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Endocrinology Vol. 141, No. 10 3898-3907
Copyright © 2000 by The Endocrine Society

ARTICLES

Comparative Effects of Neonatal Exposure of Male Rats to Potent and Weak (Environmental) Estrogens on Spermatogenesis at Puberty and the Relationship to Adult Testis Size and Fertility: Evidence for Stimulatory Effects of Low Estrogen Levels1

N. Atanassova2, C. McKinnell, K. J. Turner, M. Walker, J. S. Fisher, M. Morley, M. R. Millar, N. P. Groome and R. M. Sharpe

Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (N.A., C.M., K.J.T., M.W., J.S.F., M.M., M.R.M., R.M.S.), Edinburgh, Scotland, United Kingdom EH3 9ET; Institute of Experimental Morphology and Anthropology, Bulgarian Academy of Science (N.A.), 1113 Sofia, Bulgaria; and School of Biological and Molecular Sciences, Oxford Brookes University (N.P.G.), Headington, Oxford, United Kingdom OX3 0BP

Address all correspondence and requests for reprints to: Dr. R. M. Sharpe, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, Scotland, United Kingdom EH3 9ET. E-mail: r.sharpe{at}mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study investigated whether neonatal exposure of male rats to estrogenic compounds altered pubertal spermatogenesis (days 18 and 25) and whether the changes observed resulted in long-term changes in testis size, mating, or fertility (days 90–100). Rats were treated neonatally with a range of doses (0.01–10 µg) of diethylstilbestrol (DES; administered on alternate days from days 2–12), a high dose of octylphenol (OP; 2 mg administered daily from days 2–12) or bisphenol A (Bis-A; 0.5 mg administered daily from days 2–12), or vehicle, while maintained on a standard soy-containing diet. The effect on the same parameters of rearing control animals on a soy-free diet was also assessed as was the effect of administering such animals genistein (4 mg/kg/day daily from days 2–18). Testis weight, seminiferous tubule lumen formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to characterize pubertal spermatogenesis. Compared with (soy-fed) controls, DES administration caused dose-dependent retardation of pubertal spermatogenesis on day 18, as evidenced by decreases in testis weight, lumen formation, and spermatocyte nuclear volume per unit Sertoli cell and elevation of the germ cell apoptotic index. However, the two lowest doses of DES (0.1 and 0.01 µg) significantly increased spermatocyte nuclear volume per unit Sertoli cell. Similarly, treatment with either OP or Bis-A significantly advanced this and some of the other aspects of pubertal spermatogenesis. Maintenance of control animals on a soy-free diet also significantly advanced lumen formation and spermatocyte nuclear volume per unit Sertoli cell compared with controls fed a soy-containing diet. Administration of genistein reversed the stimulatory effects of a soy-free diet and significantly retarded most measures of pubertal spermatogenesis. In general, plasma FSH levels in the treatment groups changed in parallel to the spermatogenic changes (reduced when pubertal spermatogenesis retarded, increased when pubertal spermatogenesis advanced). By day 25, although the changes in FSH levels largely persisted, all of the stimulatory effects on spermatogenesis seen on day 18 in the various treatment groups were no longer evident.

In adulthood, testis weight was decreased dose dependently in rats treated neonatally with DES, but only the lowest dose group (0.01 µg) showed evidence of mating (3 of 6) and normal fertility (3 litters). Animals treated neonatally with OP or Bis-A had normal or increased (Bis-A) testis weights and exhibited reasonably normal mating/fertility. Animals fed a soy-free diet had significantly larger testes than controls fed a soy-containing diet, and this difference was confirmed in a much larger study of more than 24 litters, which also showed a significant decrease in plasma FSH levels and a significant increase in body weight in the males kept on a soy-free diet. Neonatal treatment with genistein did not alter adult testis weight, and although most males exhibited normal mating and fertility, a minority did not mate or were infertile. It is concluded that 1) neonatal exposure of rats to low levels of estrogens can advance the first wave of spermatogenesis at puberty, although it is unclear whether this is due to direct effects of the estrogen or to associated elevation of FSH levels; 2) the effect of high doses of OP and Bis-A on these processes is essentially benign; and 3) the presence or absence of soy or genistein in the diet has significant short-term (pubertal spermatogenesis) and long-term (body weight, testis size, FSH levels, and possibly mating) effects on males.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS GROWING interest in the role that estrogens play in male reproductive development and function, and to some extent this interest has emerged from concerns about the potential adverse effects of environmental estrogens in the male (1). The latter concerns have been supported by the demonstration of widespread expression of estrogen receptors (ERs) in the testis and reproductive tract of the male in fetal and neonatal life (2, 3, 4, 5, 6) and by the numerous studies that have shown major changes in the structure and function of these tissues after the administration of (potent) estrogens in perinatal life (7, 8, 9, 10, 11, 12, 13). However, these findings contrast starkly with the converse situation, in which estrogen production or action is reduced via the transgenic inactivation of ER{alpha} (14, 15), ERß (16), or aromatase (17, 18) in male mice. In each of these transgenics, grossly normal development of the testis and reproductive system reportedly occurs, at least up to puberty, findings that question whether estrogens play a significant physiological role in male reproductive development (1). The contrast between these two extreme situations is puzzling and presumably indicates that current understanding of estrogen action in the male is insufficient to enable the two sets of findings to be reconciled. In turn, this lack of understanding represents an obstacle to informed assessment of whether individual environmental estrogens pose any significant risk to the developing male (10, 19, 20).

