 2. Numbers of Sertoli cells per testis in neonatal, juvenile, and adult rhesus monkeys (mean SD) determined by volume fraction of seminiferous compartment calculated from data presented in Table 1. Each stage of development was significantly different from each other. logarithmically transformed. On the other hand, the variances of the three groups in study 2 were homogeneous, and transformation was therefore unnecessary. The significance of differences among groups of each study was determined by use of an analysis of variance, and, where appropriate, the significance of differences between groups was assessed by the Newman-Keuls test [21]. A difference was considered significant when p c.5. All data are expressed as mean ± SD. RESULTS Study 1: To Determine the Number of Sertoli Cells and Undifferentiated Spermatogonia in the Testes of Neonatal, Juvenile, and Adult Monkeys The testes of neonatal and juvenile monkeys were small and inguinal, whereas those of adults were larger and scrotal. Testicular weight of the juveniles was approximately 4- fold greater than that of the neonates, and the increased size of the more mature group was the result of a selective increase in seminiferous cord length (Table 1). In contrast, the large size of the adult testis was associated with an increase in both diameter and length of seminiferous tubule (Table 1). The testicular parenchyma of both groups of immature monkeys were similar in appearance, consisting of seminiferous cords surrounded by dense fibrous tissue; in contrast, the adult testis consisted of mature seminiferous tubules and a less dense interstitium (Fig. 1). The Sertoli cells in the cords of the neonatal and juvenile testes contained spherical to ellipsoid nuclei. On the other hand, in the seminiferous epithelium of adults, the Sertoli I Co X U) C).4 Co F Neonate Juvenile Adult FIG. 3. Numbers of Ad (top panel) and Ap (bottom panel) spermatogonia (mean ± SD) in neonatal, juvenile, and adult rhesus monkeys determined by either volume fraction of seminiferous compartment (solid bar) calculated from data presented in Table 1, or volume fraction of nucleus of each cell type (hatched bar) calculated from data presented in Table 2. For both cell types, the adult stage of development was significantly different from the other two stages regardless of morphometric method used for analysis. cells had pleomorphic nuclei and a single nucleolus. Both methods for calculating total number of Sertoli cells-the method that utilizes volume fraction of the seminiferous cords (Table 1) and the method that employs the volume fraction of Sertoli cell nuclei (Table 2)-yielded similar results for the neonatal monkeys (51.1 million vs million, respectively) and juvenile (331 million vs. 286 million, respectively) monkeys. Because of the pleomorphic nature of the Sertoli cell nuclei in adult testes, nuclear diameter at this stage of development could not be measured, and thus the calculation of adult Sertoli cell number was based only on the volume fraction of the seminiferous tubules. With this procedure, the mean number of Sertoli cells per testis of adults was found to be significantly greater than that from either of the other two stages of development (Fig. 2). In the case of the Ad and Ap spermatogonia, the nuclei were spherical or ellipsoid at all stages of development and, I T TABLE 2. Parameters of Sertoli cells and Ad and Ap spermatogonia (mean + SD) of neonatal, juvenile, and adult rhesus monkeys. Sertoli cells Ad spermatogonia Ap spermatogonia Developmental stage (n) Vv of nuclei %) Nuclear diameter (m) V, of nuclei (%) Nuclear diameter (m) Vv of nuclei %) Nuclear diameter (pm) Neonate Juvenile Adult * ± ±+.1 *Because of the pleomorphic nuclear shape of adult Sertoli cells, nuclear diameter can not be measured. on 3 July 218

5 1196 MARSHALL AND PLANT FIG. 4. Photomicrographs of testes from representative juvenile rhesus monkeys that were untreated (A) or had received pulsatile GnRH infusion for 5 (B) or 1 (C) wk. Premature activation of gonadotropin secretion induced by 5 wk of GnRH treatment resulted consistently in the appearance of a small number of differentiated spermatogonia. In addition, a few leptotene spermatocytes (not shown) were also observed in one of three animals treated for 5 wk. Continuation of the GnRH regimen for a further 5 wk resulted in a more mature germinal epithelium: in all 3 animals receiving 1 wk of GnRH, isolated leptotene spermatocytes were observed, and in two monkeys more mature pachytene spermatocytes (not shown) were occasionally found. Arrowheads, Ad spermatogonia; arrows, Ap spermatogonia; S, Sertoli cells; D, differentiated spermatogonia; L, leptotene spermatocytes. Solid bar = 5 pm. therefore, both methods for assessing cell number were employed throughout. The specific volume fractions, nuclear diameters, and cell number per cord/tubular cross section used for calculating stem spermatogonial number are shown in Tables 1 and 2. For a given stage of development, both methods yielded similar estimates of Ad and Ap spermatogonial numbers (Fig. 3). A small but statistically significant increase in the stem spermatogonia was observed in the neonatal-juvenile transition, and this contrasted with a marked and significant elevation in the juvenile-adult transition. Study 2: To Determine the Number of Sertoli Cells and Undifferentiated Spermatogonia Following Precocious Activation qf the Pituitary-Testicular Axis in the Monkey Activation of an adult pattern of endocrine activity in the pituitary-testicular axis in juvenile rhesus monkeys with 5 or 1 wk of uninterrupted