History and Function of the Lactate Receptor GPR81/HCAR1 in the Brain: A Putative Therapeutic Target for the Treatment of Cerebral Ischemia.

GPR81 is a G-protein coupled receptor (GPCR) discovered in 2001, but deorphanized only 7 years later, when its affinity for lactate as an endogenous ligand was demonstrated. More recently, GPR81 expression and distribution in the brain were also confirmed and the function of lactate as a volume transmitter has been suggested since then. These findings shed light on a new function of lactate acting as a signaling molecule in the central nervous system, in addition to its well-known role as a metabolic fuel for neurons. GPR81 seems to act as a metabolic sensor, coupling energy metabolism, synaptic activity, and blood flow. Activation of this receptor leads to Gi-mediated downregulation of adenylyl cyclase and subsequent reduction in cAMP levels, regulating several downstream pathways. Recent studies have also suggested the potential role of lactate as a neuroprotective agent, mainly under brain ischemic conditions. This effect is usually attributed to the metabolic role of lactate, but the underlying mechanisms need further investigation and could be related to lactate signaling via GPR81. The activation of GPR81 showed promising results for neuroprotection: it modulates many processes involved in the pathophysiology of ischemia. In this review, we summarize the history of GPR81, starting with its deorphanization; then, we discuss GPR81 expression and distribution, signaling transduction cascades, and neuroprotective roles. Lastly, we propose GPR81 as a potential target for the treatment of cerebral ischemia.

Role of non-classical effects of testosterone and epitestosterone on AMH balance and testicular development parameters.

Testosterone (T) and its 17-α epimer, epitestosterone (EpiT), are described as having non-classical effects in addition to their classical androgen actions via the intracellular androgen receptor (iAR). The actions of these androgens play an essential role in triggering factors that shift Sertoli cells from the proliferation phase to the maturation phase. This process is essential for successful spermatogenesis and normal fertility. The aim of this work was to investigate the difference between T and EpiT effects in normal and in chemically castrated Wistar rats. We also tested the effects of these hormones when the iAR-dependent pathways were inhibited by the antiandrogen flutamide. Rats were chemically castrated on postnatal day (pnd) 5 using EDS, a cytotoxic agent that promotes apoptosis of Leydig cells, reducing androgen levels. Then, animals received replacement with T or EpiT and were treated or not with flutamide from pnd 6 to pnd 13 or 20 and were euthanized on pnd 14 and 21. Animals treated with EpiT and flutamide had lower body weight overall. Epididymis weight was also reduced in animals treated with EpiT and flutamide. Flutamide per se reduced epididymis weight at both ages (pnd 14 and 21). Testicular weight and the testicular/body weight ratio were reduced in EDS animals, and flutamide further reduced this weight in animals which received T replacement. EDS administration reduced mRNA levels of both AMH (anti-Müllerian hormone) and its receptor, AMHR2, at pnd 14. In the testes of flutamide-treated animals, EpiT reduced AMH, and both T and EpiT replacement diminished AMHR2 mRNA expression also on pnd 14. EDS decreased iAR expression, and androgen replacement did not change this effect on pnd 21. In rats receiving flutamide, only those also receiving T and EpiT replacement exhibited decreased iAR expression. An increase in connexin 43 expression was observed in animals treated with EpiT without flutamide, whereas in rats treated with flutamide, both hormones were ineffective to increase connexin 43 expression reduced by EDS. Our results suggest that EpiT has an antiandrogen effect on androgen-sensitive tissues such as the epididymis. Nonetheless, the effects of T and EpiT on testicular development parameters are similar. Both hormones may act through their iAR-independent non-classical pathway, regulating AMH and AMHR2, as well as iAR expression.

Electrophysiological Responses to Different Follicle-Stimulating Hormone Isoforms on Human Cumulus Oophorus Cells: Preliminary Results.

OBJECTIVE: The aim of the present study was to provide a better understanding of the specific action of two follicle-stimulating hormone (FSH) isoforms (ß-follitropin and sheep FSH) on the membrane potential of human cumulus cells. METHODS: Electrophysiological data were associated with the characteristics of the patient, such as age and cause of infertility. The membrane potential of cumulus cells was recorded with borosilicate microelectrodes filled with KCl (3 M) with tip resistance of 15 to 25 MΩ. Sheep FSH and ß-follitropin were topically administered onto the cells after stabilization of the resting potential for at least 5 minutes. RESULTS: In cumulus cells, the mean resting membrane potential was - 34.02 ± 2.04 mV (n = 14). The mean membrane resistance was 16.5 ± 1.8 MΩ (n = 14). Sheep FSH (4 mUI/mL) and ß-follitropin (4 mUI/mL) produced depolarization in the membrane potential 180 and 120 seconds after the administration of the hormone, respectively. CONCLUSION: Both FSH isoforms induced similar depolarization patterns, but ß-follitropin presented a faster response. A better understanding of the differences of the effects of FSH isoforms on cell membrane potential shall contribute to improve the use of gonadotrophins in fertility treatments.

OBJETIVO: O objetivo do presente estudo foi fornecer uma melhor compreensão da ação específica de duas isoformas de hormônio folículo estimulante (FSH, sigla em inglês) (ß-folitropina e FSH ovino) no potencial de membrana de células do cumulus oophorus humanas. MéTODOS: Dados eletrofisiológicos foram associados às características da paciente, como idade e causa da infertilidade. O potencial de membrana das células do cumulus foi registrado com microeletrodos de borossilicato preenchidos com KCl (3 M) com uma resistência de 15 a 25 MΩ. O FSH ovino e a ß-folitropina foram administrados topicamente nas células após a estabilização do potencial de repouso durante pelo menos 5 minutos. RESULTADOS: Nas células do cumulus, o potencial médio de membrana em repouso foi de -34,02 ± 2,04 mV (n = 14). A resistência média da membrana foi de 16,5 ± 1,8 MΩ (n = 14). O FSH ovino (4 mUI/mL) e a ß-folitropina (4 mUI/mL) produziram despolarização no potencial de membrana 180 e 120 segundos após a aplicação do hormônio, respectivamente. CONCLUSãO: Ambas as isoformas de FSH induzem padrões de despolarização semelhantes, mas a ß-folitropina apresentou uma resposta mais rápida. Uma melhor compreensão das diferenças dos efeitos das isoformas do FSH no potencial da membrana celular contribuirá para aprimorar o uso das gonadotrofinas no estímulo ovariano controlado e em protocolos de maturação oocitária in vitro.

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.

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 other's electrophysiological responses suggesting crosstalk between the intracellular signaling pathways.

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.

Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin.

Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 µM), nandrolone (0.1, 0.5 and 1 µM) and the flavonoid catechin (0.1, 0.5 and 1 µM) alone and also after infusion with flutamide (1 µM), diazoxide (100 µM) or U73122 (1 µM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 µM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 µM) and nandrolone (1 µM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 µM) and catechin (1 µM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes.