Localization of oestrogen receptors alpha and beta ... - Semantic Scholar

Molecular Human Reproduction Vol.7, No.6 pp. 497–503, 2001

Localization of oestrogen receptors alpha and beta in human testis Sirpa Ma¨kinen1,2, Sari Ma¨kela¨1,3, Zhang Weihua1, Margaret Warner1, Bjo¨rn Rosenlund2, Saija Salmi3, Outi Hovatta2,4 and Jan-Åke Gustafsson1 Karolinska Institutet, 1Department of Medical Nutrition and 2Department of Clinical Science, Division of Obstetrics and Gynaecology, Huddinge University Hospital, S-141 86 Huddinge, Sweden and 3University of Turku, Institute of Biomedicine, Department of Anatomy, Kiinamyllynkatu 10, FIN-20520 Turku, Finland 4To

whom correspondence should be addressed.

Cellular localization of oestrogen receptor alpha (ERα) and beta (ERβ) proteins were studied in human testis samples using immunohistochemistry, and the expression of the corresponding mRNA was examined with reverse transcription-polymerase chain reaction (RT-PCR). Seven men, aged 28–48 years, who underwent diagnostic testicular biopsy because of azoospermia or to give spermatozoa for intracytoplasmic injection for infertility treatment, donated tissue for the study. One of them had anejaculation but normally functioning testes, and one was diagnosed as having Sertoli cell-only syndrome (SCOS). In addition, expression of ERβ protein was examined in one testis sample obtained from a man undergoing a sex change operation. Strong ERβ immunoreactivity was detected in the nuclei of spermatogonia, spermatocytes and early developing spermatids. Elongating spermatids, mature spermatozoa, Sertoli and Leydig cells were all negative for ERβ. The presence of ERβ protein was confirmed in Western analysis. With RT–PCR, both wild-type ERβ and ERβcx, the isoform which represses wild-type ER function, were easily detected. In most cases, ERβcx mRNA was more abundantly expressed than wild-type ERβ. The patient with SCOS expressed neither ERβ isoform. Neither ERα protein nor ERα mRNA was detected in any of the samples. We conclude that in the human testis, ERβ is likely to be the ER that mediates the effects of oestrogen. Key words: human/oestrogen/oestrogen receptor/spermatogenesis/testis

Introduction The role of oestrogen in the regulation of gonadotrophin secretion from the pituitary in males has been known for some time. What has been obscure is the local function of oestrogen in the testis, an organ which synthesizes oestradiol and where the concentration of oestradiol can be very high (Carreau et al., 1999). During the past decade, several experimental studies have clearly indicated an essential role for endogenous oestrogens and oestrogen receptors (ER) in the regulation of testicular function. Mice lacking a functional aromatase gene (aromatase knock-out, ArKO mice), and thus lacking endogenous oestrogen production, are initially fertile, but show severe impairment in spermatogenesis later in life, and develop Leydig cell abnormalities (Robertson et al., 1999). Oestrogen receptor α (ERα)-deficient ERKO mice also become infertile with age (Eddy et al., 1996), but morphologically show a testicular phenotype very different from ArKO mice. ERKO mice have impaired fluid absorption in the efferent ducts, which leads to fluid accumulation and back pressure in testis, and subsequently, atrophy of seminiferous tubules (Hess et al., 1997). © European Society of Human Reproduction and Embryology

Transplantation of germ cells from ERKO to wild-type mice testes has shown that the presence of ERα in sperm cells is not required for development, viability or function of spermatozoa (Mahato et al., 2000). Male mice lacking functional oestrogen receptor β (ERβ) (BERKO mice) are reported to be fertile (Krege et al., 1998), but detailed analysis of their testicular phenotype has not yet been reported. In the human male, the role of oestrogens in the testis is less clear. The young male patient with inactive ERα, the only case reported to date, had apparently normal testes, normal sperm density but decreased sperm viability (Smith et al., 1994). Patients with aromatase deficiency can have enlarged or small testes and may have oligozoospermia and immotile spermatozoa (Morishima et al., 1995; Caranini et al., 1997). Some of the variability in the phenotype of aromatase-deficient human males may be due to the presence of exogenous oestrogens such as dietary phyto-oestrogens or xeno-oestrogens in the environment. A recent study with cultured seminiferous tubules has suggested that human germ cells may respond directly to oestrogens; withdrawal of serum and hormones 497

