Molecular Human Reproduction, Vol. 7, No. 3, 227-236,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Differential expression of oestrogen receptor 
and ß proteins in the testes and male reproductive system of human and non-human primates
MRC Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET, UK
Abstract
The role(s) oestrogens play in male adult reproductive function remains uncertain. We have used antibodies specific for oestrogen receptor- 
(ER) and - ß (ERß) to investigate their distribution within the male. In testes from adult human, macaque and marmoset, ERß protein was detected in Sertoli cells, Leydig cells and peritubular myoid cells. In germ cells, the intensity of immunostaining for ERß was variable between species. Immunoexpression in preleptotene, leptotene and zygotene spermatocytes was low/absent in all species. Elongated spermatids were consistently immunonegative. No ER immunoexpression was detected in testes. ERß was detected in epithelial and stromal cell nuclei throughout the male reproductive system [efferent ductules (ED), epididymis, vas deferens, seminal vesicles] and in the bladder. ER was detected in non-ciliated epithelial cells in the ED, but rarely in epithelial and basal cells within the epididymis. Epithelial cells from seminal vesicles and bladder were immunonegative for ER. Expression of ER in stromal cells was rare in the ED, epididymis and bladder but more frequent in seminal vesicles. Expression of ER, and long and short forms of ERß, was confirmed by Western blotting. The widespread expression of ERß suggests that it is the primary target for modulation of tissue function via oestrogenic ligands in the male reproductive system.
efferent ductules/epididymis/oestrogen receptor/testis/vas deferens
Introduction
The impact of endogenous oestrogens, or exogenous ligands with oestrogenic activity, on the formation of the male reproductive system, and on reproductive function in adulthood, remain the subject of intensive research activity and debate (see reviews by Toppari et al., 1996; Sharpe, 1998a,b). In males, oestrogens are synthesized from androgens by the aromatase enzyme both within the brain and testes and levels within the male reproductive tract are generally higher than in the general circulation (reviewed by Hess, 2000). In the adult testis the major site of oestrogen synthesis appears to be the Leydig cells (Payne et al., 1976
); however, studies in a number of species including mice and rats have detected aromatase activity within mature germ cells and spermatozoa (Nitta et al., 1993; Hess et al., 1995). Oestrogen action is mediated via high affinity receptors that are shifted to a transcriptionally active state after ligand binding. Two forms of oestrogen receptor commonly known as (ER, Green et al., 1986) and ß (ERß, Kuiper et al., 1996) have been cloned. Although they exhibit significant sequence homology within both their DNA and ligand binding domains (reviewed in Saunders, 1998) they are encoded on different chromosomes (Enmark et al., 1997).
Studies over a number of years, mostly in mice, but with supporting human data, have demonstrated that exposure of pregnant females to high doses of potent oestrogens such as diethylstilboestrol (DES) can cause reproductive abnormalities in their male offspring (McLachlan and Newbold, 1975; Gill et al., 1979) which may in part result from impaired hormonal biosynthesis by fetal Leydig cells (Majdic et al., 1996). Additional data from animal studies in which oestrogens have been administered immediately after birth have shown that exposure at this time can result in abnormalities in the rete testis, efferent ductules (Aceitero et al., 1998; Fisher et al., 1999), prostate (Prins, 1992) and testes (Sharpe et al., 1995; Atanassova et al., 1999). Both DES and other oestrogens used in these in-vivo studies have been shown to bind to ER and ERß in vitro (Kuiper et al., 1997).
