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BOR - Papers in Press, published online ahead of print October 10, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.059857
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BIOLOGY OF REPRODUCTION 78, 59–67 (2008)
DOI: 10.1095/biolreprod.106.059857
© 2008 by the Society for the Study of Reproduction, Inc.

Testis; 

Activin A and Equine Chorionic Gonadotropin Recover Reproductive Dysfunction Induced by Neonatal Exposure to an Estrogenic Endocrine Disruptor in Adult Male Mice1

Katsuhiko Warita 3 5, Kazutaka Okamoto 6, Ken-ichiro Mutoh 6, Yoshihisa Hasegawa 7, Zhan-Peng Yue 8, Toshifumi Yokoyama 3 4, Yoshiki Matsumoto 5, Takanori Miki 5, Yoshiki Takeuchi 5, Hiroshi Kitagawa 3 4, Teruo Sugawara 9, and Nobuhiko Hoshi 2 3 4

Department of Bioresource and Agrobiosciences,3 Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan Department of Anatomy and Neurobiology,5 Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan Departments of Veterinary Anatomy6 and Experimental Animal Science,7 School of Veterinary Medicine and Animal Sciences, Kitasato University, Aomori 034-8628, Japan Department of Histology and Embryology,8 Veterinary College, Jilin University, Jilin 130062, China Department of Animal Science,4 Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan Department of Molecular Biochemistry,9 Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan

ABSTRACT

We aimed to elucidate the mechanism of action of estrogenic endocrine disruptors and the rescue of reproductive function, particularly the responsiveness of testes to eCG and/or activin A (ACT) after establishing reproductive disorders. Newborn male mice (n = 29) were randomly divided into an untreated group and three treatment groups that received diethylstilbestrol (DES; 100 µg per animal) subcutaneously on Postnatal Day 3 to establish reproductive disorders and daily treatment with PBS (controls: DES + PBS), eCG (eCG group: DES + eCG), or eCG + ACT (eCG + ACT group: DES + eCG + ACT) at 6–8 wk of age prior to mating. After treatment, the controls showed diminished Leydig cells in the testes and thin germ cell layers containing pyknotic germ cells and multinucleated cells. In the eCG and eCG + ACT groups, spermatids and Leydig cells increased markedly. The immunoexpression of androgen receptors in the eCG group and steroidogenic acute regulatory (STAR) protein in the eCG and eCG + ACT groups recovered to approximately the levels in the untreated group; plasma LH and testosterone levels also increased relative to those in the controls. In addition, the cell proliferation index, which is estimated from 5-bromo-2'-deoxyuridine immunoexpression in spermatogonia, increased significantly under eCG treatment, and even more with eCG + ACT. However, the numbers of germ and Leydig cells decreased at 12 wk of age. Thus, ACT and eCG help the testes to recover from the dysfunction induced by neonatal DES administration. Furthermore, the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents may be more likely to result from dysfunction of the hypothalamic-pituitary axis than from dysfunction of the lower reproductive organs.

activin, activin A, androgen receptor, diethylstilbestrol, endocrine disruptor, equine chorionic gonadotropin, Leydig cells, luteinizing hormone, male sexual function, steroidogenic acute regulatory protein