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caused germ cell apoptosis and this apoptosis could be inhibited by oestradiol (Pentika¨ inen et al., 2000). Although it is likely that oestradiol is necessary for normal testicular function, the differences in phenotypes of the ARKO, ERKO and BERKO testes and those of the patients who lack either ERα or aromatase have raised even more questions about the exact function of the two oestrogen receptors in the testis and the differences in oestrogen responsiveness between the rodent and human testis. In rats, ERα is confined to testicular interstitial Leydig cells, but ERβ is expressed in multiple cell types, Sertoli cells, peritubular cells, fetal Leydig cells and gonocytes, spermatogonia and most pachytene spermatocytes (Saunders et al., 1998). In adult rats, Leydig cells are the sites of oestradiol production and in fetal rats Sertoli cells produce oestrogen. Oestradiol limits development and growth of the Leydig cell population (Abney, 1999). Both ERα and ERβ were found in early meiotic spermatocytes and elongating spermatids (Pelletier et al., 2000). Information about the expression of ER subtypes in human testis is very limited. ERβ mRNA has been localized to spermatogenic cells, while the interstitium was negative for ERβ (Enmark et al., 1997). Recently, it was reported (Taylor and Al-Azzawi, 2000), using immunolocalization, that ERβ protein is present in all testicular cells, while ERα protein was observed in Sertoli and Leydig cells. On the other hand, another recent study (Pelletier and El-Alfy, 2000), using the same antibody, reported that only Sertoli cells and Leydig cells were immunopositive for ERβ. The role of oestrogen and ERs in male fertility remains, therefore, to be clarified. The issue of the functions of oestrogen receptors in the testis is further complicated by the presence, in the human testis, of a C-terminal truncated ERβ splice variant, ERβcx, which does not bind 17β-oestradiol, and acts as a dominant negative regulator of ERα in vitro (Ogawa et al., 1998a). The specific functions of ERβcx in human testis are not yet known. There is obviously a need for more information about ER expression patterns in diseased as well as age-matched normal human testis. In this study, ER distribution in eight human testicular biopsies was examined. Because of the difficulty in obtaining normal human testis samples, we have used biopsy material from men with infertility either of known or unknown aetiology as well as biopsy material from a patient whose testes should be normal because his infertility was secondary to neuromuscular dysfunction, multiple sclerosis. For comparison, expression patterns of ERα and ERβ in adult mouse testis were determined by immunohistochemistry in parallel with the human samples.

Materials and methods Tissue samples Human testicular tissue was obtained from seven men, aged 28– 48 years, who underwent diagnostic testicular biopsies because of azoospermia or who underwent testicular sperm extraction for intracytoplasmic sperm injection. Patient 1 proved to have Sertoli cell-only syndrome (SCOS), patient 5 had anejaculation but normal sperm production, patient 6 had obstructive azoospermia of unknown

