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a MRC Reproductive Biology Unit, Edinburgh EH3 9ET, Scotland, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the rat and other rodents, Sertoli cell replication is confined to the neonatal period, and replication ceases co-incident with the entry of the first germ cells into meiosis, i.e., the early stages of puberty [3, 5]. In domestic animals such as the bull and ram, the situation is similar, although Sertoli cell replication extends to 6–10 wk [3]. In nonhuman primates, there is limited but contrasting data. The Cebus monkey clearly exhibits Sertoli cell replication in the neonatal period [6], whereas the Rhesus monkey exhibits only a minor increase in Sertoli cell numbers at this stage of life, with the major increase occurring in the peripubertal period [7]. The limited data for the human male shows that Sertoli cell replication occurs neonatally [3, 8], but a further increase in Sertoli cell number occurs subsequent to this, probably in the peripubertal period [8]. The contrast between the Rhesus monkey on the one hand (negligible Sertoli cell replication neonatally), and the rat and the human on the other hand (major Sertoli cell replication neonatally) is made obvious by comparison of the blood levels of inhibin B, which is a specific product of the Sertoli cells and the levels of which probably reflect Sertoli cell number in prepuberty [9, 10]. Thus, both the rat [9] and human [11] show rising (and highest) levels of inhibin B neonatally, whereas the Rhesus monkey shows very low levels neonatally with the major increase occurring peripubertally, coincident with Sertoli cell replication [7, 12]. The difference in the timing of Sertoli cell replication between the Rhesus monkey and the human male is possibly related to the fact that the testes of Rhesus macaques do not descend into the scrotum until the peripubertal period, whereas testicular descent occurs in late gestation in the normal human male.
Although there are several factors identified that can affect Sertoli cell replication neonatally (for reviews, see [3, 13]), the most important appears to be FSH. Ablation of FSH secretion neonatally in the rat via a variety of approaches (e.g., administration of a GnRH antagonist) reduces Sertoli cell replication and results in a permanent ~50% reduction in Sertoli cell number [9, 14, 15] and lowering of blood levels of inhibin B [9]. As a result, in neonatally GnRH antagonist-treated male rats, adult testis size is reduced by ~40% though spermatogenesis is grossly normal and its efficiency may even be increased [9, 16]. Indirect evidence (based on final testis size and sperm counts) from human males with GnRH deficiency suggest a similar role for FSH [17–19], and it is well established that FSH levels are elevated in the neonatal period in the human male [11].
The overall aim of the present studies was to address the contrasts between species and between primates in terms of the timing and regulation of Sertoli cell replication by undertaking studies in the common marmoset (Callithrix jacchus), in which, as in the human, the testes descend before birth. In the marmoset, Sertoli cell number was measured at the end of the neonatal period and in adulthood in two groups of male co-twins treated neonatally with either vehicle or a GnRH antagonist (GnRHa). In addition, in adulthood, germ cell and Leydig cell volume and other testicular parameters were established by morphometry in the same animals to evaluate whether changes occurred similar to those observed in comparably treated rats [16]. Data for the same endpoints in the latter are provided for comparison.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Marmosets Animals were captive-bred common marmoset monkeys (Callithrix jacchus) maintained in a colony that has been closed since 1973. As marmosets exhibit considerable variation between individuals in parameters related to testis size and development, the present study utilized male co-twins, with one co-twin being administered vehicle as a control and the other co-twin being administered a long-acting GnRH antagonist (GnRHa). This design enabled pair-wise comparison of data for each control and treated co-twin, thus minimizing the number of animals required for study. The primary use of the animals from which testes were obtained for the present studies (9 sets of control/GnRHa-treated co-twins; 7 other vehicle-treated controls) was for other studies [20, 21] unconnected with those presented here. Starting on the day of birth, co-twins received s.c. injections of either 10 mg/kg GnRHa (Antide: Contraceptive development branch, Center for Population Research, National Institute of Child Health and Human Development [NICHD], Bethesda, MD) in propylene glycol:water (1:1, v:v) or with the vehicle alone (control). This treatment was repeated on Days 3 and 7 and then at weekly intervals until the last injection at Week 14. An earlier study of the present animals [21], and related studies [22] have shown that this treatment regimen effectively suppresses LH secretion and abolishes the normal neonatal rise in testosterone levels by causing regression of the Leydig cells [23]. Four sets of control/GnRHa-treated co-twins were killed at the end of the neonatal period/early infancy (18–22 wk of age), and the remaining 5 were killed in early adulthood (92+ wk); a terminal blood sample was collected from the latter animals for the measurement of testosterone levels. Testosterone levels in these animals during the neonatal period of treatment have been reported elsewhere [21]. Other control animals (n = 7) in which Sertoli cell numbers were determined were either untreated or had received vehicle treatment as controls for experiments unrelated to the present studies; they were killed at 28 wk (n = 2) or at 112–132 wk (n = 5).