INTRODUCTION

It has been suggested that a wide variety of chemicals released into the environment mimic the action of estrogens by binding to estrogen receptors (ERs). These chemicals have been demonstrated to play a role in decreased male reproductive success and reproductive failure in laboratory animals, including some wildlife species [1]. During the fetal and neonatal periods, there is a critical window during which organisms are particularly sensitive to hormonelike materials [2–4]. Rodents exposed prenatally to xenoestrogens are particularly well suited for studies on the developmental effects of these substances on the central nervous system and reproductive system, because the maturity level of rodents in the first few days of life resembles that of the human fetus at the end of the first trimester [5]. Numerous studies have shown that exposure of male rodents to estrogenic agents induces a decrease in the weight of reproductive organs and the sperm count and motility [6, 7]; it also results in structural and functional abnormalities of the testes and reproductive tract [8–15] because of widespread expression of ERs during the early developmental period [16–20]. In addition, exposure to estrogens during the critical period induces long-term changes in various organs, including persistent molecular alterations [5]. Diethylstilbestrol (DES), a synthetic nonsteroidal estrogen, exhibits high estrogenic activity by binding to ERs and is thus a useful model compound for evaluating the potential toxicity of a broad range of chemicals that affect or mimic estrogen activity [1]. Previous reports have shown that inappropriately high estrogen exposure during neonatal life impairs Sertoli cell functional maturation [12], including a decrease in the number of Sertoli cells that express estrogen receptor β (ESR2) [19, 20]. It also has been suggested that the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than Sertoli cells to neonatal reprogramming by estrogens [21]. Furthermore, it has recently been reported that coadministration of testosterone esters with DES prevents the decrease in germ cell volume per Sertoli cell induced by treatment with DES alone [22], indicating that a lack of androgen action may be a primary factor in these changes.

To investigate the working mechanisms of estrogenic endocrine disruptors, we evaluated the effects of activin and/or eCG on the testes and hormonal secretion in male mice rendered infertile by neonatal exposure to high doses of DES. Activins, which are members of the transforming growth factor-β (TGFB) superfamily, have a stimulatory effect on pituitary FSH secretion. Activins are widely expressed and have been shown to regulate a variety of cell functions, including upregulation of gonadotropin-releasing hormone receptor (GNRHR) gene expression, stimulation of GNRH release from GNRH neurons in the hypothalamus, and induction of apoptosis [23]. Equine CG is composed of {alpha}- and β-subunits, and the β-subunits of eCG and equine luteinizing hormone (eLH) are encoded by the same gene. Interestingly, eCG exhibits pronounced FSH activity in addition to its LH activity in species other than the horse [24, 25]. Biologic responsiveness to physiologically active substances, such as activin A (ACT) and eCG, is uncertain in adult mice exposed neonatally to estrogenic endocrine disruptors. Our findings not only support the hypothesis that exposure to potent estrogens during the early developmental period results in permanent infertility in male mice but also indicate that spermatogenesis and steroidogenesis can be restored in adulthood, including the expression of the steroidogenic acute regulatory (STAR) protein, which mediates the rate-limiting and acutely regulated steps in steroidogenesis.

MATERIALS AND METHODS

Animals

ICR mice were obtained from Japan SLC Inc. (Hamamatsu, Japan) and bred in our own animal facility. All mice were kept under a 14L:10D regimen at a constant temperature of 21°C–24°C and a relative humidity of 40%–60%. They were allowed access to a laboratory diet (MR-A1; Nosan Corp., Yokohama, Japan) and tap water ad libitum. This study was conducted in accordance with Japanese laws as well as rules and guidelines concerning the treatment and use of laboratory animals at Kitasato University.

Treatment of Animals

The present study included eight pregnant female mice and their male pups (n = 29). After deliveries, the litters were culled so that each dam had a litter containing three to four newborn male pups. There was no growth difference between the groups; therefore, the litters from the eight dams were pooled and redistributed. The eight dams and their pups were randomly allocated to four groups: an untreated group (n = 7) and three treatment groups (n = 7–8 in each group) in which the pups were administered DES (100 µg per animal; Sigma-Aldrich Corp., St. Louis, MO) in 10 µl sesame oil (Kanto Chemical Co. Inc., Tokyo, Japan) subcutaneously on Postnatal Day 3 to establish reproductive disorders. In the DES-exposed groups, the pups were administered PBS, eCG (Teikoku Zouki Co., Tokyo, Japan), and/or ACT [26] subcutaneously daily for 14 days beginning at 6 wk of age. The PBS group (controls; n = 7) received 200 µl PBS without eCG or ACT; the eCG group (n = 7), 200 µl PBS containing 5 units eCG; and the eCG+ACT group (n = 8), 200 µl PBS containing 5 units eCG and 20 µg ACT. All mice except those used for fertility testing (3–4 mice per test group) were killed at 8 wk of age. The animals were injected with 30 mg/kg 5-bromo-2'-deoxyuridine (BrdU) labeling reagent (Amersham Pharmacia, Tokyo, Japan) intraperitoneally 2 h before being killed. Blood samples were collected under diethyl ether anesthesia to measure LH and testosterone levels. The testes, epididymides, and seminal vesicles with coagulating glands were excised and weighed.

