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Ontogeny of Estrogen Receptor-ß Expression in Rat Testis¹

Department of Cell Biology, Medical School, University of Utrecht (A.M.M.v.P., D.G.d.R.), and the Netherlands Institute for Developmental Biology, Hubrecht Laboratory (B.v.d.B., P.T.v.d.S.), Utrecht, The Netherlands; and the Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute (J.-Å.G., G.G.J.M.K.), Huddinge, Sweden

Address all correspondence and requests for reprints to: Dr. A. M. M. van Pelt, Department of Cell Biology, Medical School, Utrecht University, AZU H02.314, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: A.Pelt{at}pobox.accu.uu.nl

	Abstract

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The recently discovered estrogen receptor-ß (ERß) is expressed in rodent and human testes. To obtain insight in the physiological role of ERß we have investigated the cell type-specific expression pattern of ERß messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERß mRNA in their cytoplasm and the ERß protein in their nucleus. In testes of 11- and 15-day-old rats, ERß mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERß mRNA as well as ERß protein was found in pachytene spermatocytes from epithelial stages VII–XIV and in round spermatids from stages I–VIII. Low ERß expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERß. In adult testes, no ERß mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERß. The expression of ERß in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.

	Introduction

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AT THE ONSET of puberty, spermatogenesis is thought to be initiated and maintained through combined direct actions of testosterone and FSH on Sertoli cells (1). Although LH, FSH, and testosterone are accepted as the predominant endocrine regulators of gonadal function, the identification of glucocorticoid receptors (2) and estrogen receptors (3, 4) in testicular cells raised the possibility that estrogens and adrenal steroids may also modulate the processes of gametogenesis and gonadal steroidogenesis. The initial response of adult rat testicular Leydig cells to stimulation by LH is an increase in testosterone secretion, followed shortly thereafter by a significant increase in estrogen production (5). The aromatase enzyme is expressed not only in Leydig cells of the rat testis (6), but also within the seminiferous tubules (7), more particularly in Sertoli cells (8, 9), and germ cells (10, 11). In addition, an estrogen-specific sulfotransferase is expressed in the Leydig cells of the adult rat testis and might play a role in controlling the biological activity of estrogen in the testicular microenvironment (12). Specific and high affinity binding of 17ß-[³H]estradiol has been demonstrated in rat testicular nuclear extracts and in nuclear and cytosolic extracts of isolated Leydig and Sertoli cells (3, 4, 6, 13, 14, 15). The synthesis and metabolism of estrogen as well as the presence of estrogen receptors indicate that the rat testis is a site of estrogen action. An important example of estrogen action is the direct inhibitory action of intratesticular estrogen on testosterone production by rat Leydig cells (6).

After cloning of the estrogen receptor- (ER), various ER-specific antibodies became available. Immunohistochemical studies with these antibodies revealed that ER is localized in the nuclei of Leydig cells in fetal and adult rodent testes, whereas no expression within the seminiferous tubules was detected (16, 17). Unexpectedly, a novel ER complementary DNA was cloned from rat prostate, named ERß (18). Expression of ERß was investigated by Northern blotting and RT-PCR; prominent expression was found in rat prostate, ovary, epididymis, testis, bladder, uterus, lung, colon, small intestine, and brain (18, 19, 20). Saturation ligand binding experiments revealed high affinity and specific binding of estrogen by ERß protein, and ERß is able to stimulate the transcription of an estrogen response element containing reporter gene in an estrogen-dependent manner (20, 21).

No data are available on the cellular localization and ontogeny of ERß expression in the rat testis. Therefore, in the present study we have investigated the cellular localization of ERß in testes of rats of different ages. Using in situ hybridization and immunohistochemical techniques, we found expression of ERß not only in Sertoli cells, but also in fetal and adult germ cells.

