Expression and regulation of the estrogen receptors in PC-3 human prostate cancer cells.

The aim of this study was to identify the expression, cellular localization and regulation of classic estrogen receptors ERα and ERß, ER-α36 isoform and GPER in the androgen-independent prostate cancer cell line PC-3. In addition, we evaluated the relative contribution of these receptors to the activation of the ERK1/2 (extracellular signal-regulated protein kinases) signaling pathway. These four estrogen receptors were detected by Western blot assays and were shown by immunofluorescence assays to localize preferentially in extranuclear regions of PC-3 cells. In addition, treatment with 17ß-estradiol (E2) (1 µM) for 24 h led to down-regulation of the classic estrogen receptors, whereas E2 at physiological concentration (0.1 nM) for 24h tended to increase the levels of ERα and ERß. Furthermore, the ERα-selective agonist PPT selectively increased the expression of ERß and the ERß-selective agonist DPN increased ERα levels. None of these treatments affected expression of the ER-α36 isoform. The unusual cytoplasmic localization of the classic estrogen receptors in these cells differs from the nuclear localization in the majority of estrogen target cells and suggests that rapid signaling pathways may be preferentially activated. In fact, treatment with selective agonists of ERα, ERß and GPER induced ERK1/2 phosphorylation that was blocked by the respective antagonists. On the other hand, activation of ERK1/2 induced by E2 may involve additional mechanisms because it was not blocked by the three antagonists. Taken together, the results indicate that there is a crosstalk between ERα and ERß to regulate the expression of each other, and suggest the involvement of other receptors, such as ER-α36, in the rapid ERK1/2 activation by E2. The identification of new isoforms of ERs, regulation of the receptors and signaling pathways is important to develop new therapeutic strategies for the castration-resistant prostate cancer.

Relaxin affects cell organization and early and late stages of spermatogenesis in a coculture of rat testicular cells.

Relaxin and its receptor RXFP1 are co-expressed in Sertoli cells, and relaxin can stimulate proliferation of Sertoli cells. In this study, we investigated a role of relaxin in spermatogenesis, using a short-term culture of testicular cells of the rat that allowed differentiation of spermatogonia to spermatids. Sertoli, germ, and peritubular myoid cells were the predominant cell types in the culture. Sertoli and germ cells expressed RXFP1. Cultures were incubated without (control) or with 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN) for 2 days. Cell organization, number, and differentiation were analyzed after 2 (D2), 5 (D5) or 8 (D8) days of culturing. Although the proportion of germ cells decayed from D2 to D5, the relative contribution of HC, 1C, 2C, and 4C germ cell populations remained constant in the control group during the whole culture. RLN did not affect the proportion of germ cell populations compared with control, but increased gene and/or protein expression of the undifferentiated and differentiated spermatogonia markers PLZF and c-KIT, and of the post-meiotic marker Odf2 in D5. RLN favored organization of cells in tubule-like structures, the arrangement of myoid cells around the tubules, arrangement of c-KIT-positive spermatogonia at the basal region of the tubules, and expression of the cell junction protein ß-catenin close to the plasma membrane region. Knockdown of relaxin with small interfering RNA (siRNA) reduced expression of ß-catenin at the cell junctions, and shifted its expression to the nucleus. We propose that relaxin may affect spermatogenesis by modulating spermatogonial self renewal and favoring cell contact.

Effects of the oestrogen receptor antagonist Fulvestrant on expression of genes that affect organization of the epididymal epithelium.

The role of oestrogens in epididymal function is still unclear. Knockout of the oestrogen receptor ESR1 (Esr1(-/-) ) or treatment with the anti-oestrogen Fulvestrant affect epididymal milieu and sperm motility. We investigated the effect of in vivo treatment of rats with Fulvestrant on: (i) expression of genes that may be important for the architecture and function of the epididymal epithelium: prominins 1 and 2, metalloproteinase 7, claudin 7, beta-catenin and cadherin 13, and (ii) levels of oestradiol and testosterone, and expression of oestrogen and androgen receptors, in the initial segment (IS), caput, corpus and cauda epididymis. Fulvestrant (i) reduced gene expression of prominin 1 (variant 1) in the caput, reduced prominin 1 protein content in the caput epididymis and in the efferent ductules, and increased the localization of prominin 1 in microvilli of the caput and corpus; (ii) reduced gene expression of prominin 2 in the corpus and cauda epididymis; (iii) increased the metalloproteinase 7 content in the apical region of principal cells from IS/caput; (iv) reduced in the corpus epididymis, but increased in the efferent ductules, the cadherin 13 mRNA level; (v) reduced testosterone but increased oestradiol levels in the corpus and cauda; (vi) increased the androgen receptor protein content in all regions of the epididymis, and the oestrogen receptor GPER in the corpus and cauda epididymis. In conclusion, treatment with Fulvestrant induced regional-specific changes in hormonal and steroid receptor content, and affected expression of proteins important for epithelial organization and absorption/secretion. The mechanisms of oestrogen action may differ among epididymal regions, which may contribute to determine region-specific sperm functions.

