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Original Article |
MRC Human Reproductive Sciences Unit (T.L.G., L.L.L.R., R.A.A., P.T.K.S.), Centre for Reproductive Biology, Edinburgh EH16 4SB; and School of Biological and Molecular Sciences (N.P.G.), Oxford Brookes University, Headington, Oxford OX3 0PB, United Kingdom
Address all correspondence and requests for reprints to: Dr. Philippa Saunders, MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh Academic Centre, 49 Little France Cresent, Edinburgh EH16 4SB, United Kingdom. E-mail: p.saunders{at}ed.ac.uk.
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Abstract |
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, ERß, and ERß2 variant) in human fetal testes at 12–19 wk gestation. ER, mRNA, and protein were not detected in any of the fetal testes. In contrast, using an antibody directed against the hinge domain of ERß expression was detected in multiple testicular nuclei. RT-PCR with primers specific for full-length wild-type ERß (ERß1) or the ERß2 variant formed by splicing of an alternative eighth exon, was performed on whole-tissue extracts and materials recovered by laser capture and revealed that mRNAs for both isoforms were expressed. Immunohistochemistry with isotype-specific monoclonal antibodies showed that ERß1 was low/undetectable in gonocytes, whereas these cells expressed the highest levels of ERß2, compared with other testicular cell types. Both ERß1 and ERß2 were detected in some but not all Sertoli cells, peritubular cells, and other interstitial cells including those tentatively identified as Leydig cells. Our immunohistochemical results demonstrate that during the second trimester, some but not all somatic cells within the human fetal testis express wild-type ERß (ERß1) protein and/or the variant isoform of ERß (ERß2) that lacks amino acids essential for binding of estradiol. ERß2 protein was readily detectable in fetal gonocytes, whereas ERß1 was not. We did not detect expression of ER. The expression of ERß2, a variant proposed act as a dominant negative receptor, might prevent estrogen action in gonocytes. We suggest that during this period of fetal life, estrogenic ligands are most likely to act on somatic cells that contain ERß1 protein. |
Introduction |
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One possible etiology for the increase in the incidence of testicular germ cell cancers, as well as male reproductive abnormalities, could be disturbances in the hormonal environment of the developing fetus, one aspect of which might be elevated exposure to estrogens (1, 15). Further support for this idea has been provided by data showing testicular cancer appears to be more common in individuals with disorders of the male reproductive tract including intersex, cryptorchidism, and androgen insensitivity syndrome (16, 17, 18). Studies on twins have suggested that abnormally high estrogen levels in women during pregnancy may increase the risk of sons developing testis cancer (19, 20). Li et al. (21) have reported that incubation of gonocytes from d 3 rat testes with estradiol activated proliferation in vitro, suggesting that in rodent models estrogens can have direct effects on the function of immature germ cells.
Estrogen action is mediated via high-affinity intracellular receptors expressed in target tissues. Following ligand binding the receptors undergo a conformational change, dimerize, bind regulator regions within genes, recruit cofactors, and thereby regulate the transcription of target genes (22, 23). Two subtypes of estrogen receptors (ERs), commonly known as ER (NR3A1) and ERß (NR3A2), have been cloned from human tissues (24, 25, 26). The two receptors are encoded by genes located on different chromosomes (27) but like other members of the steroid receptor superfamily share a common arrangement of five structure-function domains, denoted A-F (28). In 1998 a novel human ERß variant, named ERßcx, was identified in a human testis cDNA library (29) (accession AB006589). In separate experiments Moore et al. (30) identified a number of mRNAs in human tissues, including testis, which encoded hERß isoforms including one identical to hERßcx that they named hERß2. To avoid confusion, the original hERß protein identified as the homolog of the rat ERß will be referred to as hERß1 and the hERßcx/hERß2 splice variant will be referred to as hERß2 for the rest of this article.
We and others (31, 32) have shown previously that adult human testicular germ cells express ERß but not ER. In fetal rat testes, ERß protein has been localized to multiple cell types including fetal germ cells (gonocytes) (33, 34). Limited data have been presented for human fetal testes. For example, Brandenberger et al. (35) have demonstrated the presence of hERß mRNA but did not determine in which cell type the mRNA was expressed. Takeyama et al. (36) reported expression of ERß in cells within the human fetal testis when they used tissue from a single fetus obtained at 20 wk gestation.