Based on published findings it is clear that perinatal overexposure to estrogens results in adverse effects on male reproductive development, and investigators searching for potential effects of environmental estrogens have therefore focused on trying to demonstrate similar adverse effects of these compounds. In general, perinatal exposure to high doses of potent estrogens appears necessary for the induction of major adverse effects on the developing male reproductive system (8, 9, 10, 11, 12, 13, 21), although there are published studies that have shown effects (not necessarily adverse) of much lower levels of potent estrogens (22). As all identified environmental estrogens are considerably less potent than estradiol and diethylstilbestrol (DES), it is perhaps most logical to expect that little or no adverse effects of such compounds will be found on male reproductive development, even at high doses, unless their effects are not related straightforwardly to their estrogenic potency. Recently published studies that have addressed this issue have yielded conflicting results (23, 24, 25, 26, 27, 28, 29, 30, 31, 32), and although they have generated an intense debate about low dose effects of estrogens, opinion remains divided as to whether environmental estrogens are likely to induce adverse effects at other than very high doses.

From our own studies involving neonatal administration of potent estrogens such as DES, we have been puzzled by the need to administer very high doses to induce gross adverse changes to the developing male reproductive tract (13, 21). Findings from transgenic mice (ERKOs, BERKOs, and ARKOs) (14, 15, 16, 17, 18) and our own studies of adult rats treated with an aromatase inhibitor (33) have all demonstrated no gross changes to the reproductive tract (other than the efferent ducts in ERKOs) after manipulation of endogenous estrogen levels, and this caused us to question whether the end points used for these studies were the most appropriate or the most sensitive. This was reinforced by our preliminary finding that neonatal administration of high doses of environmental estrogens caused either no or minimal effects on the end points that we studied (13) (our unpublished data). This prompted us to investigate whether there were hidden (i.e. not gross) effects of low levels of estrogens that were not obvious, but were only discernable by systematic analysis. For these studies we focused on the pubertal development of the seminiferous cords/tubules of the testis for two reasons. First, Sertoli cells and most germ cells express ERß throughout early development (4, 5, 6) (our unpublished data), and we have shown that neonatal exposure to relatively low doses of DES can affect the development of Sertoli cells (11, 21). Second, isolated reports have indicated that estrogens might exert stimulatory effects on early germ cell development in the testes of several animals (34, 35, 36, 37, 38). We therefore compared the effects of neonatal exposure of male rats to high levels of two industrial environmental (weak) estrogens [octylphenol (OP) and bisphenol A (Bis-A)] with the effects of the phytoestrogen genistein (and soy-free vs. soy-containing diets) and contrasted these with the effects of the potent estrogen DES administered in doses spanning a 1000-fold range. Because a number of treatment-induced changes in early testicular development were observed, we also assessed whether these treatments resulted in any long-term consequences by measuring testis weight and assessing fertility in adulthood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, treatments, and sample collection
Wistar rats bred in our own animal house were maintained under standard conditions and unless otherwise stated were maintained on a standard diet (rat and mouse breeding diet No. 3; SDS, Dundee, Scotland, UK) that contains 15.5% soymeal flour. All-male litters of 8–12 pups were generated by cross-fostering pups on day 1 (=day of birth). Beginning on postnatal day 2, rats were subjected to one of the following treatments: 1) sc injection of DES (Sigma, Poole, UK) at a dose of 10, 1, 0.1, or 0.01 µg in 20 µl corn oil on days 2, 4, 6, 8, 10, and 12; 2) sc injection of 2 mg 4-tert-octylphenol (Aldrich Chemical Co., Inc., Poole, UK) in 20 µl corn oil on each of days 2–12; 3) sc injection of 0.5 mg Bis-A (Aldrich) in 20 µl corn oil on each of days 2–12; and 4) sc injection of the vehicle alone (20 µl corn oil).