Rats Wistar rats bred in our own animal house were maintained under standard conditions. On postnatal Days 2 and 5 (day of birth = Day 1), rats received s.c. injections of 10 mg/kg of a long-acting GnRHa (Antarelix; Europeptides, Argenteuil, France) in 20 µl 5% mannitol or with vehicle alone (controls). This GnRHa has characteristics and potency similar to those of the GnRHa (Antide) used for treatment of marmosets (which was no longer available), and the present treatment regimen using Antarelix in rats has been shown [9, 16, 24] to suppress FSH secretion to the limit of detection until the end of the neonatal period. Groups of 4–5 control and GnRHa-treated animals were then killed at the end of the neonatal period (Day 18) or in adulthood (90 days).
Tissue Collection and Processing
Marmosets Testes were dissected free of connective tissue and the epididymis, and weighed. The right testis was then immersion-fixed for 5.5 h in Bouin's fluid and the left testis in Sorenson's for 24 h before being reweighed; fixed testes from adult animals were then sliced transversely into ~2-mm-thick slices using a razor blade. The tissue was then processed and embedded in paraffin (Bouin's-fixed) or in epoxy resin (Sorenson's-fixed) using techniques comparable to those detailed previously by us for rat tissues [25, 26]. These tissues were used subsequently for cell quantification using the methods described below. Testes from another group of 5 adult marmosets were fixed in Bouin's fluid for 24 h, and one testis was then sampled in a random systematic manner, i.e., of 4 transverse slices, either slices 1 and 3 or slices 2 and 4 were sampled. These were processed through graded ethanols before infiltration with JB4 resin (TAAB, Berkshire, UK) and were used subsequently for enumeration of Sertoli cells using the optical disector method (see below).
Rats Testis tissue was either immersion-fixed for 24 h (Day 18), or perfusion-fixed for 40 min and then immersion-fixed for 5.5 h (adults) using Bouin's fluid as detailed previously [16, 25]. Fixed tissue from neonatal males was sampled and processed into JB4 resin as described for marmosets and was used subsequently for Sertoli cell counts according to the optical disector method. Fixed testicular tissue from adult animals was sliced transversely into 6 slices, which were embedded in paraffin and subsequently used for determination of Sertoli cell volume per testis as described below.
Sertoli Cell Counts and Other Morphometric Measurements
Two different methods (nucleator and optical disector) were used to count Sertoli cells in testes from marmosets [27], both utilizing an Olympus BH2 microscope fitted with an automatic stage (Prior Scientific Instruments, Cambridge, UK) and Image Proplus software (Media Cybernetics, Silver Spring, MD). The two methods yield comparable results (see [27]), although the nucleator method is inordinately more time-consuming. The choice of counting method was determined by the method of fixation and sampling of the available tissue, which largely precluded use of the optical disector method. For the nucleator method, Sertoli cell nuclear volume for each animal was first determined by measuring 4 separate radii from the nucleolus to the nuclear membrane for each of 70–100 Sertoli cell nuclei, and then a mean value was determined using the equation [27]:
Then, using a graticule with 121 points and a 40x objective, 10 fields on testicular cross sections were selected randomly and subjected to standard point-counting; the number of fields examined was based on power calculations derived from Wreford [27]. After determination of the percentage volume per testis for each of the components counted (Sertoli cell nucleus, germ cell nuclei, seminiferous epithelium, interstitium), total volume per testis was determined by multiplying the percentage value by fixed testis weight. Sertoli cell number per testis was then calculated as described by Wreford [27], using the value for mean nuclear volume derived above. Other parameters were expressed simply in terms of total volume (germ cells) or percentage volume (seminiferous epithelium, interstitium). The nucleator method was used for determination of Sertoli cell number in control and neonatally GnRHa-treated co-twin marmosets sampled in neonatal and adult life. For another group of 5 adult marmosets, the optical disector method was used to determine Sertoli cell number, as described by Wreford [27]. Comparison of values obtained using the two methods in the two separate groups of adult marmosets gave the following close agreement: Nucleator method, 24.9 ± 6.4 x 106/testis; disector method, 25.7 ± 2.9 x 106/testis; means ± SD, both N = 5.
