Abstract

Primary pituitary cultures from adult female rats were used to investigate the effects of steroidal (oestradiol and progesterone) and non-steroidal (inhibin, follistatin) ovarian hormones on the suppressive actions of the ovarian factor gonadotrophin surge-attenuating factor (GnSAF) in the control of gonadotrophin secretion. The source of GnSAF was a chromatographic preparation from follicular fluid containing four distinct protein bands as resolved on SDS–PAGE. Oestradiol and progesterone added alone had no effect on gonadotrophin secretion but had a wide range of effects on the suppression of both LH and FSH secretion caused by the non-steroidal factors. Oestradiol, progesterone and oestradiol+progesterone enhanced the suppressive actions of GnSAF on GnRH-induced LH secretion (causing 19·3 ± 5·2% (P<0·05), 41·9 ± 3·4% (P<0·001) and 32·2 ± 5·3% (P<0·001) greater suppression than GnSAF alone). Progesterone and oestradiol+progesterone completely abolished the suppression of basal FSH secretion caused by inhibin (causing 157·1 ± 22·2%, P<0·001, and 160·9 ± 11·3%, P<0·001, stimulation compared with inhibin alone). Separately the steroids had no effect on the suppression of gonadotrophin secretion caused by follistatin. However, in combination, oestradiol+ progesterone potentiated the suppressive actions of follistatin on GnRH-induced LH secretion causing 29·9 ± 5·3% (P<0·05) greater suppression than follistatin alone. In combination, high-dose follistatin and GnSAF caused 31·1 ±6·5% (P<0·01) greater suppression than GnSAF alone. Thus in combination high-dose follistatin and GnSAF have additive effects on the suppression of GnRH-induced LH secretion. Recombinant human inhibin and GnSAF added in combination had little further effect compared with either alone suggesting that they may have a similar mechanism of action at the pituitary level. These results demonstrate that while FSH secretion in vitro is mainly controlled by inhibin and follistatin, LH secretion is affected by the presence of a whole range of factors. We have demonstrated that oestradiol and progesterone potentiate the suppressive actions of GnSAF in vitro. These data are compatible with the suggestion that in the late follicular phase it is falling levels of GnSAF that allow positive feedback of the steroids on the pituitary to elicit the LH surge, rather than increases in the stimulatory effects of the ovarian steroids overcoming GnSAF. The actions of GnSAF on the pituitary may be modulated by follistatin but it is unlikely that inhibin has any modulatory effects on the GnSAF-induced suppression of LH secretion.