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origin, and the others had hypospermatogenesis. Samples of human testicular tissue were frozen immediately in cryotubes by placing them in liquid nitrogen. In addition, a sample from a normal human testis cDNA pool (Clontech, Palo Alto, CA, USA) was used for RT– PCR. One larger sample of the human testis, sufficient for sucrose gradient binding assay and Western blotting, was obtained from a 39 year old man, who underwent a sex change operation, and had been treated with oestrogen for 3 years prior to the operation. Normal adult mouse testis samples were obtained from two 4 month old mice, one testis of each was frozen immediately and the other one was embedded in paraffin wax. Before sectioning, the tissue samples were brought to optimal cutting temperature (–19°C) and fixed to the holder with Tissue Tek (Histolab, Gothenburg, Sweden). The 12 µm tissue sections were mounted on gelatine-coated slides and air-dried for 30 min. The slides were then fixed with ice-cold methanol (3 min) and acetone (3 min), air-dried for 30 min and stored in a freezer (–20°C) or immunostained immediately. We also used 5 µm sections of formalin-fixed, paraffinembedded testis tissue samples in experiments with ERα antibody in order to attain optimal morphology and staining. Paraffin sections were deparaffinized in xylene, dehydrated in graded alcohols, and antigen was retrieved by microwaving the sections in 0.01 mmol/l sodium citrate. Endogenous peroxidase was blocked, as in frozen sections (below). Antibodies used in immunohistochemistry For ERβ, a polyclonal chicken ERβ 503 antibody was used at dilutions of 1:100 and 1:500. For simplicity, this antibody is designated here as ERβ 503 antibody. The 503 protein used for immunization is human ERβ1, modified in its ligand-binding domain (LBD) by insertion of the rat 18 amino acid sequence (Ogawa et al., 1998b). This modified protein was expressed in SF9 cells and purified from the nuclear extracts. Antibodies were raised in chickens and yolk immunoglobulins (IgY) were purified by PEG precipitation and DE52 (ion exchange) chromatography. The specificity of this antibody in immunohistochemistry with rat tissue has been previously described (Saji et al., 2000). Two mouse monoclonal antibodies against human ERα (NCL-ER-6F11; Novocastra, Newcastle, UK; and ID5 clone; Dako, Glostrup, Denmark) were used at dilutions of 1:100 and 1:150. For mouse tissue, rabbit polyclonal antibody against ERα (MC-20; Santa Cruz Inc., California, USA) was used at a dilution of 1:400. As secondary antibodies, a peroxidase-conjugated rabbit anti-mouse IgG antibody (Sigma, Missouri, USA) at a dilution of 1:200 was used for ERα antibody from Dako and Novocastra, and a peroxidaseconjugated rabbit anti-chicken IgG (Sigma) at a dilution of 1:1000 was used against ERβ 503 antibody. For ERα MC-20 antibody, peroxidase-conjugated goat anti-rabbit (Sigma) at a dilution of 1:400 was used. Immunohistochemical staining The frozen tissue slides were thawed for 30 min in a closed box before fixing in 4% paraformaldehyde for 10 min. After washing with phosphate-buffered saline (PBS), endogenous peroxidase was blocked using 1% hydrogen peroxidase in 50% methanol/50% PBS for 15 min and washed with PBS. Non-specific binding of the secondary antibody was reduced by incubating the slides in 10% rabbit serum or goat serum (for MC-20 antibody). After washing with PBS, the slides were treated with 0.5% Triton X-100 in PBS for 5 min at room temperature and washed with PBS. The sections were incubated overnight in a humidified box (4°C) with the primary antibody diluted in PBS with 3% bovine serum albumin (BSA). The specificity of immunostaining for ERβ was controlled by using preadsorbed ERβ 503 antibody. Preadsorbed antibody was prepared