pulsatile GnRH treatment was associated with an approximately 5-fold increase in testicular weight (Table 3). The precocious testicular growth in juvenile macaques was associated with marked increases in the diameter and length of the seminiferous compartment (Table 3). The testicular parenchyma of juvenile monkeys treated with 5 or 1 wk of pulsatile GnRH is illustrated in Figure 4. The mean number of Sertoli cells in testes taken from juvenile monkeys in which gonadotropin secretion had been prematurely stimulated by either 5 or 1 wk of GnRH treatment was significantly greater than that in the untreated juvenile monkeys. The estimates for this cell type were similar regardless of whether seminiferous cord length or ab- TABLE 3. Testicular parameters (mean + SD) in juvenile rhesus monkeys either untreated or receiving pulsatile GnRH administration for 5 or 1 wk. Seminiferous Total Gn RH tubule/cord V, seminiferous seminiferous No. Sertoli No. Ad/ No. Ap/ treatment Specific diameter tubule/cord tubule/cord cells/cross cross cross (weeks) (n) Testis weight (g) gravity (pm)* (%) length (m) section section section ± ± ± ± ± ± ± ± ± ± ±.1.5 ±.2 *Derived values. on 3 July 218

6 PUBERTY AND SERTOLI CELL PROLIFERATION 1197 (a.ct x 2 1 T 4 Xl 8 '; 6. T T T 4, Cn wwft u Duration of GnRH (week) FIG. 5. Number of Sertoli cells per testis (mean SD) in juvenile rhesus monkeys that were untreated or had received pulsatile GnRH infusion for 5 or 1 wk. Cell number was determined by either volume fraction of seminiferous compartment (solid bar) calculated from data presented in Table 3, or volume fraction of cell nucleus (hatched bar) calculated from data presented in Table 4. Both 5 and 1 wk of premature activation of pituitary-testicular axis with exogenous GnRH resulted in a significant increment in Sertoli cell number regardless of morphometric method used for analysis. solute nuclear volume (see Tables 3 and 4) was used to calculate this parameter (Fig. 5). In the case of Ad spermatogonia, although premature activation of the juvenile testis also resulted in an increase in the number of these cells, this effect was not significant (Fig. 6). In the case of Ap spermatogonia, however, the effect was more marked, and the increase in this cell type attained statistical significance after 1 wk of GnRH stimulation. DISCUSSION The finding in the present study that the mean testicular weight of juvenile rhesus monkeys was approximately 4- fold greater than that in neonatal animals is consistent with several earlier studies of nonhuman primates [12, 22, 231. Similarly, the dramatically larger size of the adult testis compared with that of either the neonatal or juvenile monkey has also been previously described on several occasions [24-26]. Moreover, it is well established that testicular growth between the juvenile phase and adulthood is greatest during puberty [24-26]. It is therefore not surprising that we observed a striking increase in testicular size in association with a premature induction of an adult pattern of endocrine activity in the pituitary-gonadal axis of juvenile monkeys with pulsatile GnRH administration. Interestingly, the testicular growth induced prematurely in the present V 4 'o ' 2 (a a a ao r- u Duration of GnRH (week) FIG. 6. Number of Ad Itop panel) and Ap (bottom panel) spermatogonia per testis (mean SD) in juvenile rhesus monkeys either untreated or receiving pulsatile GnRH administration for 5 or 1 wk. Cell number was determined by either volume fraction of seminiferous compartment (solid bar) calculated from data presented in Table 3, or volume fraction of nucleus of each cell type (hatched bar) calculated from data presented in Table 4. With volume fraction of nucleus used to calculate respective cell number, both 5 and 1 wk of premature activation of pituitary-testicular axis with exogenous GnRH resulted in significant increment in Ap spermatogonia. study exceeded that reported by Arslan et al. [9], who, in a study of the hormonal control of spermatogenesis, treated juvenile monkeys with testosterone and FSH. The greater growth of the testis in response to exogenous pulsatile GnRH stimulation presumably reflects a more physiologic gonadotropin drive. That seminiferous cord length in juvenile testis was greater than that in neonatal animals, while cord diameter was indistinguishable at these two phases of development, suggests that the growth of the prepubertal testis is primarily due to a selective lengthening of the seminiferous cord. In contrast, the pubertal growth of the testis that occurs spontaneously or that may be induced prematurely with pulsatile GnRH treatment results from an increase in both cord length 1u T TABLE 4. Parameters of Sertoli cells and Ad and Ap spermatogonia (mean + SD) in juvenile rhesus monkeys either untreated or receiving pulsatile GnRH administration for 5 or 1 wk. GnRH Sertoli cells Ad spermatogonia Ap spermatogonia treatment (weeks) (n) Vv of nuclei %) Nuclear diameter (um) Vv of nuclei (%) Nuclear diameter (pm) Vv of nuclei %) Nuclear diameter (pm) ± ± ± ±.5 8. ±.4 on 3 July 218

