Maria Fatima M. Lazari

Receptors for androgen-binding proteins : internalization and intracellular signalling

In plasma, most steroid hormones are bound and transported by the specific binding protein, testosterone-estradiol-binding globulin (TeBG). For years, it was believed that the only function of this protein was to regulate the concentration of free steroids in plasma. However, a number of reports have provided evidence for the presence of specific TeBG receptors on plasma membranes. Furthermore, the interaction of TeBG with its receptor was shown to be inhibited when steroids are bound to TeBG, suggesting that TeBG is an allosteric protein. The purpose of this manuscript is to review the evidence that androgen-binding proteins bind to membrane receptors, and, in some cells, this binding stimulates cAMP accumulation, and transfer TeBG/ABP into tissue as a consequence of receptor mediated endocytosis. Recent studies from our laboratories have demonstrated binding and uptake of TeBG by MCF-7 breast cancer cells. The interaction of unligated rabbit TeBG with membranes from MCF-7 cells resulted in a time and concentration-dependent increase in adenylate cyclase activity. The TeBG alone also had a reproducible effect on intact cells by increasing cAMP accumulation by 30-35%. The addition of DHT to cells, after TeBG has been allowed to bind, resulted in increases in cAMP of greater than 4-fold. This effect was not blocked by antiandrogens. These data support the hypothesis that extracellular SHBG is a regulator of cellular function through a membrane receptor that is coupled to adenylate cyclase.

17β-estradiol signaling and regulation of Sertoli cell function

In this review, we will present an overview of estrogen actions in the testis from immature and adult animals, with special emphasis on signaling mechanisms involved in the 17β-estradiol regulation of Sertoli cell function from in immature rats. 17β-estradiol activates Sertoli cell proliferation in immature rats by a mechanism that involves the translocation of the estrogen receptors ESR1 and ESR2 to the plasma membrane, phosphorylation of epidermal growth factor receptor and activation of mitogen-activated protein kinase 3/1. Activation of the G protein-coupled estrogen receptor (GPER) also induces phosphorylation of mitogen-activated protein kinase 3/1 via epidermal growth factor receptor transactivation, which in turn increases expression of the antiapoptotic protein BCL2 and decreases the expression of proapoptotic protein BAX, indicating an antiapoptotic role of E2-GPER in immature rat Sertoli cells. In conclusion, ESRs and GPER can mediate rapid 17β-estradiol signaling in Sertoli cells, and modulate 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 understand the causes of male infertility.

Locally produced relaxin may affect testis and vas deferens function in rats

We have previously shown that the rat testis and vas deferens contain high levels of the relaxin receptor, RXFP1. The present study was undertaken to determine the expression of relaxin in these tissues, and the effect of exogenous relaxin on Sertoli cell proliferation and on the mRNA levels of some proteins that may contribute to epithelial secretion and tissue reorganization in the vas deferens. Relaxin mRNA levels in testis and vas deferens were much lower than in the prostate. Sertoli cells seem to be an important source of relaxin mRNA in testis. Relaxin immunoreactivity was detected in the seminiferous epithelium but not in the interstitial compartment. The relaxin precursor was expressed in the vas deferens, and relaxin immunoreactivity was detected in apical cells of the vas deferens. Castration, but not treatment with the anti-estrogen ICI 182,780, dramatically reduced relaxin mRNA levels in the prostate and vas deferens, and this effect was prevented by testosterone. Rxfp1 mRNA levels in the vas deferens and prostate were not affected by castration or treatment with ICI 182,780. Exogenous relaxin increased the incorporation of (3)H-thymidine in cultured Sertoli cells, and treatment of the vas deferens with 100 ng/ml relaxin increased the mRNA levels for the cystic fibrosis chloride channel (cystic fibrosis transmembrane regulator) about three times, and doubled mRNA levels for the inducible form of nitric oxide synthase and metalloproteinase 7. These results suggest that locally produced relaxin acts as an autocrine or paracrine agent in the testis and vas deferens to affect spermatogenesis and seminal fluid composition.

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

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

Receptors and signaling pathways involved in proliferation and differentiation of Sertoli cells

The identification of the hormones and other factors regulating Sertoli cell survival, proliferation, and maturation in neonatal, peripubertal, and pubertal life remains one of the most critical questions in testicular biology. The regulation of Sertoli cell proliferation and differentiation is thought to be controlled by cell–cell junctions and a set of circulating and local hormones and growth factors. In this review, we will focus on receptors and intracellular signaling pathways activated by androgen, follicle-stimulating hormone, thyroid hormone, activin, retinoids, insulin, insulin-like growth factor, relaxin, and estrogen, with special emphasis on estrogen receptors. Estrogen receptors activate intracellular signaling pathways that converge on cell cycle and transcription factors and play a role in the regulation of Sertoli cell proliferation and differentiation.

Receptor-mediated endocytosis of angiotensin II in rat myometrial cells.

