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Rapid signaling responses in Sertoli cell membranes induced by follicle stimulating hormone and testosterone: calcium inflow and electrophysiological changes.

This minireview describes the rapid signaling actions of follicle stimulating hormone (FSH) and testosterone in immature Sertoli cells mainly related to Ca(2+) inflow and the electrophysiological changes produced by hormones. The rapid membrane actions of FSH occur in a time frame of seconds to minutes, which include membrane depolarization and the stimulation of (45)Ca(2+) uptake. These effects can be prevented by pertussis toxin (PTX), suggesting that they are likely mediated by Gi-protein coupled receptor activation. Furthermore, these effects were inhibited by verapamil, a blocker of the L-type voltage-dependent Ca(2+) channel (VDCC). Finally, FSH stimulation of (45)Ca(2+) uptake was inhibited by the (phosphoinositide 3-kinase) PI3K inhibitor wortmannin. These results suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from pre-pubertal rats is mediated by the Gi/Gβγ/PI3Kγ pathway, independent of its effects on insulin-like growth factor type I (IGF-I). Testosterone depolarizes the membrane potential and increases the resistance and the (45)Ca(2+) uptake in Sertoli cells of the seminiferous tubules of immature rats. These actions were nullified by diazoxide (K(+)(ATP) channel opener). Testosterone actions were blocked by both PTX and the phospholipase C (PLC) inhibitor U73122, suggesting the involvement of PLC - phosphatidylinositol 4-5 bisphosphate (PIP2) hydrolysis via the Gq protein in the testosterone-mediated pathway. These results indicate that testosterone acts on the Sertoli cell membrane through the K(+)(ATP) channels and PLC-PIP2 hydrolysis, which closes the channel, depolarizes the membrane and stimulates (45)Ca(2+) uptake. These results demonstrate the existence of rapid non-classical pathways in immature Sertoli cells regulated by FSH and testosterone.

Pertussis Toxin Nullifies the Depolarization of the Membrane Potential and the Stimulation of the Rapid Phase of 45Ca2+ Entry Through L-type Calcium Channels that are Produced by Follicle Stimulating Hormone in 10- to 12-Day-Old Rat Sertoli Cells

The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca2+ into cells and to the Ca2+-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of 45Ca2+ uptake and [14C]-methyl aminoisobutyric acid ([14C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca2+ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of 45Ca2+ entry and [14C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca2+ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.

Mechanisms of Hormonal Regulation of Sertoli Cell Development and Proliferation: A Key Process for Spermatogenesis

In adulthood, the main function of the testes is the production of male gametes. In this process, Sertoli cells are essential for sustained spermatogenesis, providing the developing germ cells with the physical and nutritional support required. The total number of Sertoli cells in adulthood determines the daily gamete production, since Sertoli cells can support only a limited number of developing germ cells. Considering that Sertoli cell proliferation only occurs during the immature period, proper development and proliferation of the Sertoli cells during the proliferative phase are crucial to male reproductive health in adulthood. The proliferation process of the Sertoli cells is finely regulated by an assortment of hormonal and paracrine/autocrine factors, which regulate the rate and extent of proliferation. In the present review, we discuss the most important hormonal and paracrine factors involved in the regulation of Sertoli cell proliferation, as well as the signaling mechanisms by which they exert their effects.

Epitestosterone and testosterone have similar nonclassical actions on membrane of Sertoli cells in whole seminiferous tubules.

Epitestosterone is the 17α-epimer of testosterone. This steroid possesses antiandrogenic activities. The mechanism of action of epitestosterone has not been elucidated. The aim of this study was to investigate the nonclassical effect of epitestosterone on the membrane of Sertoli cells in proliferative phase (rats aged 15 days) and in nonproliferative phase (rats aged 21 and 35 days). The membrane potential of Sertoli cells was recorded using a standard single microelectrode technique. Epitestosterone (0.5, 1, and 2 μM) or testosterone (1 μM) was administered alone and after infusion with flutamide (1 μM), verapamil (100 μM), or U-73122 (2 μM). The testes of rats aged 12-15 days were preincubated with 45Ca2+ with or without flutamide (1 μM) and incubated with epitestosterone (1 μM) or testosterone (1 μM). Epitestosterone and testosterone produced a depolarization in the membrane potential and increased the membrane input resistance on Sertoli cells from rats of all 3 ages. The effect of epitestosterone did not change after perfusion with flutamide. Epitestosterone increased 45Ca2+ uptake within 5 min and this effect was not inhibited by flutamide. The absence of an effect by flutamide suggests that epitestosterone acts independently of the intracellular androgen receptor. The depolarizing effect was inhibited by verapamil, a voltage-dependent calcium channel blocker, and by U-73122, a phospholipase C inhibitor. These results indicate that epitestosterone acts on the membrane via a nonclassical signaling pathway; the effect was similar to the testosterone action on membrane of Sertoli cells in whole seminiferous tubules from rat testes.

