Replenishment of LH stores of gonadotrophs in relation to gene expression, synthesis and secretion of LH after the preovulatory phase of the sheep oestrous cycle

Abstract

The pattern of replenishment of LH secretory granule stores in sheep pituitary gonadotrophs, after an induced LH surge, was determined by immunogold localisation at the ultrastructural level by electron microscopy. Twentyfour Welsh Mountain ewes were initially synchronised with progestagen devices for 14 days before luteolysis was induced by a prostaglandin F 2 analogue, cloprostenol. A further 24 h later, a preovulatory LH surge was induced by intravenous injection of a GnRH agonist, buserelin. Animals were divided into four groups (n=6) and blood sampled at 2 h intervals from 4 h prior, to 18 h after, buserelin administration and then at infrequent intervals (1 to 8 h) thereafter until death. Pulse profiles of LH were also obtained by an additional collection of blood samples within a 6 h window directly preceding death. Groups of animals were killed at 24, 48, 72 or 96 h after buserelin treatment. Pituitaries were dissected and processed for transmission electron microscopy and frozen for later molecular biological analysis. A characteristic preovulatory surge of LH was observed in all animals. The cytoplasm of gonadotrophs, in animals killed 24 h after buserelin treatment, was completely empty of secretory granules. This was associated with diminutive pituitary LH content, low pituitary GnRH binding levels and an almost complete absence (one pulse in one animal) of LH pulsatile secretion. Despite the lack of apparent secretory activity, clusters of exposed LH label present within the cytoplasm at this time and constant LH mRNA expression levels irrespective of tissue collection time, suggest that the cell is actively synthesising LH . The formation of sparse numbers of small LH immuno-labelled electron-dense secretory granules was apparent at 48 h after buserelin treatment, and replenishment of LH immuno-labelled granule stores continued until total granule numbers had increased two-fold (P<0·01) by 96 h post-treatment. Affiliated with granule replenishment was a significant increase in pituitary LH content (P<0·01), pituitary GnRH binding levels (P<0·01) and the restoration of LH pulsatile secretion. Despite the replenishment of granule stores with time, cytoplasmic area did not vary. These results suggest that restoration of pulsatile LH secretion after a preovulatory LH surge is related to replenishment of LH secretory granule stores and an increase in GnRH binding levels.

No Evidence for Pituitary Priming to Gonadotropin-Releasing Hormone in Relation to Luteinizing Hormone (LH) Secretion Prior to the Preovulatory LH Surge in Ewes1

“…However, the apparent shift in distribution of numbers of granule size classes in this study after stimulation with GnRH has been observed previously during similar physiological events in both sheep [36,87] and mice [47]. The disappearance of all granules of 84-126 nm in diameter and the attenuation in numbers of granules of 127-168 nm in diameter in ewes at 1 h after GnRH administration suggests that it was these granules that underwent exocytosis and were secreted into the blood stream.…”

“…This emphasizes the capacity of gonadotrope cells to store copious amounts of both LH and FSH. Only during and after a complete preovulatory LH surge has the gonadotrope cell significantly depleted its stores of LH [8,87], after which replenishment of storage granules containing LH was first observed sometime between 24 and 48 h postsurge [87]. A reduction in LH and FSH pituitary content was evident in the few ewes that were killed in the midst of an endogenous preovulatory surge in the present study.…”

Effects of intracerebroventricular infusion of genistein on gonadotrophin subunit mRNA and immunoreactivity of gonadotrophins and oestrogen receptor-α in the pituitary cells of the anoestrous ewe

“…This could be interpreted as a rapid release of the hormone together with an enhanced synthesis of LH, possibly mediated by ERa after treatment with genistein. It has been reported that in ewes treated with GnRH agonist the cytoplasm of gonadotropes is completely depleted of secretory granules labelled for LHb by 24 h post-treatment, but maintains a constant LHb mRNA expression, which suggests an active synthesis of LHb (Crawford et al, 2000). These authors also observed the LH surge 10-14 h after GnRH agonist administration.…”

The postnatal ontogeny of gonadotroph cells in the female sheep

“…This intracellular mechanism has been suggested by Currie and McNeilly (1995) to switch non-releasing gonadotrophs into a potentially releasable state. Some LH is additionally released constitutively (McNeilly et al, 1991), possibly to maintain basal levels of LH (Crawford et al, 2000). In view of discussed above discrepancies, the findings of the current study imply the existence of the histomorphological feature of the intrahypo- Fig.…”