Early Castration in Horses Does Not Impact Osteoarticular Metabolism.

The castration of stallions is traditionally performed after puberty, at around the age of 2 years old. No studies have focused on the effects of early castration on osteoarticular metabolism. Thus, we aimed to compare early castration (3 days after birth) with traditional castration (18 months of age) in horses. Testosterone and estradiol levels were monitored from birth to 33 months in both groups. We quantified the levels of biomarkers of cartilage and bone anabolism (CPII and N-MID) and catabolism (CTX-I and CTX-II), as well as of osteoarthritis (HA and COMP) and inflammation (IL-6 and PGE2). We observed a lack of parallelism between testosterone and estradiol synthesis after birth and during puberty in both groups. The extra-gonadal synthesis of steroids was observed around the 28-month mark, regardless of the castration age. We found the expression of estrogen receptor (ESR1) in cartilage and bone, whereas androgen receptor (AR) expression appeared to be restricted to bone. Nevertheless, with respect to osteoarticular metabolism, steroid hormone deprivation resulting from early castration had no discernable impact on the levels of biomarkers related to bone and cartilage metabolism, nor on those associated with OA and inflammation. Consequently, our research demonstrated that early castration does not disrupt bone and cartilage homeostasis.

Development of a chronic focal equine arteritis virus infection of a male reproductive tract cell line.

Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating from the male reproductive tract of different species. EAV infection was fully cytopathic for 92BR (donkey cells) and DDT1 MF-2 (hamster cells) cells, and less cytopathic for PC-3 cells (human cells); ST cells (porcine cells) seemed to eliminate the virus; LNCaP (human cells) and GC-1 spg (murine cells) cells were not permissive to EAV infection; finally, TM3 cells (murine cells) were permissive to EAV infection without any overt cytopathic effects. Infected TM3 cells can be maintained at least 7 days in culture without any subculture. They can also be subcultured over 39 days (subculturing them at 1:2 the first time at 5 dpi and then every 2-3 days), but in this case, the percentage of infected cells remains low. Infected TM3 cells may therefore provide a new model to study the host-pathogen interactions and to help determine the mechanisms involved in EAV persistence in stallion reproductive tract.

Ex vivo effects of 17ß-estradiol on the prepubertal rat testis.

Although it is well established that testis produces estrogens, their precise effect is not fully documented, particularly during the prepubertal period. In a previous in vivo study, we demonstrated that an exposure of prepubertal rats (15-30 days post-partum (dpp)) to 17ß-estradiol (E2) delays the establishment of spermatogenesis. In order to characterize the mechanisms of action and the direct targets of E2 on the immature testis, we developed an organotypic culture model of testicular explants obtained from prepubertal rats (15, 20 and 25 dpp). To determine the involvement of nuclear estrogen receptors (ERs) in the effect of E2, particularly that of ESR1 which is the major ER expressed in the prepubertal testis, a pre-treatment with the full antagonist of this type of ERs (ICI 182.780) was performed. Histological analyses, gene expression studies and hormonal assays were conducted to investigate the effects of E2 on steroidogenesis- and spermatogenesis-related endpoints. Testicular explants from 15 dpp rats were unresponsive to E2 exposure while E2 effects were observed in those obtained from 20 and 25 dpp rats. An E2 exposure of testicular explants obtained from 20 dpp rats seemed to accelerate the establishment of spermatogenesis, whereas an E2 exposure of 25 dpp testicular explants induced a delay of this process. These effects could be related to the E2-induced modulation of steroidogenesis, and involved both ESR1-dependent and -independent mechanisms of action. Overall, this ex vivo study demonstrated differential age- and concentration-related effects of E2 on the testis during the prepubertal period.

Effects of bisphenol A and estradiol in adult rat testis after prepubertal and pubertal exposure.

