Abstract
Neonatal treatment of male rats with diethylstilboestrol (DES) or a gonadotrophin-releasing hormone antagonist (GnRHa) reduces final Sertoli cell number, an effect presumed to occur via suppression of follicle-stimulating hormone (FSH). As both treatments also suppress androgen action, we investigated whether androgens and oestrogens might affect Sertoli cell nuclear volume/number in the rat using single or combined treatments that differentially affected FSH, testosterone and oestrogen (DES) levels. Neonatal treatment with flutamide (50 mg/ kg) significantly reduced Sertoli cell nuclear volume/ number per testis and blood inhibin-B levels at day 18, despite elevating FSH levels; this treatment also exacerbated the reduction in Sertoli cell nuclear volume per testis induced by treatment with 0.1 μg DES without affecting FSH levels. Treatment with 0.1 μg DES on its own also reduced Sertoli cell nuclear volume per testis without affecting FSH/testosterone levels, but co-administration of 0.1 μg DES+GnRHa, which suppressed FSH and testosterone levels, resulted in a markedly greater effect. Treatment with GnRHa alone or 10 μg DES alone grossly suppressed FSH and testosterone levels and reduced Sertoli cell nuclear volume/number per testis by approximately 60%, but co-administration of the two treatments had no greater effect than either alone. Co-administration of testosterone esters with 10 μg DES partially prevented the 10 μg DES-induced reduction in Sertoli cell nuclear volume per testis, and normalized FSH levels. In all treatment groups, plasma levels of inhibin-B paralleled changes in Sertoli cell nuclear volume/number per testis, but treatments that suppressed FSH levels (GnRHa, 10 μg DES) caused a proportionately greater reduction (approximately 90%) in inhibin-B levels than in Sertoli cell nuclear volume/number (50–60%). Germ cell volume per unit Sertoli cell was reduced in animals treated with 10 μg DES alone or in those treated with 0.1 μg DES plus either flutmaide or GnRHa, but otherwise remained relatively constant between treatment groups. It is concluded that, in the neonatal rat, (1) endogenous androgens, as well as FSH, play a physiological role in increasing Sertoli cell number, (2) exogenous oestrogen exposure can decrease Sertoli cell number without altering FSH levels, (3) these effects probably share a common pathway and (4) blood inhibin-B provides a robust indicator of change in Sertoli cell number.
Introduction
It is well established that the formation of normal numbers of Sertoli cells is a key factor in determining testis size, germ cell numbers per testis and sperm-production rate in adulthood in a range of mammals, including humans (reviewed in Sharpe et al. 2003). The number of Sertoli cells per testis appears to be largely determined by their rate of proliferation during various life stages, as there are few reports to indicate that the rate of apoptosis is important. Although there are some differences between species in when Sertoli cells proliferate, in most mammals, including humans, the fetal and neonatal periods are important and probably also the immediately prepubertal period (reviewed in Sharpe et al. 2003). Studies of Sertoli cell proliferation postnatally in rodents and primates, and experimental manipulations to alter gonadotrophin levels, have generated the consensus view that follicle-stimulating hormone (FSH) is the primary factor that increases the rate of Sertoli cell proliferation in postnatal life (e.g. Marshall & Plant 1996, Meachem et al. 1996, Singh & Handelsman 1996, Sharpe et al. 2000, Plant & Marshall 2001), although thyroid hormone plays an important role in some species in determining the length of the postnatal period during which Sertoli cells can proliferate (van Haaster et al. 1993, De Franca et al. 1995, Sharpe et al. 2003). Indeed, a recent study in transgenic mice concluded that FSH action was probably sufficient to achieve normal Sertoli cell numbers (Allan et al. 2004), a conclusion that fits well with the reduced Sertoli cell numbers in mice in which FSH action has been prevented by knockout of FSH or its receptor (Wreford et al. 2001, Haywood et al. 2003, Johnston et al. 2004) or in rats and primates following the administration of gonadotrophin-releasing hormone (GnRH) antagonists to suppress FSH (and luteinizing hormone (LH)) secretion (Atanassova et al. 1999, Sharpe et al. 2000, 2003).