Histologic Analysis

The testes in each group were fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin by the routine method. Sections were cut at 5 µm thickness for histologic and immunohistochemical analyses. For general histologic analysis, the specimens were stained with hematoxylin and eosin (H&E; Merck & Co. Inc.) following the manufacturer's instructions.

Scanning Electron Microscopy

The embedded testes were deparaffinized for hydration and processed for scanning electron microscopy (SEM) by rinsing twice in 0.1 M PBS, fixing in 0.2% osmium tetroxide at 4°C overnight, and dehydrating through a gradient of ethanol [27]. The dehydrated specimens were subsequently dried by the t-butyl alcohol freeze-drying method and coated with platinum-palladium using Hitachi-1038 (Hitachi Ltd., Tokyo, Japan). The samples were examined under a scanning electron microscope (S4300; Hitachi).

Immunohistochemical Analysis

For the immunohistochemical analysis, deparaffinized sections were placed in a temperature-controllable microwave processor (MI-77; Azumaya, Tokyo, Japan) and treated with 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval [28], washed in 10 mM PBS, and then treated with 0.3% H2O2 dissolved in 100% methanol to inactivate endogenous peroxidase activity. The primary rabbit antibodies for androgen receptors (ARs; Affinity BioReagents Inc., Golden, CO) and STAR (a generous gift from Dr. Jerome F. Strauss III of the Virginia Commonwealth University, Richmond, VA) were diluted at ratios of 1:200 and 1:500, respectively, in 10 mM PBS and incubated over the sections in a humidified chamber for 14 h at 4°C. Immunohistochemical staining for ARs and STAR was carried out by the labeled streptavidin biotinylated antibody method using a Histofine SAB-PO (R) Kit (Nichirei Corp., Tokyo, Japan). Monoclonal antibodies for BrdU were purchased from Amersham and diluted 1:100 in 10 mM PBS. The antibodies for BrdU were incubated with the tissues for 2 h at room temperature. Monoclonal antibodies in mouse tissues were identified by immunohistochemical staining using a HistoMouse Plus Kit (Zymed Laboratories Inc., San Francisco, CA), according to the manufacturer's instructions. Immunoreactions were visualized with 3,3'-diaminobenzidine for ARs and STAR and with 3-amino-9-ethylcarbazole for BrdU. Androgen receptors and STAR immunoexpressions were evaluated qualitatively. Negative controls, in which the primary antibodies were replaced with nonimmunized rabbit sera, did not show nonspecific staining.

Determination of Cell Proliferation Index

All seminiferous tubules present in three randomly cut sections from a testis were scored based on the percentage of BrdU-positive nuclei in spermatogonia as follows: 4 points, >80% positive; 3 points, 50%–80% positive; 2 points, 20%–50% positive; 1 point, <20% positive; and 0 points, unstained (Fig. 1). This is a modified version of the method described by Bak and Panos [29]. In all cases, the observer was unaware of the group to which the specimen belonged.


Figure 01
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  		FIG. 1 Immunohistochemical analysis of BrdU and scoring of seminiferous tubules. Based on the percentage of BrdU-positive nuclei in spermatogonia, the seminiferous tubules in the specimen were scored as follows: (a) 4 points, >80% positive; (b) 3 points, 50%–80% positive; (c) 2 points, 20%–50% positive; (d) 1 point, <20% positive; and (e) 0 points, unstained. The cell proliferation indices were determined based on the average scores of all seminiferous tubules. Bars = 100 µm.

Plasma Hormone Measurement

Blood samples were immediately put into heparinized tubes and centrifuged at 4°C for 10 min at 800 x g. Harvested plasma was stored at –80°C until assay. Plasma hormones were measured by the time-resolved fluoroimmunoassay method [30].