Evidence for the expression of both and ß forms of ER in the testes and reproductive tracts of a number of species has been gathered using a variety of techniques (reviewed in Saunders, 1998; Hess, 2000). To date, immunohistochemical investigations on the reproductive tracts of male primates have been limited to studies on the expression of ER in the marmoset monkey (Fisher et al., 1997), rhesus and cynomolgus macaques (West and Brenner, 1990) and human (Ergun et al., 1997). Pelletier et al. (1999) investigated expression of ERß mRNA in tissues from two adult cynomolgus macaques and reported that silver grains were localized to cells within the seminiferous epithelium but not to intersitial cells; they did not identify individual cell types. Enmark et al. (1997) also reported that ERß probes localized preferentially to round spermatids in human testes but did not find evidence of expression of ERß mRNA in Sertoli cells, other germ cells, or Leydig cells. Studies on expression of ERß protein in tissues from the reproductive system of male human and non-human primates have been limited to inclusion of testicular sections in a general survey of human tissues (Taylor and Al-Azzawi, 2000) and detection of both ER and ERß in germ cells within small segments of human seminiferous tubules (Pentikainen et al., 2000).
The aim of the present study was to compare the expression of ER and ERß proteins within the testes and reproductive tracts of human and non-human primates. In addition to Western analyses of protein extracts, a detailed immunohistochemical study was undertaken. The latter was considered essential as in-vitro studies suggest that ER and ERß will form both homo- and heterodimers (Cowley et al., 1997) and that the proportions of the ER isotypes present can influence cellular responses to oestrogens (Hall and McDonnell, 1999; Saville et al., 2000).
Materials and methods
Tissue samples
Testicular tissues were obtained from men (n = 7) undergoing surgical investigations for non-obstructive azoospermia or surgical correction of vasectomy. The protocol was approved by the Lothian Reproductive Medicine Ethics committee, and the men gave informed consent. Vas deferens were obtained from men undergoing vasectomy. Additional human tissues (n = 6) were obtained from the Peterborough Hospitals NHS Trust tissue bank. Marmoset tissues were collected from adult captive bred animals (n = 5, Callthrix jacchus) maintained in a colony which has been closed since 1973. Testes with epididymides attached were also obtained from adult stump-tailed macaques (n = 3, Macaca arctoides) which were surgically castrated under general anaesthesia for colony management purposes. Samples for immunohistochemistry were fixed in Bouin's fluid for 6–7 h and processed into paraffin wax using standard methods (Millar et al., 1993). Tissues for extraction of protein for Western analysis were chopped into ~0.5 cm fragments, snap-frozen on dry ice or in liquid nitrogen and stored at –70°C before use.
Antibodies
A mouse monoclonal antibody raised against human recombinant ER was obtained from Novocastra (NCL-ER-6F11, Newcastle, UK); this antibody is specific to ER and does not bind to recombinant ERß on Western blots (Saunders et al., 2000; see also results).
A sheep polyclonal antibody specific for ERß was prepared using a peptide (P4 CAGKAKRSGGHAPRVREL) located within the hinge (D) domain of hERß (Mosselman et al., 1996) which was conjugated to keyhole limpet haemocyanin. The preparation and affinity purification of this antibody has already been reported in detail (Saunders et al., 2000); this antibody does not bind to recombinant ER on Western blots (Saunders et al., 2000; see also results).
Immunohistochemistry
Immunolocalisation of ER and ERß utilized 5 µm tissue sections which were subjected to heat-induced antigen retrieval (Norton et al., 1994) in a pressure cooker (Tefal, Nottingham, UK) containing 2 l of near boiling 0.01 mol/l glycine/EDTA pH 3.5. Sections were incubated with 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. After a wash in water, slides were transferred into Tris-buffered saline (TBS, 0.05 mol/l Tris pH 7.4, 0.85% saline) for 5 min and blocked for 30 min in normal rabbit serum (NRS; Diagnostics Scotland, Carluke, Lanarkshire, UK) diluted 1:4 in TBS containing 5% bovine serum albumin (NRS/TBS/BSA). An avidin-biotin block was performed using reagents from Vector (Peterborough, UK) as follows: eight drops of avidin were added to each 1 ml TBS used, this was incubated on sections for 15 min, which were then rinsed in TBS and washed in TBS for 5 min. Thereafter, eight drops of biotin were added per 1 ml TBS and this was incubated on sections for 15 min, rinsed in TBS and then washed in TBS for a further 15 min. Primary antibodies were diluted in NRS/TBS/BSA. Sheep anti-hERß was used at 1:800 on all tissues except testes which were incubated with a range of dilutions from 1:500 to 1:1250. The mouse anti-hER was used at 1 in 20. Incubations were allowed to proceed overnight at 4°C. Sections were washed in TBS and incubated with the appropriate biotinylated secondary antibodies, for anti-ERß, rabbit anti-sheep (Vector), and for anti-ER rabbit anti-mouse (Dako, Cambridge, UK) both of which were diluted 1:500 in NRS/TBS/BSA. Incubations lasted for 1 h and were followed by two washes in TBS (5 min each). Thereafter, sections were incubated in avidin-biotin-horseradish peroxidase complex (Dako) for 1 h, washed in TBS (2x5 min) and bound antibodies visualized by incubation with 3,3'-diaminobenzidine tetra-hydrochloride (liquid DAB cat K3468, DAKO). Sections were counterstained with haematoxylin. Images were captured using an Olympus Provis microscope (Olympus Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak Co. Rochester, NY, USA), stored on a Macintosh PowerPC computer and assembled using Photoshop 5 (Adobe, Mountain View, CA, USA).