However, the view that FSH is of prime importance in Sertoli cell proliferation in rodents has been challenged by new findings which indicate that androgens can also play an important role in determining Sertoli cell number in mice (Haywood et al. 2003, Johnston et al. 2004), and may be particularly important in the fetal period when FSH appears to be unnecessary for Sertoli cell proliferation (Baker & O’Shaughnessy 2001, Johnston et al. 2004). The latter authors showed that Sertoli cell numbers in testicular feminized (tfm) mice, which lack a functional androgen receptor (AR), exhibit an approximately 50% reduction at birth and an approximately 70% reduction in adulthood, despite the presence of normal/elevated FSH action in the same animals. We have confirmed these findings in complete AR-knockout mice but have shown that mice with selective knockout of the AR in Sertoli cells develop apparently normal numbers of Sertoli cells (de Gendt et al. 2004, and also de Gendt et al. unpublished observations). In fact evidence for positive effects of androgens on Sertoli cell number is not a new finding, as over the past decade such findings have been reported in at least three studies in which LH or human chorionic gonadotrophin was administered (with or without FSH) to rhesus monkeys during the juvenile period (Arslan et al. 1993, Schlatt et al. 1995, Ramaswamy et al. 2000). These observations were generally viewed as evidence for differences in hormonal regulation of Sertoli cell proliferation between rodents and primates (Ramaswamy et al. 2000). However, it is evident from studies in rodents involving complete suppression of FSH levels/action during the fetal and/or neonatal periods of Sertoli cell proliferation, that the maximum reduction in Sertoli cell numbers achieved is of the order of 40–60% (Atanassova et al. 1999, Wreford et al. 2001, Sharpe et al. 2003, Johnston et al. 2004), leaving scope for the influence of other factors such as androgens.
Based on the findings in tfm mice by Johnston et al.(2004), it may be that some previous studies (particularly in rodents) have overlooked androgen effects on Sertoli cell proliferation/numbers, especially in situations in which both FSH and LH (and thus androgen) action may have been reduced experimentally. This prompted us to reexamine our own studies in which we have administered either a GnRH antagonist (GnRHa) or an oestrogen (diethylstilboestrol, DES) to neonatal rats and shown that this reduces final Sertoli cell number by 40–50% in association with suppression of FSH levels in blood (Sharpe et al. 1998, 1999, Atanassova et al. 1999). We had attributed the reduction in Sertoli cell numbers in such studies to suppression of FSH levels, although the possibility of a direct effect of the DES on Sertoli cell proliferation was also considered (Sharpe et al. 1998). However, neonatal treatment with GnRHa or with DES also suppresses androgen levels by 70–90% and the DES treatment also reduces AR protein expression in the testis (McKinnell et al. 2001a, Rivas et al. 2002, 2003), raising the possibility that these changes may have played some part in the reduction in Sertoli cell numbers. It is difficult in such situations to distinguish between the effects of FSH, DES and testosterone on Sertoli cell number. Therefore, we have undertaken three studies in which we have tried to separate out these three hormonal effects in normal intact male rats by using combinations of hormonal manipulations or treatments that selectively block androgen action (flutamide) while leaving FSH levels unchanged/elevated. Our findings provide strong support for an effect of neonatal androgens on Sertoli cell number in the rat, but also point to a role for oestrogens in addition to the accepted role of FSH.
Materials and Methods
Animals, treatments, sample collection and processing
All animal studies and treatments were conducted under Project Licence approval in accordance with UK Home Office guidelines for the care and welfare of laboratory animals. Wistar rats, bred in our own animal house, were maintained under standard conditions and were maintained on a soya-free diet (rat and mouse soya-free breeding diet; Special Diet Service, Dundee, Scotland, UK). On the day after birth, rats from several litters were cross-fostered into all-male litters of 8–12 pups and these animals were then allocated to two or three different treatments (n=4–5 per group) according to the protocols described below. The purpose of the treatment regimens was to induce changes in Sertoli cell number and to combine various treatments in order to discriminate between changes induced as a consequence of direct oestrogen action, or of inhibition of FSH or androgen action; the treatment protocols that we used are of proven efficacy (Atanassova et al. 1999, 2000, Rivas et al. 2002, 2003). The present study essentially comprised three experiments.
Experiment 1 Rats were subjected to one of the following four treatments administered by subcutaneous injection. (i) 10 μg DES (Sigma Chemical Co, Poole, Dorset, UK) in 20 μl corn oil administered on days 2, 4, 6, 8, 10 and 12; this treatment is proven to cause marked suppression of Sertoli cell number as well as FSH and testosterone levels (Sharpe et al. 1998, Atanassova et al. 2000, Rivas et al. 2002). (ii) 10 mg/kg of a long-acting GnRHa (Teverelix; Ardana, Edinburgh, Scotland, UK) in 20 μl corn oil on days 2 and 5 alone. This treatment regimen has been shown previously (see references cited for treatment (i)) to effectively suppress gonadotrophin secretion until postnatal days 15–25 with consequent suppression of Sertoli cell number, FSH and testosterone levels (Atanassova et al. 1999, Sharpe et al. 1999, Rivas et al. 2002), and to induce a similar reduction in testis weight at day 18 to that induced by treatment with 10 μg DES as in (i). (iii) A combination of the treatments in (i) and (ii) to test if this would induce greater suppression of Sertoli cell number and other parameters than either treatment alone. (iv) 20 μl corn oil alone (control).