It was also evaluated whether the phytoestrogens in soy were able to affect male reproductive development. This was undertaken by comparing male offspring reared by mothers maintained on a (standard) soy-containing diet (no. 4 described above) with those 5) reared by mothers maintained on a nominally soy-free diet (rat and mouse soy-free breeding diet, SDS), in which the soy is substituted by fishmeal (maize gluten is also added, and the overall cereal content is increased to 78% compared with 64% in the standard diet). Additionally, the effect of 6) administering genistein to male pups maintained on a soy-free diet was also evaluated. Soy-free reared animals were generated as follows. Adult female rats were placed on a soy-free diet for a minimum of 3 weeks before mating and then maintained on this diet throughout mating, pregnancy, and lactation; the male pups when weaned were subsequently maintained on the same soy-free diet until they were killed. At birth, some of the male offspring born to mothers maintained on a soy-free diet were treated with the soy derivative genistein (Sigma, St. Louis, MO) by daily sc injection from days 2–18. The dose of genistein administered was 4 mg/kg·day to equate to the reported total phytoestrogen intake by 4-month-old human infants maintained on a 100% soy-formula milk diet (39, 40). The genistein was prepared by dissolving in 0.4 M KOH and then diluting 1:20 with 0.1 M PBS containing 2.5 mg/ml gelatin (Sigma) to give a dosing solution of 0.8 mg/ml, which was kept at 4 C. The control animals maintained on the same soy-free diet were treated with the vehicle alone. Vehicle- and genistein-treated pups were equally represented in each cross-fostered litter.

Rats from the various treatment groups described above were subsequently sampled on day 18, 25, or 90–100 (=adults). Animals were anesthetized with flurothane, blood was collected from the heart into a heparinized syringe, and the animals were then killed by cervical dislocation. For the rats sampled at 18 and 25 days the testes were dissected out, weighed, and fixed for approximately 5 h in Bouin’s’ solution. In excess of 300 males were used for these studies, and at least 2 separate experiments were performed for each of the treatments specified on day 18; comparable results were obtained in each experiment. Data for individual treatment groups from the separate experiments were pooled for analysis and illustration.

A more detailed comparison of males in natural litters reared to adulthood on soy-free and soy-containing diets was also made. A total of 24 litters of males reared on a soy-free diet were compared with 29 litters of males reared on the standard soy-containing diet. At 90–95 days of age, the males were killed by inhalation of rising levels of CO2, blood was collected from the posterior vena cava into a heparinized syringe, and the testes were dissected out and weighed. Values for mean testis weight and blood FSH levels for each litter were then generated.

Mating and fertility
Some of the males from the treatment groups above that were reared until adulthood were assessed for mating and fertility at 80–90 days of age. Each male was placed with an adult female rat in a mating cage floored with a wire mesh grid. Cages were checked each day for mating plugs, and when one or more plugs were found, the male was removed, and the female was caged separately until birth, when the offspring were counted. If a plug was not found after 7 days, the male was scored as not having mated and was removed from the cage; the female was monitored to confirm the absence of pregnancy.

Processing of blood samples and testis tissue
Plasma samples were stored at -20 C until used for hormone analysis. After fixation in Bouin’s solution, testis tissue was transferred into 70% ethanol before being processed for 17.5 h in an automated Shandon processor and embedded in paraffin wax. Sections of 5 µm thickness were cut, floated onto slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma), and dried at 50 C overnight before being used for cell quantification studies and visualization of apoptotic germ cells as described below.

Determination of Sertoli cell and germ cell nuclear volume per testis, the germ cell apoptotic index, and seminiferous tubule lumen formation on days 18–25
The purpose of these studies was to quantify various aspects of the first wave of spermatogenesis at puberty in the various treatment groups. Standard point counting of cell nuclei was used, as described previously (11, 21), to determine the nuclear volume per testis of Sertoli cells, germ cells (apoptotic and nonapoptotic), and seminiferous tubule lumen volume. These data were then used to determine for each animal the following: 1) the nuclear volume of viable (nonapoptotic) spermatocytes and spermatogonia per unit Sertoli cell nuclear volume as an index of spermatogenic efficiency; 2) the germ cell apoptotic index based on the ratio of the nuclear volume of apoptotic/viable germ cells per testis; and 3) seminiferous tubule lumen percent volume (=lumen formation) as an indicator of Sertoli cell functional development (41). Apoptotic germ cells were identified based on DNA fragmentation, which was detected by in situ DNA 3'-end labeling in histological sections using a nonradioactive labeling method that resulted in a high degree of specificity and low background staining, as described and validated in detail previously (11). Cross-sections of testes from 5–14 (day 18) or 5–12 (day 25) rats in each treatment group were examined under oil immersion using a Leitz x63 plan apo objective fitted to a Leitz Laborlux microscope and a 121-point eyepiece graticule (Rockleigh, NJ). Using a systematic clock-face sampling pattern from a random starting point, 16 fields were counted. Points falling over the nuclei of Sertoli cells, spermatogonia, or spermatocytes (apoptotic or nonapoptotic) or over seminiferous tubule lumens were scored and expressed as a percentage of the total points counted. For each animal, the values for percent nuclear volume were converted to absolute nuclear 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.

