Non-steroidal factors in bovine follicular fluid inhibit or facilitate the action of pulsatile administration of GnRH on LH release in the female rat. (1/111)

Using steroid-free bovine follicular fluid (bFF), we studied the action of gonadotrophin surge-inhibiting factor/attenuating factor (GnSIF/AF) on GnRH-induced self-priming in phenobarbital-blocked female rats. For the experiments we used intact rats, short-term (4 h) ovariectomized (OVX) rats and long-term (14 days) OVX rats. In the latter case the rats were injected with 17beta-oestradiol benzoate (OB, 40 micrograms) or vehicle only, 2 or 48 h before the experiment. GnRH (10-50 pmol/kg body weight) was injected intra-arterially in 5 or 15 pulses, respectively 60 or 20 min apart, starting 1 or 4 h after injection of bFF (0.5 or 1.0 ml). In response to 25 pmol/kg GnRH pulses (1/h), we observed no effect in the long-term OVX rats, a minor effect in the intact rats and an enhanced self-priming effect in the short-term OVX rats. Administration of bFF attenuated or even completely inhibited the self-priming process. However, in the case of long-term OVX rats LH release was inhibited only after long-term OB priming. Furthermore, 4 h after administration of bFF, LH release in response to 25 pmol/kg GnRH pulses (3/h) was inhibited transiently in intact rats and long-term OVX rats. The results support the hypothesis of a functional antagonistic action between GnRH and GnSIF/AF. However, when injected 1 h before, bFF facilitated the initial release of the surge-like LH pattern in intact rats in response to 3 pulses/h of GnRH. These results are consistent with an important role of GnSIF/AF and other non-steroidal ovarian factors in the control of both low LH concentrations and the generation of the LH surge. Some genomic action of oestradiol might be a prerequisite for the inhibitory effect of GnSIF/AF on GnRH-induced LH release.  (+info)

Purification of a candidate gonadotrophin surge attenuating factor from human follicular fluid. (2/111)

Gonadotrophin surge attenuating factor (GnSAF) is a new non-steroidal ovarian substance, different from inhibin, which attenuates the pre-ovulatory luteinizing hormone (LH) surge in superovulated women. Human follicular fluid (FF) was used as a source for the isolation of GnSAF, the activity of which was monitored in an in-vitro pituitary bioassay. Primary rat pituitary cells were incubated with test substances for 48 h and subsequently washed and incubated with 0.1 micromol/l gonadotrophin releasing hormone (GnRH) plus test substances for 4 h. GnSAF activity was expressed as the reduction of GnRH-induced LH secretion in the 4 h incubation. GnSAF was purified from 250 ml of FF which was heat-treated at 80 degrees C for 5 min. Heparin-sepharose chromatography, Con-A sepharose chromatography, reversed-phase high-performance liquid chromatography (HPLC) and preparative native gel electrophoresis were used for GnSAF fractionation. Using these purification steps, we have obtained an apparently homogeneous preparation that stains as a single band on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. GnSAF has an apparent molecular weight of 12.5 kDa and was identified by amino acid sequence (mass spectrometry) to be the C-terminal fragment of human serum albumin.  (+info)

The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare. (3/111)

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C-peptide (50 amino acids). These results were confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex-determination. In invertebrates, there is no other example where sex-differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti-Mullerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.  (+info)

Is GnRH self-priming an obligatory feature of the reproductive cycle? (4/111)

Insufficient suppression of LH (premature elevation) and FSH (prolonged release) give rise to blood concentrations which may cause damaging effects on oocyte viability and too many follicles respectively. During the surge, LH rises from low to high threshold values to initiate processes from initiation of the resumption of oocyte meiosis to the induction of ovulation. In general, it is thought that a dramatic increase in LH concentration is required to attain the high threshold for ovulation. A self-priming mechanism, by which gonadotrophin-releasing hormone (GnRH) enhances the LH (and FSH) responses to its own action, was thought to be responsible. However, normal LH surges in rats consist of <2-7% of the maximal pituitary releasing capacity. The physiological roles of LH and FSH favour a control mechanism that restrains their blood concentrations during most of the cycle. Ovarian proteins, e.g. inhibin and putative gonadotrophin-surge-inhibiting factor/attenuating factor (GnSIF/AF), are involved in this process. We argue that the increased pituitary LH responsiveness during the mid-cycle surge is not the result of a self-priming process that 'dramatically' increases the LH releasing capacity of the pituitary gland. This is probably due to elimination by GnRH of the inhibitory action of the putative ovarian proteins GnSIF/AF.  (+info)

Relationship between follicle size and gonadotrophin surge attenuating factor (GnSAF) bioactivity during spontaneous cycles in women. (5/111)

BACKGROUND: We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries. METHODS: To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined. RESULTS: Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations. CONCLUSIONS: The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.  (+info)

Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles. (6/111)

