Crosstalk between FSH and relaxin at the end of the proliferative stage of rat Sertoli cells.

Follicle-stimulating hormone (FSH) stimulates the proliferation of immature Sertoli cells through the activation of PI3K/AKT/mTORC1 and MEK/ERK1/2 pathways. Mature Sertoli cells stop proliferating and respond to FSH by stimulating cAMP production. To gain insight into possible mechanisms involved in this switch as well as the impact of paracrine factors that stimulate cell proliferation, we analyzed the effects of FSH and relaxin on intracellular signaling pathways involved with proliferation and differentiation in Sertoli cells from 15-day-old rats, which are close to the transition between the two stages. FSH stimulated 3H-thymidine incorporation and cyclin D1 expression, changes associated with proliferation. In contrast, FSH inhibited AKT and ERK1/2 phosphorylation, activated cAMP production and induced changes in several cell cycle genes that were compatible with differentiation. Relaxin also stimulated 3H-thymidine incorporation but increased phosphorylation of ERK1/2 and AKT. When both hormones were added simultaneously, relaxin attenuated FSH-mediated inhibition of ERK1/2 and AKT phosphorylation and FSH-mediated activation of cAMP production. FSH but not relaxin increased CREB phosphorylation, and relaxin but not FSH shifted NF-κB expression from the cytoplasm to the nucleus. Relaxin did not inhibit the effects of FSH on inhibin α and Bcl2 expression. We propose that at this time of Sertoli cell development, FSH starts to direct cells to differentiation through activation of cAMP/CREB and inhibition of ERK1/2 and AKT pathways. Relaxin counteracts FSH signaling through the inhibition of cAMP and activation of ERK1/2, AKT and NF-κB, but does not block the differentiation process triggered by FSH.

Estrogen receptor beta (ERß) mediates expression of ß-catenin and proliferation in prostate cancer cell line PC-3.

The aim of the present study was to characterize the mechanism underlying estrogen effects on the androgen-independent prostate cancer cell line PC-3. 17ß-estradiol and the ERß-selective agonist DPN, but not the ERα-selective agonist PPT, increased the incorporation of [methyl-(3)H]thymidine and the expression of Cyclin D2, suggesting that ERß mediates the proliferative effect of estrogen on PC-3 cells. In addition, upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by 17ß-estradiol and DPN were blocked by the ERß-selective antagonist PHTPP in PC-3 cells. Upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by DPN were also blocked by PKF118-310, a compound that disrupts ß-catenin-TCF (T-cell-specific transcription factor) complex, suggesting the involvement of ß-catenin in the estradiol effects in PC-3 cells. A diffuse immunostaining for non-phosphorylated ß-catenin was detected in the cytoplasm of PC-3 cells. Low levels of non-phosphorylated ß-catenin immunostaining were also detected near the plasma membrane and in nuclei. Treatment of PC-3 cells with 17ß-estradiol or DPN markedly increased non-phosphorylated ß-catenin expression. These effects were blocked by pretreatment with the ERß-selective antagonist PHTPP, PI3K inhibitor Wortmannin or AKT inhibitor MK-2206, indicating that ERß-PI3K/AKT mediates non-phosphorylated ß-catenin expression. Cycloheximide blocked the DPN-induced upregulation of non-phosphorylated ß-catenin, suggesting de novo synthesis of this protein. In conclusion, these results suggest that estrogen may play a role in androgen-independent prostate cancer cell proliferation through a novel pathway, involving ERß-mediated activation of ß-catenin.

Estrogen receptor ESR1 mediates activation of ERK1/2, CREB, and ELK1 in the corpus of the epididymis.