Insulin and IGF-I actions on IGF-I receptor in seminiferous tubules from immature rats

Insulin and insulin-like growth factor 1 (IGF-I) are capable of activating similar intracellular pathways. Insulin acts mainly through its own receptor, but can also activate the IGF-I receptor (IGF-IR). The aim of this study was to investigate the involvement of the IGF-IR in the effects of insulin and IGF-I on the membrane potential of immature Sertoli cells in whole seminiferous tubules, as well as on calcium, amino acid, and glucose uptake in testicular tissue of immature rats. The membrane potential of the Sertoli cells was recorded using a standard single microelectrode technique. In calcium uptake experiments, the testes were pre-incubated with (45)Ca(2+), with or without JB1 (1 μg/mL), and then incubated with insulin (100 nM) or IGF-I (15 nM). In amino acid and glucose uptake experiments, the gonads were pre-incubated with or without JB1 (1 μg/mL) and then incubated with radiolabeled amino acid or glucose analogues in the presence of insulin (100 nM) or IGF-I (15 nM). The blockade of IGF-IR with JB1 prevented the depolarising effects of both insulin and IGF-I on membrane potential, as well as the effect of insulin on calcium uptake. JB1 also inhibited the effects of insulin and IGF-I on glucose uptake. The effect of IGF-I on amino acid transport was inhibited in the presence of JB1, whereas the effect of insulin was not. We concluded that while IGF-I seems to act mainly through its cognate receptor to induce membrane depolarisation and calcium, amino acid and glucose uptake, insulin appears to be able to elicit its effects through IGF-IR, in seminiferous tubules from immature rats.

Non-classical effects of androgens on testes from neonatal rats

The intratesticular testosterone concentration is high during the early postnatal period although the intracellular androgen receptor expression (iAR) is still absent in Sertoli cells (SCs). This study aimed to evaluate the non-classical effects of testosterone and epitestosterone on calcium uptake and the electrophysiological effects of testosterone (1μM) on SCs from rats on postnatal day (pnd) 3 and 4 with lack of expression of the iAR. In addition, crosstalk on the electrophysiological effects of testosterone and epitestosterone with follicle stimulating hormone (FSH) in SCs from 15-day-old rats was evaluated. The isotope (45)Ca(2+) was utilized to evaluate the effects of testosterone and epitestosterone in calcium uptake. The membrane potential of SCs was recorded using a standard single microelectrode technique. No immunoreaction concerning the iAR was observed in SCs on pnd 3 and 4. At this age, both testosterone and epitestosterone increased the (45)Ca(2+) uptake. Testosterone promoted membrane potential depolarization of SCs on pnd 4. FSH application followed by testosterone and epitestosterone reduced the depolarization of the two hormones. Application of epitestosterone 5 min after FSH resulted in a delay of epitestosterone-promoted depolarization. The cell resistance was also reduced. Thus, in SCs from neonatal Wistar rats, both testosterone and epitestosterone act through a non-classical mechanism stimulating calcium uptake in whole testes, and testosterone produces a depolarizing effect on SC membranes. Testosterone and epitestosterone stimulates non-classical actions via a membrane mechanism, which is independent of iAR. FSH and testosterone/epitestosterone affect each others electrophysiological responses suggesting crosstalk between the intracellular signaling pathways.

Epitestosterone- and testosterone-replacement in immature castrated rats changes main testicular developmental characteristics

Epitestosterone is the 17α-epimer of testosterone and has been described as an anti-androgen, since it inhibits the effects produced by testosterone and dihydrotestosterone via the nuclear androgen receptor (nAR). However, epitestosterone also displays an effect which is similar to the non-classical effect of testosterone, depolarizing the membrane potential of Sertoli cells and inducing a rapid Ca2+ uptake. This study aimed to investigate the effects of a treatment with epitestosterone on developmental parameters of immature rats. Animals were chemically castrated by using the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix and then received a replacement of 7 days with epitestosterone or testosterone. Replacement with either epitestosterone or testosterone restored the anogenital distance (AGD) and testicular weight which had been reduced by chemical castration. The immunocontent of nAR and the nAR-immunoreactivity were reduced by epitestosterone treatment in the testis of both castrated and non-castrated animals. Furthermore, testosterone was unable of changing the membrane potential of Sertoli cells through its non-classical action in the group of animals castrated and replaced with epitestosterone. In conclusion, in relation to the level of protein expression of nAR epitestosterone acts as an anti-androgen. However, it acts in the same way as testosterone when genital development parameters are evaluated. Moreover, in castrated rats epitestosterone suppressed the non-classical response of testosterone, changing the pattern of testosterone signalling via a membrane mechanism in Sertoli cells.