The events involved in the processing of the angiotensin II (Ang II)-receptor complex were studied in primary cultures of rat myometrial cells. Ang II bound to rat myometrial cells in a specific, time- and temperature-dependent fashion. Pretreatment with cycloheximide did not interfere with binding up to 3 hr, but inhibited increases in binding observed over longer periods. The [3H]Ang II binding to intact cells was inhibited by dithiothreitol (DTT), and the rank order of potency of Ang II and nonpeptide antagonists to inhibit the [3H]Ang II binding was Ang II > Losartan >> PD 123319 or CGP 42112B, indicating the presence of the AT1 receptor type. Whereas most of the [3H]Ang II binding at 4 degrees was susceptible to acid or pronase treatment, binding at 35 degrees was resistant to both treatments, suggesting an internalization of the Ang II-receptor complex. Phenylarsine oxide (PAO) and N-ethylmaleimide (NEM) caused a concentration-dependent inhibition when the binding assay was performed at 35 degrees, but no effect was observed at 4 degrees, indicating that these agents did not alter cell-surface binding but actually prevented the internalization process. Simultaneous treatment with 1 mM DTT or beta-mercaptoethanol prevented the inhibitory effect of NEM, but only DTT could prevent the inhibition caused by PAO, indicating that two closely located sulfhydryl groups must be involved in the internalization process. Chloroquine (100 microM) inhibited the [3H]Ang II dissociation from cells, and monensin (25 microM) induced a 30% inhibition of [3H]Ang II binding (35 degrees, 3 hr), suggesting endosomal processing of the Ang II-receptor complex with receptor recycling to the cell surface. These results indicate that Ang II binding to AT1 receptors in rat myometrial cells is followed by internalization of the Ang II-receptor complex and recycling of the receptor to the cell surface.

Some functional aspects of the renin-angiotensin system in the snake Bothrops jararaca

Abstract Vasopressor and steroidogenic effects of synthetic angiotensin [Asn 1 , Val 5 ] AII and Bothrops jararaca crude renin were compared in anesthetized Bothrops jararaca snakes. While the intravenous infusion of [Asn 1 , Val 5 ] AII (2.0 μg/kg/min) promoted an arterial blood pressure elevation of 18.2 ± 2.7 mmHg but had no detectable steroidogenic effect, Bothrops jararaca crude renin (0.4 ml/kg/min) induced an increase of both, mean arterial blood pressure (12.8 ± 2.0 mmHg) and plasma corticosterone concentration (85.8 ± 20.1 ng/ml). In vitro experiments indicated that the relative potencies of synthetic angiotensins were [Asn1, Val5] AII 1 , Ile 5 ] AII = (Asp 1 , Val 5 ] AII in B. jararaca uterus, but [Asn 1 , Val 5 ] AII > (Asp 1 Ile 5 ] AII in the guinea-pig ileum. The results indicate that the renin-angiotensin system may be important for the control of blood pressure and steroidogenesis in the snake, although some differences in the structural requirements for angiotensin action occur in this animal when compared to mammals.

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.

CORRECTION: Expression and Signaling of G Protein-Coupled Estrogen Receptor 1 (GPER) in Rat Sertoli Cells

FIG. 5. Signaling pathways involved in MAPK3/1 phosphorylation induced by G-1 in the Sertoli cells. Cells were incubated in the absence (C, control) and presence of G-1 (1 nM). Cells were untreated or pretreated with the SRC family of protein tyrosine kinases inhibitor (PP2, 5 nM, 30 min; A); metalloprotease inhibitor GM6001 (GM200 nM, 30 min; A), MAP2K1/2 inhibitor (U0126, 20 lM, 30 min; A), pertussis toxin (PTX, 100 lM, 16 h; B), and EGFR kinase inhibitor (AG 1478, 50 lM, 15 min; B). Afterwards, cells were stimulated with G-1 (1 nM, 10 min). Total cell lysates (60 lg of protein/lane) were resolved in 10% SDS/PAGE, transferred to PVDF membrane, and probed with antibody specific for phosphorylated MAPK3/1 (top panels) or with antibody that recognizes total (phosphorylation state-independent) MAPK3/1 proteins (bottom panels). The relative positions of phosphorylated MAPK3/1 and total MAPK3/1 proteins are shown at the right. The data shown are representative of four independent experiments. Right panels: bars represent the densitometric analysis of the Western blot assays. Solid bars1⁄4MAPK3; open bars1⁄4MAPK1. Results were normalized to total MAPK3/1 expression in each sample and plotted (mean 6 SEM) in relation to control, C (1⁄41). *MAPK3/1 activation significantly greater than that of control (P , 0.05, Student t-test).

Effects of relaxin in a co-culture of Sertoli and germ cells.

Spermatogenesis is controlled by FSH, testosterone and paracrine factors produced by Sertoli cells. The knockout of relaxin decreases sperm maturation in mice. Studies from our laboratory have shown that relaxin and its receptor RXFP1 are expressed in rat Sertoli cells, and exogenous relaxin stimulates Sertoli cell proliferation. Relaxin receptors are also detected in the rat germ cells at specific stages of development. Relaxin could therefore affect spermatogenesis either indirectly, by stimulating Sertoli cell proliferation, or directly, by affecting germ cells. The aim of the present study was to explore a role of relaxin at specific stages of spermatogenesis using a co-culture of rat Sertoli and germ cells. Relaxin seems to increase the number of pre-meiotic and meiotic cells.