For rat studies, the optical disector method was used for determination of Sertoli cell number at 18 days of age, whereas only data for Sertoli cell volume and germ cell volume per testis was determined in adulthood, using standard point-counting methods described elsewhere [16]. Our previous studies have established that relative values for Sertoli cell volume measured in adulthood using point-counting [16] equate closely to those measured using the optical disector at Day 18 ([16], unpublished data).
Determination of Leydig Cell Volume Per Testis
Leydig cell volume per testis in adult marmosets and rats was determined using point-counting methods similar to those outlined above for Sertoli cells, on sections of Bouin's-fixed testes that had been immunostained for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) as described previously [28]. A total of 15 (marmosets) or 12 fields (rats) were examined under oil immersion using either an Olympus BH2 microscope fitted with an automatic stage (marmosets) or a Leitz (New York, NY) 63x plan apo objective fitted to a Leitz laborlux microscope (rats), both fitted with a 121-point eyepiece graticule. Points falling over the nuclei of cells with 3ß-HSD-positive cytoplasm were scored and expressed as a percentage of the total points possible. For each animal, the values for percentage volume were then converted to absolute volumes per testis by reference to testis volume (= weight), as shrinkage was minimal, i.e., testis weights before and after fixation were comparable in each treatment group.
Plasma Levels of Testosterone
Plasma levels of testosterone were measured using an ELISA adapted from an earlier RIA method [29] as detailed elsewhere [16]. The limit of detection was 12 pg/ml. All samples were assayed together in one run.
Statistical Analysis
The paired t-test was used for comparison of data from control and GnRHa-treated co-twin marmosets, whereas Student's unpaired t-test was used for comparison of data for control and GnRHa-treated rats.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cell number Neonatal (Weeks 1–14) treatment of marmosets with GnRHa resulted in an average ~35% reduction (P = 0.002) in Sertoli cell number, compared with that of vehicle-treated co-twins, when measured at 18–22 wk of age (Fig. 1). This was matched by a similar decrease (P = 0.016) in testis weight (Table 1). These changes were evident in each of the GnRHa-treated animals when compared with their co-twin controls (Fig. 1). Comparable treatment of rats with GnRHa resulted in a 45% reduction in Sertoli cell number when measured on Day 18 (Fig. 1).
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Testicular morphology Gross morphology of the testes in control and GnRHa-treated co-twins at 18–22 wk of age was similar (Fig. 2). During point-counting, Sertoli cell nuclei exhibiting 0, 1, or 2+ nucleoli were recorded to provide a possible indicator of dividing cells (2+ nucleoli). Analysis of this data revealed a significant difference (P = 0.05) between control and GnRHa-treated animals in the numbers of Sertoli cells with 2 or more nucleoli (control 5.1 ± 0.7% of Sertoli cell nuclei; GnRHa 3.5 ± 1.4%; means ± SD).
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Effect of Neonatal GnRHa Treatment on the Testis in Adulthood
Sertoli cell number Sertoli cell number (Fig. 3) and testis weight (Table 1) in adult marmosets treated neonatally with GnRHa were not significantly different from values in their corresponding co-twins; however, in 4 out of 5 sets of co-twins, Sertoli cell number (Fig. 3) and testis weight (not shown) were higher in the GnRHa-treated co-twin. In contrast, Sertoli cell volume in rats treated neonatally with GnRHa was reduced significantly compared to that of controls, a decrease that matched the difference in Sertoli cell number observed at the end of the neonatal period (compare Figs. 1 and 3).