As significant changes in Sertoli cell number occur in some of the treatment groups used in the present studies (21), account had to be taken of this difference when comparing relative volumes of germ cells per testis. Therefore, data for spermatocytes and spermatogonia have been expressed as nuclear volumes per testis relative to Sertoli cell nuclear volume per testis to indicate the relative (nuclear) volume of each germ cell type supported per unit Sertoli cell nuclear volume. Cell nuclear volume can be equated to numbers of cells per testis, assuming no change in nuclear diameter of the target cell in the different treatment groups. Measurement of spermatocyte nuclear diameter in selected treatment groups on day 18 in the present studies confirmed no major difference (data not shown). 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, although we have shown previously that point count measurement of Sertoli cell nuclear volume per testis equates broadly to Sertoli cell number determined by the disector method (21) (see also Results). It was therefore not possible to convert the present data to absolute numbers of germ cells per Sertoli cell.

In accord with previous studies of the first wave of spermatogenesis at puberty (41), it was noted that most apoptotic germ cells in the present studies appeared to be spermatocytes. However, as apoptotic germ cells cannot be identified with complete certainty, the apoptotic index reported in the present studies was derived by expressing the relative nuclear volume per testis of apoptotic germ cells to the combined nuclear volumes per testis of spermatogonia and spermatocytes. As the latter cell types do not have identical nuclear diameters, derivation of the index in this way may introduce a small error. However, as changes in spermatogonia and spermatocyte nuclear volumes per testis individually paralleled the change in total germ cell nuclear volume per testis in the various treatment groups, expression of apoptotic germ cell nuclear volume relative to that of viable spermatocytes (or spermatogonia) yielded results of comparable significance to those presented below for the apoptotic index (not shown).

Measurement of plasma hormone levels
Plasma levels of FSH were measured by RIA using materials generously supplied by the NIDDK. Results have been expressed in terms of the rat FSH RP-2 standard. In the FSH assay, plasma levels measured in hypophysectomized rats range from 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 that uses a capture antibody directed against the C-terminal portion of the human ßB-subunit and the F(ab) fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the inhibin {alpha}-subunit conjugated to alkaline phosphatase (42, 43). The assay has been previously validated for measurement of inhibin B in the rat (44, 45), 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 castrate adult male rat plasma were reduced to undetectable levels (<60 pg/ml).

Statistics
Comparison of the different parameters for the various treatment groups was made using ANOVA, as all data were normally distributed. Where significant differences between groups were indicated, subgroup comparisons also used ANOVA, but employed the variance from the experiment as a whole (for that parameter) as the measure of error.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Seminiferous tubule lumen formation and germ cell apoptotic index on day 18
On day 18 in control rats maintained on a standard soy-containing diet, lumen formation was only evident in about 25% of seminiferous tubules, and changes to this incidence were evident in the various treatment groups (Fig. 1Go). In general, the more advanced that lumen formation was, the fewer apoptotic germ cells were evident (Fig. 1Go). Point counting was used to quantify the changes in these two parameters and revealed that across all treatment groups, lumen formation, as determined by percent lumen volume per testis, and the germ cell apoptotic index were inverse images of each other (Fig. 2Go). Lumen volume varied from 0.7–6.2%/testis in vehicle-treated control rats and was associated with a low, but consistent, apoptotic rate in germ cells. Neonatal treatment with DES doses greater than 0.01 µg caused dose-related increases in germ cell apoptosis and a corresponding decrease in lumen volume, although the latter decrease only achieved statistical significance with the 10-µg dose of DES (Fig. 2Go). In contrast, neonatal treatment with Bis-A or OP significantly advanced lumen formation and slightly decreased the apoptotic rate of germ cells (Figs. 1Go and 2Go). Maintenance of control rats on a soy-free diet did not significantly affect lumen formation or germ cell apoptosis, but, compared with this group, administration of genistein significantly retarded lumen formation and increased the germ cell apoptotic rate (Figs. 1Go and 2Go); values in the genistein-treated group were also significantly different (P < 0.01) from those in the control group maintained on a soy-containing diet (Fig. 2Go).



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  		Figure 1. Effect of neonatal treatment with vehicle (Control), DES (10 µg), Bis-A, or OP; maintenance of rats on a soy-free diet (SF control); or the administration of genistein on testicular morphology, lumen formation (asterisks), and germ cell apoptosis (brown-staining nuclei) on day 18. For quantitative data on these end points see Figs. 2Go and 3Go. Note that with the exception of SF control and genistein groups, all animals were maintained on a standard soy-containing diet. Magnification, x250.

 


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  		Figure 2. Effect of neonatal treatment with DES, Bis-A, or OP maintenance of rats on a soy-free diet (SF control) or the administration of genistein on the germ cell apoptotic index and lumen formation in the seminiferous tubules in rats, aged 18 days. Each column is the mean ± SEM for 7–14 rats/group from two separate experiments, except for DES (0.01 µg), where the data derive from five animals from a single experiment. Note that with the exception of SF control and genistein groups, all animals were maintained on a standard soy-containing diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with the appropriate control).