Oestrogen receptors in human testis by incubating the antibody for 12 h at 4°C with ER-β protein coupled to activated Sepharose. A molar excess of ERβ protein was added to 1 ml activated Sepharose (50 µmol active group/ml). As another control for the adsorption, BSA was also coupled to Sepharose and used for preadsorption of the antibody. The sections were washed in PBS and then incubated with the secondary antibody diluted in PBS with 3% human serum albumin (HSA) for human tissue or 2% mouse serum for mouse tissue for 1 h at room temperature, and washed in PBS. Colour developing was carried out using 3,3⬘-diaminobenzidine tetra-hydrochloride (DAB, Liquid; Dako A/S, Glostrup, Denmark) according to the manufacturer’s protocol. After colour developing and rinsing with distilled water, the sections were counterstained slightly with Mayer’s haematoxylin, dehydrated in graded alcohols, cleared in xylene and mounted using Pertex (Histolab). Sections of frozen and paraffin-embedded human and mouse prostate were used as positive controls for ERβ and detection without primary antibody was used as another negative control. For ERα, formalin-fixed paraffin sections (5 µm) of human uterus and mouse uterus were used as positive controls. Detection of ERβ isoform (wild-type, ERβWT, and C-terminal truncated, ERβcx) and ERα mRNA by RT–PCR Total RNA from individual patients was isolated by RNA WIZ (Ambion, Austin, TX, USA) according to the protocol provided by the company. For the RT reaction, 5 µg of RNA together with 2.2 µmol/l oligo (dT) was denatured at 70°C for 5 min. The RNA was added to a mixture of RT reaction buffer (Gibco), dNTPs (each 1 mmol/l), 50 units of RNASIN, and 20 units of SuperScript II reverse transcriptase (Gibco) in a final volume of 30 µl. The reaction was allowed to proceed for 1 h at 46°C, after which the enzyme was inactivated at 95°C for 10 min. The reverse-transcribed RNA was stored at –20°C for further use. Two negative controls were included: one without the RNA sample and one without the reverse transcriptase. The PCR was run on a Gene Amp PCR system 2400 (Perkin Elmer, Norwalk, CT, USA) and amplified in 35 cycles by incubation at 95°C for 30 s, 56°C for 30 s, 72°C for 60 s, and final incubation at 72°C for 3 min. The reaction mixture consisted of 3 µl of cDNA, PCR buffer, 0.2 mM dNTPs, primers (each 1 pmol/l) and 0.5 units of Taq polymerase in a total volume of 50 µl. The PCR products were loaded on 1.5% agarose gel with 1⫻tris acetate EDTA (TAE) as buffer. The PCR product for ERβ wild-type (ERβWT) was 268 bp, for ERβcx 214 bp, and for ERα 291 bp. The primers used were: forward primer shared by both ERβWT and ERβcx, 5⬘CGATGCTTTGGTTTGGGTGAT-3⬘; reverse primer for ERβWT, 5⬘GCCCTCTTTGCTTTTACTGTC-3⬘; reverse primer for ERβcx, 5⬘CTTTAGGCCACCGAGTTGATT-3⬘. Primers used for ERα were: forward, 5⬘- AATTCAGATAATCGACGCCAG-3⬘; reverse, 5⬘GTGTTTCAACATTCTCCCTCCTC-3⬘. Preparation of cytosol for sucrose density gradient centrifugation Tissue, frozen in liquid nitrogen, was pulverized in a dismembrator (Braun Melsungen) for 45 s at 1800 rpm. Pulverized tissue was added to a buffer composed of 10 mmol/l Tris chloride, pH 7.5, 1.5 mmol/l EDTA and 5 mmol/l sodium molybdate. For MCF-7 cells the suspension was 100 µg and for testis it was 1 g tissue per ml buffer. Cytosol was obtained by centrifugation of the homogenate at 75 000 g for 1 h in a 70Ti rotor at 4°C Sucrose density gradient assay and Western blotting Gradients were done as previously described (Jensen et al., 1968). Testis extracts were incubated for 2 h at 0°C with 10 nmol/l [3H]oestradiol, either in the presence or absence of an excess of radioinert

oestradiol and the bound and unbound steroids were separated with dextran-coated charcoal. Sucrose density gradients [10-30% (w/v) sucrose] were prepared in a buffer containing 10 mmol/l Tris–HCl, 1.5 mmol/l EDTA, 1 mmol/l α-monothioglycerol (Sigma), 10 mmol/ l KCl. Samples of 100 µl were layered on 4 ml gradients and centrifuged for 16 h at 300 000 g in an SW-60Ti rotor (Beckman Instruments, Palo Alto, CA, USA) at 4°C in a Beckman L-70K ultracentrifuge. Successive 100 µl fractions were collected from the bottom by paraffin oil displacement, using a collector of our own design, and assayed for radioactivity by liquid scintillation counting. For Western blotting, fractions were first precipitated with TCA, and the precipitate was resuspended in methanol. Samples were placed on dry ice for 30 min and the protein recovered by centrifugation. Pellets were dissolved in sodium dodecyl sulphate (SDS) sample buffer and proteins resolved by SDS-polyacrylamide gel electrophoresis in gradient gels 4–20% (Novex), with a Tris-glycine buffer system. Transfer to PVDF membranes was done in a Tris-glycine buffer. After 1 h blocking with blocking buffer (10% skim milk in PBS with 0.1% NP-40) at room temperature, the membranes were incubated with the primary antibody diluted 1:3000 in blocking buffer at 4°C, overnight. This was followed by 1 h washing in blocking buffer, and then incubation of HRP-conjugated secondary goat anti-rabbit IgG (Dako) diluted 1:10 000 in blocking buffer for 1 h at room temperature. After sequential washing with blocking buffer, PBS with 0.1% NP40 and PBS, signals were developed using ECL plus (Amersham Pharmacia). The primary antibody was 1633, a polyclonal rabbit antibody, raised in this laboratory, against the complete ERβ ligandbinding domain (LBD), and is designated here as ERβ LBD antibody. The use of this antibody in Western blotting has been previously described (Saji et al., 2000).