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Germ cell volume per Sertoli cell In adulthood, germ cell volume per Sertoli cell was not significantly different between control and neonatally GnRHa-treated marmosets, though it was noticeable that animals with the highest Sertoli cell numbers had the lowest germ cell volume per Sertoli cell, and vice-versa (Fig. 3). A similar, but more pronounced, relationship was evident in neonatally GnRHa-treated rats with reduced Sertoli cell numbers, as germ cell volume per Sertoli cell in these animals was significantly increased by ~20% (Fig. 3). Morphometric analysis revealed that the percentage of the adult testis occupied by seminiferous epithelium was consistently lower (P = 0.018) in GnRHa-treated marmosets than in their co-twin controls (controls, 83.4 ± 15.2%; GnRHa, 77.8 ± 30.0%; means ± SD). It was also noteworthy that the average germ cell volume per Sertoli cell in control rats (i.e., the efficiency of spermatogenesis) was ~5-fold higher than the corresponding values in control marmosets (Fig. 3); in the latter, high-power examination of seminiferous tubule cross sections consistently revealed relatively small cohorts of germ cells per Sertoli cell in both control and GnRHa-treated animals (Fig. 4).
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Interstitial and Leydig cell volume and testosterone levels In adult marmosets treated neonatally with GnRHa, interstitial tissue volume per testis was consistently and significantly (P = 0.04) increased when compared with values in their co-twin controls (Fig. 5). This was due in part to increased Leydig (3ß-HSD-positive) cell volume per testis (P = 0.01; Figs. 5 and 6). A similar increase in Leydig cell volume per testis was evident in adult rats treated neonatally with GnRHa (Figs. 5 and 6). It was also noteworthy that the percentage of interstitial tissue and Leydig cell volume in the testis of adult control marmosets was ~3-fold greater than in control rats (Fig. 5; see also Fig. 6). Testosterone levels in control and GnRHa-treated co-twins were not significantly different (P = 0.43) in adulthood (controls, 5.4 ± 4.7 ng/ml; GnRHa, 9.7 ± 7.2 ng/ml; mean ± SD).
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Testicular morphology With the exception of changes in the interstitium already noted, there were no obvious differences in gross morphology of the testes in adult marmosets that had been treated neonatally with vehicle or GnRHa (Fig. 4).
Sertoli Cell Number Versus Age in the Marmoset
Data for Sertoli cell number in control twins from the two experiments described above, together with data from a further 7 control animals unconnected with the main studies, are plotted against age in Figure 7. This illustrates that in the normal marmoset, most, if not all, of adult Sertoli cell numbers are already present by the end of the neonatal period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present studies show that major proliferation of Sertoli cells occurs in the neonatal period (or earlier) in the marmoset and that adult numbers of Sertoli cells are present at the start of infancy. In this respect, the marmoset is similar to the Cebus monkey [6], which is also a new world monkey, but is radically different from the rhesus macaque, in which Sertoli cell proliferation in the neonatal period is minimal, with most replication occurring peripubertally [7]. Similarly, the cynomolgus macaque also appears to lack a neonatal "surge" in Sertoli cell replication [31]. The available, though limited, evidence for the human shows a clear peak in Sertoli cell replication in the neonatal period with further replication occurring subsequently, probably in the peripubertal period [3, 8]. The difference between the human and rhesus monkey is highlighted by measurement of the blood levels of inhibin B, which, before adulthood, has been shown in rats [9] and the rhesus monkey [10] to provide a reliable index of Sertoli cell numbers within the testes. In the human, blood levels of inhibin B rise rapidly in the neonatal period to reach their highest levels at any period in life [11]. In contrast, blood levels of inhibin B in the rhesus monkey remain low in the neonatal period and only rise rapidly before puberty coincident with Sertoli cell replication [7, 12]. Unfortunately, the available assay is unable to detect inhibin B in the marmoset, precluding comparison with this species.