Western analysis
Recombinant human ERß corresponding to `short' (~53 kDa) and `long' (~59 kDa) forms of the receptor (Mosselman et al., 1996; Ogawa et al., 1998) and recombinant hER were all obtained from Pan Vera (Madison, WI, USA). Tissues were kept frozen whilst they were ground to a fine powder, then rapidly homogenized in homogenization buffer. Homogenization buffer contained 10 mmol/l HEPES pH 7.9, 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l dithiothreitol, 0.5 mmol/l PMSF, 1xprotease complete (cat no. 1836170; Roche Diagnostics, Lewes, Sussex, UK). Samples (400 µg or 100 µg total protein) and aliquots (0.5 µg) of recombinant proteins were denatured and run on minigels containing an acrylamide gradient from 4 to 20% (w/v) polyacrylamide (InVitrogen, Groningen, The Netherlands) and blotted onto PVDA membranes (Millipore, Watford, UK). Membranes were washed twice in TBS containing 0.05% Tween-20 (TBST), then blocked in TBST containing 5% (w/v) skimmed milk powder (Marvel) for 2–3 h at room temperature. Primary antibodies were diluted in TBST (anti-hER, 1:200; anti-hERß, 1:2000) and incubated overnight at 4°C. The next day membranes were washed in TBST (2x15 min then 4x5 min). Peroxidase-conjugated secondary antibodies (donkey anti-sheep for ERß (Diagnostics Scotland) or sheep anti-mouse for ER (Amersham Pharmacia Biotech UK, Little Chalfont, Bucks, UK) were diluted 1 in 4000 in TBST and incubated on the membranes for 2 h at room temperature. Membranes were washed thoroughly in TBST (2x15 min then 4x5 min) and bound antibodies were detected using the ECL detection kit (Amersham) and visualized using X-ray film (Kodak).
Results
Immunolocalization of ER within human and primate testes
ERß
Results for the pattern of expression of ERß within the seminiferous epithelium were based on evaluation of immunostaining in the three species at a range of antibody dilutions and for completeness they were compared to those we have observed in the rat (Saunders et al., 1997; Turner et al., 2001). Sertoli cell nuclei express ERß at a relatively high level when compared to expression in most germ cell types (Figure 1, arrows). This is particularly true in the macaque (Figure 1c), marmoset (Figure 1d) and rat (not shown), but in the human (Figure 1a, a', b) levels of protein in some Sertoli cell nuclei appeared lower than in some germ cell nuclei (Figure 1a, a'). ERß was present in most, and probably in all, A- and B-type spermatogonia including both A pale and A dark in the human (Figure 1, arrowheads). In many instances, expression in spermatogonia was higher than in other germ cell types (e.g. macaque, Figure 1c, marmoset, Figure 1d) though this differential was not so evident in the human (Figure 1a, b). ERß immunoexpression in spermatocytes showed a consistent pattern in the three species (and in the rat) with low/negative immunoexpression in preleptotene, leptotene and zygotene spermatocytes (e.g. marmoset Figure 1d). Thereafter there was a progressive increase in the level of expression in pachytene spermatocytes (P) as these cells advanced in development and size, until nuclear immunoexpression was lost at the diplotene/secondary spermatocyte stage. The differential in ERß immunoexpression level between pre-pachytene and late pachytene spermatocytes was most marked in marmosets and macaques and least marked in human. There was also considerable variation in the magnitude of this differential between specimens from different individuals of the same species. Immunoexpression of ERß in spermatids (Figure 1, labelled R) was evident in round spermatids shortly after their formation and until initiation of nuclear condensation when immunoexpression was lost. All later spermatids were negative for ERß immunoexpression in all three species (Figure 1, asterisks). Within the interstitium, immunopositive nuclei were present in both Leydig cells (L) and peritubular myoid cells (e.g. Figure 1b).