	Materials and Methods

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Animals
Wistar rats U:WU (CPB) (Central Laboratory Animal Institute Utrecht, Utrecht University, Utrecht, The Netherlands) were used and maintained according to regulations provided by the animal ethical committee of the University of Utrecht Medical School. Testes (six animals of each age) from fetal rats 16 days postcoitum; from postnatal rats on days 4, 11, and 15; and from adult (10-week-old) rats were used. Adult animals were killed by inhalation of carbon dioxide, all other animals were killed by cervical dislocation. Testes were removed and fixed for further examination of ERß expression.

Preparation of digoxigenin-labeled probes
The sense and antisense complementary RNA probes used for in situ hybridization were synthesized by in vitro transcription from plasmids in which DNA fragments of the 5'-untranslated region (nucleotides 0–391, plasmid 24) and A/B domain (nucleotides 391–738, plasmid 74) of the rat ERß complementary DNA (18) were cloned. For preparation of the sense and antisense riboprobes, the plasmids were linearized using AccI and EcoRI, respectively, for plasmid 24, and PstI and AccI, respectively, for plasmid 74. Linearization was checked by agarose-gel electrophoresis. Labeled complementary RNA probes were synthesized by incubating the linearized plasmids with digoxigenin (DIG)-labeled UTP in the presence of T3 or T7 RNA polymerase according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany).

In situ hybridization
Tissues obtained from three animals of each age were fixed in 4% paraformaldehyde overnight at 4 C and embedded in paraffin Stemcowax (Adamas Instrument BV, Amerongen, The Netherlands). Tissue sections of 5 µm were made and transferred to 3-aminopropyl triethoxysilane-coated slides (Sigma Chemical Co., St. Louis, MO). Sections were dewaxed in xylene, rehydrated, fixed in 4% paraformaldehyde, treated with proteinase K, again fixed, and incubated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Hybridization was performed for about 44 h at 55 C in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 10% dextran sulfate, 1 x Denhardt’s solution, 10 mM NaH₂PO₄, and 0.5 mg/ml yeast transfer RNA) containing a combination of the DIG-labeled ERß (sense or antisense) riboprobes, each at 5 ng/ml hybridization buffer. After hybridization, the coverslips were removed in 5 x SSC (standard saline citrate). Sections were washed under high stringency in 50% formamide-2 x SSC at 65 C for 30 min, ribonuclease treated, and again washed under high stringency. Then the sections were washed in 2 x SSC at room temperature for 15 min and in 0.1 x SSC for 30 min at 60 C.

The hybridized DIG-labeled probes were detected with anti-DIG monoclonal antibodies. Endogenous peroxidase was blocked by incubating the sections in 0.35% H₂O₂. To reduce aspecific background staining, sections were incubated for 1 h at room temperature in 10% horse serum before incubation overnight at 4 C in 100 ng/ml mouse monoclonal anti-DIG (Boehringer Mannheim) and 10% horse serum in Tris-buffered saline (TBS; 0.1 M Tris, pH 7.6, and 0.15 M NaCl). After washing, the sections were incubated for 1 h at room temperature in 1:200 biotinylated antimouse IgG (avidin-biotin peroxidase complex staining kit, Vector Laboratories, Inc., Burlingame, CA) and 10% horse serum in TBS. The avidin-biotin complex reaction was performed according to the manufacturer’s protocol (ABC peroxidase staining kit, Vector Laboratories). To visualize the complex, sections were covered with 0.5 mg/ml diaminobenzidene (Dako Corp., Carpintera, CA) in 0.05 M Tris-HCl (pH 7.6) containing 0.01% H₂O₂. Sections were counterstained with Mayer’s hematoxylin.

Antibody preparation
The rat ERß ligand-binding domain protein was expressed in Escherichia coli and purified to homogeneity as previously described (22). Two laying hens were immunized with five injections of 25 µg ERß-LBD protein at 2-week intervals. Chicken Igs (IgY) were purified from the egg yolk according to the method of Song et al. (23). As preimmune control, IgY was also purified from five preimmunization eggs. The specificity of the anti-ERß antibodies was tested on Western blots containing nuclear extracts of insect Sf9 cells, overexpressing human ER and rat ERß protein, respectively. No cross-reaction with the ER protein was detected, whereas the antibody specifically detected a protein with an apparent molecular mass of approximately 55 kDa in nuclear extracts of cells expressing ERß (not shown).