Expression of the estrogen receptor ESR1 is higher in the corpus than it is in the initial segment/caput and cauda of the epididymis. ESR1 immunostaining in the corpus has been localized not only in the nuclei but also in the cytoplasm and apical membrane, which indicates that ESR1 plays a role in membrane-initiated signaling. The present study investigated whether ESR1 mediates the activation of rapid signaling pathways by estradiol (E2) in the epididymis. We investigated the effect of E2 and the ESR1-selective agonist (4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) on the activation of extracellular signal-regulated protein kinases (ERK1/2), CREB protein, and ETS oncogene-related protein (ELK1). Treatment with PPT did not affect ERK1/2 phosphorylation in the cauda, but it rapidly increased ERK1/2 phosphorylation in the initial segment/caput and corpus of the epididymis. PPT also activated CREB and ELK1 in the corpus of the epididymis. The PPT-induced phosphorylation of ERK1/2, CREB, and ELK1 was blocked by the ESR1-selective antagonist MPP and by pretreatment with a non-receptor tyrosine kinase SRC inhibitor, an EGFR kinase inhibitor, an MEK1/2 inhibitor, and a phosphatidylinositol-3-kinase inhibitor. In conclusion, these results indicate that the corpus, which is a region with high expression of the estrogen receptor ESR1, is a major target in the epididymis for the activation of rapid signaling by E2. The sequence of events that follow E2 interaction with ESR1 includes the SRC-mediated transactivation of EGFR and the phosphorylation of ERK1/2, CREB, and ELK1. This rapid estrogen signaling may modulate gene expression in the corpus of the epididymis, and it may play a role in the dynamic microenvironment of the epididymal lumen.

Differential role of the estrogen receptors ESR1 and ESR2 on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats.

The aim of the present study was to investigate the role of each estrogen receptors on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats. Activation of ESR1 by 17ß-estradiol (E2) and ESR1-selective agonist PPT increased CCND1 expression, and this effect was dependent on NF-kB activation. E2 and the ESR2-selective agonist DPN, but not PPT, increased, in a PI3K and CREB-dependent manner, the expression of CDKN1B and the transcription factors GATA-1 and DMRT1. Analyzing the expression of ESR1 and ESR2 in different stages of development of Sertoli cells, we observed that the ESR1/ESR2 ratio decreased with age, and this ratio seems to be important to determine the end of cell proliferation and the start of cell differentiation. In Sertoli cells from 15-day-old rats, the ESR1/ESR2 ratio favors the effect of ESR1 and the activation of this receptor increased [Methyl-(3)H]thymidine incorporation. We propose that in Sertoli cells from 15-day-old rats E2 modulates Sertoli cell proliferation through ESR1/NF-kB-mediated increase of CCND1, and cell cycle exit and differentiation through ESR2/CREB-mediated increase of CDKN1B, GATA-1 and DMRT1. The present study reinforces the important role of estrogen for normal testis development.

Physiopathological aspects of the Wnt/ß-catenin signaling pathway in the male reproductive system.

The Wnt/ß-catenin signaling pathway controls several biological processes throughout development and adult life. Dysregulation of Wnt/ß-catenin signaling underlies a wide range of pathologies in animals and humans, including cancer in different tissues. In this review, we provide an update of the Wnt/ß-catenin signaling pathway and the possible roles of the Wnt/ß-catenin signaling in the biology of testis, epididymis and prostate. Data from our laboratory suggest the involvement of 17ß-estradiol and estrogen receptors (ERs) on the regulation of ß-catenin expression in rat Sertoli cells. We also provide emerging evidences of the involvement of Wnt/ß-catenin pathway in testis and prostate cancer. Our understanding of the role of Wnt/ß-Catenin signaling in male reproductive tissues is still evolving, and several questions are open to be addressed in the future.

Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate signaling in the hippocampus from rats in proestrous and estrous phases.

The aim of the present study was to investigate the involvement of estrogen receptors in the activation of phospholipase C (PLC)-phosphoinositide hydrolysis in the hippocampus from rats in estrous and proestrous phases. 17ß-Estradiol (E2) and ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, induced a rapid increase on total [³H]-inositol phosphate accumulation in the hippocampus from both rats. These effects are mediated by PLC activation, since the inhibition of this protein decreased the total [³H]-inositol phosphate accumulation. The pretreatment with ESR1 and ESR2 antagonist ICI 182,780, but not with GPER antagonist G-15, blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT, confirming that ESR1 is upstream component regulating this rapid effect. SRC family of protein tyrosine kinases inhibitor PP2 blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT in hippocampus, suggesting that ESR1 undergoes translocation from the nuclei to the plasma membrane region via SRC to activate rapid signaling pathways. Furthermore, the magnitude of the response to E2 and PPT was higher in hippocampus from rats in proestrous than in estrous. On the other hand, the expression of the ESR1 is higher in hippocampus from rats in estrous than in proestrous, indicating that the regulation of this receptor by estrous cycle does not play a role in the magnitude of the response to E2 and PPT in hippocampus. In conclusion, our results indicate that E2 activates SRC-mediated translocation of ESR1 to the plasma membrane, which results in the activation of PLC-inositol phosphate signaling pathway in rat hippocampus. Thus, these rapid estrogen actions in hippocampus might be a key step mediating cellular events important for learning and memory.