|
ER
We were unable to detect ER
in any cell type within the testis of any of the three species examined using Bouins-fixed material (not shown) although in some cases intense immunopositive staining of efferent ductules (see Figure 2) was noted on the same sections. Leydig cells in sections from adult rat testes run at the same time were consistently immunopositive (not shown).
|
Differential expression of ER
and ERß within the male reproductive system detected using immunohistochemistryERß
Expression of ERß in the nuclei of epithelial and stromal cells was detected in all portions of the male reproductive tract from all three species examined (Figures 2 and 3
), including the ED, epididymis, vas deferens, seminal vesicle and bladder. In contrast to the pattern of expression of ER (see below), the transition from the ED to the epididymis was not marked by a loss of ERß immunoexpression (e.g. macaque, Figure 2c, arrow). Although the intensity of immunostaining was generally highest within the epithelial cell populations it was variable between cells even within the same epithelium (e.g. Figure 3g) and immunonegative cells were also observed (e.g. Figure 2k, blue arrows). A proportion of cells within the stroma were immunonegative (stained blue by the counterstain) in all tissues examined.
|
ER
ER
was immunolocalized to epithelial cell nuclei in the efferent ductules (ED) of the human (Figure 2b) macaque (Figure 2e, f) and marmoset (not shown), using Bouins-fixed tissues. Within the epithelium of the ED not every nucleus was stained (see Figure 2f, blue arrows); in the stump-tailed macaque, immunopositive staining was detected only within non-ciliated cells in agreement with previously published data (West and Brenner, 1990). In the macaque, the transition between the ED and the tall columnar epithelium of the initial segment of the epididymis was marked by loss of expression of ER (Figure 2e, arrow); similar results have been obtained in the marmoset (Fisher et al., 1997; and not shown). In all of the regions of the epididymis, in the vas deferens and in the bladder, ER-positive cells were extremely rare (Figures 2 and 3). In the epithelium of the epididymides from macaques fixed rapidly at the time of surgical castration, a few immunopositive basal cells were detected (Figure 2l, black arrowheads) as were very occasional cells with elongated nuclei (Figure 2j, arrowhead) the identity of which was uncertain. No immunostaining was seen in tissue that was fixed post mortem (human). Faint immunostaining of occasional stromal cells(s) was observed in the bladder and epididymides from marmoset and macaque (Figure 3). In contrast, within the seminal vesicles, although no ER-positive cells were detected in the epithelium using tissue from the marmoset (Figure 3f) or human (not shown), multiple cells within the stroma were immunopositive (e.g. marmoset Figure 3f).
