The LH receptor (LHR) activates several families of heterotrimeric G proteins, but only the activation of Gs and subsequent generation of cAMP are universally accepted as important mediators of LH actions. To examine the involvement of the Gq/11 family on the actions of LH, we crossed Cyp19Cre and Gαq(f/f);Gα11(-/-) mice to generate mice with a granulosa cell-specific deletion of Gαq in the context of a global deletion of Gα11. Granulosa cells from Gαq(f/f);Gα11(-/-);Cre(+) mice have barely detectable levels of Gαq/11, have a normal complement of LHR, and respond to LHR activation with a transient increase in cAMP accumulation, but they fail to respond with increased inositol phosphate accumulation, an index of the activation of Gαq/11. The LHR-provoked resumption of meiosis, cumulus expansion, and luteinization are normal. However, the Gαq(f/f);Gα11(-/-);Cre(+) mice display severe subfertility because many of the oocytes destined for ovulation become entrapped in preovulatory follicles or corpora lutea. Because follicular rupture is known to be dependent on the expression of the progesterone receptor (Pgr), we examined the LHR-induced expression of Pgr and 4 of its target genes (Adamts-1, Ctsl1, Edn2, and Prkg2). These actions of the LHR were impaired in the ovaries of the Gαq(f/f);Gα11(-/-);Cre(+) mice. We conclude that the defect in follicular rupture is secondary to the failure of the LHR to fully induce the expression of the Pgr. This is the first conclusive evidence for the physiological importance of the activation of Gq/11 by the LHR and for the involvement of Gαq/11 in ovulation.

MAPK kinase (MEK)1 and MEK2 were deleted from Leydig cells by crossing Mek1(f/f);Mek2(-/-) and Cyp17iCre mice. Primary cultures of Leydig cell from mice of the appropriate genotype (Mek1(f/f);Mek2(-/-);iCre(+)) show decreased, but still detectable, MEK1 expression and decreased or absent ERK1/2 phosphorylation when stimulated with epidermal growth factor, Kit ligand, cAMP, or human choriogonadotropin (hCG). The body or testicular weights of Mek1(f/f);Mek2(-/-);iCre(+) mice are not significantly affected, but the testis have fewer Leydig cells. The Leydig cell hypoplasia is paralleled by decreased testicular expression of several Leydig cell markers, such as the lutropin receptor, steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 17α-hydroxylase, and estrogen sulfotransferase. The expression of Sertoli or germ cell markers, as well as the shape, size, and cellular composition of the seminiferous tubules, are not affected. cAMP accumulation in response to hCG stimulation in primary cultures of Leydig cells from Mek1(f/f);Mek2(-/-);iCre(+) mice is normal, but basal testosterone and testosterone syntheses provoked by addition of hCG or a cAMP analog, or by addition of substrates such as 22-hydroxycholesterol or pregnenolone, are barely detectable. The Mek1(f/f);Mek2(-/-);iCre(+) males show decreased intratesticular testosterone and display several signs of hypoandrogenemia, such as elevated serum LH, decreased expression of two renal androgen-responsive genes, and decreased seminal vesicle weight. Also, in spite of normal sperm number and motility, the Mek1(f/f);Mek2(-/-);iCre(+) mice show reduced fertility. These studies show that deletion of MEK1/2 in Leydig cells results in Leydig cell hypoplasia, hypoandrogenemia, and reduced fertility.

Activation of the LH receptor (LHR) in Leydig cells results in the phosphorylation of ERK1/2 by cAMP-dependent and cAMP-independent pathways. Here we examine the mechanisms by which cAMP stimulates ERK1/2 phosphorylation. We show that the stimulation of steroidogenesis is not necessary or sufficient to stimulate the phosphorylation of ERK1/2 but that other cAMP-dependent mitochondrial functions are involved. Using MA-10 cells as a model, we showed that cAMP analogs increase reactive oxygen species (ROS) formation and that an uncoupler of oxidative phosphorylation and a ROS scavenger prevent this increase. These two compounds also inhibit the increase in ERK1/2 phosphorylation provoked by cAMP analogs, thus suggesting that the cAMP-induced phosphorylation of ERK1/2 is mediated by mitochondrial ROS. In agreement with this hypothesis we also show that a reduction in glutathione levels, which alters the redox state of MA-10 cells, potentiates the effect of cAMP on ERK1/2 phosphorylation. Measurements of the dephosphorylation of ERK and the activation of Ras showed that the ROS scavenger prevents the cAMP-provoked activation of Ras and that cAMP, with or without a ROS scavenger, has little or no effect on the dephosphorylation of ERK. Lastly, we show that the uncoupler of oxidative phosphorylation and the ROS scavenger also prevent the ability of cAMP analogs to increase ERK1/2 phosphorylation in primary cultures of mouse Leydig cells. We conclude that, in Leydig cells, cAMP enhances the phosphorylation of ERK1/2 via a mitochondria-derived, ROS-dependent activation of Ras.

We used proliferating primary cultures of immature rat Leydig cells expressing the recombinant human LH/choriogonadotropin (CG) receptor (LHR) to test the hypothesis that activation of this receptor inhibits apoptosis. We also compared the effects of LH/CG with epidermal growth factor (EGF) and IGF-I because these have been previously shown to stimulate proliferation and/or inhibit apoptosis in Leydig cells. Human CG (hCG), EGF, and IGF-I stimulated the phosphorylation of ERK1/2 and Akt in primary cultures of immature rat Leydig cells. These three hormones also robustly stimulated thymidine incorporation and inhibited drug-induced apoptosis. Using selective inhibitors of ERK1/2 (UO126) or Akt phosphorylation (LY294002), we show that the ERK1/2 and Akt cascades are both involved in the hCG- and EGF-dependent proliferation of Leydig cells, but only the ERK1/2 cascade is involved in their antiapoptotic actions. The same strategy showed that the proliferative and antiapoptotic actions of IGF-I are mediated entirely by the Akt pathway. These results show that activation of the LHR inhibits apoptosis in Leydig cells and that it does so through stimulation of the ERK1/2 pathway.