7 1198 MARSHALL AND PLANT and diameter. Of these two parameters, cord length appears to be proportionately the more important determinate of growth during this period of development. Since the juvenile phase of development in primates is a hypogonadotropic state [13], it is tempting to speculate that longitudinal growth of the seminiferous cord is relatively independent of gonadotropin. Also, it is interesting to note that the foregoing postnatal ontogeny of seminiferous compartmental growth is different from that of the rodent, where circumferential growth is initiated at birth or soon thereafter [ The present findings that Sertoli cell number was 6-fold greater in the adult than in the juvenile, and that precocious activation of the pituitary-testicular axis in juveniles resulted in a similar fold increase in the number of this cell type leads to the conclusion that puberty in primates is associated with a pronounced proliferation of this somatic testicular cell. This view is supported by the earlier data in humans that postmortem Sertoli cell number in testes of adult trauma victims was greater than that in corresponding prepubertal tissue [8]. On the other hand, the idea that primate puberty is associated with Sertoli cell proliferation is at odds with current dogma that states that the proliferation of these cells is completed by the time of puberty [7]. The latter view, however, probably reflects the extensive use of the rat as an experimental paradigm to study ontogeny of testicular function. In this rodent, the ontogeny of Sertoli cell proliferation, which begins on Day 16 of gestation and is completed by approximately 21 days of life, is in accord with the dogma [1, 21. That the pubertal proliferation of Sertoli cells is an early and rapid event in sexual maturation in primates is suggested by the observation that an adult population of these cells was attained within 5 wk of activating the pituitarytesticular axis in juvenile monkeys. Moreover, since testicular weight in the juveniles after 5 wk of GnRH stimulation was still only a fraction of the adult parameter, it seems that amplification of the germinal epithelium at puberty in this species follows in the wake of the early wave of Sertoli cell proliferation, rather than in association with a slow and progressive increase in Sertoli cell number. Although it is not possible from the present study to identify the relative role of the gonadotropins in the pubertal proliferation of Sertoli cells, Arslan et al. [9] reported that in juvenile monkeys administration of either FSH or testosterone alone was as effective as combined treatment in stimulating proliferation of this somatic cell. This finding suggests that there may be some redundancy in the gonadotropin drive to Sertoli cell proliferation at puberty in the primate. In this regard, it is interesting to note that in the rat, FSH has been reported to be obligatory and sufficient for the mitotic activity of the Sertoli cell prior to puberty [2]. In addition to the activation of gonadal function at the time of puberty, a brief neonatal phase of heightened endocrine activity is also a characteristic of the ontogeny of the pituitary-testicular axis in the primate [13]. In view of the finding that a pubertal proliferation of Sertoli cells may be induced prematurely by an appropriate gonadotropin stimulus, it is perhaps surprising that there was not a greater number of these somatic cells in the unstimulated juveniles, because their testes had presumably been exposed for the first few months of neonatal life to LH and FSH levels very similar to those observed in the adult [3]. Since neonatal Leydig cells respond to the endogenous LH drive with robust testosterone production [311, the absence of marked Sertoli cell proliferation in the infant is unlikely to be accounted for by a deficit in androgen production. It would seem reasonable to conclude, therefore, that the mitotic potential of the Sertoli cell of this stage of development is relatively limited. However, the apparent failure of the neonatal Sertoli cell to respond to appropriate gonadotropin stimulation does not represent a generalized deficit in function, since circulating immunoreactive inhibin concentrations are elevated during this phase of development [141. As in the case of Sertoli cells and as previously reported [23, 32-34], the number of undifferentiated or stem spermatogonia, Ad and Ap, were found to moderately increase during the neonatal-juvenile transition, with a marked augmentation between the juvenile and adult phases of development. The foregoing findings are consistent with the previous finding in the monkey that in vitro thymidine labeling of Ad and Ap spermatogonia is largely restricted to the adult testis [5]. Although undifferentiated spermatogonia were also increased after premature activation of the pituitarygonadal axis with pulsatile GnRH, the magnitude of the increase was not as marked as that in the Sertoli cells. Similarly, small increments in undifferentiated spermatogonia were observed in juvenile monkeys after FSH and testosterone administration [9]. All of the foregoing findings taken together suggest that the populations of the stem spermatogonia are indeed amplified at the time of puberty, but that the effect seems to be delayed relative to the early and robust proliferation of the Sertoli cells, underlining the role of these somatic cells in mediating the action of the gonadotropins on the stem cells [35]. ACKNOWLEDGMENTS The authors wish to acknowledge the expert technical assistance of) Debbie Bolette and the support of the Primate, Histology and Assay Cores of the Center for Research in Repro ductive Physiology. GnRH was provided by the NIDDK through the National Hormone and Pituitary Program, University of Maryland, School of Medicine REFERENCES 1. Steinberger A, Steinberger E. Replication pattern of Sertoli cells in maturing rat testis in vivo and in organ culture. Biol Reprod 1971; 4: Orth JM. Proliferation of Sertoli cells in fetal and postnatal rats Anat Rec 1982: 2) on 3 July 218

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