Experiment 2 Rats were subjected to one of the following five treatments administered by subcutaneous injection. (i) 10 μg DES in 20 μl corn oil administered on days 2, 4, 6, 8, 10 and 12. (ii) 200 μg testosterone esters (TE; Sustanon; Organon Labs, Cambridge, UK) in 20 μl corn oil on days 2, 4, 6, 8, 10 and 12. (iii) A combination of the treatments in (i) and (ii) to test whether prevention of the DES-induced reduction in testosterone levels was able to prevent the DES-induced reduction in Sertoli cell number; the dose of TE used has been shown previously to prevent most of the adverse effects of DES treatment on the developing male reproductive tract (McKinnell et al. 2001a, Rivas et al. 2003). (iv) 50 mg/kg of the AR antagonist flutamide (Sigma) in 20 μl corn oil on days 2, 4, 6, 8, 10 and 12. This dose was chosen based on studies by Imperato-McGinley et al.(1992) who showed that it caused major reproductive tract abnormalities in male offspring when administered to pregnant rats; we have shown that administration of this dose neonatally retards normal development of the reproductive tract (Rivas et al. 2002). (v) 20 μl corn oil alone (control). This experiment was repeated twice and data from the three experiments combined.
Experiment 3 Rats were subjected to one of the following four treatments administered by subcutaneous injection. (i) 0.1 μg DES in 20 μl corn oil administered on days 2, 4, 6, 8, 10 and 12; this treatment has been shown previously to cause a small reduction in Sertoli cell numbers but to have no effect on testosterone levels (Atanassova et al. 1999, Rivas et al. 2002) or on FSH levels (RM Sharpe, M Walker, C McKinnell, JS Fisher & NN Atanassova, unpublished observations). (ii) 0.1 μg DES in combination with 50 mg/kg flutamide in 20 μl corn oil on days 2, 4, 6, 8, 10 and 12. This was to test whether suppression of androgen action in combination with DES exposure will further reduce Sertoli cell number/nuclear volume per testis. (iii) 0.1 μg DES in 20 μl corn oil administered on days 2, 4, 6, 8, 10 and 12 in combination with 10 mg/kg of a long-acting GnRHa (Teverelix) in 20 μl corn oil administered on days 2 and 5. This is to confirm that suppression of both FSH and testosterone levels (GnRHa) in concert with DES exposure will suppress Sertoli cell number/nuclear volume per testis to the lowest level achievable. (iii) 20 μl corn oil alone (control).
Rats from these treatment groups were killed on day 18, a time by which final Sertoli cell number per testis has been determined in the rat (Sharpe et al. 1999, 2003). The treatment-induced changes in blood hormone levels that are detectable at this age are similar to those that occur during the period of treatment (Sharpe et al. 1998, and RM Sharpe, M Walker, C McKinnell, JS Fisher & NN Atanassova, unpublished observations); although treatments had stopped on day 12 the long-acting nature of the compounds (GnRHa, TE) and/or their administration in corn oil means that treatment effects are detectable until approximately day 25 (Sharpe et al. 1998, Atanassova et al. 2000 and RM Sharpe, M Walker, C McKinnell, JS Fisher & NN Atanassova, unpublished observations). Animals were anaesthetized with flurothane, blood collected into a heparinized syringe by cardiac puncture and the animals then killed by cervical dislocation. The testes were dissected out, weighed and then fixed for approximately 5 h in Bouins; in some treatment groups the left testis was fixed in this way while the right testis was fixed for 24 h in Bouins and then used for Sertoli cell enumeration using the optical disector method, as outlined below. Plasma was separated by centrifugation and stored at −20 °C until used for hormone assays as described below.
Measurement of Sertoli cell and germ cell nuclear volume per testis and determination of Sertoli cell number
Testes that had been fixed for 5 h were transferred to 70% ethanol before being processed for 17.5 h in an automated Leica TP1050 processor and embedded in paraffin wax. Sections of 5 μm thickness were cut and floated onto slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma) and dried at 50 °C overnight. They were then stained with haematoxylin and eosin and used for quantification of Sertoli cell and germ cell nuclear volume per testis using standard point-counting of cell nuclei as described previously (Atanassova et al. 1999, 2000). In brief, cross-sections of testes from every rat in each treatment group were examined under oil immersion using a Leitz 63× plan apo objective fitted to a Leitz Laborlux microscope and a 121-point eyepiece graticule. Using a systematic clock-face sampling pattern from a random starting point, 16 fields were counted. Points falling over the nuclei of Sertoli cell or germ cell nuclei were scored and expressed as a percentage of the total points counted. For each animal, the values for percentage nuclear volume were converted to absolute nuclear volumes per testis by reference to testis volume (equivalent to weight) as shrinkage was minimal; that is, testis weights before and after fixation were comparable in each treatment group. However, because of the complex shape of the Sertoli cell nucleus it was not possible in the present studies to determine average Sertoli cell nuclear size, but we have shown previously that point-count measurement of Sertoli cell nuclear volume per testis yields similar results to Sertoli cell number determined by the disector method (Atanassova et al. 1999, 2000). This was shown also in the present studies by using the disector method (see below) to confirm key findings that emerged from the measurement of Sertoli cell nuclear volume per testis.
