Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after treatment of ewes with bovine follicular fluid during the luteal phase of the cycle

Abstract

The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3-11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32 h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-beta, LH-beta and common alpha subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P less than 0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P less than 0.01) increased after cessation of treatment, with maximum secretion being reached 18-22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P less than 0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P less than 0.01) reduced FSH-beta mRNA levels in the luteal phase. Increased levels of FSH-beta, LH-beta and alpha subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…Total RNA was extracted from frozen pituitary tissue and LH mRNA levels were quantified in 15µg samples by Northern analysis using a 32 P-labelled cDNA probe for LH (Brooks et al 1992). Uniformity of loading was measured by reprobing the membrane with a rat 18S ribosomal RNA oligonucleotide probe.…”

“…Pituitary LH content and GnRH binding levels were measured as previously described (Brooks et al 1992). The sensitivity of the LH radioimmunoassay used to determine pituitary LH content was 2 ng/ml and the intra-assay coefficient of variation was 11·6%.…”

Gene expression and secretion of LH and FSH in relation to gene expression of GnRH receptors in the brushtail possum (Trichosurus vulpecula) demonstrates highly conserved mechanisms

“…This parallel, if somewhat delayed, pattern between gene expression and plasma levels of FSH in possums is similar to that observed in most eutherian species studied (Crawford & McNeilly 2002, Crawford et al 2002, whereby levels decline during the late follicular stage of the oestrous cycle presumably due to the increasing negative feedback effects of oestradiol (Baird et al 1981) and inhibin-A (Mann et al 1992, Padmanabhan & McNeilly 2001) from the growing preovulatory follicle, which also subsequently suppresses the activin gene (Gregg et al 1991, Nett et al 2002. Elevation of gene expression levels and secretion of FSH observed in possums in the luteal stages of the oestrous cycle were probably due to the removal of these negative feedback effects (Wallace & McNeilly 1986, Wallace et al 1988, Brooks et al 1992, together with augmented progesterone levels (Mann & Barraclough 1973, Rao & Mahesh 1986, Attardi & Fitzgerald 1990, Crawford et al 2006 and possibly a reduced GnRH pulse frequency (Molter-Gérard et al 1999). The absence of pulses of FSH in this study is similar to that observed in other mammalian species and reinforces the dogma that FSH is mainly released via a constitutive pathway.…”

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

“…The preparation of all the 32 P-labeled cDNA probes has previously been described [8,46,49,50]. Uniformity of loading was measured by reprobing the membrane with rat 18s ribosomal RNA.…”

“…Plasma concentrations of inhibin-A were measured by ELISA as described in detail previously [49,59], with a 30-pg/ml sensitivity and intra-and interassay coefficients of Ͻ10%. Pituitary LH and FSH content and GnRH binding levels were measured exactly as previously described [50].…”