Detection of ER proteins in tissue extracts from male reproductive tissues by Western blotting
A single protein band which migrated with an identical molecular size (~66 kDa) to human recombinant ER was detected in extracts prepared from human, macaque and marmoset tissues (Figure 4, upper panel). The anti-ER antibody did not bind to recombinant hERß run on the same gels, in agreement with previous data (Saunders et al., 2000; Williams et al., 2000). ER was detected in the human vas deferens (lanes 7, 8), macaque epididymis (lane 9), marmoset seminal vesicle (lane 10), marmoset bladder (lane 11) and marmoset (lane 12) and human (Saunders et al., 2000) prostate. The total amount of ER in the extracts was low and a specific signal was only obtained when 400 µg of total protein was loaded in each lane of the gel and exposure of films allowed to proceed overnight. In comparison, a similar signal was obtained using only 100 µg of total protein extracted from marmoset uterus or human placenta (Figure 4, lanes 13 and 14 respectively). However, the amount of ER protein in all the samples obtained from males appeared similar, which was at odds with the apparent variation in numbers of ER immunopositive cells seen in the tissue sections (e.g. between seminal vesicle and bladder). We have obtained similar results in rodents (unpublished observations). These findings may reflect levels of ER in individual cells which are too low to be detected by immunohistochemistry, although we do not believe this to be the case. Alternatively, the use of large amounts of protein and extended exposure times may have reduced the dynamic range of the assay, resulting in the capturing of similar levels of signal by the X-ray film.
|
ER
protein was detected in one sample extracted from the human testis (lane 6) but even when exposure times were extended we did not find evidence of expression of ER protein in a second human testicular sample 5 (lane 5) or in samples extracted from macaque (lanes 3 and 4) and marmoset testes (lanes 1 and 2). These results were in agreement with results of the immunohistochemistry in which no ER was detected on sections of fixed testicular tissues.
An antibody directed against a peptide within the hinge domain of hERß bound specifically to both forms of recombinant hERß (long, ßL, ~59 kDa, arrowhead; short, ßs, ~53 kDa, small arrow (Mosselman et al., 1996; Ogawa et al., 1998) but not to recombinant hER (Figure 4, lower panel). Two sizes of ERß protein, migrating with an apparent molecular size similar to that of the two forms of recombinant ERß, were detected in all the tissues examined including the testes of all three species (human, lanes 5 and 6; macaque, lanes 3 and 4; marmoset, lanes 1 and 2). The larger ERß isoform (~60 kDa) appeared to be the more abundant isoform (Figure 4, lower panel, arrowhead). A smaller protein (molecular size ~30 kDa) was detected only in samples from the macaque tissues tested (testes, vas deferens). In the samples extracted from the epididymis of the macaque (lane 9) and the marmoset prostate (lane 12), marmoset bladder (lane 11), marmoset seminal vesicle (lane 10) and marmoset uterus (lane 13), the majority of the ERß protein detected co-migrated with the hERßL recombinant marker although with extended exposures some ERß short (ERßs) was identified (not shown).
Discussion
In the present study, we have made use of material from human and two non-human primates to compare the pattern of expression of the and ß subtypes of ER in the testes and reproductive ducts of the male. Studies on the pattern of expression of ERß in these species have not previously been reported in detail and provide us with valuable information on the cellular targets for the action of natural and synthetic oestrogens and anti-oestrogens.
The expression of ERß in multiple cell types, including germ cells, in the testes of all three species is in agreement with the results reported for adult rodents both by ourselves (Saunders, 1998; Saunders et al., 1998) and others (van Pelt et al., 1999). Using human testes fixed in formol saline, Taylor and Al-Azzawi (2000) reported that they were able to localize ERß protein to Sertoli cells, Leydig cells, spermatocytes and spermatogonia but made no mention of round and elongating spermatids which did not seem to be present in the sections shown in their paper. In the current study, we noted that there were significant differences in the relative levels of ERß protein detected by immunohistochemistry in the different testicular cell types both between species and between individuals from the same species. The wide range in the levels of expression of protein presented particular difficulties in obtaining consistent immunohistochemical results and a comprehensive comparison of different species was only possible when a range of dilutions of the primary ERß antibody were employed. Less variation in levels of expression of ERß was noted in other tissues from the male reproductive system. In larger tissue blocks, such as those from the testes of adult macaque monkeys, we found differences in the intensity of immunostaining in the individual cell types across the tissue section with higher levels at the edges of the block. We have also found that the intensity of the positive staining within cell nuclei can be influenced by the choice of pH of the retrieval medium; in testes we have found that the use of glycine buffer at pH 3.5 is optimal whereas in many other tissues, including the ovary and prostate (Saunders et al., 2000; Williams et al., 2000), we have routinely made use of citrate buffer at pH 6. Our conclusion is that studies on expression of ERß within testicular tissue will need to pay particular attention to choice of fixative and the duration of fixation and will need to perform immunohistochemistry at a range of antibody dilutions on several individuals to avoid bias.