 
Testis weight, Sertoli cell nuclear volume, and plasma levels of inhibin B on day 18
In general, data for testis weight closely paralleled those for lumen formation, with groups that showed increased lumen volume (and decreased germ cell apoptosis) exhibiting significantly higher testis weights and vice versa for groups with low lumen volumes (compare Fig. 2Go with Table 1Go). The exception was the genistein-treated group in which the relationship of lumen volume to testis weight did not match closely as it did in both soy-fed and soy-free controls. Changes in Sertoli cell nuclear volume per testis and plasma inhibin B levels paralleled each other (Fig. 3Go), as reported previously for rats of this age (45) and also largely paralleled changes in testis weight (Table 1Go) and lumen volume (Fig. 2Go) across the different treatment groups. However, in the genistein-treated group the high Sertoli cell nuclear volume matched testis weight, but did not match lumen volume per testis. In DES-treated rats a significant decrease in Sertoli cell nuclear volume was only observed in the group treated with 10 µg DES, whereas a recent study of ours (21) showed a dose-dependent decrease in Sertoli cell number measured at 18 days of age. This suggests that measurement of Sertoli cell nuclear volume (Fig. 3Go) may slightly underestimate changes in Sertoli cell number per testis on day 18 in rats treated with lower doses of DES. The increase in Sertoli cell nuclear volume per testis in rats treated neonatally with Bis-A or OP or maintained on a soy-free diet (Fig. 3Go) may indicate an increase in Sertoli cell number in these groups.


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  		Table 1. Effect of neonatal treatment with diethylstilbestrol (DES), bisphenol A (Bis-A), or octylphenol (OP) or maintenance of rats on a soy-free diet (SF control) or administration of genistein on testis weight in rats aged 18 days

 


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  		Figure 3. Effect of neonatal treatment with DES, Bis-A, or OP maintenance of rats on a soy-free diet (SF control) or administration of genistein on plasma levels of inhibin B and Sertoli cell nuclear volume per testis in rats, aged 18 days. Each column is the mean ± SEM for 7–14 rats/group from two separate experiments, except for DES (0.01 µg) where the data derive from five animals from a single experiment. Note that with the exception of SF control and genistein groups, all animals were maintained on a standard soy-containing diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with the appropriate control).

 
Pubertal spermatogenesis and plasma FSH levels on day 18
Pubertal spermatogenesis, as indicated by the nuclear volume of spermatocytes per unit Sertoli cell nuclear volume, showed major changes according to treatment (Fig. 4Go). Animals treated with 10 µg DES showed a significant reduction in spermatocyte nuclear volume per Sertoli cell nuclear volume. In contrast, animals treated with either 0.1 or 0.01 µg DES showed a significant increase in this parameter, as did animals treated with either Bis-A or octylphenol (Fig. 4Go). Control rats maintained on a soy-free diet also exhibited a major increase in spermatocyte nuclear volume per Sertoli cell nuclear volume, compared with controls kept on a soy-containing diet, and this increase was abolished by treating rats with genistein while they were fed a soy-free diet (Fig. 4Go). Comparison of spermatogonia nuclear volume per Sertoli cell nuclear volume in the different treatment groups showed changes that paralleled those shown for spermatocytes (data not shown).



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  		Figure 4. Effect of neonatal treatment with DES, Bis-A, or OP maintenance of rats on a soy-free diet (SF control) or administration of genistein on plasma levels of FSH and spermatocyte nuclear volume per unit Sertoli cell nuclear volume in rats, aged 18 days. Each column is the mean ± SEM for 7–14 rats/group from two separate experiments, except for DES (0.01 µg) where the data derive from five animals from a single experiment. Note that with the exception of SF control and genistein groups, all animals were maintained on a standard soy-containing diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with the appropriate control).

 
The treatment-related pattern of spermatocyte nuclear volume per unit Sertoli cell nuclear volume showed similarities to the treatment-related pattern for other parameters of pubertal onset of spermatogenesis, such as testis weight (Table 1Go), lumen volume (Fig. 2Go), and germ cell apoptotic index (inverse relationship; Fig. 2Go). However, plasma FSH levels also showed significant changes among the various treatment groups, and, in general, decreases or increases in FSH levels (relative to controls) were associated with a parallel change in spermatocyte nuclear volume per unit Sertoli cell nuclear volume (Fig. 4Go). The findings suggested that low levels of neonatal estrogen exposure are associated with elevation of FSH levels on day 18, the exception again being genistein-treated animals, in which FSH levels were significantly suppressed compared with those in soy-free controls (Fig. 3Go).

