Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes.

Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT1 and MT2. The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[125I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive Gi/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the ß-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Beta-nerve growth factor stimulates spontaneous electrical activity of in vitro embryonic mouse GnRH neurons through a P75 mediated-mechanism.

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (ß-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca ß-NGF (aß-NGF) and its 3D structure and compared it with human recombinant ß-NGF (hß-NGF). We showed that the ß-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that ß-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells.

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Gonadotropin-releasing hormone stimulates the biosynthesis of pregnenolone sulfate and dehydroepiandrosterone sulfate in the hypothalamus.

The sulfated neurosteroids pregnenolone sulfate (Δ(5)PS) and dehydroepiandrosterone sulfate (DHEAS) are known to play a role in the control of reproductive behavior. In the frog Pelophylax ridibundus, the enzyme hydroxysteroid sulfotransferase (HST), responsible for the biosynthesis of Δ(5)PS and DHEAS, is expressed in the magnocellular nucleus and the anterior preoptic area, two hypothalamic regions that are richly innervated by GnRH1-containing fibers. This observation suggests that GnRH1 may regulate the formation of sulfated neurosteroids to control sexual activity. Double labeling of frog brain slices with HST and GnRH1 antibodies revealed that GnRH1-immunoreactive fibers are located in close vicinity of HST-positive neurons. The cDNAs encoding 3 GnRH receptors (designated riGnRHR-1, -2, and -3) were cloned from the frog brain. RT-PCR analyses revealed that riGnRHR-1 is strongly expressed in the hypothalamus and the pituitary whereas riGnRHR-2 and -3 are primarily expressed in the brain. In situ hybridization histochemistry indicated that GnRHR-1 and GnRHR-3 mRNAs are particularly abundant in preoptic area and magnocellular nucleus whereas the concentration of GnRHR-2 mRNA in these 2 nuclei is much lower. Pulse-chase experiments using tritiated Δ(5)P and DHEA as steroid precursors, and 3'-phosphoadenosine 5'-phosphosulfate as a sulfonate moiety donor, showed that GnRH1 stimulates, in a dose-dependent manner, the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants. Because Δ(5)PS and DHEAS, like GnRH, stimulate sexual activity, our data strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.

Endocrine characterization of the reproductive axis in highly prolific lacaune sheep homozygous for the FecLL mutation.

A prolific allele named FecL(L) is known to segregate in the meat breed of the French Lacaune sheep on the basis of ovulation rate record. To gain more knowledge about the physiological effects of FecL(L), ewes homozygous for FecL(L) (L/L) were compared to wild-type ewes (+/+) for ovarian phenotype and reproductive endocrine profiles. At the ovarian level, the increased ovulation rate in L/L ewes was associated with an increased number of antral follicles of greater than 3 mm and with preovulatory follicles being, on average, 1 mm smaller. Intrafollicular estradiol and testosterone concentrations were not significantly different between the two genotypes. In contrast, L/L large follicles (>or=6 mm) had lower intrafollicular progesterone concentration. At the molecular level, expressions of ovarian markers, such as CYP19A1, CYP11A1, CYP17A1, LHR, and INHA, were not significantly different between the two genotypes. In contrast, FSHR and STAR mRNA levels increased in granulosa cells from L/L ewes. Plasma concentrations of estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone measured across a synchronized estrous cycle revealed a significant increase in estradiol levels during the follicular phase, a precocious LH surge, and an increase in progesterone level during the luteal phase of L/L ewes compared to +/+ ewes. Circulating concentrations of FSH were not different between the two genotypes. The precocious LH surge was associated with an increase in frequency of LH pulsatility during the follicular phase. At the pituitary level, mRNA levels for LHB, FSHB, GNRHR, and ESR1 were not significantly different between the two genotypes. In contrast, ESR2 mRNA expression was decreased in L/L ewes compared to +/+ ewes. Based on ovarian phenotype and endocrine profiles, these findings suggest that the mutation in the FecL gene affects ovarian function in a different way compared to other known prolific mutations affecting the bone morphogenetic protein signaling system in the ovine species.

Activin signaling pathways in ovine pituitary and LbetaT2 gonadotrope cells.

In the pituitary, activin stimulates the synthesis and release of FSH. However, the activin receptor signaling pathways that mediate these effects are poorly known. We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LbetaT2 gonadotrope cell line. POP cells and LbetaT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7). In both POP and LbetaT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt. Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min. Activin also increased the association of activin receptor IIB with PI3K. Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways. In both POP and LbetaT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LbetaT2 cells. These results indicate that activin activates several signaling pathways with different time courses in both POP and LbetaT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LbetaT2 cells.