Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation.

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.

Molecular mechanisms involved in Sertoli cell adaptation to glucose deprivation.

Sertoli cells provide the physical support and the necessary environment for germ cell development. Among the products secreted by Sertoli cells, lactate, the preferred energy substrate for spermatocytes and spermatids, is present. Considering the essential role of lactate on germ cell metabolism, it is supposed that Sertoli cells must ensure its production even in adverse conditions, such as those that would result from a decrease in glucose levels in the extracellular milieu. The aim of the present study was to investigate 1) a possible effect of glucose deprivation on glucose uptake and on the expression of glucose transporters in rat Sertoli cells and 2) the participation of different signal transduction pathways in the above-mentioned regulation. Results obtained show that decreasing glucose levels in Sertoli cell culture medium provokes 1) an increase in glucose uptake accompanied by only a slight decrease in lactate production, 2) an increase in GLUT1 and a decrease in GLUT3 expression, and 3) an activation of AMP-activated protein kinase (AMPK)-, phosphatidylinositol 3-kinase (PI3K)/PKB-, and p38 MAPK-dependent pathways. Additionally, by using specific inhibitors of these pathways, a possible participation of AMPK- and p38MAPK-dependent pathways in the regulation of glucose uptake and GLUT1 expression is shown. These results suggest that Sertoli cells adapt to conditions of glucose deprivation to ensure an adequate lactate concentration in the microenvironment where germ cell development occurs.

A novel short splice variant of the tumour suppressor LKB1 is required for spermiogenesis.

LKB1 was discovered as a tumour suppressor mutated in Peutz-Jeghers syndrome, and is a gene involved in cell polarity as well as an upstream protein kinase for members of the AMP-activated protein kinase family. We report that mammals express two splice variants caused by alternate usage of 3'-exons. LKB1(L) is the previously described form, while LKB1(S) is a novel form in which the last 63 residues are replaced by a unique 39-residue sequence lacking known phosphorylation (Ser(431)) and farnesylation (Cys(433)) sites. Both isoforms are widely expressed in rodent and human tissues, although LKB1(S) is particularly abundant in haploid spermatids in the testis. Male mice in which expression of Lkb1(S) is knocked out are sterile, with the number of mature spermatozoa in the epididymis being dramatically reduced, and those spermatozoa that are produced have heads with an abnormal morphology and are non-motile. These results identify a previously undetected variant of LKB1, and suggest that it has a crucial role in spermiogenesis and male fertility.

Expression of caveolin-1 in rat Leydig cells.

Caveolin-1, the first member of caveolin family reported, is recognized as the structural component of caveola, a plasma membrane invagination or vesicles that are a subcompartment distinct from clathrin-coated pits. This protein is also known to be involved in cholesterol trafficking. The aim of this study was to determine the expression of caveolin-1 in adult rat Leydig cells. Testis sections incubated with an antibody to caveolin-1 showed, by immunohistochemistry, a moderate number of Leydig cells with different degrees of immunoreaction and a strong reaction in endothelial cells and in the lamina propia of seminiferous tubules. Caveolin- 1 was detected in the cell cytoplasm with a granular pattern and on the cell surface of Leydig cells cultured 24 h on uncoated, laminin-1 or type IV collagen coated coverslips. We also observed a milder reaction in 3 h cultures. Immunoreaction was also detected in Leydig cells with an antibody to tyrosine-phosphorylated caveolin-1. By double immunofluorescent technique, we observed co-localization of caveolin- I and 313-hydroxysteroid dehydrogenase. Western blot analysis revealed a band of about 22 kDa molecular weight that was recognized with both caveolin-1 and tyrosine-phosphocaveolin-1 antibodies. Caveolin-l is one of a few proteins with a demonstrated ability to bind cholesterol in vivo. In this context, the presence of caveolin- in Leydig cells may be related to cholesterol traffic--a rate limiting step in steroid biosynthesis.

Type IV collagen induces down-regulation of steroidogenic response to gonadotropins in adult rat Leydig cells involving mitogen-activated protein kinase.

We have previously shown that type IV collagen (alpha1 (IV) and alpha2 (IV) collagen chains) (Col-IV) inhibits testosterone (T) production by Leydig cells (LC). The aim of this study was to analyze mechanism/s by which Col-IV exerts this effect. No significant differences in the specific binding of hCG to LH/hCG receptors in LC cultured on uncoated or Col-IV coated plates were observed. An inhibition of cAMP production in hCG-stimulated LC cultured on Col-IV was detected. The inhibition exerted by Col-IV on T production in response to hCG was also observed when cells were stimulated with 8Bromo-cAMP. In addition, conversion of steroid precursors to T in LC cultured on uncoated and Col-IV coated plates was similar. On the other hand, we detected an increase of ERK1/2 phosphorylation in hCG-stimulated LC cultured on Col-IV. Genistein added to LC cultures reduced the ability of Col-IV to increase ERK1/2 phosphorylation and reverted the inhibitory effect of Col-IV on T production. An inhibitor of MEK, PD98059 added to LC cultures also reverted the inhibitory effect of Col-IV on T production. A decrease of steroidogenic acute regulatory protein (StAR) expression in hCG-stimulated LC cultured on Col-IV coated plates that could be reverted by addition of PD98059 to the cultures was also demonstrated. All together these results suggest that Col-IV inhibits T production in LC by binding to integrins, activating ERK1/2, decreasing cAMP production and decreasing StAR expression.