The aim of the present study was to establish whether human fetal germ cells are a potential target of estrogen action. To address this issue, we used RT-PCR and immunohistochemistry to examine pattern of expression of ERs in human fetal testes obtained from 13–19 wk gestation. Because we have recently demonstrated that both wild-type ERß1 and the variant isoform, ERß2, are expressed in adult human germ cells (37), the initial study was extended using laser capture microscopy and immunohistochemistry with isoform-specific monoclonal antibodies to determine whether one or both isoforms were also expressed in the fetal germ cells.
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Materials and Methods |
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Human fetal testes were obtained following medical termination of pregnancy during the second trimester (up to 19 wk). Women gave consent according to national guidelines (38), and the study was approved by the Lothian Pediatrics/Reproductive Medicine Research Ethics Subcommittee. Termination of pregnancy was induced by treatment with mifepristone (200 mg orally), followed by prostaglandin E1 analog (Gemeprost, Beacon Pharmaceuticals, Tunbridge Wells, UK) 1 mg given every 3 h in the vagina. None of the terminations were for reasons of fetal abnormality, and all fetuses appeared morphologically normal. Gestational age was determined by ultrasound and confirmed by subsequent direct measurement of foot length; 30 specimens were examined.
Testes were either fixed for immunohistochemical analysis and laser capture microdissection or snap frozen and stored at -70 C before recovery of RNA. Fixation was carried out in Bouin’s fluid for 5 h, followed by transfer to 70% ethanol before processing into paraffin using standard methods (39).
Antibodies
A rabbit polyclonal antibody directed against anti-Mullerian hormone (AMH) (40) was a kind gift from Drs. Rodolfo Rey and Natalie Josso (INSERM 493, Montrouge, France). Mouse monoclonal antihuman ER was purchased from DAKO Corp. (clone 1D5, Cambridge, UK). ERß proteins were detected using three different antibodies. In preliminary experiments immunohistochemistry was carried out with an affinity-purified sheep polyclonal antiserum directed against hERß peptide P4 (hinge domain) prepared as described (41). In subsequent studies two specific mouse monoclonal antibodies directed against ERß1 (peptide P7) and ERß2 (peptide P8) were used. The monoclonal antibodies were prepared as described (37, 41). The specificity of all antibodies used was initially confirmed by Western blotting using recombinant proteins (31, 37, 41). When proteins are extracted from human tissues (endometrium, placenta, prostate) two sizes of each ERß protein can be detected corresponding to the use of alternative start sites within the mRNAs of both isotypes (Refs. 41 and 42 ; Scobie, G. A., and P. T. K. Saunders, unpublished observations).
Isolation of RNA and synthesis of cDNA
Intact testicular tissues. Total RNA was extracted from snap-frozen samples of fetal testis (12–19 wk gestation) using the RNeasy minikit (QIAGEN, Crawley, UK). RNA was treated with Dnase (Life Technologies, Inc., Paisley, UK) and RT performed using a first-strand cDNA synthesis kit (Roche Diagnostics, Lewes, UK). One microgram of total RNA was incubated with oligo (dT)18 primer for 10 min at 65 C and then placed on ice. A reaction mix comprising buffer, 1 mM each deoxynucleotide triphosphate, ribonuclease inhibitor, and 50 U reverse transcriptase (Life Technologies, Inc.) was added to each tube in a total volume of 50 µl, and the tubes were then incubated at 40 C for 2 h.
Cells isolated by laser capture microscopy. Sections (5 µm) were cut from paraffin wax-embedded 13- and 19-wk-old human fetal testes and mounted onto plain, uncharged glass slides. Sections were dewaxed, rehydrated, and then subjected to immunostaining. To visualize Sertoli cells within the seminiferous cords, rabbit anti-AMH was used at a concentration of 1:200 and staining performed as detailed below, except that antigen retrieval was for 30 sec and incubation times were reduced to 10 min at each stage. RNase inhibitor (Promega Corp., Madison, WI) was included in all reagents to minimize RNA degradation. After color development with diaminobenzidine, the sections were dehydrated through graded alcohols and finally xylene. Sections were stored in a vacuum desiccator containing silica gel for at least 30 min before capture. Care was taken throughout to avoid RNase contamination of sections, and all aqueous solutions were prepared with diethylpyrocarbonate-treated water.