Results Immunohistochemical localization of ERβ protein in human testis Intensive staining for ERβ was seen in the nuclei of spermatogonia, spermatocytes and early developing spermatids, but not in elongating spermatids (Figure 1A, B). Sertoli and Leydig cells were negative (Figure 1A-D). The very weak staining in some Sertoli cells (Figure 1D) was not regarded as specific after comparison with the control sections. The lack of staining in Sertoli cells was very clearly seen in the subject with SCOS (Figure 1C). Positive controls were frozen and paraffin sections of human prostate showed intense nuclear staining in the epithelium (Figure 1E, F). Preabsorption of the antibody with the antigen used for immunization completely abolished staining (Figure 1G). Controls from which the primary antibody had been omitted were also negative. Immunohistochemical localization of ERα protein in human testis No staining for ERα was observed in any cells of human testis (Figure 2A). The antibodies and the immunohistochemical methods used were not responsible for the negative results with the ERα antibodies since there was a strong nuclear staining in human uterus (Figure 2C). Negative controls from which the primary antibody had been omitted showed only the counterstain (Figure 2B, D). 499

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Figure 1. Immunohistochemical localization of oestrogen receptor β (ERβ) protein in human testis. A frozen section (A, B) stained with ERβ 503 antibody (dilution 1:100). A positive reaction (brown colour, indicated with black arrows) is seen in the nuclei of spermatogonia (SG), spermatocytes (SC), and early developing spermatids (SP). Sertoli cells (SRC, open arrowheads), Leydig cells (LC) and elongated spermatids (ESP) are negative. A frozen section from patient 1 with Sertoli cell-only syndrome (C) shows no immunoreaction in testis, except for very few possible germ cells. The Sertoli cells are clearly negative. Paraffin section of human testis (D) shows same staining pattern as frozen sections. Positive nuclear staining can be seen in frozen (E) and paraffin (F) sections of human prostate. The negative control of human testis (G) with preabsorbed ERβ 503 antibody shows no immunoreaction. Original magnifications: A, C-G ⫻400; B ⫻100.

Immunohistochemical localization of ERα and ERβ protein in adult mouse testis In the mouse testis, nuclear staining for ERβ was found in Sertoli cells and early spermatogenic cells to the spermatocyte stage (Figure 3A, B). For ERα, a distinct positive immunoreaction was seen in Leydig cell nuclei, while the other cells were negative (Figure 3E, F). Appropriate nuclear staining was observed in positive controls, mouse prostate (ERβ) and mouse uterus (ERα) (Figure 3C and G). Negative controls showed the counterstain only (Figure 3D, H). RT–PCR analysis of ERβWT and ERβcx mRNA In order to test whether the expression of the ERβ protein was paralleled by corresponding expression of the mRNA, RT– PCR was done on RNA extracted from the same samples. The RT–PCR results are shown in Figure 4. With the primers that can amplify both ERβWT and ERβcx, both ERβ isoforms were detected. ERβcx was observed in all but one case, while ERβWT was detected in only three cases. Patient 5, who had normal but quantitatively reduced spermatogenesis, showed 500

intense bands for both ERβ isoforms, similarly to the normal testis cDNA (Clontech) used as a control. In patient 1, who had SCOS, neither ERβWT nor ERβcx mRNA was detected. In patient 4, there was a very low level of ERβcx mRNA, and ERβWT was not observed. He had almost SCOS, but had some spermatogonia and patchy spermatogenesis. We could not detect ERα mRNA in any of these testicular samples by RT–PCR (data not shown). Detection of ERβ protein in cytosols from human testis by sucrose density gradient centrifugation and Western blot analysis In low-salt sucrose gradients, the cytosol receptor of the human testis showed a peak of tritiated oestradiol binding (Figure 5A) at the 4S region. As expected, the cytosols prepared from MCF-7 cells, in which the receptor is essentially all ERα, showed a peak at the 8S region (Figure 5A). Western blot analysis of the testis cytosol confirmed the presence of ERβ protein in fractions representing the 4S peak (Figure 5B). In addition, we have detected another variant of ERβ, which does

Oestrogen receptors in human testis

Figure 2. Immunohistochemical localization of oestrogen receptor α (ERα) protein in human testis. No immunoreaction is seen in any testicular cell type using mouse monoclonal antibody against ERα (A), whereas a strong positive immunoreaction is seen in human uterus (C) stained with the same antibody. The negative controls, from which primary antibody has been omitted, show only the blue counterstain (B, testis; D, uterus). Original magnifications: A, B ⫻200; C, D ⫻400.

not bind oestradiol, but is recognized by our antibody directed against ERβ LBD. This variant was also detected using another antibody directed against ERβins (data not shown). ERβins is a variant with an 18 amino acid insertion in the LBD, and earlier reported to be defective in oestradiol binding (Maruyama et al., 1998).