Over the past few decades, male fertility has been decreasing worldwide. Many studies attribute this outcome to endocrine disruptors exposure such as bisphenol A (BPA), which is a chemical compound used in plastics synthesis and exhibiting estrogenic activity. In order to assess how the window of exposure modulates the effects of BPA on the testis, prepubertal (15 dpp to 30 dpp) and pubertal (60 dpp to 75 dpp) male Sprague-Dawley rats were exposed to BPA (50 µg/kg bw/day), 17-ß-estradiol (E2) (20 µg/kg bw/day) as a positive control, or to a combination of these compounds. For both periods of exposure, the rats were sacrificed and their testes were collected at 75 dpp. The histological analysis and the quantification of the gene expression of testis cell markers by RT-qPCR confirmed the complete spermatogenesis in all groups for both periods of exposure. However, our results suggest a deleterious effect of BPA on the blood-testis barrier in adults after pubertal exposure as BPA and BPA+E2 treatments induced a decrease in caveolin-1 and connexin-43 gene expression; which are proteins of the junctional complexes. As none of these effects were found after a prepubertal exposure, these results suggested the reversibility of BPA's effects. Caution must be taken when transposing this finding to humans and further studies are needed in this regard. However, from a regulatory perspective, this study emphasizes the importance of taking into account different periods of exposure, as they present different sensitivities to BPA exposure.

Investigation of equine testis contribution to vitamin D bioactivation.

Although vitamin D acts in various biological processes, it plays a critical role in the maintenance of bone health, and regulates calcium homeostasis. In humans and rodents, the main tissues involved in vitamin D metabolism are the liver and the kidneys, however it has been shown that the testis has strongly participated in its bioactivation. Indeed, in these different species, enzymes metabolizing vitamin D (CYP27A1, CYP27B1 and CYP2R1) have been demonstrated in this tissue. Moreover, men with hypogonadism have shown a decrease in circulating levels of vitamin D. In equine species, the castration of males is a regular practice to reduce the behavior of stallions deemed too aggressive. Castration is carried out at various ages: in foals during their growth or in adulthood once they have reached their optimum size. Although horses exhibit atypical vitamin D metabolism with low circulating levels of vitamin D, it was suggested that testis may contribute to its activation as has been described in rodents and humans; castration could therefore be likely to affect its metabolism. In this study, blood levels of bioactive form of vitamin D (1 α,25[OH] 2 vitamin D 3 ) were measured before and after castration at different ages: 1 wk, after puberty (2 yr) and at adulthood (6 yr). The gene expression of enzymes involved in vitamin D metabolism has been sought in the testis of different experimental groups. No change in bioactive vitamin D3 levels was observed after castration regardless of the age at the time of surgery. The exceptional status of equine species is confirmed with a low or a lack of testis contribution to vitamin D metabolism, regardless of testicular development. This is demonstrated by a low or a lack of signal from enzymes involved in vitamin D bioactivation. Therefore, horses constitute a unique model in comparative endocrinology.

Estradiol and 1α,25(OH)2 vitamin D3 share plasma membrane downstream signal transduction through calcium influx and genomic activation in immature rat testis.

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

Acute effect of bisphenol A: Signaling pathways on calcium influx in immature rat testes.

We investigated the acute effect of low concentrations of BPA on calcium influx and the mechanism of action of BPA in this rapid response in the rat testis. BPA increased calcium influx at 1 pM and 1 nM at 300 s of incubation, in a similar manner to that of estradiol. At 1 pM, BPA stimulated calcium influx independently of classical estrogen receptors, consistent with a G-protein coupled receptor. This effect also involves the modulation of ionic channels, such as K+, TRPV1 and Cl- channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses.

Interactions between oestrogen and 1α,25(OH)2-vitamin D3 signalling and their roles in spermatogenesis and spermatozoa functions.

Oestrogens and 1α,25(OH)2-vitamin D3 (1,25-D3) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D3 are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D3 in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.

L'œstradiol et la 1α,25(OH)2-vitamin D3 (1,25-D3 ou calcitriol) sont respectivement la forme la plus active des œstrogènes et la forme hormonalement active de la vitamine D. Ces stéroïdes peuvent exercer leurs effets biologiques après fixation à des récepteurs localisés dans le cytoplasme et le noyau (récepteurs dit nucléaires) ou par fixation à des récepteurs localisés à la membrane plasmique (récepteurs membranaires) à l'origine d'effets appelés génomiques et non génomiques respectivement. Bien que les œstrogènes aient longtemps été considérés comme uniquement des hormones féminines, de nombreux travaux ont permis de montrer leur importance dans le bon déroulement de la spermatogenèse et la qualité des gamètes. De même, la 1,25-D3 est capable de réguler les fonctions testiculaires suggérant son importance dans la fertilité. Les études réalisées sur leurs mécanismes d'action, bien qu'ils ne soient pas complètement élucidés, ont permis de mettre en évidence des interactions entre les voies de signalisation de ces deux hormones. Cette revue est centrée sur les évènements régulés par les œstrogènes et la 1,25-D3 dans les testicules et les spermatozoïdes et les interactions entre leurs voies de signalisation.