Plasma LH levels were measured following a modified version of the method described in our previous study [30] using Europium-labeled rat LH and diluted anti-LH rabbit serum. Testosterone levels were measured following our previously reported procedure [31] using Europium-labeled rabbit antibodies against BSA conjugated with testosterone.

Fertility Testing

At 8 wk of age, three to four males from each DES-exposed group were mated with untreated females of proven fertility for 2 wk to determine the fertility of the males. The reproductive behavior of the male mice was confirmed by the presence of a vaginal plug in the female mice. The males were killed at 12 wk of age for histologic analysis of their testes.

Statistical Analysis

Statistical analyses were performed using StatView for Windows (version 5.0; SAS Institute Inc., Cary, NC). The data of organ weight and hormone levels were analyzed by one-way ANOVA and the Tukey-Kramer multiple comparison test. Analyses were considered to be statistically significant at P < 0.05.

RESULTS

Necropsy Findings

No abnormal pathologic findings were noted in any of the DES-exposed groups, except for slight shrinkage of the testes and accessory reproductive organs.

Body and Organ Weights

The body weights of mice at 8 wk of age, including the absolute and relative (percentage of body weight) weights of the testes, epididymides, and seminal vesicles with coagulating glands, are summarized in Table 1. The body weights in all DES-exposed groups were significantly lower than that in the untreated group (P < 0.01), although a significant difference was not detected among the control, eCG, and eCG + ACT groups. Statistically significant decreases in the absolute weights of testes, epididymides, and seminal vesicles with coagulating glands were detected between the untreated group and all DES-exposed groups (P < 0.01); however, there was no significant difference among the control, eCG, and eCG + ACT groups. Statistically significant decreases also were noted in the relative weights of testes in all DES-exposed groups compared with the untreated group (P < 0.05). The relative weights of epididymides in the control and eCG + ACT groups were significantly lower than that in the untreated group (P < 0.01 and P < 0.05, respectively). There was a significant decrease in the relative weights of seminal vesicles with coagulating glands in all DES-exposed groups compared with the untreated group (P < 0.01). However, no significant difference in relative organ weights was detected among the control, eCG, and eCG + ACT groups.


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  		TABLE 1 Body and organ weights at 8 wk of age (mean ± SD).*

Histologic and SEM Findings

In the untreated group, the testes showed normal cell arrangement in the seminiferous tubules and normal spermatogenesis at 8 wk of age (Fig. 2a). In the control group, the testes showed thin germ cell layers and enlarged tubular lumen due to a decrease in the number of germ cells, and pyknotic germ cells and multinucleated cells were noted in the seminiferous tubules (Fig. 2b). Furthermore, diminished Leydig cells were observed in the control group relative to the untreated group. In the eCG group, the germ cell layers in the testes were thicker than those in the control group, and the formation of multinucleated cells with pyknotic nuclei was observed (Fig. 2c). The number of spermatids and Leydig cells increased remarkably under eCG treatment. Both germ cell layers and Leydig cells in the eCG + ACT group were normal (Fig. 2d), and the testicular histology in this group resembled that in the untreated group. Representative SEM images of the seminiferous tubules in the control and eCG + ACT groups are shown in Figure 2, e and f, respectively. In the control group, the tubular lumens were enlarged with a marked decrease in round spermatids. In contrast, vigorous spermatids and spermatozoa were observed in the seminiferous tubules in the eCG + ACT group.