The cell-specific pattern of expression of ER and ERß within the human seminiferous epithelium that we have observed using Bouins-fixed tissue is different to that reported by Pentikaninen et al. (2000) who performed immunohistochemistry on squash preparations of segments of human seminiferous epithelium. These investigators found both ER and ERß protein within early meiotic germ cells and in early elongating spermatids but not in Sertoli cells or other germ cells. We did not detect ER protein in any of the human testes examined, although when efferent ductules were present within the same tissue block, these were strongly immunopositive. We also failed to detect ER in sections of testes from stump-tailed macaque or common marmoset. Fisher et al. (1997) reported the presence of very occasional Leydig cells with immunopositive ER staining in the marmoset, but we were not able to confirm this in the present study. Using identical methods we always detected significant levels of expression of ER in nuclei of both fetal- and adult-type Leydig cells in both rats and mice (Fisher et al., 1997 and unpublished observations) pointing to a clear difference between these species and primates in expression of ER. West and Brenner (1990) have also failed to detect ER in testes from cynomolgus and rhesus macaques using a mixture of monoclonal antibodies. On Western blots, we also failed to detect ER co-migrating with the recombinant standard in all but one of the extracts from testes of the three species being examined in the current study.
High levels of expression of both ER and ERß in nuclei of ED in both human and non-human primates is in agreement with previously published data from rat (Fisher et al., 1997; Hess et al., 1997b), goat (Goyal et al., 1997), monkey (West and Brenner, 1990) and human (Ergun et al., 1997). In mice, expression of ER by germ cells is not required for their normal development or for fertility (Mahato et al., 2000), consistent with results showing that the loss of fertility in the male oestrogen receptor knock-out (ERKO) mice (Eddy et al., 1996) is due to a defect in fluid resorption within the efferent ductules (Hess et al., 1997a). It is notable that the maintenance of expression of ERß in the ED of ERKO males (Rosenfeld et al., 1998) is not able to compensate for loss of ER. Furthermore as the fertility of male mice in which ERß is disrupted appears unaffected (Krege et al., 1998; Dupont et al., 2000), fluid resorption in the ED may be normal in the absence of a functional ERß protein. The significance of the expression of ERß in the ED is uncertain and will require additional in-vitro studies such as those carried out on the ERKO mice (Hess et al., 1997a, 2000).
In regions of the male reproductive tract other than the ED, a clear difference in the pattern of expression of ER and ERß was noted with most epithelial cells in the epididymis, vas deferens, seminal vesicle and bladder containing cell nuclei which were immunopositive for ERß but not for ER. We did not find evidence of cytoplasmic staining of epididymal epithelial cells such as that mentioned by Taylor and Al-Azzawi (2000). The expression of ERß protein in both epithelial and stromal cells throughout the length of the primate epididymides and in the vas deferens is in agreement with studies in the rat (Hess et al., 1997b; Atanassova et al., 2000; Williams et al., 2000). West and Brenner (West and Brenner, 1990) did not detect ER in epididymides of rhesus and cynomolgus macaques using immunohistochemistry and likewise Fisher et al. (1997) did not record immunopositive staining in the marmoset. Studies on epididymides from mouse (Jefferson et al., 2000) and rat (Fisher et al., 1997; Atanassova et al., 2000) suggest that expression of ER may be developmentally regulated and increased following exposure to oestrogens early in life. Morphological changes have been noted in the epididymides of ERKO mice (Hess et al., 2000). In the present study on adult primates, cells immunopositive for ER were rare in epididymides consistent with detection of low levels of ER mRNA in rodent and human epididymides by Northern blotting or following reverse transcription-polymerase chain reaction amplification (Ergun et al., 1997; Hess et al., 1997b).