These findings suggest that there is a fundamental difference between macaques, and the human and marmoset in terms of when Sertoli cell replication normally occurs, and in this regard the human and marmoset show similarities with most other species (e.g., rodents, farm animals) in which the neonatal period is an important period for Sertoli cell replication [3]. However, the present findings also demonstrate that the marmoset is still capable of replicating its Sertoli cells beyond the neonatal period, as occurred in neonatally GnRHa-treated males. The exact period when this additional proliferation occurred was not established, but it can be speculated that it occurred in the peripubertal period when Sertoli cell replication occurs in the rhesus monkey [7] and probably also in the human [8]. The lack of major Sertoli cell replication in the neonatal period in rhesus and cynomolgus macaques is unexplained, but may be related in some way to the fact that the testes of macaques do not descend into the scrotum until puberty, in contrast to the human and marmoset, in which the testes have usually descended by birth.
Although the present findings demonstrate that adult numbers of Sertoli cells are present at the start of infancy in the marmoset, they do not rule out the possibility that significant loss of Sertoli cells occurs normally during infancy, necessitating further Sertoli cell replication in the peripubertal period. This would be most easily studied by methods such as incorporation of bromodeoxyuridine (BrdU), but our studies using single injections of BrdU in the marmoset 1 h before death have so far yielded levels of cell labeling too low to permit definitive evaluation of this possibility (unpublished data). Similarly, the small number of marmoset testes that we have so far examined, their limited age range, and the scarcity of degenerating cells that we have observed precludes any informed speculation on whether or not significant degeneration of Sertoli cells occurs during infancy.
Irrespective of when Sertoli cell replication occurs, the available evidence suggests strongly that ~50% of the increase in Sertoli cell numbers is dependent on FSH [3]. Approaches such as neonatal administration of a GnRH antagonist result in ~45% reduction in Sertoli cell numbers in the rat [9, 15, 16, 24], and similar effects occur when FSH action is suppressed by other means [1, 3]. Indirect evidence from the study of human males with deficiencies in GnRH production, based on testis size and sperm counts, suggest that development of normal Sertoli cell numbers in the human is also FSH-dependent [17–19], as is Sertoli cell replication in the rhesus monkey [7]. The present findings show a similar situation in the marmoset, as administration of GnRHa in the neonatal period resulted in an average 35% reduction in Sertoli cell numbers shortly after cessation of treatment. Unfortunately, because currently available assays are unable as yet to detect marmoset FSH, it is not possible to prove definitively that this reduction was associated with suppression of FSH levels.
Marmosets treated neonatally with a GnRH antagonist exhibited grossly normal spermatogenesis in adulthood and are normally fertile [20]. However, comparison of individual control and treated co-twins revealed a tendency toward a decrease in germ cell volume per Sertoli cell in those GnRHa-treated co-twins in which Sertoli cell number exceeded that in their control co-twin (4 out of 5). Although this failed to achieve statistical significance, there was a significant difference in percentage of seminiferous epithelium between control and GnRHa-treated animals, suggesting that a change had indeed occurred. This conclusion is consistent with findings in rhesus monkeys, in which an experimentally induced decrease in Sertoli cell number (by unilateral castration) was associated with a significant increase in round spermatids per Sertoli cell in the remaining testis, coincident with elevation of FSH levels [10]. Similarly, experimental elevation of Sertoli cell numbers in the rat, by induction of neonatal hypothyroidism, results in reduced numbers of spermatids supported by each Sertoli cell, coincident with a reduction in FSH levels [32]. The converse change can also occur: experimental reduction of Sertoli cell numbers in rats, via neonatal administration of a GnRH antagonist, results in a significant increase in germ cell volume per Sertoli cell (Fig. 3; [16]). A possible explanation for these changes is alteration in FSH levels [9, 10, 16, 32], which are increased when Sertoli cell numbers are reduced and reduced when Sertoli cell numbers are increased; however, more direct studies are required to address the possible relationship between FSH levels and germ cell volume per Sertoli cell.