Individual cell fragments were captured from the stained sections using the PixCell II LCM system (Arcturus Engineering Inc., Mountain View, CA) according to the manufacturer’s instructions. Briefly, each section was overlaid with a thermoplastic membrane mounted on optically transparent caps, and cell fragments were captured by focal melting of the membrane because of laser activation. The parameters of the laser shot used in this study were: 7.5 µm diameter spot, 45 mW power, and 0.5 msec duration of laser pulse. The same parameters and number of laser shots (
600) were used for each cell type captured. Figure 1
shows the appearance of a section of human fetal testis that had been stained with anti-AMH antibodies before (Fig. 1A) and after (Fig. 1B) laser capture. The purity of the captured populations was assessed by examining the cell fragments on the cap-mounted membrane; populations substantially enriched for gonocytes (Fig. 1C) or Sertoli cells (Fig. 1D) were obtained.
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PCR was performed using either 1 µl (whole-issue extracts) or 5 µl (samples from LCM) cDNA as a template in a total volume of 25 µl containing 0.2 mM of each deoxynucleotide triphosphate, 50 pmol each forward and reverse primer and RedTaq DNA polymerase (Sigma, St. Louis, MO). Positive and negative (no template) reactions were also included in each set of reactions. PCR amplification consisted of an initial denaturation step at 94 C for 2 min, followed by 35 cycles (whole tissue) or 55 cycles (LCM samples) of denaturation at 94 C for 30 sec, annealing at 58–60 C (depending on primer pair) for 30 sec, and extension at 72 C for 45 sec; a final extension period at 72 C for 10 min completed the amplification. To visualize AMH mRNAs in LCM samples, a second round of amplification using nested primer sets was required. One microliter of the primary PCR reaction was used as template, and the cycle described above was repeated 20 times. All primer pairs were designed to span introns to avoid amplification from contaminating genomic DNA. Primer sequences are detailed in Table 1
. Note that ER cDNA was amplified with primers identical to ER-specific set 1 (exon 1–3) published previously by Lau et al. (44) (amplified product 650 bp). PCR products were analyzed on 1.0% (wt/vol) agarose gels containing ethidium bromide and visualized using a UV transilluminator.
Immunocytochemistry
Sections (5 µm) of Bouin’s fixed, paraffin-embedded fetal testis were mounted on charged glass slides, dewaxed, rehydrated, and subjected to heat-induced antigen retrieval for 5 min in 100 mM glycine buffer (pH 3.5). Endogenous peroxidase activity was blocked by incubation in 3% (vol/vol) H2O2 in methanol for 3 min. After a wash in water, slides were transferred into Tris-buffered saline [TBS; 0.05 M Tris, 0.85% NaCl (pH7.6)] for 5 min and blocked for 30 min in normal rabbit serum (NRS) (Diagnostics Scotland, Carluke, UK) diluted 1:4 in TBS containing 5% BSA (NRS/TBS/BSA). Antibodies were diluted (ER 1:50, ERß 1:800, ERß1 1:50, ERß2 1:50) in NRS/TBS/BSA and applied to the sections at 4 C overnight. Negative controls using either the preimmune serum of the sheep used to raise the anti-ERß P4 antibody or normal mouse serum were included in all runs. Sections were washed in TBS and then incubated for 30 min with biotinylated rabbit antimouse IgG (DAKO Corp.) diluted 1:500 in NRS/TBS/BSA. Following washes in TBS, sections were incubated with avidin-biotin-horseradish peroxidase-linked complex (DAKO Corp.) according to the manufacturer’s instructions. Bound antibody was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAKO Corp.).