Testes that had been fixed in Bouins for 24 h were sampled in a random systematic manner by slicing them transversely into four pieces using a razor blade and then processing either slices 1 and 3 or slices 2 and 4 through graded ethanol solutions before infiltrating with JB4 resin (TAAB, Laboratories Equipment Ltd, Aldermaston, Berks, UK). After polymerization, 20 μm sections were cut on a Reichart 2050 microtome using a Diatome Histoknife, mounted onto glass slides and stained with Harris’ haematoxylin. Sertoli cells were then counted using the optical disector method as described by Wreford (1995) and reported previously by us (Atanassova et al. 1999, Sharpe et al. 1999).
Measurement of plasma hormone levels
Plasma levels of FSH were measured by RIA using materials generously supplied by National Institute of Diabetes and Digestive Diseases, Bethesda, MD, USA. Results have been expressed in terms of the rFSH RP-2 standard. In the FSH assay, plasma levels measured in hypohysectomized rats are in the range 1.2–2.3 ng/ml and values in this range are therefore considered to be baseline. Plasma levels of inhibin-B were measured using a two-site enzyme-linked immunoassay which utilizes a capture antibody directed against the C-terminal portion of the human βB-subunit and the F(ab) fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the inhibin-αsubunit conjugated to alkaline phosphatase (Illingworth et al. 1996). The assay has been validated previously for measurement of inhibin-B in the rat (Woodruff et al. 1996, Sharpe et al. 1999), and in the present studies it was confirmed that rat plasma diluted in parallel with the inhibin-B standard and that levels of inhibin-B in plasma from castrated adult male rats were reduced to non-detectable levels (< 8 pg/ml).
Plasma levels of testosterone were measured using an ELISA which was adapted from an earlier RIA method (Corker & Davidson 1981). Plasma, to which was added trace amounts of [3H]testosterone (Amersham Biosciences, Little Chalfont, Bucks, UK), was extracted twice with 10 vol. hexane/ether (4:1, v/v) and the organic phase dried down under N2 at 55 °C. The average efficiency of extraction was 75%. The second antibody was immobilized on to an ELISA plate by addition of 100 μl acid-purified donkey anti-goat/sheep IgG (250–350 mg/ml) diluted in 0.1 M sodium carbonate buffer, pH 9.6. The plate was sealed and incubated overnight at 4 °C. The wells were then washed twice with 0.1% Tween-20 and incubated for 10 min at room temperature with 0.2 ml of the same solution to block non-specific binding sites. Samples in duplicate (50 μl) were assayed after dilution in 0.1 M PBS, pH 7.4, containing 0.1% gelatin (Sigma) and incubated overnight at 4 °C with 50 μl sheep anti-testosterone-3-(O-carboxymethyl)oxime (CMO)/BSA diluted 1:100 000 plus 50 μl testosterone-3-cmo labelled with 1:20 000 horseradish peroxidase (Amersham Biosciences). The plate was then washed several times with 0.1% Tween-20 before addition of 0.2 ml substrate (5 mM O-phenylenediamine; Sigma) and 0.03% hydrogen peroxide diluted in 0.1 M citrate/phosphate, pH 5.0, to each well. The plate was then incubated in the dark for 10–30 min until the colour reaction was optimal. The reaction was stopped by addition of 50 μl 2 M sulphuric acid to each well and the optical density then read at 492 nm in a plate reader. The limit of detection was 12 pg/ml.
Statistics
Comparison of the different parameters for the various treatment groups was made using analysis of variance with or without logarithmic transformation of the data to obtain a normal distribution. Where significant differences between groups were indicated, sub-group comparisons also utilized analysis of variance but used the variance from the experiment as a whole (for that parameter) as the measure of error.
Results
Effect of neonatal treatment with 10 μg DES with or without GnRHa on Sertoli cell number/nuclear volume per testis and hormone levels (experiment 1)
Treatment with either 10 μg DES or GnRHa alone resulted in comparable reductions in testis weight at day 18 of age (Table 1), and both treatments on their own resulted in a 49–62% reduction in Sertoli cell number and nuclear volume per testis (Fig. 1). These changes were associated with suppression of both FSH (29% suppression for DES, 54% for GnRHa) and testosterone (60% suppression for DES, 75% for GnRHa) levels in blood (Fig. 1). Plasma levels of inhibin-B were suppressed by 90% in both treatment groups (Fig. 1). Combined treatment with 10 μg DES+GnRHa had no greater effect than either treatment alone on any of these parameters (Table 1, Fig. 1), consistent with both treatments having a shared mechanism of action, e.g. suppression of FSH and/or testosterone.