An important finding from the quantitative comparison of spermatogenesis in the marmoset and rat in the present study was that the efficiency of spermatogenesis in the marmoset, measured as the germ cell volume supported per Sertoli cell, was only ~20% of that observed in the rat. The low efficiency of spermatogenesis in the marmoset is reminiscent of that in the human [3, 4, 30] and again contrasts with that of the rhesus monkey, in which relatively high efficiency of spermatogenesis is observed [3, 33]. These differences are most likely explained by differences in the organization of spermatogenesis [3], which in the rat and rhesus monkey shows a segmental/radial distribution of stages and a wave of spermatogenesis [3, 33], but in the human shows a semi-helical organization of stages and only a partial wave [3, 34, 35]. Recent evidence from the marmoset suggests that this species shows similarities in organization of spermatogenic stages to that in the human ([36], unpublished observations). The basis for these fundamental differences in organization remain unclear.
An unexpected finding from the present studies was that neonatal GnRHa treatment resulted in a significant increase in Leydig cell volume per testis in adulthood in both the marmoset and rat. As these two species otherwise showed differences in Sertoli cell number and testis size after neonatal GnRHa treatment, it is concluded that the common triggering factor is the neonatal suppression of gonadotropin levels. This results in delayed puberty in both species, manifested in the rat by delayed onset of spermatogenesis [24] and in the marmoset by attenuation of the pubertal rise in testosterone levels [20–22]. It may be that these delays trigger some form of compensatory change that leads to increased Leydig cell number in adulthood. In this regard, studies in the rat have shown that neonatal induction of hypothyroidism leads to an increase in both Sertoli and Leydig cells coincident with delayed maturation of both cell types [37, 38]. It may be that this delay allows greater proliferation of adult Leydig cells or their precursors, resulting in a permanent change in Leydig cell numbers in adulthood. Irrespective of the mechanism involved, it is clear that the increase in Leydig cell volume after neonatal GnRHa treatment does not lead to any detectable change in blood levels of testosterone in adulthood in either the rat [16] or the marmoset (this study, [21, 22]). Further study of the ontogeny of the increase in Leydig cell numbers per testis may provide insight into fundamental factors regulating Leydig cell development.
In conclusion, the present data add further support to the evidence ([36], unpublished observations) that the marmoset represents a useful model for the human in terms of the timing of Sertoli cell proliferation and the poor efficiency of spermatogenesis in adulthood. The present data also indicate that these parameters are potentially manipulable via experimental treatments. The marmoset thus offers considerable opportunities for studies of therapeutic manipulation of spermatogenesis with fairly direct relevance to the human.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported in part by the European Consortium for the Ecotoxicology of Chemicals (ECETOC). NA was in receipt of a Fellowship from the International Atomic Energy Agency. 
2 Correspondence: P.T.K. Saunders, MRC Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9ET, Scotland, UK. FAX: 44 131 228 5571; p.saunders{at}ed-rbu.mrc.ac.uk
Accepted: January 4, 2000.
Received: October 12, 1999.
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R.M. Sharpe, H.M. Fraser, M.F.H. Brougham, C. McKinnell, K.D. Morris, C.J.H. Kelnar, W.H.B. Wallace, and M. Walker Role of the neonatal period of pituitary-testicular activity in germ cell proliferation and differentiation in the primate testis Hum. Reprod., October 1, 2003; 18(10): 2110 - 2117. [Abstract] [Full Text] [PDF]
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D.D. Lunstra, T.H. Wise, and J.J. Ford Sertoli Cells in the Boar Testis: Changes During Development and Compensatory Hypertrophy after Hemicastration at Different Ages Biol Reprod, January 1, 2003; 68(1): 140 - 150. [Abstract] [Full Text] [PDF]
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R. M. Sharpe, B. Martin, K. Morris, I. Greig, C. McKinnell, A. S. McNeilly, and M. Walker Infant feeding with soy formula milk: effects on the testis and on blood testosterone levels in marmoset monkeys during the period of neonatal testicular activity Hum. Reprod., July 1, 2002; 17(7): 1692 - 1703. [Abstract] [Full Text] [PDF]
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