ABSTRACT

Oxytocin is synthesized in the granulosa-derived large cells of the ruminant corpus luteum from a gene which is dramatically up-regulated in the first few days after ovulation. In this work, the regulation of granulosa and luteal cells by prostaglandins and insulin (or insulin-like growth factor-I; IGF-I) has been explored by comparing their effects on oxytocin and progesterone production in cell culture. In granulosa cells, chronic exposure to insulin (17 nmol/l) stimulated luteinization as indicated by increased release of oxytocin and progesterone. Prostaglandin F_2α (PGF_2α) alone had little effect, but synergized with insulin (or IGF-I) to increase the release of both these hormones. In direct contrast, insulin-stimulated oxytocin production by luteal cells was inhibited by PGF_2α. The half-maximal dose (EC₅₀) for PGF_2α action in both cell preparations was similar (10–100 nmol/l). Dose–response studies revealed that PGF_2α increased the potency of insulin in granulosa cells (EC₅₀ for insulin-stimulation of oxytocin release reduced from 141 to 13 nmol/l by 1 μmol PGF_2α/l), but not in luteal cells. Insulin-stimulated oxytocin release from granulosa cells was also synergistically increased by PGE₁, PGE₂ and forskolin, suggesting this effect to be mediated by adenylate cyclase-coupled PGE receptors. The results reveal that the effects of prostaglandins on oxytocin release are dependent on both the developmental stage of the target tissue and on the presence of other regulators of cellular differentiation. Moreover, they suggest that the increase in responsiveness to insulin and IGF-I, which appears to accompany luteinization in the cow, may be an effect of prostaglandins produced locally during the peri-ovulatory period.

Journal of Endocrinology (1990) 126, 245–253

Abstract

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca²⁺ and the plateau responses are attributed, in part, to Ca²⁺ influx via voltage-sensitive Ca²⁺ channels. In recent years, store-dependent Ca²⁺ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca²⁺ influx, has emerged as a major form of Ca²⁺ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca²⁺]_i, in αT3–1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca²⁺ pool to test the possible role of SDCI in GnRH action.

In Ca²⁺-containing medium, GnRH caused a biphasic increase in [Ca²⁺]_i whereas in Ca²⁺-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca²⁺-free medium was reduced by >95% (demonstrating that Ca²⁺ pool depletion had occurred) and was recovered after brief exposure to Ca²⁺-containing medium (which enables refilling of the pool). Ionomycin (a Ca²⁺ ionophore) and thapsigargin (which inhibits the Ca²⁺-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca²⁺]_i, in Ca²⁺-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca²⁺-containing medium because addition of ionomycin and Ca²⁺-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca²⁺]_i rather than the transient increase seen without GnRH. To deplete intracellular Ca²⁺ pools, cells were pretreated in Ca²⁺-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca²⁺-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca²⁺]_i seen on return to Ca²⁺-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca²⁺ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca²⁺-containing medium. Return of GnRH-treated cells to Ca²⁺-containing medium caused an increase in [Ca²⁺]_i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca²⁺-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl₂ (used as a reporter of Ca²⁺ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca²⁺ influx via Mn²⁺ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca²⁺ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca²⁺-free medium did not enhance LH release on return to Ca²⁺-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in αT3–1 cells via nicardipine-insensitive Ca²⁺ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca²⁺ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca²⁺ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Journai of Endocrinology (1996) 149, 155–169

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both αT3-1 and LβT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in αT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone α-subunit promoter in LβT2 cells but not in αT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.