Effect of neonatal treatment with 10 μg DES with or without testosterone or with flutamide on Sertoli cell number/nuclear volume per testis and hormone levels (experiment 2)
As in the first experiment, treatment with 10 μg DES alone resulted in significant suppression of testis weight at day 18 (Table 1), as well as suppression of Sertoli cell nuclear volume per testis and suppression of FSH, testosterone and inhibin-B levels in blood (Fig. 2). When TE was administered in combination with 10 μg DES it elevated testosterone levels in blood above normal control levels, and this resulted in significant (P< 0.01), but only partial, attenuation of the DES-induced reduction in Sertoli cell nuclear volume per testis (32% reduction in DES+TE versus 61% reduction in DES) and of the DES-induced reduction in blood levels of inhibin-B (23% reduction in DES+TE versus 69% reduction in DES; Fig. 2). These changes were associated with normalization of FSH levels in rats treated with 10 μg DES+TE (Fig. 2). At face value, the comparison between rats treated with 10 μg DES and those treated with 10 μg DES+TE implies that the reduction in Sertoli cell nuclear volume induced by treatment with 10 μg DES alone may be due partly to suppression of FSH and/or testosterone levels and partly to a direct oestrogenic effect of the DES. When TE was administered on its own, it resulted in significant suppression of Sertoli cell nuclear volume and blood inhibin-B levels (36 and 42% suppression, respectively), changes that are presumed to result from suppression of FSH levels (48% suppression; Fig. 2). Administration of the anti-androgen, flutamide, resulted in a 34% suppression of Sertoli cell nuclear volume (15% reduction in Sertoli cell number) and a 17% reduction in inhibin-B levels despite significantly elevating FSH levels (testosterone levels were unchanged; Fig. 2), thus clearly implicating reduced androgen action as the cause of the reduction in Sertoli cell number/nuclear volume in this group. In all treatment groups, testis weight (Table 1) paralleled the observed changes in Sertoli cell number/nuclear volume per testis (Fig. 2).
Effect of neonatal treatment with 0.1 μg DES alone or in combination with GnRHa or with flutamide on Sertoli cell number/nuclear volume per testis and hormone levels (experiment 3)
The above findings suggest that FSH, androgens and oestrogens (DES) may all independently affect Sertoli cell number. To test this conclusion further, rats were treated neonatally with a 100-fold lower dose of DES (0.1 μg), which previous studies had shown did not suppress testosterone levels (Rivas et al. 2002). In the present study, this treatment did not cause any significant suppression of FSH or testosterone levels but did cause a significant reduction (21%) in both Sertoli cell nuclear volume per testis and blood levels of inhibin-B (Fig. 3). When treatment with 0.1 μg DES was combined with flutamide, a significantly greater suppression (P< 0.05) of Sertoli cell nuclear volume (40% reduction) and inhibin-B levels (41% reduction) was induced than by treatment with the 0.1 μg DES alone, without any significant suppression of FSH or testosterone levels (Fig. 3). Combined treatment with 0.1 μg DES+ GnRHa caused a markedly greater suppression (P< 0.001) of Sertoli cell nuclear volume (53% reduction) and blood inhibin-B levels (90% reduction) than did treatment with 0.1 μg DES alone, and significantly greater than that caused by treatment with 0.1 μg DES+flutamide (P< 0.05 and P< 0.001, respectively). These changes were associated with major suppression (P< 0.001) of FSH levels and a more minor reduction (P< 0.05) in testosterone levels in rats treated with 0.1 μg DES+GnRHa (Fig. 3). Testis weight in the treatment groups in experiment 3 changed in parallel with the change in Sertoli cell nuclear volume per testis (Table 1).
Treatment-induced changes in germ cell nuclear volume per testis or per Sertoli cell
Germ cell nuclear volume per testis, in the various treatment groups, showed the same pattern of change (not shown), as did testis weight (Table 1) and largely paralleled the changes in Sertoli cell nuclear volume per testis. Because of the major changes in Sertoli cell volume/ number induced by treatment, germ cell nuclear volume per testis was expressed relative to unit Sertoli cell nuclear volume per testis, as this should provide a measure of the capacity of each Sertoli cell to support germ cells. In most treatment groups, this capacity remained fairly constant, but in animals treated neonatally with 10 μg DES alone there was a 42–46% reduction in the volume of germ cells per unit Sertoli cell nuclear volume compared with controls, and this decrease was prevented by co-treatment with TE (Table 1). Additionally, co-administration of flutamide, and to a smaller extent GnRHa, with 0.1 μg DES also caused significant decreases in the volume of germ cells per unit Sertoli cell nuclear volume, compared with controls, whereas treatment with 0.1 μg DES alone had only a marginal, non-significant effect (Table 1).
