An in-vitro superfusion system was used to study age-dependent changes in the functional activity of LH releasing hormone (LHRH) neurones terminating in the mediobasal hypothalamus (MBH) of the cockerel. Fragments of MBH tissue were obtained from cockerels killed at 8, 12, 16 and 20 weeks of age and both the rate of release of LHRH in vitro and the residual content of LHRH were determined by radioimmunoassay. Blood was collected from a similar group of cockerels of the same age for determination of plasma LH and testosterone concentrations. Superfused cockerel MBH showed both basal and depolarization-induced release of LHRH and the calcium-dependency of the release process was demonstrated. The viability of MBH in vitro was indicated by the observation that the rate of CO2 production by the tissue remained constant during a 3-h period of superfusion. Both the basal rate of release of LHRH (7·24 ±0·63 pg/2MBH per h; mean ± s.e.m., n = 4) and the residual content of LHRH (1·10 ± 0·24 ng/2MBH) were lowest in the 8-week-old cockerel and increased progressively to reach levels three (P<0·001) and 13 (P<0·001) times greater, respectively, by 20 weeks of age. Concentrations of LH and testosterone in plasma did not increase significantly until 16 weeks of age when the respective values were 5·86 ±0·37 μg/l and 1·88 ± 0·31 nmol/l (n = 11). Whereas plasma testosterone increased further to 5·76±0·42 nmol/l (n = 11) at 20 weeks of age, plasma LH fell significantly (P<0·02) to a concentration of 4·08 ± 0·41 μg/l (n = 11). These observations support the conclusion that the onset of sexual maturation in the cockerel is primarily determined by a central neural mechanism which results in an increased activity of LHRH neurones in the hypothalamus.
S Muttukrishna and P G Knight
To investigate the extent to which the direct actions of inhibin, activin and oestradiol on pituitary output of FSH and LH are dependent on the presence of functional gonadotrophin-releasing hormone (GnRH) receptors, we have compared the effects of these agents on cultured ovine pituitary cells derived from control and GnRH agonist-suppressed ewes.
Chronic treatment with GnRH agonist reduced plasma LH and FSH levels (P<0·01) and abolished GnRH-induced release of LH and FSH both in vivo and in vitro. As expected, basal LH release and LH cell content in vitro were drastically reduced in GnRH agonist-suppressed cells (P<0·001). However, basal FSH release and FSH cell content were approximately twofold higher than in control cells (P<0·001).
Irrespective of whether the cells had been desensitized to GnRH, inhibin and oestradiol were both found to suppress basal FSH release and FSH cell content in a dose-dependent fashion (P<0·001). Although inhibin had no effect on basal release of LH from control cells, it markedly enhanced GnRH-induced release (P<0·001). In contrast, inhibin increased (P<0·001) basal LH release from GnRH agonist-suppressed cells (which were unresponsive to the GnRH challenge). Inhibin had no overall effect on total LH content/well for either control or GnRH agonist-suppressed cells. Treatment with oestradiol, on the other hand, reduced total LH content/well, an effect which was more pronounced with GnRH agonist-suppressed cells (−44%; P<0·001) than with control cells (−14%, P<0·01). Whereas in control cells activin had no significant effect on any aspect of FSH production examined, in GnRH agonist-treated cells activin enhanced basal FSH release, residual cell content and total FSH content/well (P<0·001). Altering GnRH receptor status also modified the LH response to activin. With control cells activin increased basal release (P<0·001), decreased GnRH-induced release (P<0·001) and increased total LH content/well (P<0·001). With GnRH agonist-treated cells, however, activin had a uniform inhibitory effect on each aspect of LH production examined (P<0·001 in each case).
It was concluded that desensitization of ovine gonadotrophs to GnRH by chronic agonist treatment results in a paradoxical enhancement of FSH output in vitro but has little effect on the responsiveness of the cells (in terms of gonadotrophin release and content) to either inhibin or oestradiol. In contrast, GnRH agonist treatment leads to qualitative changes in cellular reponsiveness to activin.
Journal of Endocrinology (1994) 140, 483–493
S. Muttukrishna and P. G. Knight
Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified M r 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated.
As in rats, both crude and highly purified inhibin preparations promoted a dose (P < 0·001)- and time (P < 0·001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P< 0·001) increased by the presence of either bFF (+ 75%) or highly-purified inhibin (+ 64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 μl specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17β (1 pmol/l–10 nmol/l) nor monomeric α-subunit of bovine inhibin (2·5–40 ng/ml) significantly affected basal or GnRH-induced release of LH.