Experimental varicocoele in rats affects mechanisms that control expression and function of the androgen receptor.

Varicocoele is an important cause of male infertility. Normal male reproductive function and fertility depends on a delicate balance between androgen receptor (AR) and the classic oestrogen receptors ESR1 (ERα) and ESR2 (ERß). Using a model of surgically induced varicocoele in rats, this study aimed to investigate the effects of varicocoele on the expression of AR, ESR1, ESR2 and G-protein coupled oestrogen receptor (GPER). Varicocoele did not affect the mRNA and protein expression of ESR1 and ESR2 in both testes. Varicocoele did not affect the mRNA and protein expression of GPER in the right testis, but slightly reduced the mRNA and increased the protein levels in the left testis. Varicocoele did not affect the mRNA for AR, but reduced the protein levels in both testes. A proteomic approach was used in an attempt to find differentially expressed targets with possible correlation with AR downregulation. Varicocoele caused the differential expression of 29 proteins. Six proteins were upregulated, including the receptor for activated C kinase 1 (RACK1), and 23 were downregulated, including dihydrolipoamide dehydrogenase, alpha-enolase and pyrophosphatase 1. Western blot analysis confirmed that varicocoele upregulated the expression of RACK1, a protein involved with tyrosine phosphorylation and regulation of AR transcriptional activity, AR metabolism and dynamics of the blood-testis barrier. In conclusion, this study suggests that varicocoele affects mechanisms that control AR expression and function. This regulation of AR may play an important role in the varicocoele-induced testicular dysfunction. Furthermore, varicocoele downregulates several other proteins in the testis that may be useful markers of spermatozoa function and male infertility.

Relaxin in the male reproductive system

xin (RLN) belong s to a family of hormones structurally related to insulin and presents a broad spectrum of actions. H umans have three forms of RLN , encoded by three different genes ( RLN1 , RLN2 and RLN3 ) , but nonprimate vertebrate s have only two forms of relaxin (RLN1 and RLN3) . RLN1 of these animals is encoded by Rln1 , orthologous to the h uman RLN 2 gene , and both genes , Rln1 and human RLN2 , encode the major form of relaxin found in the male reproductive system . In the reproductive tract of human male s , RLN is mainly produced by the prostate and secreted into the seminal fluid, where it seems to play a role in sperm function. R LN may also play a role in prostate cancer progression. A lack of RLN in animal models impairs male fertili ty , and RLN knockout mice display decreased sperm maturation . T he precise role of RLN in the male reproductive system , however , is still far from clear. RLN actio n is due to its interaction with the G - protein coupled receptor RXFP1. Studies from our labora tory have shown that RLN and RXFP1 are e x pressed in rat Sertoli cells, and e x ogenous RLN stimulates Sertoli cell proliferation. RLN receptors can also be detected in rat germ cells at different stages of development, suggesting that RLN may play a direct r ole in spermatogenesis. The distribution of RLN/RXFP1 , however , appears to be species - dependent, because i n the boar testis RLN production seems restricted to the Leydig cells, whereas RXFP1 is found in Leydig, Sertoli and germ cells. The co - expression of RLN and RXFP1 in several regions of the male reproductive system suggest s that the peptide may act in an autocrine/paracrine fashion.

Relaxin in the male reproductive system

xin (RLN) belong s to a family of hormones structurally related to insulin and presents a broad spectrum of actions. H umans have three forms of RLN , encoded by three different genes ( RLN1 , RLN2 and RLN3 ) , but nonprimate vertebrate s have only two forms of relaxin (RLN1 and RLN3) . RLN1 of these animals is encoded by Rln1 , orthologous to the h uman RLN 2 gene , and both genes , Rln1 and human RLN2 , encode the major form of relaxin found in the male reproductive system . In the reproductive tract of human male s , RLN is mainly produced by the prostate and secreted into the seminal fluid, where it seems to play a role in sperm function. R LN may also play a role in prostate cancer progression. A lack of RLN in animal models impairs male fertili ty , and RLN knockout mice display decreased sperm maturation . T he precise role of RLN in the male reproductive system , however , is still far from clear. RLN actio n is due to its interaction with the G - protein coupled receptor RXFP1. Studies from our labora tory have shown that RLN and RXFP1 are e x pressed in rat Sertoli cells, and e x ogenous RLN stimulates Sertoli cell proliferation. RLN receptors can also be detected in rat germ cells at different stages of development, suggesting that RLN may play a direct r ole in spermatogenesis. The distribution of RLN/RXFP1 , however , appears to be species - dependent, because i n the boar testis RLN production seems restricted to the Leydig cells, whereas RXFP1 is found in Leydig, Sertoli and germ cells. The co - expression of RLN and RXFP1 in several regions of the male reproductive system suggest s that the peptide may act in an autocrine/paracrine fashion.(AU)

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models.