Discussion
The primary purpose of the present studies was to reinvestigate whether treatments that we have shown previously to affect Sertoli cell number in the neonatal male rat may have caused this reduction in part by suppression of androgen action or by direct oestrogen action, rather than just via the suppression of FSH levels, as we had presumed. This reanalysis was prompted by findings in the literature that have demonstrated a role for androgens in Sertoli cell proliferation in both mice (Haywood et al. 2003, Johnston et al. 2004) and monkeys (Arslan et al. 1993, Schlatt et al. 1995, Ramaswamy et al. 2000). By using single and combined treatments we have generated situations in which blood levels of FSH and testosterone have been differentially manipulated and/or in which androgen or oestrogen (DES) action has been altered. The overall conclusion from these studies is that androgens can exert positive effects and oestrogens negative effects on Sertoli cell number/nuclear volume per testis in addition to the established effects of FSH on this parameter. This conclusion is tempered by the fact that in some situations it is impossible to distinguish the underlying hormonal cause for a reduction in Sertoli cell number. For example, treatment with a high dose of DES (10 μg), which causes a major reduction in Sertoli cell number, elevates oestrogen action at the same time as suppressing both FSH and androgen action. In such situations the reduction in Sertoli cell number most probably results from a combination of these hormonal changes, but their relative contribution to the reduction is impossible to dissect; moreover, the effects of each hormone might occur within different time windows during the treatment phase of this study (Fragale et al. 2001, Buzzard et al. 2003). The recognition that androgens can have positive effects on Sertoli cell number has important human health and cell-biological implications, as is discussed below.
For the present studies we have used treatments of intact neonatal rats and cell-measurement techniques that are of proven effectiveness and validity. Although the optical disector approach is widely accepted as being the definitive method for unbiased quantification of Sertoli cells, with their irregularly shaped nuclei (Wreford 1995), we have shown previously that standard stereological methods yield similar results for Sertoli cell nuclear volume per testis to those obtained using the optical disector (Atanassova et al. 1999, Sharpe et al. 2000). Although we have used the standard stereological method for all of the present studies, we also used the optical disector to confirm several of the important changes, as well as measuring blood levels of inhibin-B, which has been shown to reflect Sertoli cell number (Sharpe et al. 1999, Anderson & Sharpe 2000). A potential drawback of the present studies is that blood hormone levels were only measured at the time of death (day 18), several days after cessation of treatment. However, the treatment modalities used in the present studies were all designed to be long-acting and we have shown that the effects of GnRHa, DES and TE are all still evident at around day 25 (Sharpe et al. 1998, 1999, Atanassova et al. 2000) and the present study shows that flutamide treatment is still exerting an effect at day 18 (elevation of FSH levels) as is testosterone treatment (elevated blood testosterone levels). We have also shown (Sharpe et al. 1998, and RM Sharpe, M Walker, C McKinnell, JS Fisher & NN Atanassova, unpublished observations) that the pivotal treatment-induced hormone changes (i.e. suppression of FSH and testosterone levels) evident at day 18 are evident also at days 8–10 when treatment is still in progress.
In agreement with previous studies in mice and primates (see references cited above), our present studies provide strong evidence that endogenous androgens may exert a positive effect on Sertoli cell number/nuclear volume per testis in the neonatal rat. This was most clearly demonstrated by the ability of neonatal flutamide treatment to reduce Sertoli cell number/nuclear volume per testis despite concomitant elevation of FSH levels, which would be expected to increase, rather than decrease, Sertoli cell number (Simorangkir et al. 1995, Meachem et al. 1996). Other supporting evidence was that when flutamide was co-administered to rats with 0.1 μg DES, it caused an additional, significant reduction in Sertoli cell nuclear volume per testis compared with that in rats treated with the 0.1 μg DES alone, an effect that occurred without reducing FSH or testosterone levels. Other evidence for a positive effect of androgens on Sertoli cell number comes from the ability of testosterone co-treatment to partially reverse the negative impact of 10 μg DES treatment on Sertoli cell nuclear volume per testis, although this is equivocal as this combined treatment also prevented the DES-induced suppression of FSH levels. To what extent reduced androgen action accounts for the major reduction in Sertoli cell number/nuclear volume per testis in rats treated with 10 μg DES or with GnRHa is impossible to discern, due to the concomitant suppression of FSH levels, and the elevation of oestrogen action in DES-treated animals.