Journal of Endocrinology (1996) 150, 413–422

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

Abstract

Ovine and rat pituitary bioassays for gonadotrophin surgeattenuating factor (GnSAF) were utilized to determine whether the level of GnSAF bioactivity in pooled human follicular fluid (hFF) from superovulated women varied according to follicle diameter (≤11 mm, 12–15 mm and 16–21 mm follicles examined using the ovine bioassay, or ≤10 mm, 11–13 mm, 14–17 mm, 18–20 mm, 21–24 mm and ≥ 25 mm follicles examined using the rat bioassay). When tested using dispersed ovine pituitary cells, GnSAF bioactivity, expressed in terms of the reduction in gonadotrophin-releasing hormone (GnRH)-induced LH secretion, was inversely related to follicle diameter (P<0·01). In response to 5 μl hFF/well from follicles of ≤ 11, 12–15 and 16–21 mm diameter, GnRH-induced LH secretion was reduced to 40·5±6·6.9%, 65·2±6·6% and 83·7±7·9% of control respectively. A similar inverse relationship was observed when a second batch of hFF samples from different sized follicles was tested using rat pituitary cell monolayers. Expressing GnSAF bioactivity in terms of the dose required to suppress GnRH-induced LH secretion by rat pituitary cells to 50% of the maximal suppression observed (ED₅₀), the three smallest follicle size pools contained the most GnSAF (ED₅₀ values of 0·13, 2·79 and 5·36 μl hFF/well from follicles of ≤ 10, 11–13 and 14–17 mm respectively). The ED₅₀ values for follicles of 18–20, 21–24 and ≥25 mm were 8·81, 27·1 and 60·0 μl hFF/well respectively. Thus hFF from follicles ≤ 11 mm was over 450 times more potent than hFF from follicles ≥ 25 mm in suppressing GnRH-induced LH release. The ED₅₀ values for inhibin bioactivity (measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity (between 0·85 and 0·13 μl hFF/well). Inhibin immunoreactivity, measured by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follicles (41·96 ng/ml) and highest concentrations in the three largest follicle size groups (56·48–64·48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant human inhibin at doses of 0·025–25 ng/well. In both the sheep and rat pituitary monolayers, basal FSH was suppressed (ED₅₀=0·02 and 0·16 ng/well respectively). However, while inhibin markedly stimulated GnRH-induced LH secretion from ovine pituitary monolayers (ED₅₀=0·04 ng/well), it suppressed GnRH-induced LH secretion from rat pituitary monolayers (ED₅₀=0·31 ng/well) by 13%. The divergent effects of inhibin on GnRH-induced LH secretion in the two culture systems, and the relative insensitivity of GnRH-induced LH secretion to recombinant human inhibin in the rat system, indicates that the inverse relationship between GnSAF concentrations and follicular diameter cannot be an artefact of inhibin bioactivity. In addition, when hFF was fractionated by hydrophobic interaction chromatography using phenyl Sepharose, fractions which contained the greatest amounts of GnSAF bioactivity differed from those which contained peak levels of bioactive or immunoreactive inhibin. These results support in vivo observations that small follicles are important regulators of gonadotrophin secretion in superovulated women. Concentrations of GnSAF fall as the follicles approach an ovulatory size which enables positive steroid feedback on pituitary responses to hypothalamic GnRH, leading to the preovulatory LH surge.

Journal of Endocrinology (1994) 143, 33–44

Abstract

A penfusion system based on ovine pituitary tissue explants was used to investigate the effects of follicular fluid (hFF) and serum from superovulated women on pituitary responsiveness to gonadotrophin-releasing hormone (GnRH). The specific aims of the study were to determine both if gonadotrophin surge-attenuating factor (GnSAF) bioactivity is present in the peripheral circulation as well as in the follicles of superovulated women and if GnSAF suppresses GnRH self-priming in vitro. Two pulses of GnRH, 1 h apart, produced marked peaks in LH secreted from control chambers, with GnRH self-priming evident in the significant difference between the first (134·4±1·7–232·1±24·0% of basal secretion) and second (183·9±15·8–313·9±14·0% of basal secretion) LH peaks. Both follicular fluid and serum pooled from two different groups of women produced marked suppression of the first (unprimed) and second (primed) LH peaks. The hFF reduced the first LH peak to 69·6±7·8 and 60·2±9·7% and the second LH peak to 57·4±6·7 and 42·6±6·5% of control LH secretion. Overall, the serum reduced the first and second LH peaks to 76·8±4·2 and 62·9±3·6% of control respectively. These results demonstrated that GnSAF bioactivity suppresses GnRH self-priming, and is present in both the peripheral circulation and hFF. The same material administered to dispersed ovine pituitary monolayers produced similar marked suppression of GnRH-induced LH secretion, with approximately 50-fold less GnSAF bioactivity in serum compared with hFF. Combined doses of oestradiol and progesterone, or hFF from large follicles containing little GnSAF, produced stimulation of GnRH-induced LH secretion and GnRH self-priming (second peaks 78·1±38·9 and 27·4±15·7% respectively higher than first peaks). Thus, in conclusion, GnSAF in hFF and serum markedly attenuated both unprimed and primed pituitary response to GnRH, virtually abolishing the GnRH self-priming effect.

Journal of Endocrinology (1994) 143, 45–54