Maternal Nutrition during Pregnancy Affects Testicular and Bone Development, Glucose Metabolism and Response to Overnutrition in Weaned Horses Up to Two Years.

INTRODUCTION: Pregnant mares and post-weaning foals are often fed concentrates rich in soluble carbohydrates, together with forage. Recent studies suggest that the use of concentrates is linked to alterations of metabolism and the development of osteochondrosis in foals. The aim of this study was to determine if broodmare diet during gestation affects metabolism, osteoarticular status and growth of yearlings overfed from 20 to 24 months of age and/or sexual maturity in prepubertal colts. MATERIAL AND METHODS: Twenty-four saddlebred mares were fed forage only (n = 12, group F) or cracked barley and forage (n = 12, group B) from mid-gestation until foaling. Colts were gelded at 12 months of age. Between 20 and 24 months of age, all yearlings were overfed (+140% of requirements) using an automatic concentrate feeder. Offspring were monitored for growth between 6 and 24 months of age, glucose homeostasis was evaluated via modified frequently sampled intra veinous glucose tolerance test (FSIGT) at 19 and 24 months of age and osteoarticular status was investigated using radiographic examinations at 24 months of age. The structure and function of testicles from prepubertal colts were analyzed using stereology and RT-qPCR. RESULTS: Post-weaning weight growth was not different between groups. Testicular maturation was delayed in F colts compared to B colts at 12 months of age. From 19 months of age, the cannon bone was wider in B vs F yearlings. F yearlings were more insulin resistant at 19 months compared to B yearlings but B yearlings were affected more severely by overnutrition with reduced insulin sensitivity. The osteoarticular status at 24 months of age was not different between groups. CONCLUSION: In conclusion, nutritional management of the pregnant broodmare and the growing foal may affect sexual maturity of colts and the metabolism of foals until 24 months of age. These effects may be deleterious for reproductive and sportive performances in older horses.

Implication of the estrogen receptors GPER, ESR1, ESR2 in post-testicular maturations of equine spermatozoa.

Estrogen receptors ESR1, ESR2 and GPER are present on mature ejaculated horse spermatozoa, suggesting these cells as putative targets for estrogens. Indeed, spermatozoa are exposed to high level of estrogens during the transit in the male and female genital tracts but their roles are not investigated. So, we evaluated in vitro the role of 17ß-estradiol during post-testicular maturations: regulation of motility, capacitation and acrosome reaction. Moreover according to the pseudo-seasonal breeder status of the stallion, we analyzed the putative seasonal variations in the presence of ESRs in spermatozoa. We showed that ESRs are more present on stallion sperm during the breeding season. We showed that capacitation and acrosome reaction are independent of estradiol action in horse. Estradiol can weakly modulate the motility and this effect is strictly associated with GPER and not with ESR1 and ESR2. The subcellular localization of GPER in the neck on stallion sperm is coherent with this effect. It seems that estrogens are not major regulators of sperm maturations associated to mare genital tract, so they could act during the epididymal maturations.

Differential effects of bisphenol A and estradiol on rat spermatogenesis' establishment.

Several studies have highlighted the negative effects of bisphenol A (BPA), a chemical compound with estrogenic activity, on reproductive health. To elucidate the impact of BPA on spermatogenesis' establishment and mechanisms of action of BPA and 17ß-estradiol (E2), as both can be found in the environment, we exposed rats to BPA (50µg/kg bw/day of BPA), E2 (20µg/kg bw/day of E2) and BPA+E2 from 15 to 30days post-partum. Histological and gene expression studies revealed that BPA and BPA+E2 exposures promoted spermatogenesis establishment whereas E2 alone delayed it. Then, a decrease in gene expression of blood-testis-barrier (BTB) proteins was observed in all treated groups. Therefore, our study has demonstrated a differential effect of BPA and E2 exposures on spermatogenesis establishment in prepubertal rats and a deleterious effect of these chemicals on BTB establishment. Thus, the effects of BPA seem to be mediated by receptors other than estrogen receptors.

