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General and Comparative Endocrinology 201 (2014) 16–20
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Short Communication
Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells Pedro F. Oliveira a,⇑, Marco G. Alves a, Ana D. Martins a, Sara Correia a, Raquel L. Bernardino a, Joaquina Silva b, Alberto Barros b,c, Mário Sousa b,d, José E. Cavaco a, Sílvia Socorro a,⇑ a
CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal Centre for Reproductive Genetics Alberto Barros, 4100-009 Porto, Portugal Department of Genetics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal d Department of Microscopy, Laboratory of Cell Biology and Biomedical Research Multidisciplinary Unit (UMIB-FCT), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 16 December 2013 Revised 23 February 2014 Accepted 26 February 2014 Available online 25 March 2014 Keywords: Estrogen receptors G protein-coupled receptor 30 Sertoli cells Germ cells Estrogens
a b s t r a c t The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERa and ERb) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The male sexual function is highly dependent on hormonal regulation, particularly by sex steroid hormones. Indeed, in the last few years, it has been shown that, besides androgens, estrogens also play an important role on several aspects of the male reproductive biology, including testicular cell differentiation, proliferation, metabolism, and apoptosis (Alves et al., 2013b; Carreau et al., 2008; Rato et al., 2012b; Simões et al., 2013). Most of the estrogenic actions described so far are mediated by the classical nuclear estrogen receptors (ER), for which two different subtypes exist, ERa and ERb (reviewed by Heldring et al. (2007)). Previous work of our team, using testicular biopsies with distinct spermatogenic phenotypes, showed that both ERa and ERb are expressed in several cell types of human testis. ERa was described in Leydig cells, Sertoli cells (SCs), spermatogonia, spermatocytes, round ⇑ Corresponding authors. E-mail addresses:
[email protected] (P.F. Oliveira),
[email protected] (S. Socorro). http://dx.doi.org/10.1016/j.ygcen.2014.02.022 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.
spermatids and elongated spermatids/spermatozoa, while ERb was found to be present in all cell types except SCs and spermatogonia (Cavaco et al., 2009). For a long time some authors have suggested that estrogens exhibit effects inconsistent with its known genomic mechanisms of action (Simoncini and Genazzani, 2003). Indeed, several reports confirmed the existence of a G protein-coupled estrogen receptor (GPR30) mediating the non-genomic effects of 17b-estradiol (E2) (reviewed by Hammes and Levin (2007)). Yet, there are important gaps concerning ligand specificity, expression pattern and in vivo function of GPR30. Nevertheless, GPR30 expression was found to be regulated by gonadotrophic hormones in several cells of mammalian reproductive system, highlighting a possible role for this receptor in the reproductive function (Pang and Thomas, 2009), although GPR30-deficient mice are fertile and exhibit normal reproductive functions (Otto et al., 2009). In the human testis, it was shown that GPR30 was overexpressed in neoplastic and nonneoplastic human testicular tissue and was able to trigger in vitro proliferation of seminoma cells (Chevalier et al., 2012; Rago et al., 2011). Moreover, GPR30 mRNA expression has been
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suggested in human SCs cultured in vitro (Alves et al., 2012b). In the present short communication, using sections of human testicular biopsies with conserved spermatogenesis, the distribution of GPR30 in human testis was evaluated. Furthermore, using human testicular cell fractions and SCs cultured in vitro, we further confirmed the presence and localization of GPR30 in specific human testicular cells, by means of RT-PCR and immunoblot analyses. To the best of our knowledge, this is the first exhaustive description of the cellular distribution of GPR30 in normal human testicular tissue, opening new research prospects for the exploitation the role estrogens and GPR30 in spermatogenesis.
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2. Material and methods
cells. The resultant fluid fraction was washed twice (5 min at 300 g) in Sperm Preparation Medium (SPM) (Medicult, Mollehaven, Denmark) and incubated (5 min at 37 °C) in erythrocyte-lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 2 mM EDTA, pH 7.2 with KOH). The suspension was then washed twice in SPM (5 min, 500 g). The resultant cellular suspension was then enzymatically digested (20 min, 25 lg/mL crude DNase, 1000 units/mL collagenase-IV) in SPM and separated (5 min, 50 g) into supernatant (haploid germ cell fraction) and pellet (diploid germ cell fraction). The fractions were washed twice with SPM (5 min, 1000 g) and the final cellular pellets, correspondent to the distinct germ cell fractions, were resuspended in in vitro fertilization medium (IVF; without HEPES) (Medicult, Mollehaven, Denmark).