Sections were counterstained with hematoxylin, dehydrated, mounted, and visualized by light microscopy. Images were captured using a Provis microscope (Olympus Corp., London, UK) equipped with a DCS330 camera (Eastman Kodak Co., Rochester, NY), stored on a Macintosh Power PC computer, and assembled using Photoshop 6.0 (Adobe, Mountain View, CA).
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and ERß in human fetal testes
Although ER-specific PCR products were detected in a cDNA prepared from human endometrium (positive control) (45), no specific signal was present in the cDNA pools prepared from fetal testes obtained on 13–19 wk gestation (Fig. 2, top). In contrast, ERß-specific cDNAs were amplified from the same set of testicular cDNAs using primers specific for exons 1 and 4 of the receptor (Fig. 2, middle). All samples tested were positive for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (Fig. 2, bottom).
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and 3, B and D, arrows). Sertoli cell nuclei were more numerous than those of the gonocytes and were usually more oval than round (Sc) (Fig. 3). Consistent with the data obtained by RT-PCR, we failed to detect expression of ER protein within the nuclei of any of the cell types within the human fetal testis at any age examined (Fig. 3, A and B); immunopositive staining was present within nuclei of efferent ductules adjacent to the gonad (ED) (Fig. 3A). Using a polyclonal antibody raised against a peptide within the hinge domain (D) of hERß (41), immunopositive staining was detected in the nuclei of multiple cell types including gonocytes (arrows), Sertoli cells (Sc), and interstitial cells (In) (Fig. 3, C and D).
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By RT-PCR, a single band corresponding to the expected size was amplified from fetal testes at all gestational ages examined (13–19 wk) using primers specific for ERß1 and ERß2 and GAPDH (Fig. 4).
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Using RT-PCR, both ERß1- and ERß2-specific cDNAs were detected in amplified cDNA pools prepared from RNA extracted from fetal gonocytes and Sertoli cells isolated by LCM (Fig 5). Both ERß1 and ERß2 cDNAs were detected in all samples. We have previously demonstrated the expression of the germ cell-specific marker c-kit by RT-PCR in LCM-generated fetal germ cell samples (46). In the present study, samples were analyzed by RT-PCR using primers for the Sertoli cell-specific protein AMH (47); the results confirmed that the cDNA pool prepared from gonocytes isolated by LCM was not significantly contaminated with Sertoli cell cytoplasm (Fig. 5). RT-PCR was performed using GAPDH primers to check the quality of cDNA generated from the laser-captured samples (Fig. 5).
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Both ERß1 and ERß2 proteins were immunolocalized to cell nuclei in all specimens examined (Fig. 6). The patterns of expression of the two proteins were distinct but overlapping and were identical in samples from all ages examined (13–19 wk) in samples fixed in Bouins (present study), neutral buffered formalin, or Methacarn (Millar, M. R., unpublished observations). Intense immunopositive staining for ERß1 was detected in some (Fig. 6, C and E, arrowheads) Sertoli cell nuclei. Immunopositive staining was also detected in interstitial cells including peritubular myoid cells and the surface epithelium but gonocytes were immunonegative (Fig. 6, A, C, and E, arrows). In contrast, the most intense immunopositive reaction for ERß2 was detected in the fetal gonocytes (Fig. 6, B, D, and F, arrows) with apparently lower levels of expression within Sertoli cells (Fig. 6F, arrowheads), peritubular cells, and intersitial cells (In, Fig. 6F). With both antibodies, Sertoli cell nuclei that appeared to be immunonegative (Fig. 6, E and F, asterisks) were present.
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Discussion |
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In agreement with our previous studies on the fixed adult human testis (31), we did not detect expression of ER in any testicular cell type, although expression was evident in cell nuclei lining the efferent ductules at all ages examined. These results are in contrast to those using rodent testes in which ER mRNA and protein were easily detected in both fetal and adult Leydig cells (33, 50). Using a polyclonal antibody directed against the hinge domain of ERß, immunopositive staining appeared to be present in the majority of cell types and most intense in the fetal germ cells; ERß mRNA was detected in testicular extracts by RT-PCR. The results are in agreement with those of Brandenberger et al. (35), who reported a strong signal from human fetal testis using a primer set directed against exons 1 and 2 of ERß. Because the primers and antibody we used in these initial investigations could cross-react not only with wild-type ERß (hERß1) but also with the ERß2 receptor splice variant (37), all further investigations were undertaken using either primers or antibodies that were isotype specific.