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro.

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.

Possible role of arachidonic acid in the regulation of lactate production in rat Sertoli cells.

The aim of the study was to determine whether arachidonic acid (AA) is involved in the regulation of Sertoli cell lactate production and if this fatty acid participates in follicle-stimulating hormone (FSH) regulation of Sertoli cell function. In a first set of experiments the effect of AA and porcine pancreas phospholipase A2 (PLA2) on lactate production, glucose uptake, lactate dehydrogenase (LDH) activity and LDH A mRNA levels in Sertoli cell cultures obtained from 20-day-old rats was evaluated. In a second set of experiments the effect of two PLA2 inhibitors--quinacrine (Q) and AACOCF3--on FSH stimulation of the above-mentioned parameters of Sertoli cell function was investigated. Treatment with PLA2 and AA increased Sertoli cell lactate production. The observed action of exogenously added PLA2 involved its catalytic properties responsible for AA release. PLA2 and AA treatments also stimulated Sertoli cell glucose uptake, LDH activity and LDH A mRNA levels. In order to determine whether AA participates in FSH regulation of Sertoli cell lactate production cells were incubated with FSH in the absence or presence of the PLA2 inhibitors Q and AACOCF3. Both drugs partially inhibited the ability of FSH to stimulate lactate production, glucose uptake and LDH activity. The present investigation suggests that AA is involved in the regulation of lactate production, glucose transport, LDH activity and LDH A mRNA levels. In addition, these results suggest that cytosolic PLA2 and AA may participate in FSH-regulation of Sertoli cell energetic metabolism.

Prolactin effects on testicular interstitial cells in primary cultures

En el presente trabajo se determinaron los efectos de la prolactina (PRL) "in vitro" sobre la esteroidogénesis testicular, utilizando un cultivo primario de células intersticiales de rata inmadura. Las células fueron cultivadas en un medio químicamente definido, con el agregado de insulina y transferrina durante 2 días a 34§C. La producción de andrógenos durante el segundo día de cultivo resultó ser significativamente mayor que en el primer día (3 alfa-Androstandiol (3 alfa-Diol) 5.14 ñ 0.46 vs. 3.74 ñ 0.10 ng/microng ADN, p < 0.001); Testosterona (T) + Dihidrotestosterona (DHT) 0.40 ñ 0.04 vs. 0.34 ñ 0.03 ng/microng ADN, p < 0.001). La curva dosis respuesta para distintas dosis de hCG mostró un ED50= 2.5 mUI/ml. El efecto agudo de la PRL (10, 100 y 1000 ng/ml) sobre la producción basal de 3 alfa/Diol se evaluó luego de 45 h de cultivo. Sólo la dosis de PRL de 1000 ng/ml reveló diferencias significativas con respcto al control y dicho efecto pordría ser debido a su contaminación con LH. En otra serie de experimentos se evaluaron los efectos de menores dosis de la hormona a tiempos prolongados. La PRL (10 ng/ml), agregada durante todo el tiempo de cultivo, causó una inhibición significativa en la producción basal de 3 alfa-Diol, mientras que la respuesta a un estímulo máximo con hCG no varió. Cuando se determinó la producción de T+DHT se observó un cambio notorio en la relación T+DHT/Diol, tanto en condiciones basales (Control: 0.09 vs. PRL: 0.38) como ante el estímulo con hCG...

Prolactin effects on testicular interstitial cells in primary cultures

En el presente trabajo se determinaron los efectos de la prolactina (PRL) "in vitro" sobre la esteroidogénesis testicular, utilizando un cultivo primario de células intersticiales de rata inmadura. Las células fueron cultivadas en un medio químicamente definido, con el agregado de insulina y transferrina durante 2 días a 34ºC. La producción de andrógenos durante el segundo día de cultivo resultó ser significativamente mayor que en el primer día (3 alfa-Androstandiol (3 alfa-Diol) 5.14 ñ 0.46 vs. 3.74 ñ 0.10 ng/microng ADN, p < 0.001); Testosterona (T) + Dihidrotestosterona (DHT) 0.40 ñ 0.04 vs. 0.34 ñ 0.03 ng/microng ADN, p < 0.001). La curva dosis respuesta para distintas dosis de hCG mostró un ED50= 2.5 mUI/ml. El efecto agudo de la PRL (10, 100 y 1000 ng/ml) sobre la producción basal de 3 alfa/Diol se evaluó luego de 45 h de cultivo. Sólo la dosis de PRL de 1000 ng/ml reveló diferencias significativas con respcto al control y dicho efecto pordría ser debido a su contaminación con LH. En otra serie de experimentos se evaluaron los efectos de menores dosis de la hormona a tiempos prolongados. La PRL (10 ng/ml), agregada durante todo el tiempo de cultivo, causó una inhibición significativa en la producción basal de 3 alfa-Diol, mientras que la respuesta a un estímulo máximo con hCG no varió. Cuando se determinó la producción de T+DHT se observó un cambio notorio en la relación T+DHT/Diol, tanto en condiciones basales (Control: 0.09 vs. PRL: 0.38) como ante el estímulo con hCG... (AU)