2.1. Chemicals
2.5. Sertoli cell culture
Taq DNA Polymerase was purchased from Fermentas Life Sciences (Ontario, Canada). Random primers and M-MLV RT were purchased from NZYTech (Lisboa, Portugal). Polyclonal Antibodies were purchased from Invitrogen (Carlsbad, CA, USA), dNTPs were purchased from GE Healthcare (Buckinghamshire, UK) and Fetal Bovine Serum (FBS) was obtained from Biochrom AG (Berlin, Germany). All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Human SCs were obtained by previously described methods (Alves et al., 2012b; Oliveira et al., 2011). Culture purity was assessed by immunoperoxidase detection of SCs-specific markers such as vimentin as described elsewhere (Rato et al., 2012a). Only cultures with cell contaminants below 5%, as examined by phase contrast microscopy after 96 h, were used.
2.2. Patient selection, ethical issues and human testicular biopsies
RT-PCR was performed to analyse GPR30 mRNA expression as described by Simões et al. (2013). Specific intron-spanning primer sets (Sense: TCC ATT CTC CGT TT CAC AGC; Antisense: CCG GTG TTA TAG CCG AAC TG) were used, with an annealing temperature of 55 °C and 35 amplification cycles for amplification of a 145 bp DNA fragment. Every set of RT-PCR included a negative (no-template) and a positive (human mammary gland cells, MCF-7 cells) control.
The clinical study of the patients was performed at the Centre for Reproductive Genetics Alberto Barros (Porto, Portugal) in accordance with the Guidelines of the Local, National and European Ethical Committees. The testicular biopsies with conserved spermatogenesis (patients with anejaculation psychological, vascular or neurologic and vasectomy or traumatic section of the vas deferens) were obtained from infertile patients under treatment for recovery of male gametes and used after informed written consent. Studies have been performed according to the Declaration of Helsinki for Medical Research involving Human Subjects. Human testicular cells were isolated from five testicular biopsies and, after selection, each biopsy was collected in sperm preparation medium (SPM-Hepes buffer) (Medicult, Mollehaven, Denmark) and kept at 32 °C with 5% CO2 in air until use as previously described (Oliveira et al., 2009). 2.3. Immunohistochemistry Immunohistochemical analysis of the testicular sections was performed as described previously (Laurentino et al., 2011). Sections were incubated with a rabbit anti-GPR30 primary monoclonal antibody (1:50, sc-48524-R, Santa Cruz Biotechnology Heidelberg, Germany) diluted in PBS with 1% (w/v) BSA (PBA). Sections were then incubated with goat anti-rabbit secondary biotinylated antibody (1:200, ab64256, AbCam, Cambridge, UK) diluted in PBA, followed by incubation with ExtrAvidin Peroxidase (1:20, SigmaAldrich, St. Louis, USA) diluted in PBA. Antibody binding was detected using HRP substrate solution (Dako, Glostrup, Denmark). Sections were slightly counter-stained with Harris’ hematoxylin (Merck, Darmstadt, Germany), dehydrated, cleared, and mounted. Specificity of the staining was assessed by the omission of primary antibody. 2.4. Germ cell fractions isolation Human haploid and diploid germ cell fractions were isolated according to a method described previously by our team (Pinheiro et al., 2010, 2012; Sá et al., 2008). Briefly, seminiferous tubules were squeezed under a heated stereomicroscope to release luminal
2.6. RT-PCR
2.7. Western blot Western blot procedure was performed as previously described (Rato et al., 2013). In brief, total proteins were isolated from human SCs and germ cell fractions, fractionated in 12% polyacrylamide gels, transferred to polyvinylidene difluoride membranes and blocked for 2 h at room temperature in TBS-T (NaCl 150 mM, Tris-base 50 mM, 0.05% Tween 20) with 5% non-fat milk. The membranes were then incubated with a rabbit anti-GPR30 primary monoclonal antibody (1:500, sc-48524-R). The immune-reactive proteins were detected with goat anti-rabbit IgG-AP secondary antibody (1:5000, sc-2007, Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were reacted with ECF detection system (GE, Healthcare, Weßling, Germany) and visualized with the BioRad FX-Pro-plus (Bio-Rad, Hemel Hempstead, UK). 3. Results 3.1. Staining pattern of GPR30 in human testicular tissue sections A specific GPR30 antibody was used to stain paraffin-embedded testicular tissue derived from men with conserved spermatogenesis. Expression of GPR30 was found to be strongly associated with several cellular types of the human testicular tissue (Fig. 1). The predominant staining pattern of GPR30 was cytoplasmic as typical of seven-transmembrane receptors and as already described for this receptor in cultured breast cancer cell lines. Furthermore, staining for GPR30 occurred both in the cells of the interstitial space (Leydig cells) and in the several cells of the seminiferous tubules (Fig. 1, Panel A). When analyzing exclusively the cells present inside the seminiferous tubules, SCs (Fig. 1, Panel B, red arrows)