Figure 02
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  		FIG. 2 Representative histology of the testes in the untreated (a), control (b), eCG (c), and eCG + ACT (d) groups and SEM images of the seminiferous tubules of the control (e) and eCG+ACT (f) groups. In the control group, the testis shows diminished Leydig cells and thin germ cell layers containing pyknotic germ cells (arrow) and multinucleated cells (arrowhead); in the eCG group, the testis shows a remarkable increase in Leydig cells and thicker germ cell layers containing multinucleated cells with pyknotic nuclei (arrowhead). In the eCG + ACT group, a normal cell arrangement is present in the seminiferous tubules, and Leydig cells also appear normal. Hematoxylin and eosin staining. In the control group (e), the lumens of the seminiferous tubules are enlarged and show a marked decrease in round spermatids. Vigorous spermatids and spermatozoa are observed in the seminiferous tubules of the eCG+ACT group (f). a–d, Bars = 100 µm; e and f, Bars = 50 µm.

Immunohistochemical Findings

Androgen receptor immunoexpression was detected in the nuclei of Leydig cells, Sertoli cells, and peritubular myoid cells in the testes (Fig. 3). Qualitative assessment revealed that the number of AR-positive cells was decreased in the control group compared with the untreated group. In addition, AR immunoreactivity was slightly diminished in the Leydig cells. In the eCG group, there was an increase in the number of AR-positive Leydig cells, and increased intensity of AR immunoexpression was observed. In the eCG + ACT group, the AR immunoexpression in Leydig cells was lower than that in the untreated and eCG groups.


Figure 03
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  		FIG. 3 The nuclei of Leydig cells (arrowheads), Sertoli cells, and peritubular myoid cells in the testes are positive for ARs in the untreated (a), control (b), eCG (c), and eCG + ACT (d) groups. Androgen receptor immunoexpression in Leydig cells is lower in the control group than in the untreated group. Following treatment with eCG, the number of AR-positive cells and AR immunoreactivity increased remarkably. In the eCG + ACT group, the AR immunoexpression in Leydig cells decreased compared with that in the untreated and eCG groups. Bars = 100 µm.

STAR immunoreactivity was localized in the cytoplasm of Leydig cells (Fig. 4). The intensity of STAR immunoexpression and the immunoreactive area were decreased in the control group. However, under eCG or eCG + ACT treatment, both STAR-positive cells and STAR immunoexpression increased markedly; no difference was observed between the eCG and eCG + ACT groups.


Figure 04
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  		FIG. 4 STAR immunoreactivity is observed in the cytoplasm of Leydig cells (arrowheads) in the untreated (a), control (b), eCG (c), and eCG + ACT (d) groups. The intensity of STAR immunoexpression and immunoreactive area in the control group decreased compared with those in the untreated group. A marked increase in STAR immunoexpression was detected in the eCG and eCG + ACT groups compared with the control group. There was no difference in the intensity of STAR immunoexpression between the eCG and eCG + ACT groups. Bars = 50 µm.

Cell Proliferation Index

The cell proliferation index of spermatogonia is presented in Figure 5. The index of the control group (1.57 ± 0.01) was significantly decreased relative to that of the untreated group (1.93 ± 0.06; P < 0.01). Following eCG or eCG + ACT treatment, the indices of the eCG and eCG + ACT groups (1.85 ± 0.04 and 1.98 ± 0.03, respectively) significantly increased in comparison with that of the control group (P < 0.01) and were restored to approximately the level of the untreated group. In addition, the index of the eCG + ACT group was significantly higher than that of the eCG group (P < 0.01).


Figure 05
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  		FIG. 5 Effect of neonatal DES administration and subsequent treatment with eCG or eCG + ACT on the cell proliferation index. All seminiferous tubules present in three randomly cut sections from a testis were scored based on the percentage of BrdU-positive nuclei in spermatogonia. Each column represents the mean ± SD value of four mice per group at 8 wk of age. The Tukey-Kramer multiple comparison test was performed to compare the indices of the four groups. aP < 0.01.

Plasma Hormone Levels

A significant decrease in plasma LH level was detected in the control group compared with the untreated group (P < 0.05; Fig. 6). Under eCG or eCG + ACT treatment, the LH levels increased significantly relative to those in the control and untreated groups (P < 0.01 for both). The testosterone level in the control group was significantly lower than that in the untreated group (P < 0.01), whereas in the eCG and eCG + ACT groups, the testosterone levels increased significantly in comparison with that in the control group (P < 0.01) and were restored to approximately the level of the untreated group.