RT-PCR using isoform-specific primers detected expression of both ERß1 and ERß2 mRNAs in whole testicular extracts of samples obtained between 13 and 19 wk gestation. The isoform-specific primers were then used to interrogate cDNA generated from laser capture microdissected germ cells and Sertoli cells obtained from fixed sections of fetal testis. We confirmed that both ERß1 and ERß2 mRNAs could be detected in extracts from both cell pools. RT-PCR with specific AMH primers was used to verify that the germ cell cDNA pool was not significantly contaminated with Sertoli cell material. To ensure that the mRNAs were translated into receptor proteins, we also performed immunohistochemistry on fixed-tissue sections with isotype-specific monoclonal antibodies that have been used previously on adult tissue sections (37, 51). We found that the expression patterns of ERß1 and ERß2 were distinct but overlapping and did not alter over the gestational range tested. The same pattern of ERß1 expression was seen in tissues fixed with neutral buffered formalin or Methacarn, although background staining tended to be higher in the formalin-fixed samples (Gaskell, T. L., unpublished observations). Both isoforms of ERß were detected in some, but not all, Sertoli cell nuclei. The immunoexpression of ERß1 appeared to be more intense than that of ERß2. It is notable that the opposite was observed using fixed sections of adult human testes, i.e. adult Sertoli cells were more intensely stained with anti-ERß2 than with anti-ERß1 (37), although we should be cautious of making direct comparisons because the intensity of staining may also be influenced by antibody affinity. In the interstitium both ERß1 and ERß2 were detected in peritubular cells and other intersitial cells some of which may be fetal Leydig cells; immunostaining for ERß2 was generally more widespread and intense than ERß1.
The most striking difference in the pattern of expression of the two isoforms was observed within the fetal germ cell population in which, although mRNA for ERß1 was detected in isolated cell fragments recovered using LCM using a sensitive RT-PCR assay, we failed to detect the protein using fixed-tissue sections with a range of antibody dilutions. In contrast, both ERß2 mRNA and protein were readily detectable in most gonocytes at all ages examined. A recent study by Takeyama et al. (36) made use of a polyclonal antibody raised against synthetic peptides from the C-terminal region of ERß, which would be expected to be specific to ERß1. The results presented were from a single 20-wk human fetus in which the testis was fixed in neutral buffered formalin, embedded in paraffin, and subjected to antigen retrieval with citrate buffer, which in our experience results in a higher background level of staining than when glycine buffer is used (Millar, M. R., and T. L. Gaskell, unpublished observations). ERß1 immunoreactivity in Sertoli cells, interstitial cells, and very weakly within germ cells was detected. In the present study, it was notable that the pattern of expression of ERß detected with the polyclonal antibody that cross-reacts with both ERß variants appeared to represent the sum of the expression of ERß1 and ERß2 (compare Figs. 3 and 6). Examination of adult testes with the same isotype-specific antisera has shown that ERß1 is the predominant isoform expressed in the germ cell lineage and that with the exception of some spermatogonial cells (possibly A type), expression of ERß2 protein was largely confined to somatic cells (37). The present study was limited to the examination of tissues recovered in the second trimester of pregnancy during the period when we are able to recover testes following elective termination of pregnancy. We had hoped to extend the current study to include fetuses from later stages of gestation but have found that because these tissues were obtained after fetal death/stillbirth, tissue preservation is compromised and cell-specific patterns of expression difficult to interpret.