Stallion spermatozoa: putative target of estrogens; presence of the estrogen receptors ESR1, ESR2 and identification of the estrogen-membrane receptor GPER.

Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysis mainly showed a flagellar localization for both receptors. Immuno-TEM experiments revealed that they are mostly located near the membranes, which are classically associated with rapid, non-genomic, effects. Moreover, we evidenced the expression of the seven transmembrane estradiol binding receptor GPER in colt testis. The protein was also localized at the connecting piece in mature spermatozoa. In conclusion, our results suggest that horse spermatozoa are a target for estrogens, which could act on several receptors either during the epididymal transit and/or in the female genital tract.

1α,25(OH)2-Vitamin D3 stimulates rapid plasma membrane calcium influx via MAPK activation in immature rat Sertoli cells.

It was characterized that the rapid response to 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) on (45)Ca(2+) influx in rat Sertoli cells was mediated by voltage-dependent Ca(2+) channels (VDCCs), PKC, ERK1/2 and p38 MAPK pathways. In primary culture of 10 day-old rat Sertoli cells as well as in the whole testis, the time-course of (45)Ca(2+) influx did not change significantly in basal conditions. However, 1,25D(3) showed stimulatory effect on (45)Ca(2+) influx from 10(-15) to 10(-8) M after 60 s of incubation. The maximum effect was around 140% at 10(-12) M on purified Sertoli cells showing a steady state on (45)Ca(2+) influx between 10(-11) and 10(-9) M. Under this experimental condition, 1,25D(3) stimulated (45)Ca(2+) influx from 73% to 106% and no effect was observed at 10(-16), 10(-8) and 10(-7) M in whole testis. VDCC activities are mandatory for a full and complete stimulatory effect of 1,25D(3) in these approaches. K(+) and Cl(-) channels also are strongly involved in this rapid response coordinated by 1,25D(3). The participation of some selected kinases, points to PKC and ERK1/2 upstream activity to p38 MAPK activation suggesting an intracellular cross-talk between rapid (45)Ca(2+) influx and nuclear events. In addition, the comparative effect of microtubule disassembles and ClC-3 channel blocker on (45)Ca(2+) influx provides evidence of secretory activity of Sertoli cells triggered by 1,25D(3). Our results suggest that 1,25D(3) activates p38 MAPK and reorganizes microtubules, involving Ca(2+), PKC and ERK1/2 as upstream regulators and that extracellular Ca(2+) have a central role to rapidly start hormone-induced gene transcription and/or the secretory activity of Sertoli cell.

Role of estrogens in spermatogenesis.

Aromatase converts irreversibly androgens into estrogens and is present in the endoplasmic reticulum of various cells of mammalian testes ; at least in rodents, all testicular cells except peritubular cells express aromatase. In testis, high affinity estrogen receptors, ERalpha and/or ERbeta, together with a membrane rapid effect recently described, mediate the effects of estrogens. From the experimental models, in vitro studies and data collected in patients, it is now demonstrated that estrogens play an important role in the testis of vertebrates. At least it is supported by the widespread distribution of estrogen receptors in the testicular cells and the simultaneous presence of a biologically active aromatase in all germ cells (especially in meiotic and post-meiotic stages). Thus the role of estrogens (intracrine, autocrine and / or paracrine) in spermatogenesis (proliferation, apoptosis, survival and maturation) and more generally, in male reproduction is now evidenced, and much more complex than previously predicted.

17ß-Estradiol regulates cyclin A1 and cyclin B1 gene expression in adult rat seminiferous tubules.

