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Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCbetaI and PKCepsilon). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.

The role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCG10 and c-fos expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCG10 mRNA levels. Induction involved a protein synthesis-dependent mechanism and was maximal within 12-24 h of exposure. Chronic treatment of the cells with PMA for up to 8 days resulted in a substantial decrease (approximately 90%) in total PKC activity in the continued presence of PMA. PKC depletion did not affect NGF- or bFGF-stimulated SCG10 mRNA induction and bFGF-stimulated c-fos mRNA induction. However, NGF-stimulated c-fos mRNA induction was attenuated. In addition, induction of neurite outgrowth was not abolished in PKC-depleted cells. The results imply that PKC is not involved in NGF- and bFGF-stimulated SCG10 mRNA induction and neurite outgrowth. Furthermore, while the effect of bFGF on c-fos mRNA induction is PKC-independent, that of NGF is mediated by PKC-dependent and -independent pathways.

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.

The action of gonadotropin-releasing hormone (GnRH) upon luteinizing hormone (LH) secretion is calcium-dependent, but is not mediated by cyclic AMP, cyclic GMP, or prostaglandins. The role of calcium-mediated phospholipid turnover in GnRH action was investigated in 2-day cultured pituitary cells, in which the production and target cell effects of arachidonic acid were analyzed in relation to GnRH-stimulated LH release. Addition of 10(-8) M GnRH, which stimulated LH release 5-fold, caused a 35% increase in the rate of [3H]arachidonic acid release from prelabeled phospholipids. The effects of GnRH on arachidonic acid and LH release, which occurred within 10 min, were calcium-dependent and were not accompanied by a change in [3H]prostaglandin formation. The phospholipase A2 inhibitors, chloroquine and quinacrine, prevented the effect of GnRH on arachidonic acid formation and LH release. Addition of exogenous arachidonic acid or phorbol myristate acetate (which increases endogenous arachidonic acid) caused a dose-related release of LH, whereas prostaglandin E2 had no effect. Although GnRH action was highly calcium-dependent, stimulation of LH release by arachidonic acid was unaltered in the absence of extracellular calcium. The effect of arachidonic acid on LH release did not depend on conversion to prostaglandins and was not additive with the action of a maximal stimulating concentration of GnRH. These results demonstrate that GnRH increases arachidonic acid formation from the phospholipids of pituitary gonadotrophs, and indicate that the fatty acid or its metabolites can initiate LH release.

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.