The ability of a range of ligands to bind to ERß1 and activate reporter gene expression has been widely reported (52, 53, 54, 55). The receptor has been shown to bind to estrogen-responsive elements (EREs) or other promoter sites such as activator protein-1 elements either as a homodimer or heterodimer with ER (54, 55, 56). The ligand-binding domains of ER and ERß have been crystallized in the presence of a range of ligands, and key amino acids involved in ligand-receptor interaction have been identified (57, 58, 59). Binding of ligand agonists such as estradiol and diethylstilbestrol induces a conformational change in the ligand-binding domain resulting in realignment of the 12th helical domain of the protein (58). Notably, helix 12 adopts a different conformation when the ligand-binding pocket is occupied by an antiestrogen such as raloxifene (58, 59). Analysis of steroid receptor gene activation following steroid binding has demonstrated that it is substantially enhanced or diminished by recruitment of coactivator or corepressor proteins, respectively (60, 61). Mutation studies have shown that amino acids within helix 12 are essential for recruitment of cofactors containing the LXXLL motif, and this region is termed the AF-2 domain of the receptor (62).
In contrast, studies using ERß2 transfected into COS cells showed that the protein was unable to bind estradiol and did not induce expression of an ERE-chloramphenicol acetyl transferase reporter construct in the presence or absence of estradiol and was incapable of interacting with the coregulator TIF1a (29). Recombinant ERß2 has been shown to bind a 32P-labeled ERE in gel shift experiments both as a homo- and heterodimer with ERß1 (30). These findings are consistent with sequence analysis showing that ERß2 protein is formed from an mRNA containing an alternative exon at its 3' end. The net result of this change is the loss of 61 amino acids from the wild-type ERß1 sequence and replacement with a unique 26 amino acid sequence (29). Helix 12 and the AF-2 domain of ERß1 consist of amino acids encoded by the last exon of ERß1, but only one of the amino acids identified as essential to ER dimerization is found in this region (58, 63). Therefore, it is not surprising that ERß2 is capable of forming dimmers but is unable to recruit the cofactor TIF 1a (29, 30). Tremblay et al. (64) showed that the absence of a functional AF-2 domain in one partner of a steroid receptor dimer is sufficient to impair transcriptional activity of the full-length heterodimeric partner. Consistent with this, when ERß2 was cotransfected with ER the receptor acted as a dominant negative inhibitor of ER-induced transactivation (29). We speculated that the expression of ERß2 in fetal cells such as gonocytes may, therefore, prevent them responding to endogenous or exogenous estrogens especially because the expression of wild-type ERß1 protein in these cells was below the limit of detection of our immunohistochemical assay.
This study was undertaken to determine whether ERs were expressed within the fetal human germ cells because exposure to estrogens had been suggested to contribute to the abnormal differentiation of gonocytes and the development of testicular cancer. We found that the functional wild-type hERß1 and the hERß2 C-terminal splice variant are both expressed in the human fetal testis from 13–19 wk gestation. Expression of ER was not detected in the same samples. Although expression of hERß1 and hERß2 mRNAs could be detected in highly amplified cDNA pools prepared from fetal gonocyte material recovered by LCM, immunohistochemistry consistently revealed expression of ERß2 but not ERß1 in the fetal germ cells, suggesting that either ERß1 protein had not been transplated from the mRNA or the amount of protein was very low. ERß1 protein was easily detectable by immunohistochemistry in some but not all somatic cells including Sertoli cells adjacent to the immunonegative germ cells within the seminiferous cords. We, therefore, suggest that the human fetal germ cells may not respond directly to estrogenic ligands during the second trimester but that estrogens could act via Sertoli, peritubular, or Leydig cells at this time of life.
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Acknowledgments |
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and ERß (common) primers, Lee Evans for preparation of monoclonals, and Mike Millar and Sheila McPherson for assistance with immunohistochemistry. |
Footnotes |
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Received May 28, 2002.
Accepted September 16, 2002.
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References |
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in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
and ß proteins in the testes and male reproductive system of human and non-human primates. Mol Hum Reprod 7:227–236 and ß in human testis. Mol Hum Reprod 7:497–503 (ER-) and ß (ER-ß) mRNA in the midgestation human fetus. J Clin Endocrinol Metab 82:3509–3512 and ß in the human fetus. J Clin Endocrinol Metab 86:2258–2262 and -ß and androgen receptor in the ovaries of marmoset and human. Biol Reprod 63:1098–1105 and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc Natl Acad Sci USA 96:5722–5727, is present in the vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 86:1370–1378 (ER) within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood. J Endocrinol 153:485–495[Abstract]
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