Western analysis has confirmed the specificity of the antisera used for the different ER isotypes (Saunders et al., 2000; Williams et al., 2000; and the current study). The tissue extracts in which we detected the highest levels of ER were from the seminal vesicle and the `top' of the epididymis (including the efferent ductules); however, the differential between the level of protein in the tissue extracts was not as marked as expected from the immunohistochemistry data. In all the protein extracts the ER detected migrated with the same apparent molecular size to that of the recombinant ER and we saw no evidence of expression of variants (Pentikainen et al., 2000). During both the current investigation and other studies (Saunders et al., 2000; Williams et al., 2000), Western analysis of ERß has been complicated by the inability of the either the recombinant protein, or the ERß present in tissue extracts, to withstand freeze-thawing. In the present study, protein extracts were always aliquoted immediately after extraction and snap-frozen to avoid proteolytic degradation, resulting in detection of low molecular weight proteins. In all the samples examined, we detected protein which co-migrated on the gels with the full length `long' form of recombinant ERß prepared by PanVera from a cDNA which includes the ATG start site identified by Ogawa et al. (1998). Although in all the tissue extracts the long form of ERß appeared more abundant, evidence of the use of alternative start sites for translation was obtained with the identification of an ERß protein which co-migrated with the short form of recombinant ERß (Mosselman et al., 1996) in all three species. Sequencing of ERß cDNA isolated from marmoset (Accession number Y09372) and macaque (G.Scobie, Human Reproductive Sciences Unit, Edinburgh) has confirmed that the positions of translational start sites are conserved between these species and the human.
The role(s) of ERß in maintenance of normal fertility in human and non-human primate is not known, as individuals with mutations in ERß have not as yet been identified. Studies on mice in which the functional integrity of the ERß gene has been disrupted (Krege et al., 1998; Dupont et al., 2000) have not suggested that ERß is essential for normal spermatogenesis or maintenance of fertility. Although the fertility of male mice lacking both ER and ERß (Couse et al., 1999; Dupont et al., 2000) is compromised, their phenotype appears to resemble that of the ERKO. In male mice lacking the ability to form oestrogens due to disruption of the aromatase gene (ARKO; Fisher et al., 1998), male infertility is only observed in adult animals from 4.5 months of age and is associated with loss of germ cells. The results from these mutant mice may not really reflect the importance of oestrogens in intact males because of disturbances in the hormonal status of the animals throughout their lives (Couse and Korach, 1999) and/or the ability of other factors such as testosterone or prolactin to compensate lack of oestrogen action (Sharpe et al., 2000). Ebling et al. (2000) have treated hpg mice that lack endogenous steroids with oestradiol implants and observed that spermatogenesis was restored and FSH concentrations elevated in the treated males compared with controls. Pentikainen et al. (2000) have treated isolated human seminiferous tubules with oestradiol and found reduced levels of germ cell apoptosis as a result of treatment. Studies on rats in vivo have revealed that gene expression within the fetal testis (Majdic et al., 1996), immature testis (Sharpe et al., 2000), epididymis and prostate (Williams et al., 2000) can all be modified by exogenous treatment with oestrogens and that in part these effects may be mediated by binding of oestrogens to ERß (Williams et al., 2000).
In conclusion, the present study provides conclusive evidence for differences in the pattern of expression of the two isoforms of ER in the male reproductive system of human and non-human primate and highlights the widespread expression of ERß.
Acknowledgments
The authors thank Dr M.Hair, K.Morris and Colin Penny for their assistance in obtaining tissue samples and Drs H.Fraser and R.Hess for helpful comments.
Notes
1 To whom correspondence should be addressed. E-mail: p.saunders{at}ed.ac.uk 
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Submitted on October 5, 2000; accepted on December 27, 2000.
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