Immunohistochemistry
Tissues obtained from three animals of all ages were fixed in 3.7% formaldehyde (Klinipath, Duiven, The Netherlands) for 6 h and postfixed in a diluted Bouin solution [71% picric acid (0.9%), 24% formaldehyde (37%), and 5% acetic acid] for 18–20 h. Fixed tissues was processed for 17 h in a tissue processor (Microm, Heidelberg, Germany) and embedded in paraffin (Stemcowax, Adamas Instrument BV). Tissue sections of 5 µm were made and transferred to 3-aminopropyl triethoxysilane-coated slides (Sigma Chemical Co.). Sections were dewaxed in xylene, rehydrated, and blocked for endogenous peroxidase by incubation with 0.35% H₂O₂ in PBS for 10 min. After washing in water, slides were incubated in 0.1% trypsin (Worthington Biochemical Corp., Freehold, NJ) in 0.05 M Tris (pH 7.6) and 0.1% CaCl₂ for 10 min at 37 C. Slides were washed in TBS (50 mM Tris, pH 7.6, and 0.85% NaCl) and blocked with 10% goat serum (Aurion, Wageningen, The Netherlands) and 5% BSA (Sigma Chemical Co.) in TBS for 1 h to prevent nonspecific binding of the antibodies. Subsequently, the slides were incubated overnight at 4 C with the primary antibody diluted 1:250 in TBS containing 10% goat serum. After washing in TBS, sections were incubated for 1 h at room temperature with a biotinylated goat antichicken Ig (Rockland, Gilbertsville, PA) diluted 1:250 in TBS containing 10% goat serum. The avidin-biotin complex reaction and counterstaining were performed as described above. The specificity of immunostaining was checked by using IgY purified from eggs obtained from the same chicken before immunization as a preimmune control.

	Results

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Detection of ERß messenger RNA (mRNA) expression in rat testes by in situ hybridization
ERß mRNA expression was studied in testes of rats of various ages using in situ hybridization with DIG-labeled riboprobes. In testes of fetal rats 16 days postcoitum (dpc) and in testes from 4-day-old rats (onset of spermatogenesis with division of gonocytes), a hybridization signal for ERß was seen in gonocytes, Sertoli cells, and interstitial cells (Fig. 1, a and c). However, at 4 days the signal in gonocytes was much stronger than at 16 dpc. In testes of 11-day-old rats (formation of adult-type Leydig cells) and 15-day-old rats (differentiation of Sertoli cells), a weak hybridization signal was found, evenly distributed over the cytoplasm of Sertoli cells and A spermatogonia. In the seminiferous tubules of testes from 60- to 70-day-old rats (complete spermatogenesis), the hybridization signal was most clear in pachytene spermatocytes from stages VII–XIV and in round spermatids (Fig. 1e). A weak hybridization signal was found in the basal compartment of the seminiferous tubules, in the cytoplasm of Sertoli cells, and in A spermatogonia. In the interstitial tissue no hybridization for ERß was found.

View larger version (149K): [in this window] [in a new window]   Figure 1. Rat testicular sections after in situ hybridization with the antisense (a, c, and e) and sense probes for ERß (b, d, and f); successive sections were used. At 16 dpc (a and b), a weak hybridization signal was seen in gonocytes (some indicated by arrowheads) and Sertoli cells. In the 4-day-old rat (c and d), hybridization signal was seen in gonocytes (indicated by arrowheads) and Sertoli cells. In adult rats (e and f), a clear hybridization signal was seen in pachytene spermatocytes (some indicated by asterisks) and round spermatids (some indicated by open arrows), whereas Sertoli cells and A spermatogonia show a weak hybridization signal. Bars = 25 µm.