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expressions were assessed in cells derived from primary cultures. We were able to demonstrate the expression of GPR30 in SCs at mRNA level, by means of the RT- PCR technique (Fig. 2, Panel A), using as positive control cDNA derived from MCF-7 cells (Fig. 2, Panel C), which are known to widely express this receptor. Using a specific GPR30 antibody we were able to further confirm the expression of GPR30 in these cells by detecting a single band with an apparent molecular weight of 38 kDa in the Western blot analysis, which corresponds to the intact receptor protein (Fig. 2, Panel E). 3.3. GPR30 is expressed in human diploid but not in haploid germ cells Various types of germ cells are present in the human seminiferous tubules, corresponding to the different stages of progression of spermatogenesis. The simplest division of these developing germ cells concerns their chromosomic content, tagging them as diploid or haploid germ cells. In our experiments, using methods previously described by our team, were able to separate diploid from haploid germ cells and evaluate the mRNA and protein expression of GPR30 on both fractions. Indeed, concurrently to what was described in the immunohistochemistry analysis, we observed that the GPR30 mRNA transcripts were present in the diploid fraction of germ cells, while no expression was detected in the haploid germ cells (Fig. 2, Panel B). These results were corroborated when GPR30 protein expression was evaluated using a specific antibody. Diploid germ cells expressed the GPR30 protein, while no signal was detected when we evaluated the protein expression in the haploid germ cell fraction (Fig. 2, Panel D). 4. Discussion
Fig. 1. Immuno-localization of GPR30 in human testicular biopsies with conserved spermatogenesis. Representative microphotographs showing positive GPR30 immuno-reactivity in human seminiferous epithelium with 100 (Panel A) and 400 magnification (Panel B) are provided. Red, green and blue arrows indicate, respectively, Sertoli cells, diploid germ cells and haploid germ cells. Negative controls are provided as insert panels. Scale bars = 200 lm (Panel A), 20 lm (Panel B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and germ cells closest to the basal lamina (diploid germ cells), particularly spermatogonia, (Fig. 1, Panel B, green arrows) showed the distinctive staining correspondent to the presence of GPR30, while germ cells in more advanced stages, of development (haploid germ cells), particularly spermatocytes and elongated spermatids (Fig. 1, Panel B, blue arrows), showed no positive reaction for GPR30 with the antibody used. 3.2. GPR30 is expressed in human Sertoli cells To more accurately examine the cellular specificity of the expression pattern of GPR30 in human SCs, its mRNA and protein
Spermatogenesis is a complex event controlled by several endocrine factors, including the multiple sex steroid hormones. Among those, E2 (and estrogens, in general), which is produced within testicles from androgens by the enzyme aromatase (for review see Lambard et al. (2005)), plays important roles in the development and physiology of male reproductive tract of mammals. Its specific functions are still a matter of debate, even though there is a growing body of evidence suggesting that E2 play an important role via their specific receptors (Alves et al., 2013a; Hess et al., 1997). Indeed, ER knockout animal models present compromised spermatogenesis, steroidogenesis and male fertility (Carreau and Hess, 2010). Estrogens exert their numerous effect by binding, with high affinity, ERa and ERb, classic members of nuclear receptors superfamily, which are selectively expressed in testicular cells (Cavaco et al., 2009). More recently, the existence of an alternative ER, whose action is not blocked by the