Discussion We observed a distinct expression of ERβ protein in human germ cells, from spermatogonia to round spermatids, while Sertoli cells and interstitial cells were negative. Immunohistochemical localization of ERβ protein reported here is similar to that of expression of ERβ mRNA, shown earlier in our laboratory. With in-situ hybridization, ERβ mRNA was localized in primary spermatocytes and round spermatids (Enmark et al., 1997). In the current study, we found that, in addition, spermatogonia expressed ERβ protein. This difference is probably because the histological identification of spermatogonia is easier in immunohistochemistry than in-situ hybridization. On the other hand, our results differ considerably from those reported by another group (Taylor and Al-Azzawi, 2000). They used two polyclonal antibodies developed against Nand C-terminal regions of human ERβ, and showed positive immunoreaction in practically all testicular cells. This difference could be due to different antibodies used, or the postmortem tissue that they used may already have undergone biochemical changes. A more recent report (Pelletier and El-Alfy, 2000) using the same antibody against the N-terminal region of human ERβ shows completely different results. They observed few positive cells inside the tubuli, interpreted as Sertoli cells, while germ cells were negative. Immunopositive cells, interpreted as Leydig cells, were observed in interstitium.

Figure 3. Immunohistochemical localization of oestrogen receptor β (ERβ) and ERα in adult mouse testis. A frozen section shows strong staining with ERβ 503 antibody in Sertoli cells and early spermatogenic cells, while Leydig cells were negative (A, B). Positive control staining from mouse prostate (C) shows intense nuclear staining. No staining can be seen in mouse testis using preabsorbed ERβ-503 antibody (D). ERα protein is seen only in mouse Leydig cells (E, F). Positive control staining from mouse uterus (G) shows strong nuclear immunoreaction. Negative controls show the blue counterstain only (D, frozen section of mouse testis incubated with preabsorbed ERβ-503 antibody; H, paraffin section of mouse uterus with the omission of primary antibody). Original magnifications: A, C, D, E, G, H ⫽ ⫻200, B, F ⫽ ⫻400.

We have confirmed our immunohistochemical findings using sucrose density gradient centrifugation and Western blotting was used to confirm the presence of ERβ protein, which binds oestradiol in human testis. The expression pattern in adult human testis was only partially similar to that described in adult rodent testis. In the mouse testis, we found ERβ in Sertoli cells and early spermatogenic cells. With an antibody directed against the LBD of rat ERβ, adult rat Sertoli cells, pachytene spermatocytes, round spermatids and type A spermatogonia, but not type B spermatogonia and early spermatocytes, have been shown to express ERβ protein (van Pelt et al., 1999). Similar findings have been reported (Saunders et al., 1998) with a polyclonal antibody raised against a peptide from rat ERβ 501

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Figure 4. Reverse transcription–polymerase chain reaction analysis of oestrogen receptor β (ERβ)WT (A) and ERβcx (B) mRNA in human testicular tissue samples. Patients 1–7 are indicated by P1P7 respectively. P1 is the subject with Sertoli cell-only syndrome. P4 has patchy spermatogenesis. NT is normal testis cDNA from Clonetech, used as a positive control, and showing intense bands for both ERβWT and ERβcx.

Figure 5. Sedimentation analysis of tritiated oestradiol binding by receptors in cytosol from human testis (A). Human testis showed a peak at the 4S region, while MCF-7 human breast cancer cells show a peak at 8S. In Western blot analysis of testicular cytosol (B) oestrogen receptor β (ERβ) protein (size 60–62 kDa, indicated by an arrow on the right) was detected with ERβ LBD antibody from fractions representing the 4S peak. The numbers of the fractions are indicated above the lanes. The positive control was recombinant ERβ 503 protein (first lane on the left), giving an intense band of 55 kDa (indicated by an arrow on the left).

