Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.

Dichlorodiphenyltrichloroethane impairs follicle-stimulating hormone receptor-mediated signaling in rat Sertoli cells.

Any toxicant that affects Sertoli cell development can potentially disturb male fertility. So far, the effects of organochlorine compounds have been poorly investigated in male. Here, we studied the effects of dichlorodiphenyltrichloroethane (DDT), an organochloride pesticide, on Sertoli cells. DDT inhibited the cAMP response to follicle-stimulating hormone (FSH), the major endocrine control of Sertoli cell development, and to a beta2-agonist, isoproterenol. DDT exposure decreased the level of FSH binding sites. Direct adenylyl cyclase activation by Forskolin was unaltered by DDT, while the activation of Galphas by cholera toxin was decreased by DDT. The DDT inhibitory effect on the FSH response was also observed in Ser W3 cells, a Sertoli cell-derived immortalized cell line. All these effects were reproduced by the lipophilic aromatic bisphenol A but not by structurally unrelated CisPlatin. In conclusion, these results are a first step in understanding the molecular basis of DDT deleterious effects in spermatogenesis.

G protein-coupled receptor kinase 2 and beta-arrestins are recruited to FSH receptor in stimulated rat primary Sertoli cells.

FSH-receptor (FSH-R) signaling is regulated by agonist-induced desensitization and internalization. It has been shown, in a variety of overexpression systems, that G protein-coupled receptor kinases (GRKs) phosphorylate the activated FSH-R, promote beta-arrestin recruitment and ultimately lead to internalization. The accuracy of this mechanism has not yet been demonstrated in cells expressing these different molecules at physiological levels. Using sucrose gradient fractionation, we show that FSH induces the recruitment of the endogenous GRK 2 and beta-arrestin 1/2 from the cytoplasm to the plasma membrane of rat primary Sertoli cells. As assessed by ligand binding, the FSH-R was found expressed in the fractions where GRK 2 and beta-arrestins were recruited upon FSH treatment. In addition, the endogenous beta-arrestin 1 was found dephosphorylated in an agonist-dependent manner. Finally, a significant FSH-binding activity was co-immunoprecipitated with the endogenous beta-arrestins from agonist-stimulated but not from untreated Sertoli cell extracts. This FSH-R interaction with beta-arrestins was sustained for up to 30 min. In conclusion, our data strongly suggest that the GRK/beta-arrestin machinery plays a physiologically relevant role in the regulation of the FSH signaling.

A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation.

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.

Extracellular signal-regulated kinases (ERK) 1, 2 are required for luteinizing hormone (LH)-induced steroidogenesis in primary Leydig cells and control steroidogenic acute regulatory (StAR) expression.

The luteinizing hormone (LH) plays a critical role in steroidogenesis, by stimulating cAMP-dependent protein kinase A (PKA) and phospholipase A2 activity, and by mobilizing calcium and chloride ions. In contrast, whether the ERK 1, 2 mitogen-activated protein (MAP) kinases are involved in LH-induced steroidogenesis is less obvious. Here, we sought to clarify this point in rat primary Leydig cells, naturally bearing the LH receptor (LH-R) in male, and in the mouse tumoral Leydig cell line (MLTC 1). Pre-incubation of both cell types with the mitogen-activated protein kinase kinase (MEK) inhibitors U0126 and PD98059 reduced LH-induced steroidogenesis, and tonically enhanced the expression of the StAR protein. Furthermore, ERK1, 2 were inducibly phosphorylated following LH exposure of MLTC 1 cells. Altogether, our results indicate that in primary as well as in tumoral Leydig cells, inhibiting MEK dampened LH-induced steroidogenesis but enhanced basal as well as LH-induced StAR expression, suggesting that ERK1,2 could be involved in these responses.

Follicle-stimulating hormone activates p70 ribosomal protein S6 kinase by protein kinase A-mediated dephosphorylation of Thr 421/Ser 424 in primary Sertoli cells.

FSH is a major hormonal input that drives Sertoli cells to their fully differentiated function in male reproduction. It is a physiologically important issue to define how FSH mediates its effects at the cellular level to regulate gene expression. FSH biological activities are transduced via a seven-spanned transmembrane receptor, the FSH-R, primarily leading to cAMP-dependent protein kinase A (PKA) activation and cAMP response element binding protein-mediated transcriptional responses. Nevertheless, the intracellular mechanisms interacting with PKA to control Sertoli cell differentiation by FSH are still incompletely defined. Here, we report that, in primary cultures of Sertoli cells isolated from prepubertal rats, FSH enhanced p70S6K enzymatic activity, in a PKA-dependent manner. p70S6K was constitutively phosphorylated on Thr 389, in a manner sensitive to inhibitors of phosphatidyl-inositide-3 kinase and mammalian target of rapamycin. But FSH could not enhance p70S6K phosphorylation on Thr 389. Rather, the hormone induced the dephosphorylation of Thr 421/Ser 424, located in the autoinhibitory domain of p70S6K, in a PKA-dependent manner. Consistently, FSH-induced phosphorylation of the S6 ribosomal protein, a cellular substrate of p70S6K, required PKA activity. In conclusion, these results show that FSH triggers unexpected regulations of p70S6K by dephosphorylation of Thr 421/Ser 424 mediated by PKA, and stimulates S6 phosphorylation, in Sertoli cells.

A mechanistic overview on male infertility and germ cell cancers.

The testis is devoted to two important tasks: haploid cell production and sexual steroid synthesis. A number of highly sophisticated and unique strategies operate during spermatogenesis, a process crucial for reproduction, heredity and evolution. It is particularly important to decipher the underlying molecular mechanisms whose function can be perverted in pathological situations, such as infertility and testicular cancers, which represent an increasing biomedical issue today. This review summarises the currently available data concerning some key molecular components that are altered or potentially involved in male infertility and testicular tumors, with the aim of defining some common "hot spots". We particularly focused on genetically engineered in vivo models in which testicular functions are altered and we pinpointed to the potential involvement of the targeted genes in testicular pathologies. Those molecular mechanisms peculiar to the male gonad can be envisioned as a basis for the design of novel drugs potentially dedicated to testicular dysfunction.

G protein-coupled receptor kinases and beta arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells.

The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and beta-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/beta-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and beta-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and beta-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and beta-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/beta-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.