Detection of ERß protein expression in rat testes
The cell type-specific distribution of ERß protein in rat testis was studied by immunohistochemistry with an ERß-specific chicken polyclonal antibody. In control experiments with this antibody, ERß immunoreactivity was detected in nuclei of the secretory epithelial cells of rat prostate (not shown), in agreement with the previously described high expression of ERß in these cells (18).

In fetal testes (16 dpc), a clear immunostaining for ERß was found in the nuclei of gonocytes, whereas immunostaining in nuclei of Sertoli cells and fetal Leydig cells was weak (Fig. 2a). In testes of 4-day-old rats, the immunostaining in the nuclei of gonocytes was even stronger (Fig. 2b). In testes of 11- and 15-day-old rats, only weak immunostaining was found in the nuclei of Sertoli cells and in A spermatogonia (Fig. 2, c and d). Other germ cells showed no immunostaining. Finally, in the adult testis, ERß was clearly immunolocalized in the nuclei of pachytene spermatocytes from epithelial stages VII–XIV and in round spermatids from stages I–VIII. In all epithelial stages, Sertoli cells and A spermatogonia had weak immunopositive nuclei (Fig. 2e), whereas no immunostaining was found in the nuclei of In and B spermatogonia, early spermatocytes, and adult Leydig cells.

View larger version (164K): [in this window] [in a new window]   Figure 2. Rat testicular sections after immunostaining for ERß (a–e) or with the preimmune IgY extract (f). At 16 dpc (a) and day 4 after birth (b), gonocytes (some indicated by arrowheads) and Sertoli cells (some indicated by solid arrows) showed nuclear staining for ERß. In 11-day-old (c) and 15-day-old rats (d), only A spermatogonia (some indicated by arrowheads) and Sertoli cells (some indicated by solid arrows) showed immunostaining for ERß in their nuclei. No staining was found in other germ cells. In testes of adult rats (e and f), immunostaining was found in A spermatogonia (indicated by arrowheads), Sertoli cells (some indicated by solid arrows), pachytene spermatocytes (some indicated by asterisks), and round spermatids (some indicated by open arrows). No immunostaining was found in In and B spermatogonia and early spermatocytes. Bars = 25 µm.

	Discussion

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In this report the cellular localization of ERß mRNA and protein is described during testicular development. Both the in situ hybridization and immunohistochemistry experiments revealed ERß expression in gonocytes, all type A spermatogonia, some pachytene spermatocytes, round spermatids, and, although weakly, in Sertoli cells of rat testes at all ages. It is important to note that ER is only expressed in fetal and adult type Leydig cells of the rat testis (16), and therefore, it appears that the ER subtypes are differentially expressed in rat testis (Table 1).

View this table: [in this window] [in a new window]   Table 1. ER, ERß, and aromatase activity in various cell types in the testis

At the start of spermatogenesis, gonocytes give rise to A spermatogonia (25). At all ages, the A spermatogonia are weakly positive for ERß in both the in situ hybridization and the immunohistochemical studies. Cell types in between A spermatogonia and pachytene spermatocytes show no ERß expression. From pachytene spermatocytes at epithelial stage VII up to round spermatids at stage VIII, the ERß mRNA and protein are clearly present. Interestingly, in mouse and rat testis, P450 aromatase is expressed from pachytene spermatocytes through round spermatids (10, 11), indicating that germ cells, in addition to Leydig cells, are a site of estrogen synthesis in adult rodent testis. The expression of ERß and aromatase in spermatocytes and round spermatids suggests a role for estrogens in early spermatid maturation. Indirect evidence supporting this hypothesis comes from studies showing reduced spermatid maturation after the injection of an aromatase inhibitor into rats, dogs, and monkeys (26).