Figure 06
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  		FIG. 6 Effect of neonatal DES administration and subsequent treatment with eCG or eCG+ACT on the plasma LH and testosterone levels. Each column represents the mean ± SD value of three to four mice per group at 8 wk of age. The Tukey-Kramer multiple comparison test was performed to compare the plasma LH and testosterone levels among the four groups. aP < 0.05; bP < 0.01.

Fertility Test

All males in the untreated group (n = 3) were fertile, whereas those in the control (n = 3) and eCG (n = 3) groups were infertile or did not mate. In the eCG + ACT group, one of the four males mated successfully, and a vaginal plug was observed in a female mouse; however, the mating did not result in the birth of a litter.

Histologic Findings at 12 Wk of Age

In the eCG and eCG + ACT groups, the testicular histology at 12 wk of age was clearly different from that at 8 wk of age. A decrease in both germ cells and Leydig cells was observed in the eCG + ACT group, and a marked decrease was observed in the eCG group. The testicular histology of these two groups at 12 wk of age resembled that of the control group at 8 wk of age.

DISCUSSION

Exposure to estrogenic agents during the early developmental period induces various adverse effects in male mammals, such as demasculinization of the central nervous system and androgen-dependent growth of the reproductive organs, and these effects are reported to result in long-term histologic and endocrine changes [21, 32–34], including molecular alterations [5, 35–37]. These studies suggest that endocrine disruptors play an important role in imprinting genes through the process of epigenetic change (persistently altered gene expression in the absence of gene mutation).

Activins, whose subunits are structurally related to the TGFB family of peptides, were first identified as gonadal proteins that regulate the production and release of FSH from the anterior pituitary gland in a classic feedback loop [38]. It has been reported that activins have many reproductive and nonreproductive functions, such as hormonal regulation, cell differentiation, and neuronal survival. On the other hand, eCG is known to induce the growth of seminiferous epithelium through both FSH-like and LH-like actions; indeed, we confirmed a remarkable proliferation of germ cells and Leydig cells in the hypoplastic testes of adult hypogonadal (hpg) mice, which lack hypothalamic GNRH, by pre-experimental eCG administration (data not shown). Evaluation of testicular responsiveness to these hormones allowed us to clarify the mechanism of action of estrogenic agents that induce permanent reproductive disorders in male mice exposed to these chemicals neonatally.

Clear short-term and long-term inhibitory effects of potent estrogens such as DES on testicular development have been demonstrated previously [10, 39]. In the present study, pronounced adverse effects on the testes and hormonal secretion at 8 wk of age also were detected in mice that received high doses of DES neonatally. We have confirmed a remarkable increase in testicular weight in adult hpg mice by ACT administration for 2 wk (Kaneko et al., unpublished data). In addition, sperm formation was observed in these mice following eCG administration (data not shown). The present histologic analyses in the eCG and eCG + ACT groups showed a remarkable proliferation/activation of germ cells and Leydig cells. Furthermore, following eCG + ACT treatment there was a marked increase in spermatozoa, which recovered to the levels observed in the untreated group. This result is confirmed by the cell proliferation indices, which indicate the proliferation potency of spermatogenesis. The activin signal to the intracellular signaling pathways is transmitted by activin type II receptors (ACVR2A and ACVR2B). In rat testes, in situ hybridization analyses have revealed the presence of an ACVR2A signal in round spermatids and pachytene spermatocytes and a weak ACVR2B signal in interstitial and Leydig cells [40]. Previous in vitro studies have shown that ACT regulates mitotic spermatogonial DNA synthesis [41–43]. The present histologic findings indicate that adult testes impaired by DES neonatal treatment may respond to ACT and/or eCG; however, spermatogenesis and the number of Leydig cells tend to decrease after the treatments are stopped. This implies that the permanent reproductive disorder induced by neonatal DES administration occurs indirectly through dysfunction in the hypothalamic-pituitary axis.