Changes to the various parameters on day 25
With one or two exceptions, the pronounced treatment-related changes evident on day 18 were no longer evident on day 25. For example, apart from animals maintained on a soy-free diet, no treatment group exhibited any significant increase in spermatocyte nuclear volume per Sertoli cell nuclear volume compared with controls (Fig. 5Go), although it should be noted that the spermatocyte nuclear volume per Sertoli cell nuclear volume ratio increased more than 6-fold in controls from day 18 (Fig. 4Go) to day 25 (Fig. 5Go). The latter change occurred despite no increase in FSH levels in controls between days 18 and 25, and in the various treatment groups the blood FSH profile still showed treatment-related changes (mainly increases compared with control) similar to those seen on day 18 (Figs. 4Go and 5Go). The only treatment group that showed a more or less identical (comparative) profile to that on day 18 was the group treated neonatally with 10 µg DES, in which there was an elevated germ cell apoptotic index, decreased spermatocyte nuclear volume per Sertoli cell nuclear volume, and reduced FSH levels (Fig. 5Go) and retarded lumen formation (not shown). Comparison of control rats reared on a soy-free diet with those reared on a soy-containing diet showed echoes of the changes evident on day 18, but only one of these parameters was marginally statistically significant (Figs. 4Go and 5Go). Moreover, on day 25 genistein-treated animals showed no significant differences from soy-free controls for any of the parameters measured (Fig. 5Go). It is emphasized that treatment with genistein continued up until day 18, in contrast to the other treatments (DES, Bis-A, and OP), which had ceased on day 12.



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  		Figure 5. Effect of neonatal treatment with DES, Bis-A, or OP maintenance of rats on a soy-free diet (SF control) or administration of genistein on the germ cell apoptotic index, plasma levels of FSH, and spermatocyte nuclear volume per unit Sertoli cell nuclear volume in rats, aged 25 days. Each column is the mean ± SEM for 4–12 rats/group. Note that with the exception of SF control and genistein groups, all animals were maintained on a standard soy-containing diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with the appropriate control).

 
Effects of neonatal treatments on adult testis morphology, testis weight, and fertility
Apart from changes in testicular morphology reported previously in adult rats treated neonatally with high doses of DES (21), all other treatment groups exhibited normal gross morphology of the testis (not shown). As we have shown previously (21) that testis weight in adult rats provides a reliable guide to total germ cell numbers per testis, this end point was used as a quantitative measure of any changes to spermatogenesis. In adulthood, rats treated neonatally with DES showed a more or less dose-dependent reduction in testis weight, with a dose of 0.01 µg being without significant effect (Table 2Go). No decrease in testis weight was observed in any of the other treatment groups relative to controls, and in fact the trend was toward heavier testes, a trend that was significant for groups treated with bisphenol A or reared on a soy-free diet. Notably, rats treated with genistein neonatally had testis weights comparable to control rats maintained on a soy-free diet (Table 2Go). Preliminary assessment of mating (plugging) and fertility was made for the various treatment groups. Animals treated with DES neonatally did not mate, although at the lowest dose of DES tested (0.01 µg), three of six treated males did mate, and each of the three matings resulted in the birth of litters (although one litter was cannibalized; Table 2Go). Mating and fertility were reasonably normal in rats treated neonatally with either OP or Bis-A, although animals treated neonatally with genistein exhibited inconsistent results, with two of nine males not mating, and one of seven of the matings failing to result in pregnancy (Table 2Go). These changes did not reach statistical significance.


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  		Table 2. Effect of neonatal treatment with diethylstilbestrol (DES), bisphenol A (Bis-A), or octylphenol (OP) or maintenance of rats on a soy-free diet (SF control) or administration of genistein on testis weight, mating, and fertility in adulthood

 
Effect of life-long rearing on soy-free vs. soy-containing diets
In view of the various changes observed in animals reared on a soy-free, compared with a conventional soy-containing, diet, a more thorough comparison was made using the litter means for males as the unit of comparison (Table 3Go). These findings indicated that rats reared on a nominally soy-free diet had significantly larger testes and significantly lower FSH levels in adulthood and also had significantly greater body weights than males maintained on a standard soy-containing diet (Table 3Go); however, the scale of the differences was only in the range of 6–11%, on average, for the parameters measured.


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  		Table 3. Effect of rearing rats throughout life on a soy-free diet or a standard soy-containing laboratory diet on body weight, testis weight, and plasma FSH levels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present findings provide important new information on the effects of potent and weak estrogens on male reproductive development and the first wave of spermatogenesis at puberty in rats. They demonstrate that whereas high doses of potent estrogens, such as DES, are clearly inhibitory to testicular development in both the short and long term, as we have demonstrated previously (11, 21), as the dose is titrated downward, (short-term) stimulatory effects begin to emerge. Moreover, neonatal administration of very high doses of either of two weak environmental estrogens was able to mimic these stimulatory effects. Manipulation of the phytoestrogen exposure of male rats during development also exerted significant short- and long-term effects on testicular development and final size (and possibly mating), although in many respects the short-term effects were more similar to those induced by higher doses of DES, as they were generally opposite those induced by neonatal exposure to weak/low doses of estrogens; these findings have implications for all studies of estrogens and endocrine disrupters in laboratory animals fed soy-containing diets. Regardless of the influence of the various treatments on pubertal spermatogenesis and testicular development, our findings are generally reassuring, showing that only exposure to high doses of a potent estrogen such as DES is able to induce any major long-term adverse change in testis size, mating, or fertility, although our findings also suggest that further studies of the effects of genistein are warranted.