At all ages, fetal as well as adult, Sertoli cells showed weak expression of ERß. Hence, proliferating as well as terminally differentiated Sertoli cells contain ERß. The expression of an ER in Sertoli cells has been previously suggested by Nakla et al. (13), who found binding of radiolabeled estrogen in Sertoli cell lines as well as in primary Sertoli cells, and by Saunders et al. (27), who found ERß-like immunoreactivity in Sertoli cells of adult rats. These and our observations indicate the possibility that estrogens play a role in the development of Sertoli cells and/or via their influence on Sertoli cell function in the regulation of the spermatogenic process. It is known that Sertoli cells in the neonatal as well as in the adult animal have aromatase activity (8, 9), which indicates that, as in the case of pachytene spermatocytes and spermatids, these cells can also synthesize estrogens locally from testosterone.

In mice injected with diethylstilbestrol or other xenoestrogens between days 9–16 of gestation, there is a increased risk of intraabdominal testes, sterility, and abnormalities of the urogenital tract in the male offspring (28, 29, 30, 31). In detailed studies, it was shown that exposure to estrogens or xenoestrogens alters the expression of steroidogenic factor I and cytochrome P450 17-hydroxylase/C17–20 lyase in the fetal rat testis (32, 33). A wide range of xenoestrogens interacts weakly with the ER and ERß protein and stimulates the transcriptional activity of ER and ERß in a trans-activation assay system (21). The expression of ER and/or ERß in various cell types, including germ cells and Sertoli cells, of the fetal rat testis indicates that both ER subtypes could mediate the aberrant developmental and reproductive effects of diethylstilbestrol or other xenoestrogens. Careful comparison of the effects of xenoestrogens on testis development and function in currently available ER knockout mice (34, 35, 36) and ERß knockout mice, under construction in various laboratories, could be very informative in this regard.

Finally, preliminary analysis of mice deficient in aromatase (ArKO) because of targeted disruption of the P450-aromatase gene revealed enlargement of the male accessory sex glands and reduced rate of litter siring with advancing age (37). Detailed analysis of the morphology and function of the testes from these mice might provide additional insights into the role of estrogens in the testis.

	Note Added in Proof

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While this manuscript was under editorial review, a paper was published that described the immunohistochemical localization of ERß in the nuclei of Leydig cells, Sertoli cells, and gonocytes in the fetal rat testis and in the nuclei of Sertoli cells, spermatogonia, and pachytene spermatocytes in the immature and adult rat testis (Saunders PTK, Fisher JS, Sharpe RM, Millar MR 1998 Expression of oestrogen receptor beta (ERß) occurs in multiple cell types, including some germ cells, in the rat testis. J Endocrinol 156:R13–R17). Another recent paper described the detection of ERß-like immunoreactivity in the cytoplasm and nuclei of Leydig cells in the adult mouse testis, while Sertoli cells and spermatocytes were negative (Rosenfeld CS, Ganjam VK, Taylor JA, Yuan X, Stiehr JR, Hardy MP, Lubahn DB 1998 Transcription and translation of estrogen receptor-ß in the male reproductive tract of estrogen receptor- knock-out and wild-type mice. Endocrinology 139:2982–2987).

	Acknowledgments

 
We are grateful to Y. Ekendahl (Department of Medical Nutrition) for immunization and care of the laying hens, to H. J. G. van de Kant (Department of Cell Biology) for skillful technical assistance, to R. Scriwanek and A. N. van Rijn (University of Utrecht, Utrecht, The Netherlands) for preparing the photographs, and to O. Engström, T. Bonn, and M. Carlquist (KaroBio AB, Huddinge, Sweden) for rat ERß ligand-binding domain protein.

	Footnotes

 
¹ This work was supported by grants from the Swedish Medical Research Council (MFR K98–04P-12596–01A) and the Loo och Hans Ostermans Stiftelse (to G.G.J.M.K.) and a grant from the European Union (EU-PL95–1223; to B.v.d.B., P.T.v.d.S., and J.-Å.G.).

Received April 21, 1998.

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