Relaxin and Sertoli cell proliferation.

Immature Sertoli cells proliferate and several factors affect their number, including the follicle stimulating hormone (FSH), testosterone, estradiol and several paracrine growth factors. Using a primary culture of Sertoli cells isolated from 15-day old Wistar rats we have shown that relaxin stimulates Sertoli cell proliferation through the activation of MEK/ERK1/2 and PI3K/AKT pathways. In contrast, FSH inhibited both ERK1/2 and AKT phosphorylation. Furthermore, FSH strongly increased cAMP production, whereas relaxin inhibited basal cAMP production. Our results indicate that in rat Sertoli cells from 15-day old rats relaxin and FSH affect the same signaling pathways in opposite directions. Interplay between both hormones may be important to control the proliferation and differentiation of Sertoli cells.

Na+/K+-ATPase α1 isoform mediates ouabain-induced expression of cyclin D1 and proliferation of rat sertoli cells.

Novel roles for the interaction of cardiotonic steroids to Na(+)/K(+)-ATPase have been established in recent years. The aim of this study was to investigate the intracellular signaling events downstream the action of ouabain on Na(+)/K(+)-ATPase in Sertoli cell obtained from immature rats. Treatment of Sertoli cells with ouabain (1 µM) induced a rapid and transient increase in the extracellular signal-regulated kinase (ERK1/2 or MAPK3/1) and phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT) phosphorylation. Also, ouabain upregulated the expression of cyclin D1 and incorporation of [methyl-(3)H]thymidine, both of which were dependent on MAPK3/1 but not AKT intracellular cascade, as shown by pretreatment with MEK (MAP2K1/2) inhibitor U0126 and PI3K inhibitor wortmannin respectively. Moreover, the effect of ouabain on these proliferation parameters was completely prevented by phospho-cAMP response element-binding protein (CREB)/CREB-binding protein complex inhibitor KG501 and only partially by nuclear factor κB nuclear translocation inhibitor SN50. Pretreatment with estrogen receptor antagonist ICI 182780 showed that MAPK3/1 activation by ouabain does not involve this receptor. The Na(+)/K(+)-ATPase α1 isoform, but not α4, was detected in Sertoli cells, suggesting that ouabain effects in Sertoli cells are mediated via α1. Taken together, these results show a rapid ouabain action in the Sertoli cells, which in turn can modulate nuclear transcriptional events essential for Sertoli cell proliferation in a critical period of testicular development. Our findings are important to understand the role of ouabain in the testis and its possible implications in male infertility.

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells.

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.

17Beta-estradiol signaling and regulation of proliferation and apoptosis of rat Sertoli cells.