BACKGROUND: Up to 13% of IVF cancellations are due to poor responses during down-regulated cycles. Because premature luteinization occurs more frequently in older or "poor responder" patients, defective production of gonadotrophin surge-attenuating factor (GnSAF) may be involved. METHODS: Nine women with normal previous IVF response (NORM) and 9 with previous poor IVF response (POOR) were monitored in a spontaneous cycle (blood samples: days 2, 7, 11, 15 and 20) and then stimulated with recombinant human FSH (rFSH) under GnRH agonist (blood samples: treatment days GnRH agonist + 2, GnRH agonist + 7, day of HCG administration and days HCG + 1 and HCG + 8). LH, FSH, estradiol, progesterone and inhibin-A and -B were assayed in individual samples while GnSAF bioactivity was determined in samples pooled according to day, cycle and IVF response. RESULTS: During spontaneous cycles LH, steroids and inhibins were similar between NORM and POOR women, FSH was elevated in POOR women (4.9 +/- 0.3 versus 6.7 +/- 0.6 mIU/l, P < 0.01) and GnSAF bioactivity was detectable on days 2, 7 and 11 in NORM women only. During IVF cycles inhibin-A and -B rose more markedly in NORM than POOR women. Similarly GnSAF production peaked on day GnRH agonist + 7 in NORM women, but on the day of HCG administration in POOR women. CONCLUSIONS: Defects in ovarian responsiveness to FSH include reduced GnSAF production. This suggests that GnSAF should be investigated as a marker of ovarian reserve once an immunoassay becomes available.  (+info)

Inhibin B, follicle stimulating hormone, luteinizing hormone, and estradiol and their relationship to the regulation of follicle development in girls during childhood and puberty. (7/111)

Inhibin B, produced by granulosa cells in the ovary, is a heterodimeric glycoprotein suppressing synthesis and secretion of the follicle stimulating hormone (FSH). The aim of the present study was to determine hormone profiles of inhibin B, FSH, luteinizing hormone (LH), and estradiol in girls during childhood and puberty and to evaluate whether inhibin B is a marker of follicle development. We examined the correlation between inhibin B and gonadotropins and estradiol during the first two years and across the pubertal development. Using a specific two-side enzyme-linked immunosorbent assay (ELISA), inhibin B levels were measured in the serum of 53 healthy girls divided into 8 groups according to age. In addition, serum FSH, LH, and estradiol were measured by chemiluminescent immunoassay in all serum samples. A rise in serum levels of inhibin B (55.2+/-7.3 ng/l, mean +/- S.E.M.) and FSH (1.78+/-0.26 UI/l), concomitant with a moderate increment of serum LH (0.36+/-0.09 UI/l) and estradiol (45.8+/-12.2 pmol/l) concentrations was observed during the first three months of life and declined to prepubertal concentrations thereafter. A strong positive correlation between inhibin B and FSH (r = 0.48, p<0.05), LH (r = 0.68, p<0.001) and estradiol (r = 0.59, p<0.01) was demonstrated during the first 2 years of life. A rise in serum levels of inhibin B, FSH, LH, and estradiol was found throughout puberty. Inhibin B had a strong positive correlation with FSH (stage I of puberty: r = 0.64, p<0.05; stage II of puberty: r = 0.86, p<0.01), LH (I: r = 0.61, p<0.05; II: r = 0.67, p<0.05), and estradiol (II: r = 0.62, p<0.05) in early puberty. From pubertal stage II, inhibin B lost this relationship to gonadotropins and estradiol. Serum inhibin B and FSH levels increased significantly during pubertal development, with the highest peak found in stage III of puberty (133.5+/-14.3 ng/l), and decreased thereafter. In conclusion, inhibin B is produced in a specific pattern in response to gonadotropin stimulation and plays an important role in the regulation of the hypothalamic-pituitary-gonadal axis during childhood and puberty in girls. Inhibin B is involved in regulatory functions in developing follicles and seems to be a sensitive marker of ovarian follicle development.  (+info)

Demonstration of a non-steroidal, non-inhibin factor in the ovine corpus luteum of pregnancy that reduces pituitary responsiveness to GnRH-induced LH secretion in vitro. (8/111)

The decline in pulsatile LH secretion and pituitary responsiveness to GnRH as pregnancy advances may be due to non-steroidal factors secreted by the ovine corpus luteum of pregnancy. Corpora lutea from ten ewes on days 70-80 of gestation were homogenized, charcoal-treated and, together with charcoal-treated follicular fluid from superovulated women, were subjected to inhibin immunoaffinity chromatography, reducing dimeric inhibin A and B by >90% and abolishing inhibin bioactivity. These preparations were investigated using cultures of rat pituitary cells. GnRH-induced LH and FSH secretion in vitro was reduced by ovine corpus luteum extract and human follicular fluid by 47+/-5% and 42+/-5% of control LH and by 37+/-5% and 50+/-10% of control FSH, respectively (P<0.001). Extracts prepared from corpora lutea and placentae that were collected on days 50, 90 and 120 of pregnancy (five ewes per stage of pregnancy) showed increased GnRH-induced LH-suppressing bioactivity, particularly in the case of the placental extracts, with a threefold increase in activity. When partially purified by pseudochromatofocusing, GnRH-induced LH-suppressing bioactivity in extracts of ovine corpora lutea was identified at pH 5.40 and 5.77. Although these values are similar to published gonadotrophin surge-attenuating factor (GnSAF) bioactivity pI values, a GnSAF-blocking antiserum had no consistent effect on ovine corpus luteum extract GnRH-induced LH-suppressing bioactivity. It was concluded that the ovine corpus luteum of pregnancy contains a non-steroidal, non-inhibin factor, probably not GnSAF, that has the ability to reduce pituitary responsiveness to GnRH in vitro.  (+info)