classic ER antagonists, has been demonstrated (for review see Prossnitz et al. (2008)). Studies have shown that estrogens stimulate second messenger signaling characteristic of the seven transmembrane-spanning receptors and linked it to GPR30, which mediates estrogen effects (Ariazi et al., 2010; Chevalier et al., 2012). GPR30 has been detected in multiple human tissues, including ovary and prostate (for review see Prossnitz et al. (2008)). In addition, GPR30 also has been described in human (Rago et al., 2011) and rodent testis (Otto et al., 2009), although no exhaustive study concerning its expression in specific human testicular cellular types has been done, to the best of the authors knowledge. In the present work we were able to detect GPR30 protein in human testicular somatic (SCs and Leydig cells) and diploid germ cells, by immuno-histochemical analysis using a GPR30 specific antibody. Germ cells in more advanced stages of differentiation (haploid germ cells) did not exhibit any staining correspondent to the expression of GPR30. Interestingly, our results also indicated
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Fig. 2. Expression of GPR30 in cellular fraction and cell types of human seminiferous tubules with conserved spermatogenesis. Panels A and B are representative images of RT-PCR products by agarose gel electrophoresis showing the presence of a GPR30 135 bp amplicon in Sertoli cells (SCs), diploid germ cells (DGc) and haploid germ cells (HGc). Panel C: Representative agarose gel electrophoresis image of a RT-PCR showing positive (PtC, MCF-7 cells cDNA) and negative control (NtC, no-template control) of GPR30 amplification. Panel D: Representative immunoblot analysis of the expression of GPR30 protein in diploid germ cells (DGc) and haploid germ cells (HGc). Panel E: Representative immunoblot image showing the 38 kDa band correspondent to the GPR30 protein in SCs.
that GPR30 displays a intracellular staining pattern predominantly cytoplasmic, which is consistent with prior reports describing a similar distribution pattern in human cells lines, which refer to a endoplasmic reticulum localization of this receptor (Otto et al., 2008; Ye et al., 2011). Furthermore, the present study confirmed, by using cellular fractions correspondent to the distinct cellular types within the human seminiferous tubules, that GPR30 was expressed in SCs and diploid germ cells. It has been shown that these somatic cells (SCs) are key targets of the hormonal regulation that is exerted by sex steroid hormones in the testicular environment (for review see Alves et al. (2013b), Rato et al. (2012b)). It was previously shown by our group that SCs derived from human adult testicular biopsies express ERa (mRNA and protein) (Cavaco et al., 2009), but the expression of GPR30 has only been suggested by immunohistochemical means (Rago et al., 2011). In our work, we were able to detect both mRNA and protein expression of GPR30 in isolated human SCs, by means of molecular biology techniques, suggesting that this receptor plays a role in mediating estrogenic actions in those somatic cells. Furthermore, both mRNA and protein expression of GPR30 were detected in diploid germ cells and not in haploid germ cells. Diploid germ cells directly contact with the circulating constituents of the plasma, while the more differentiated haploid cells are located beyond the blood-testis barrier, one of the tightest blood-tissue barriers, and are not in direct contact with the interstitial fluid, depending almost entirely on SCs to provide them with necessary metabolites and factors for the completion of spermatogenesis (for review see Alves et al. (2012a)). The results here presented clearly demonstrate a differential testicular expression pattern of GPR30, suggesting that this receptor may play a role on estrogen signaling in germ cell differentiation and that its role may be dependent on the progression stage of the spermatogenic event. Nevertheless, these results were obtained using testicular biopsies of men undergoing fertility treatment, and although only samples from individuals exhibiting conserved spermatogenesis (e.g. individuals with anejaculation psychological, vascular or neurologic and vasectomy or traumatic section of the vas deferens) were utilized, further studies are needed to fully enlighten the involvement of GPR30 on spermatogenesis and, hence, on the male reproductive potential.