These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat.
Journal of Endocrinology (1990) 127, 149–159
P G Knight and S Muttukrishna
Several years ago we developed a novel two-site immunoradiometric assay (IRMA) for dimeric inhibin. However, relative to the purified 32 kDa bovine inhibin standard used at that time, the immunopotencies of crude inhibin-containing samples were much less than their biopotencies estimated by pituitary cell bioassay. In attempting to improve assay performance and resolve this discrepancy we recently discovered that introduction of a preassay oxidation step to the IRMA results in a dramatic increase in the immunopotencies of inhibin-containing test samples (e.g.: bovine, human, porcine follicular fluid (FF)) and of a new (purified in 1993) 32 kDa bovine inhibin standard. However, the oxidation step did not affect the immunopotency of our original standard (purified in 1987), indicating that this material had undergone spontaneous oxidation during long-term storage, thus accounting for its higher immunopotency in our original IRMA and providing an explanation for the discrepancy between immunoactivity and bioactivity referred to above.
These findings, together with other observations on the behaviour of oxidized and non-oxidized samples of inhibin, related peptide fragments and inhibin-containing samples in the IRMA and α subunit radioimmunoassay (RIA), indicate that the anti-βA 82–114 monoclonal antibody (E4) used as tracer in the IRMA binds selectively to the oxidized (Met O 89,91,108) form of the peptide. This property of the antibody can be exploited to advantage by incorporating simple modifications to existing inhibin/activin immunoassays to ensure that all samples and standards are fully oxidized before antibody addition.
Dimeric inhibin levels in individual bovine FF samples (n=105) estimated by the 'oxidized' IRMA (9·66 ± 0·50 ng/μl) were highly correlated (r=0·90; P<0·001) with values derived using the original IRMA (0·75 ± 0·05 ng/μl) but were 13-fold higher. Levels of total immunoreactive (ir)-β (estimated by RIA) and αβ dimer in bovine FF were highly correlated (r=0·89; P<0·001) whereas no correlation existed between levels of total ir-α (estimated by RIA) and either total ir-β or αβ dimer. This observation indicates that availability of β rather than α subunit regulates the amount of dimeric inhibin produced by each follicle. Preliminary observations indicate that the modified IRMA can detect serum levels of dimeric inhibin in rats and women undergoing ovarian hyperstimulation with exogenous gonadotrophin but not in normal women or cattle.
Journal of Endocrinology (1994) 141, 417–425
P. G. Knight and R. J. Castillo
Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist d-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2–3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P <0·05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630. These observations indicate that charcoal-treated bovine FF, a rich source of inhibin, can directly suppress pituitary FSH secretion in vivo, irrespective of whether a functionally intact hypothalamo-pituitary-ovarian axis is present.
J. Endocr. (1988) 117, 431–439
J H M Wrathall and P G Knight
Primary monolayer cultures of bovine theca cells isolated from pooled ovarian follicles (3–10 mm diameter) were used to examine the effects of various granulosa cell-derived substances on basal and luteinizing hormone (LH)-induced androgen and progesterone secretion. After an overnight pretreatment period, cells were incubated with a range of treatments including LH, oestradiol-17β, inhibin, activin and follistatin. Media were collected after 48 h and assessment of androstenedione and progesterone secretion made by radioimmunoassay.