An elegant series of studies by Russell and colleagues (41, 46, 47) has demonstrated that the first wave of spermatogenesis at puberty in the male rat is characterized by several interrelated temporal changes involving formation and progressive expansion of the seminiferous tubule lumen, a progressive increase in germ cell volume and numbers, and a progressive decrease in the number of degenerating germ cells (48). We have therefore used these three end points to characterize pubertal spermatogenesis in rats treated neonatally with DES, OP, or Bis-A or in which exposure to soy/genistein was manipulated. The trends observed in the three parameters from 18–25 days of age in control rats in the present studies were comparable to those reported by Russell and colleagues in their studies. However, we recognized that significant variation occurs between (control) males from different litters (due to litter size, genetic differences, maternal competency, etc.) with regard to the precise onset of the temporal trends in spermatogenic parameters, raising the possibility of detecting changes in these parameters due simply to chance rather than as a consequence of treatment. This possibility was minimized by 1) cross-fostering male pups from several litters at birth into control and experimental groups and by restricting litter size to 8–12 pups, 2) repeating each experiment on day 18 at least twice and only accepting an effect as treatment induced if a similar change was found in both experiments, 3) pooling data from the different experiments and from the several control groups to fully represent the spectrum of changes due to chance, and 4) in statistical evaluation, using the pooled variance for each parameter from the study as a whole (i.e. from all control and treatment groups, with n = >120 for day 18 animals) to minimize the possibility of false detection of treatment-induced changes.

Our findings on the effects of the different estrogenic compounds on the first wave of spermatogenesis at puberty have shown that all three of the evaluated parameters changed more or less in concert, although the treatment-induced changes ranged from severe retardation of spermatogenesis in males exposed to the highest dose (10 µg/injection) of DES, as reported in our earlier studies (11), to stimulatory effects by low doses of DES or by treatment with OP or Bis-A. The latter findings suggest that low levels of potent estrogens or a high dose of weak estrogens have similar effects and that these can result in significant advancement of testicular/spermatogenic development at puberty. In contrast, the present data suggest that the presence of soy (and thus phytoestrogens) in the rats’ diet or the direct administration of genistein to neonates results in retardation of several aspects of pubertal spermatogenesis, a change that is opposite to that induced by the weak estrogens OP and Bis-A or by low doses of DES. Thus the rearing of control (vehicle-injected) animals on a soy-free diet substantially increased spermatocyte and spermatogonia nuclear volume per unit Sertoli cell nuclear volume compared with that in controls reared on a standard soy-containing diet, and administration of genistein to rats kept on a soy-free diet reversed this change and also retarded lumen formation, suppressed FSH levels, and increased the germ cell apoptotic index. These effects of genistein were broadly equivalent to a dose of approximately 1 µg DES, although it is emphasized that, unlike DES, genistein did not uniformly suppress all aspects of pubertal spermatogenesis, as it had only a minor effect on testis weight and was associated with high, rather than low, Sertoli cell nuclear volume per testis. The apparent selectivity of action of soy/genistein and its surprisingly high (apparent) estrogenic potency could perhaps be related to the reported relatively high affinity of genistein for ERß (49), which is expressed in both Sertoli and germ cells (4, 5, 6). As the dose of genistein administered in our studies was nominally based on the total phytoestrogen exposure of human infants fed a 100% soy-formula milk diet (39), our findings reinforce the conclusion from this study (39) that such a diet is likely to have biological effects (see also below). As outlined below, soy and genistein also express significant biological activity in addition to their estrogenicity, and this should be kept in mind when considering the possible pathways of their actions on the testis.

There are several possible mechanisms that could explain the present findings. Most obviously, plasma FSH levels on day 18 showed changes that broadly paralleled the changes in pubertal spermatogenesis, i.e. higher FSH levels associated with advancement, and lower FSH levels associated with retardation, of spermatogenesis. However, this relationship was not absolutely consistent from group to group, and the fact that on day 25 the FSH changes persisted whereas the (positive) changes to spermatogenesis were generally no longer evident raises doubts as to whether all of the changes observed can be explained simply by changes in FSH. As testosterone is also a key player in spermatogenesis at puberty (48), and elevated FSH levels can advance (48, 50, 51), whereas estrogen can retard (48, 52), pubertal Leydig cell development, it will be important to explore whether changes in Leydig cell development/testosterone levels might also underlie some of the changes in some of the treatment groups. Such studies are in progress. Regardless of what these findings may show, the present demonstration that neonatal exposure to low doses of potent estrogens/high doses of weak estrogens can elevate FSH levels in blood in early puberty (after treatment has ceased) is of interest, especially as we have shown that such a change may persist into adulthood (21). This may indicate some resetting of hypothalamic-pituitary control of FSH secretion.