Acknowledgments This work was supported by FCT – Foundation for Science and Technology (Portugal) (PTDC/QUI-BIQ/121446/2010 and PEst-C/ SAU/UI0709/2011) co-funded by Fundo Europeu de Desenvolvimento Regional – FEDER via Programa Operacional Factores de Competitividade – COMPETE/QREN. M.G. Alves (SFRH/BPD/
80451/2011) and S. Correia (SFRH/BD/60945/2009) were funded by FCT (Portugal). P.F. Oliveira was funded by FCT (Portugal) through FSE and POPH funds (Programa Ciência 2008). R.L. Bernardino was funded by Santander/Totta – UBI (Portugal) protocol. UMIB was funded by national funds through FCT (Portugal) under the Fcomp-01-0124-FEDER-015893 project. References Alves, M.G., Oliveira, P.F., Socorro, S., Moreira, P.I., 2012a. Impact of diabetes in blood-testis and blood–brain barriers: resemblances and differences. Curr. Diabetes Rev. 8, 401–412. Alves, M.G., Socorro, S., Silva, J., Barros, A., Sousa, M., Cavaco, J.E., et al., 2012b. In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17beta-estradiol and suppressed by insulin deprivation. Biochim. Biophys. Acta (BBA) – Mol. Cell Res. 1823, 1389–1394. Alves, M.G., Rato, L., Carvalho, R.A., Moreira, P.I., Socorro, S., Oliveira, P.F., 2013a. Hormonal control of Sertoli cell metabolism regulates spermatogenesis. Cell. Mol Life Sci. 70, 777–793. Alves, M.G., Rato, L., Carvalho, R.A., Moreira, P.I., Socorro, S., Oliveira, P.F., 2013b. Hormonal control of Sertoli cell metabolism regulates spermatogenesis. Cell. Mol. Life Sci. 70, 777–793. Ariazi, E.A., Brailoiu, E., Yerrum, S., Shupp, H.A., Slifker, M.J., Cunliffe, H.E., et al., 2010. The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells. Cancer Res. 70, 1184–1194. Carreau, S., Hess, R.A., 2010. Oestrogens and spermatogenesis. Philos. Trans. R. Soc. B: Biol. Sci. 365, 1517–1535. Carreau, S., Silandre, D.e., Bois, C., Bouraima, H.l., Galeraud-Denis, I., Delalande, C., 2008. Estrogens: a new player in spermatogenesis. Folia Histochem. Cytobiol. 45, 4–5. Cavaco, J., Laurentino, S., Barros, A., Sousa, M., Socorro, S., 2009. Estrogen receptors and in human testis: both isoforms are expressed. Syst. Biol. Reprod. Med. 55, 137–144. Chevalier, N., Vega, A.l., Bouskine, A., Siddeek, B.n., Michiels, J.-F.ß., Chevallier, D., et al., 2012. GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation. PLoS One 7, 34672. Hammes, S.R., Levin, E.R., 2007. Extranuclear steroid receptors: nature and actions. Endocr. Rev. 28, 726–741. Heldring, N., Pike, A., Andersson, S., Matthews, J., Cheng, G., Hartman, J., et al., 2007. Estrogen receptors: how do they signal and what are their targets. Physiol. Rev. 87, 905–931. Hess, R.A., Gist, D.H., Bunick, D., Lubahn, D.B., Farrell, A., Bahr, J., et al., 1997. Estrogen receptor (a and b) expression in the excurrent ducts of the adult male rat reproductive tract. J. Androl. 18, 602–611. Lambard, S., Silandre, D., Delalande, C., Denis-Galeraud, I., Bourguiba, S., Carreau, S., 2005. Aromatase in testis: expression and role in male reproduction. J. Steroid Biochem. Mol. Biol. 95, 63–69. Laurentino, S., Gonçalves, J., Cavaco, J.E., Oliveira, P.F., Alves, M.G., de Sousa, M., et al., 2011. Apoptosis-inhibitor Aven is downregulated in defective spermatogenesis and a novel estrogen target gene in mammalian testis. Fertil. Steril. 96, 745–750. Oliveira, P.F., Sousa, M., Barros, A., Moura, T., Rebelo da Costa, A., 2009. Intracellular pH regulation in human Sertoli cells: role of membrane transporters. Reproduction 137, 353–359. Oliveira, P.F., Alves, M.G., Rato, L., Silva, J., Sá, R., Barros, A., et al., 2011. Influence of 5a-dihydrotestosterone and 17b-estradiol on human Sertoli cells metabolism. Int. J. Androl. 34, e612–e620. Otto, C., Rohde-Schulz, B., Schwarz, G., Fuchs, I., Klewer, M., Brittain, D., et al., 2008. G protein-coupled receptor 30 localizes to the endoplasmic reticulum and is not activated by estradiol. Endocrinology 149, 4846–4856.
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