Addition of LH (5–50 ng/ml) to the cells resulted in a dose-dependent stimulation of both androstenedione (2·5-to 3-fold rise; P<0·01) and progesterone (∼ 1·6-fold rise; P<0·001) production. Secretion of androstenedione was also raised (up to 5-fold; P<0·001) by addition of oestradiol-17β (0·3–300 ng/ml), whilst levels of the androgen in the presence of both LH (20 ng/ml) and oestradiol (300 ng/ml) were up to 12-fold higher (P<0·001) than control values. In contrast, oestradiol treatment inhibited by up to 50% both basal (P<0·001) and LH-stimulated (P<0·001) secretion of progesterone. Exposure of cells to purified bovine inhibin (5–125 ng/ml) consistently raised androstenedione secretion by up to 42% over basal levels (P<0·001). Inhibin also enhanced both LH-stimulated (∼20%; P<0·001) and oestradiol-stimulated (∼20%; P<0·05) secretion of androstenedione. In direct contrast, treatment of theca cells with human recombinant activin-A (1–50 ng/ml) inhibited both LH-stimulated (∼50%; P<0·001) and oestradiol-stimulated (∼30%; P<0·005) androstenedione secretion. Activin also reversed the positive effect of inhibin on basal (P<0·01), LH-stimulated (P<0·001) and oestradiol-stimulated (P<0·001) androstenedione secretion, though activin alone did not affect basal steroid output. Simultaneous addition of human recombinant follistatin reversed the inhibitory effects of activin on LH- and oestradiol-induced androstenedione secretion but did not modify the effects of inhibin. Follistatin alone did not alter either basal or LH-stimulated androstenedione output. Neither basal nor LH-stimulated secretion of progesterone were consistently affected by inhibin, activin or follistatin.
As well as confirming the stimulatory effects of both LH and oestradiol on bovine thecal cell androgen production, these observations are indicative of opposing intrafollicular paracrine roles for granulosa cell-derived inhibin and activin in modulating thecal cell responses to gonadotrophins and steroids in the bovine ovary. Though inhibin and oestradiol had qualitatively similar effects in promoting thecal androgen secretion, the magnitude of the response to oestradiol was much greater. The results also support an intrafollicular role of follistatin as a binding protein capable of neutralizing the effect of activin, but not inhibin, on thecal androgen production.
Journal of Endocrinology (1995) 145, 491–500
P G Knight, S Muttukrishna and N P Groome
The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and α2 macroglobulin (α2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit ∼10 pg/well), precise (mean within- and between-plate coefficients of variation 4·9 and 9·1% respectively) and accurate (activin-A recovery values of 102 ± 3 and 96 ± 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and α2M (100 μg/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all <0·5%), bovine pro-αC and follistatin (both <0·1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (M r ∼25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent M r values of >700 and 60–200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n=76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r=+0·54; P<0·001) and total β subunit immunoreactivity (r=+0·32; P<0·005) but not with total α subunit immunoreactivity (r= −0·09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total β subunit levels were highest in oestrogen-inactive follicles (P<0·01) whereas total α subunit levels were lowest in these follicles (P<0·001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and ∼ 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy.
In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man.
Journal of Endocrinology (1996) 148, 267–279
T M Lovell, P G Knight and R T Gladwell
Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor β (TGFβ) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24–25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n=8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnRH-R), LH β subunit, FSH β subunit and GAPDH. Levels of mRNA for inhibin/activin βA and βB subunits, inhibin α subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (<2 amol/reaction). Significant changes in expression (P<0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P<0.001) and beta-glycan (r=0.45; P<0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P<0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH β and FSH β subunit were both lowest (P<0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.
P.G. Knight, N. Groome and A.J. Beard
A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the α subunit and the C-terminal region (82-114) of the βA subunit of Mr ∼30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (< 0.1 and < 2% respectively). Although the detection limit of the IRMA (∼ 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin α subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA. It is suggested that this quantitative discrepancy between inhibin contents estimated by IRMA and bioassay may be due to (1) loss of bioactivity of the inhibin standard during its purification and/or storage, (2) failure of the IRMA to detect high Mr forms of bioactive inhibin and/or (3) cross reaction of follistatin and other FSH-suppressing substances in the in vitro bioassay.
Claire Glister, Nigel P Groome and Philip G Knight
The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isoforms of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follicles (n=146) from 2–20 mm diameter were dissected from ovaries of ~40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quantify the relative abundance of different FS isoforms. Follicle growth from 2–6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2–10 mm. From 6–2 mm, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2–6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2–6 mm before falling 2-fold from 6 to 17–20 mm. These findings imply a marked increase in relative activin ‘tone’ around the stage at which dominant follicle selection occurs. When larger follicles (13–20 mm) were subdivided according to E/P ratio, those with high ( > 5) E/P ratio had lower (2-fold; P < 0.001) levels of inh-A and act-A in comparison to follicles with low ( < 5) E/P ratio, but there were no significant differences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26, 13 and 17% respectively of total FS. During growth from 2–20 mm the proportion of total FS represented by 65, 41 and 37 kDa isoforms increased ~2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1.6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin ‘tone’ accompanies bovine follicle growth from 3–6 mm, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.