The present demonstration that neonatal treatment with 0.1 μg DES was able to significantly reduce Sertoli cell number/nuclear volume per testis, without affecting FSH or testosterone levels, provides reasonable evidence that oestrogens can directly, and negatively, regulate Sertoli cell number. Nevertheless, if FSH and androgen action were both suppressed in the rats exposed to 0.1 μg DES, by co-treatment with GnRHa, considerably greater suppression of Sertoli cell nuclear volume per testis resulted than with 0.1 μg DES treatment alone. However, if elevation of oestrogen action is able to directly reduce Sertoli cell number, it is puzzling that in animals treated with 10 μg DES+GnRHa, in which androgen and FSH action were strongly suppressed at the same time as massive elevation of oestrogen action, there was no greater suppression of Sertoli cell number per testis than in animals treated with GnRHa or 10 μg DES treatment alone. This implies that FSH, androgens and oestrogens affect a common pathway in Sertoli cells in the neonatal period in the rat and that this pathway is only responsible for determining 50–60% of final Sertoli cell number. It is presumed that the direct effects of DES on Sertoli cell number are mediated via oestrogen receptor-β in Sertoli cells (Saunders et al. 1998), as oestrogen receptor-α is not expressed in this cell type at any stage in the rat (Fisher et al. 1997).
In the present studies, measurement of inhibin-B levels in plasma provided strong supporting evidence for the measured changes in Sertoli cell number/nuclear volume per testis. In all instances in which the latter was altered by treatments, inhibin-B levels showed a similar, and significant, change. However, it was also apparent that the magnitude of reduction in inhibin-B levels following some of the treatments was noticeably greater than was the reduction in Sertoli cell number/nuclear volume per testis. For example, inhibin-B levels were reduced by approximately 90% in all animals treated with GnRHa (alone or in combination) compared with a 50–60% reduction in Sertoli cell number/nuclear volume per testis. This is almost certainly explained by the parallel suppression of FSH by GnRHa treatment, as in every situation in the present studies in which FSH levels were suppressed there was a disproportionately greater suppression of inhibin-B levels than of Sertoli cell number/nuclear volume per testis. This is a logical finding, because at the age of 18 days in the rat the negative-feedback effect of inhibin-B on FSH secretion does not yet operate whereas the positive effect of FSH on inhibin-B secretion does (Sharpe et al. 1999, Anderson & Sharpe 2000).
Although the focus of the present studies was on hormonal determination of Sertoli cell number, it also provided (indirect) evidence that Sertoli cell function may have been differentially affected by some of the treatments. This emerged from comparison of the capacity of each Sertoli cell (equivalent to unit nuclear volume) to support germ cells (i.e. germ cell nuclear volume per unit Sertoli cell nuclear volume). This revealed that treatment with 10 μg DES alone reduced the volume of germ cells supported by > 42%, whereas other single treatments had no or only small effects. Based on earlier findings this decrease probably results from increased germ cell apoptosis (Sharpe et al. 1998, Atanassova et al. 1999, 2000). The present finding that co-administration of TE with 10 μg DES completely prevented the decrease in germ cell volume per Sertoli cell induced by treatment with 10 μg DES alone points to lack of androgen action as a primary factor in these changes. However, treatments that reduced testosterone production (GnRHa alone) or action (flutamide alone) did not induce such a change, though co-administration of either of these treatments with 0.1 μg DES did induce a decrease in germ cell volume per Sertoli cell. Taken together, these findings suggest that it is alteration of the androgen/oestrogen balance (reduced androgen action and elevated oestrogen action) that may provide a unifying explanation for these changes, consistent with other recent findings related to development of the male reproductive system (Rivas et al. 2002, 2003).
The present data showing a role for androgens in regulation of Sertoli cell number in the rat adds to the growing evidence that both the timing and hormonal regulation of Sertoli cell proliferation in rodents, domestic animals and primates are very similar and differ only in detail (see Sharpe et al. 2003 for review). However, the evidence for a role for androgens in Sertoli cell proliferation also raises a conundrum. For all of fetal life and either some (rats, mice) or all (primates) of neonatal life, which are arguably the most important periods of Sertoli cell proliferation, the Sertoli cell is not an androgen target as it does not express ARs (McKinnell et al. 2001b, Sharpe et al. 2003; reviewed in Sharpe 2004). Therefore, any effect of androgens on Sertoli cell proliferation at such times is presumed to occur via the peritubular myoid cells which express ARs at all of these times (Sharpe 2004), and which are known to exert important regulatory effects on the developing Sertoli cells (Skinner 1991, Verhoeven et al. 1992). This effect could occur, for example, by peritubular myoid cells determining the extent of the basement membrane for which Sertoli cells compete, thus delimiting Sertoli cell proliferation (Schlatt et al. 1996). Such an arrangement, by which proliferation of Sertoli cells is regulated indirectly via androgen action on neighbouring peritubular cells, would provide an intriguing parallel with the rest of the developing male reproductive tract in which proliferation and differentiation of epithelial cells is regulated indirectly via androgen action on neighbouring mesenchymal/stromal cells (Cunha et al. 1995, Kurzrock et al. 1999, Marker et al. 2003).