Androgen receptor immunoexpression is found in the nuclei of Leydig cells, Sertoli cells, and peritubular myoid cells in the testes [44]. In the present study, neonatal DES administration caused a slight decrease in AR immunoexpression in Leydig cells. However, AR immunoexpression in the eCG group increased markedly. The decreased AR immunoexpression, which was considered to occur due to a decrease in the amount or activity of ARs resulting from neonatal DES treatment, may have been responsible for the observed reproductive dysfunction; however, eCG administration in adulthood appears to restore the AR immunoexpression levels.

In the present study, the plasma LH and testosterone levels in the control group were significantly lower than those in the untreated group at 8 wk of age; these hormones showed remarkable resilience under eCG + ACT treatment and an even greater resilience under treatment with eCG alone. Previous studies have suggested that ACT inhibits testosterone secretion by Leydig cells [45, 46]. In the present study, testosterone secretion may have been inhibited by ACT. Although a significant increase in plasma LH following eCG administration is required to determine the biologic significance of this result, both the LH-like action of eCG and the increase in plasma LH appear to stimulate Leydig cells; this stimulation is mediated by the LH receptor at the first step of the signaling pathway of steroidogenesis. It has been recognized that proteins are required for the translocation of sterol substrates to inner mitochondrial membranes where the P450 enzymes that catalyze these reactions reside [47–49]. STAR, which has been identified in a mouse Leydig cell tumor line (MA-10 cells), is known to be such a protein [50]. LH stimulates Star mRNA expression through intercellular cAMP increases in MA-10 cells [50, 51], resulting in the production of the STAR protein that mediates the rate-limiting and acutely regulated step in steroidogenesis [52–57]. In the present study, STAR immunoexpression was detected in the cytoplasm of Leydig cells and was slightly decreased in the control group compared with the untreated group. The STAR immunoexpression in Leydig cells was restored under ACT and/or eCG treatment, and the testosterone levels then correlated approximately with the STAR immunoexpression. This finding suggests that the STAR protein in the adult testes decreases due to neonatal DES exposure; however, this decrease in STAR is not irreversible, because the ability to produce STAR is maintained in adulthood. In the present study, the testes in the eCG + ACT group appeared to be histologically normal at 8 wk of age. One of the four males in the eCG + ACT group mated successfully, and a vaginal plug was observed in a female mouse; however, the mating did not result in the birth of a litter. The full spermatogenic cycle in mice is longer than the dosing period in this study. Therefore, the fertility may have recovered by the continued eCG + ACT administration. Alternatively, there exists the possibility of decreased sperm count, decreased sperm motility, or altered sperm transit; these factors may hamper the process of fertilization of the oocyte, leading to postovulatory overripeness ovopathy and defective implantation in animals, transitory retardation in the rate of development, and increased prenatal loss [58]. With respect to the infertile males in the eCG and eCG + ACT groups, no vaginal plugs were detected in the female mice. The gonadal steroids play a role in the differentiation of neurons and, subsequently, in the sexual dimorphisms of the brain structure and behavior [59, 60]. It has also been reported that estrogen influences cell migration, cell survival, cell death, and synaptic neuron plasticity [61–63], thereby affecting subsequent behavioral and cognitive function [64]. Neonatal DES exposure may have irreversibly altered the part of the brain that is most responsible for reproductive behavior.

The present findings strongly suggest that the testes maintain the ability to recover from the dysfunction induced by neonatal DES administration. However, this restoration by physiologically active substances, such as ACT and eCG, is a short-term response, indicating that the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents is more likely to result from a dysfunction of the hypothalamic-pituitary axis than from a dysfunction of the lower reproductive organs.

FOOTNOTES

1Supported in part by Grants-in-Aid for Scientific Research (C) (12836014) and (B) (15390510) and for Scientific Research on Priority Areas (1) (14042260) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to N.H. Back

Correspondence: 2FAX: 81 78 803 5811; e-mail: nobhoshi{at}kobe-u.ac.jp

Received: 15 January 2007.

First decision: 12 March 2007.

Accepted: 20 September 2007.

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