The aim of the present study was to investigate the intracellular signaling events downstream of the classical estrogen receptors (ESRs) and G protein-coupled estrogen receptor 1 (GPER) involved in regulation of proliferation and apoptosis of rat Sertoli cells, in which we have previously described ESR1, ESR2, and GPER. ESRs play a role in Sertoli cell proliferation, and GPER, but not ESRs, plays a role modulating gene expression involved with apoptosis. The present study shows that 17beta-estradiol (E2) and the GPER-selective agonist G-1 rapidly activate phosphatidylinositol 3-kinase (PIK3)/serine threonine protein kinase (AKT) and cyclic AMP response element-binding (CREB) phosphorylation. E2 and the ESR1-selective agonist 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) increase the expression of cyclin D1 (CCND1), whereas the ESR2-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and G-1 do not change the expression of this protein, suggesting that ESR1 is the upstream receptor regulating Sertoli cell proliferation. E2- or PPT-ESR1, through activation of epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase 3/1 (MAPK3/1) and PIK3 pathways, induces upregulation of CCND1. KG-501, the compound that disrupts the phospho-CREB/CREB binding protein (CBP) complex, does not change E2- or PPT-ESR1-mediated CCND1 expression, suggesting that phospho-CREB/cyclic AMP response element/CBP is not involved in the expression of this protein. E2- or G-1-GPER, through activation of EGFR/MAPK3/1 and PIK3 pathways, may be involved in the upregulation of antiapoptotic proteins BCL2 and BCL2L2. E2- or G-1-GPER/EGFR/MAPK3/1/phospho-CREB decreases BAX expression. Taken together, these results show a differential effect of E2-GPER on the CREB-mediated transcription of proapoptotic and antiapoptotic genes of the same BCL2 gene family. ESR1 and GPER can mediate the rapid E2 actions in the Sertoli cells, which in turn can modulate nuclear transcriptional events important for Sertoli cell function and maintenance of normal testis development and homeostasis. Our findings are important to clarify the role of estrogen in a critical period of testicular development, and to direct further studies, which may contribute to better understanding of the causes of male infertility.

Estrogen receptors mediate rapid activation of phospholipase C pathway in the rat endometrium.

The aim of the present study was to investigate the activation of rapid signaling events by 17ß-estradiol in the rat uterus. 17ß-Estradiol induced a rapid increase of total [3H]-inositol phosphate accumulation in the whole uterus and endometrium, but not in the myometrium. The effect of 17ß-estradiol in the endometrium was blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [(3)H]-inositol phosphate accumulation in the endometrium. The G-1 effects were blocked by GPER antagonist (G-15). 17ß-Estradiol and G-1 promoted an additive effect on total [3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17ß-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormone stimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

Estrogen receptors mediate rapid activation of phospholipase C pathwayin the rat endometrium

The aim of the present study was to investigate the activation of rapid signaling events by 17b-estradiolin the rat uterus. 17b-Estradiol induced a rapid increase of total [3H]-inositol phosphate accumulation inthe whole uterus and endometrium, but not in the myometrium. The effect of 17b-estradiol in the endometriumwas blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [3H]-inositol phosphate accumulation in the endometrium.The G-1 effects were blocked by GPER antagonist (G-15). 17b-Estradiol and G-1 promoted an additive effect on total [3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17b-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormonestimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

17beta-estradiol induces the translocation of the estrogen receptors ESR1 and ESR2 to the cell membrane, MAPK3/1 phosphorylation and proliferation of cultured immature rat Sertoli cells.

The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study.

The aim of the present work was to study the effect of muscarinic agonist on cell proliferation and muscarinic acetylcholine receptors (mAChRs) regulation in rat Sertoli cells. Primary cultures of Sertoli cells were obtained from 8-day and 15-day old male Wistar rats. In proliferation assays, [methyl-3H]thymidine incorporation in Sertoli cells from 8-day and 15-day old rats reached a plateau after 60 min of carbachol incubation and decreased after 120 min of agonist incubation. Binding studies with [N-Methyl-3H]scopolamine ([3H]NMS) indicated a rapid loss of cell surface mAChRs when Sertoli cells from 15-day old rats were incubated with carbachol at 35 degrees C for 2 min. This effect was temperature-dependent. When the incubation of the cells was prolonged at 35 degrees C or at 4 degrees C, after the agonist had been washed away, 94% of mAChRs were present in the cell surface after 120 min incubation at 35 degrees C. At 4 degrees C, however, a low percentage of mAChRs was detected in the cell surface. In the presence of cycloheximide, the recycling of mAChRs to the cell surface was not changed, suggesting that the appearance of mAChRs on cell surface was not dependent on de novo receptor synthesis. In conclusion, our studies indicate that the activation of mAChRs may play a role in rat Sertoli cell proliferation. These receptors may be under regulation (internalization and recycling) when cells are exposed to muscarinic cholinergic agonist.