The emerging evidence for a role for androgens in determination of Sertoli cell number in primates and rodents also has human health implications. Recent concerns about falling sperm counts and the increasing incidence of testicular germ cell cancer and of other male reproductive developmental disorders have been crystallized into the hypothesis that these form a ‘testicular dysgenesis syndrome’ (TDS), which stems from dysfunction of the Leydig and Sertoli cells during fetal development (Skakkebaek et al. 2001). There is strong cell biological and epidemiological evidence to support this hypothesis (Sharpe & Skakkebaek 2003), and one of its central tenets is that impaired androgen production/action is a major factor in development of this syndrome. The present study in neonates, together with other published data showing that deprivation of normal androgen action during fetal development results in fewer Sertoli cells (Johnston et al. 2004), and thus reduced capacity to produce sperm in adulthood (Sharpe et al. 2003), provides a further pathway via which the reduced sperm counts in TDS patients might occur.
The number of animals used for the studies reported in Figs 1–3, their testis weights and the capacity of their Sertoli cells to support germ cells, as measured by germ cell nuclear volume per unit Sertoli cell nuclear volume (means±s.d.). The final column shows nuclear volume per testis of germ cells divided by nuclear volume per testis of Sertoli cells
| n | Testis weight (mg) | Germ cell volume/Sertoli cell volume | |
|---|---|---|---|
| *P<0.02, †P<0.01, ‡P<0.001, in comparison with the respective control group. | |||
| aP<0.05, bP<0.01, cP<0.001, in comparison with the respective DES-only treated group. | |||
| Neonatal treatment | |||
| Experiment 1 | |||
| Control (corn oil) | 5 | 71.2±10.4 | 0.78±0.18 |
| 10 μg DES | 5 | 24.3±3.1‡ | 0.46±0.13* |
| GnRHa | 5 | 19.9±0.9‡ | 0.60±0.13 |
| 10 μg DES+GnRHa | 5 | 22.7±1.3‡ | 0.55±0.19* |
| Experiment 2 | |||
| Control (corn oil) | 13 | 77.2±10.6 | 0.85±0.17 |
| 10 μg DES | 11 | 22.9±2.9‡ | 0.46±0.05‡ |
| TE | 7 | 41.9±5.1‡ | 0.73±0.22 |
| 10 μg DES+TE | 8 | 45.9±6.6‡c | 0.74±0.10b |
| Flutamide | 14 | 51.8±12.5‡ | 0.79±0.31 |
| Experiment 3 | |||
| Control (corn oil) | 5 | 74.3±4.3 | 0.93±0.16 |
| 0.1 μg DES | 4 | 46.7±7.8‡ | 0.79±0.18 |
| 0.1 μg DES+flutamide | 4 | 32.3±1.3‡b | 0.47±0.04†a |
| 0.1 μg DES+GnRHa | 5 | 25.4±1.1‡c | 0.65±0.11* |
Effect of neonatal treatment with 10 μg DES or with a GnRHa, alone or in combination, on Sertoli cell nuclear volume per testis (bottom panel) in relation to the blood levels of testosterone, FSH and inhibin-B. Inset in the bottom panel shows data for Sertoli cell number per testis determined by the optical disector method (n=5 per group). Values are means± s.e.m. (n values are given in Table 1). *P< 0.05, **P< 0.01, ***P< 0.001, in comparison with the respective control value.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05884
Effect of neonatal treatment with 10 μg DES or TE, alone or in combination, or treatment with the anti-androgen flutamide on Sertoli cell nuclear volume per testis (bottom panel) in relation to the blood levels of testosterone, FSH and inhibin-B. Inset in the bottom panel shows data for Sertoli cell number per testis determined by the optical disector method (n=5 per group). Values are means± s.e.m. (n values are given in Table 1). *P< 0.05, **P< 0.01, ***P< 0.001, in comparison with the respective control value; aP< 0.05, bP< 0.01, cP< 0.001, in comparison with the respective value for the DES-only treated group.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05884
Effect of neonatal treatment with 0.1 μg DES alone or in combination with either a GnRHa or with flutamide (Flut), on Sertoli cell nuclear volume per testis (bottom panel) in relation to the blood levels of testosterone, FSH and inhibin-B. Values are means± s.e.m. (n values are given in Table 1). *P< 0.05, **P< 0.01, ***P< 0.001, in comparison with the respective control value. aP< 0.05, bP< 0.01,cP< 0.001, in comparison with the respective value for the DES-only treated groups.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05884
We thank Catrina Kivlin and Arantza Esnal for technical assistance and Denis Doogan and Mark Fisken for expert animal husbandry. N N A was supported by a Wellcome International Research Training Fellowship. This study was supported in part by contracts QLK4-1999-01422 and QLK4-CT-2002-00603 from the European Union. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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