Spermatogenesis, which is the fundamental mechanism allowing male gamete production, is controlled by several factors, and among them, estrogens are likely concerned. In order to enlighten the potential role of estrogen in rat spermatogenesis, seminiferous tubules (ST) from two groups of seminiferous epithelium stages (II-VIII and IX-I) were treated with either 17ß-estradiol (E(2)) agonists or antagonists for estrogen receptors (ESRs). In this study, we show that cyclin A1 and cyclin B1 gene expression is controlled by E(2) at a concentration of 10(-9) M only in stages IX-I. This effect is mimicked by a treatment with the G-protein coupled estrogen receptor (GPER) agonist G1 and is abolished by treatment with the ESR antagonist ICI 182 780. Moreover, using letrozole, a drug that blocks estrogen synthesis, we demonstrate that these genes are under the control of E(2) within rat ST. Thus, germ cell differentiation may be regulated by E(2) which acts through ESRs and GPER, expressed in adult rat ST.

Estrogens: new players in spermatogenesis.

Aromatase that irreversibly transforms androgens into estrogens is present in the smooth endoplasmic reticulum of nearly all cell types in the mammalian testis. In rodents, all testicular cells except for myoid cells express aromatase activity. We have demonstrated the presence of the functional aromatase (transcript or protein, and biological activity) in adult rat germ cells including pachytene spermatocytes and round spermatids. We have also demonstrated estrogen output from these cells equivalent to that of Leydig cells. Unlike androgen receptors, which are localized mainly in testicular somatic cells, estrogen receptors are present in both somatic and germ cells in the testis. Moreover, we have recently described the rapid membrane effects of estrogens (via G protein-coupled receptor [GPER]) in purified rat germ cells. On the basis of various experimental models, in vitro studies and clinical data, it can be concluded that estrogens play an essential role in male reproduction, specifically in the development of spermatozoa.

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.

Regulation of aromatase expression by 1α,25(OH)2 vitamin D3 in rat testicular cells.

It is well known that the vitamin D endocrine system is involved in physiological and biochemical events in numerous tissues, especially gut, bone and kidney but also testis. Therefore, in this study the effect and mechanisms of action of 1α,25(OH)(2) vitamin D(3) (1,25D) on aromatase gene expression in immature rat Sertoli cells were evaluated. Vitamin D receptor transcripts were present in immature Sertoli cells as well as in adult testicular germ cells and somatic cells. The treatment of immature Sertoli cells with 100 nM 1,25D increased the amount of aromatase transcript, mainly in 30-day-old rats. The protein kinase A (PKA) blocker, H89, partially inhibited the 1,25D effect. The stimulation of aromatase gene expression in 30-day-old Sertoli cells by the agonist 1α,25(OH)(2) lumisterol(3), and the suppression of the 1,25D effect by the antagonists 1ß,25(OH)(2) vitamin D(3) and (23S)-25-dehydro-1α (OH)-vitamin D(3)-26,23-lactone suggested, besides a genomic effect of 1,25D, the existence of non-genomic activation of the membrane-bound vitamin D receptor involving the PKA pathway.

Nongenomic and genomic effects of 1α,25(OH)2 vitamin D3 in rat testis.

The steroid hormone 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) regulates gene transcription through a nuclear receptor (VDRnuc) and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). It has been described that successful mating and fertility rates are significantly decreased in vitamin D deficient male rats and a VDR null mutant rodent has decreased sperm count and motility and expresses rare spermatogenesis. Although the Sertoli cells are pointed as the major target of 1,25D(3) in the testis the mechanism of 1,25D(3) action, particularly in Sertoli cells, remains unclear. Several studies undertaken in the testicular cells showed that 1,25D(3) can produce both genomic and nongenotropic actions in those cells. 1,25D(3) can modulate kinase activities and ionic fluxes (Ca(2+) and Cl(-)) at the plasma membrane resulting in the regulation of secretory processes in Sertoli cells. The enormous complexity of the nongenomic actions of 1,25D(3) implies that specific receptor or specific ligand-binding sites located on the plasma membrane or in the nucleus are believed to initiate specific cell responses. Apparently the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface receptors is directly related with the physiological action to be better accomplished. The demonstration that 1,25D(3) can regulate both Sertoli cell and sperm function may be useful for the study and development of new therapeutic strategies to the treatment of male reproductive disorders. This review summarizes recent research on the rapid actions of 1,25D(3) and identifies questions that remain to be answered in this area.