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D-region, with the exception that type B spermatogonia were also positive. In a recent study (Pelletier et al., 2000) with an antibody directed against amino acids 54–71 of rat ERβ, Sertoli cells, but not spermatogenic cells, were positive in adult rat testis. In our study, and all three previously published studies, adult rat Leydig cells were negative for ERβ. We found no ERα protein or mRNA in human testis. Furthermore, in sucrose density gradient centrifugation assay, no peak of tritiated oestradiol binding at 8S characteristic of ERα was seen. This is clearly different from rodent testis, where Leydig cells have been found to express ERα (Saunders et al., 1998). Other investigators have demonstrated this for adult rat tissue (Fisher et al., 1997; Pelletier et al., 2000), and we observed the same for adult mouse tissue in the present study. However, ERα has also been detected in round spermatocytes and spermatids of adult rat testis (Pelletier et al., 2000). These discrepancies could be due to the many problems associated with ER immunohistochemistry, as has been discussed in the context of rat brain (Butler et al., 1999; Leng, 1999). The differences in the cellular distribution patterns of ER subtypes between human and rodent testis may partly be technical, i.e. due to use of different antibodies and different fixatives in the various laboratories. Results can also be influenced by the method used for tissue preparation, i.e. paraffin-embedded versus frozen sections. There may also be real differences in the ER expression patterns between rodents and humans. Furthermore, there might also be variation between species in expression of different ERβ isoforms (Petersen et al., 1998). Our RT–PCR analysis demonstrated the presence of both ERβWT and a splice variant, ERβcx mRNA, in human testis. ERβcx was detected in all but one sample, while ERβWT was detected only in three out of seven samples. Furthermore, in most of our cases, ERβcx mRNA appeared to be more abundant than ERβWT. At present, we have no explanation why the ERβWT mRNA was not observed in all samples. In theory, this could be related to some underlying pathology within these samples. The only case in which both ERβ mRNA were undetectable was a case with Sertoli cell-only syndrome, i.e. lacking spermatogenic cells. This may suggest that ERβ gene expression could indeed be associated with spermatogenesis. ERβcx has been previously described in the normal human testis cDNA libraries (Moore et al., 1998; Ogawa et al., 1998a) and our study indicates that this isoform is expressed in diseased as well as normal testis. It has been shown that ERβcx works as silencer for ERα, when these receptors are co-expressed (Ogawa et al., 1998a). However, we did not detect ERα in any of our samples, and futher studies on the role of ERβcx in human testis are warranted. The role of ERα in the development and function of spermatozoa in the rodent and human testis is uncertain. ERα knockout mice (ERKO mice) are infertile because of disrupted spermatogenesis. This, however, is secondary, and is a result of abnormal fluid absorption in the efferent ducts. Continuous secretion of fluid by Sertoli cells leads to accumulation in the efferent ducts, rete testis and finally in the seminiferous tubules, causing pressure within the testis. This pressure results in disruption of spermatogenesis (Hess et al., 1997). Transplanted

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germ cells from infertile ERKO mice developed into functional spermatozoa within the reproductive system of wild-type recipient males (Mahato et al., 2000). This supports the theory that ERα does not have a significant role in spermatogenic cells. It has been shown recently that oestrogen has a dosedependent mitogenic effect on gonocytes isolated from 3 day old rats and cultured without Sertoli cells. The effect of oestrogen on proliferation was inhibited by an ER antagonist, which suggests that the effect was mediated by binding of oestrogen to its receptor (Li et al., 1997). There is ERβ but no ERα in rat gonocytes. This has led to the suggestion that the mitogenic effects of oestrogen are mediated by ERβ (van Pelt et al., 1999). Based on reports regarding aromatase deficiency in men (Caranini et al., 1997; Faustini-Fustini et al., 1999), oestrogens appear to have a role in spermatogenesis. Two brothers with aromatase deficiency had severe disturbance in sperm production. However, an ERα-deficient male has apparently normal testes and normal sperm density (Smith et al., 1994), indicating that ERα may not be critical for spermatogenesis in humans. This is consistent with our observation of the lack of ERα expression in adult human testis. Therefore, it is more likely that ERβ, which is widely distributed in spermatogenic cells, could be the receptor mediating the effects of oestrogen in human testis.

Acknowledgements This study was supported by grants from the Swedish Cancer Fund and from KaroBio AB. We thank Nicholas Bolton for revising the language. We are extremely grateful to Christina Thulin-Andersson for skilful technical assistance, and Dr Victoria Keros from the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine for one of the testis samples.

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