An entirely different explanation for the stimulatory effect of low levels of estrogens on pubertal spermatogenesis in the present studies is that they are the result of a direct effect of estrogen on the Sertoli and/or germ cells. Sertoli cells, spermatogonia, and most spermatocytes express ERß at all stages of normal pubertal development (4, 5, 6) (our unpublished data), and we have produced several lines of evidence that suggest direct effects of neonatal estrogen treatment on Sertoli cells (11, 21). There are also data in the literature for a number of species that demonstrate stimulatory effects of estrogens on gonocytes (37), spermatogonial stem cells (38), and spermatogonia (34, 35) as well as precocious activation of spermatogenesis in the human testis associated with an estrogen-secreting tumor (36). In the present studies most of the treatments ceased on day 12 (soy and genistein manipulations extended to day 18), so if the effects observed on pubertal spermatogenesis are attributable to direct stimulation of Sertoli or germ cells by estrogen, then clearly these effects are able to persist for several days after cessation of treatment. The absence of these stimulatory effects on day 25, compared with controls, might then reflect the longer period since withdrawal of exogenous estrogen. With such an explanation in mind, we extended treatment with one of the stimulatory low doses of DES (0.1 µg/injection) up to day 18 of neonatal life, but found that the stimulatory effects on pubertal spermatogenesis in these rats was no greater than that shown for the comparable group in the present studies when treatment was terminated on day 12 (unpublished data). It is obviously difficult in the present studies to disentangle possible direct effects of estrogens on Sertoli/germ cells from effects due to altered FSH levels (and possibly altered testosterone levels). Nevertheless, in view of the other pieces of data cited from the literature, further study of the direct effect of estrogens on germ cells are clearly warranted.

The present data suggest that the effects of OP and Bis-A on reproductive development of the neonatal male rat are benign, athough it is emphasized that only a single high dose of either compound was tested. However, in almost every respect the effects of these two compounds were comparable to those of the lowest doses of DES. Logically, it therefore seems unlikely that lower doses of these compounds will induce adverse effects (because of their estrogenicity), although the presently observed stimulatory effects will presumably be lost at some lower dose. These data, and especially the conclusions, are somewhat at odds with other published studies of the effects of Bis-A or OP/nonylphenol in rats or mice, in which either no effects (28, 29, 30, 31, 32) or adverse effects on reproductive development/function (23, 24, 25, 26, 27) were reported. However, these studies used different aged animals and/or treatment regimens and did not study the end points used in the present studies. Based on the limited data that we present here, it would appear unlikely that human exposure to OP or Bis-A during neonatal life/puberty would result in any detectable adverse effect on spermatogenesis or fertility, because the level of human exposure is unlikely to be as high as that used in our treatments.

The present data concerning the effects of male exposure to soy/genistein are not so reassuring with regard to the likely consequences of human exposure. Removal of soy from the diet of our rats had positive (and life-long) consequences in terms of pubertal spermatogenesis and final testis and body size, implying that phytoestrogens or other components of the soymeal may exert mild retardation of these end points. The fact that administration of genistein, to rats fed a soy-free die, reversed these changes (with the exception of change in testis size) is consistent with these effects being due to phytoestrogens, although clearly there are other possible interpretations that are unrelated to the estrogenicity of soy/genistein, e.g. inhibition of tyrosine kinase activity in testicular cells. For example, in vitro studies have shown that genistein is able to inhibit differentiation of immature Sertoli cells (53) and to inhibit the growth and proliferation of various testicular cell lines (54), and at least some of these effects have been attributed to the tyrosine kinase inhibitory activity of genistein (53). Similar effects in vivo could certainly account for some of the presently described inhibitory effects of genistein and a soy-containing diet on spermatogenesis at puberty.

There is a substantial literature on the effects of soy-derived phytoestrogens on rodent reproductive development (55, 56, 57, 58), although none has investigated the end points described here in early male puberty, and many of the earlier studies may not have taken account of the presence or absence of soymeal in the diet. Our studies have shown detectable effects of neonatally administered genistein on reproductive development of male rats, although this did not result in any major detrimental long-term consequence in terms of testis size. Furthermore, although the data for mating and fertility were largely normal in genistein-treated rats, a proportion of the treated animals exhibited evidence of impaired mating and/or fertility. It is cautioned that the numbers of animals involved in the latter studies was low, and more detailed studies, using a range of doses of genistein, are required to establish whether this is a real or a chance finding. The fact that most laboratory animal colonies are maintained on a diet containing high levels of genistein and other soy phytoestrogens with no evidence of widespread breeding problems is relevant in this regard. However, our finding that the continuous presence of soy in the diet mildly retards spermatogenic development and results in lifelong alterations in body weight, testis weight, and FSH levels does have significant implications for all studies of male reproductive development and function. Although the effects observed were not huge and were probably of minimal biological consequence, it is clear that variation in such effects due to variation in the isoflavone content of soymeal (59) could be an important contributor/creator of "noise" in studies of endocrine disruption. Our findings add to the conviction (59) that such effects may explain some of the controversy and inconsistency of findings in this area.


   Acknowledgments
 
We are grateful to Jim MacDonald for technical assistance.


   Footnotes
 
1 This work was supported in part by the European Center for the Ecotoxicology of Chemicals and AstraZeneca plc. Back

2 Recipient of a Royal Society/North Atlantic Treaty Organization postdoctoral fellowship. Back

Received March 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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