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.
T M Lovell, P G Knight and R T Gladwell
Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-β (TGFβ) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (~40 mm).
Ovaries were collected (n=16) from mid-sequence hens maintained on a long-day photoschedule (16 h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of mRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0.05) in 8–9.9 mm follicles, whereas ActRIIA rose significantly from 6–7.9 mm to 8–9.9 mm, before falling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan mRNA expression rose 3-fold from 4–5.9 mm to 8–9.9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4–7.9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0.05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 mm to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActRIIA increased 2-fold from F2 to F1 (P < 0.05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before falling ~50% in the F1.
In all follicles studied expression of betaglycan and ActRI (granulosa: r=0.65, P < 0.001, n=144/group; theca: r=0.49, P < 0.001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI, ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11.4-fold; ActRIIB, 5.1-fold; ActRI, 3.8-fold; ActRIIA, 2.8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.
S. C. Wilson, R. T. Gladwell and F. J. Cunningham
Diurnal changes of LH secretion in sexually immature hens of 9, 11, 13 and 15 weeks of age consisted of 25–40% increases in the mean concentrations of LH in plasma between 15.00 and 18.00 h, i.e. between 2 h before and 1 h after the onset of darkness. During this time there was a tendency for the mean contents of LHRH-I in the anterior hypothalamus and posterior hypothalamus to increase by 21–74% and 20–56% respectively. In hens of 9 and 15 weeks, diurnal changes in the plasma concentration of LH closely paralleled those of LHRH-I content in the posterior hypothalamus. In contrast, the diurnal rhythm of LH secretion in hens of 11 and 13 weeks was more marked and plasma concentrations of LH continued to rise steeply between 18.00 and 21.00 h, i.e. between 1 and 4 h after the onset of darkness. At 11 weeks, this was associated with a reduction (P<0·01) in the contents of LHRH-I and LHRH-II, particularly in the anterior hypothalamus. In laying hens, a diurnal decline (P<0·01) in the plasma concentration of LH between 1 and 4 h after the onset of darkness was preceded by a fall (P<0·05) in the content of LHRH-I in the posterior hypothalamus and in the total hypothalamic content of LHRH-II (P<0·01). In all groups of hens, irrespective of the times of day at which tissue was taken, significant (P<0·05–<0·001) correlations between the contents of LHRH-I and LHRH-II in the anterior hypothalamus were observed.
It is concluded that a diurnal rhythm of release of LHRH-I may drive the diurnal rhythm of LH secretion. Thus, in sexually immature hens of 9 and 15 weeks and laying hens in which diurnal changes in plasma LH were small there were parallel changes in the content of LHRH-I in the posterior hypothalamus. However, where the plasma concentration of LH was increased substantially, as at 11 weeks, there was a decline in the hypothalamic contents of LHRH-I. A simultaneous fall in the hypothalamic content of LHRH-II raises the possibility of a causal relationship between the activities of LHRH-II, LHRH-I and the release of LH.
Journal of Endocrinology (1991) 130, 457–462
S. C. Wilson, R. T. Gladwell and F. J. Cunningham
Changes in the hypothalamic contents of LHRH-I and LHRH-II were determined in intact and castrated cockerels injected i.m. with gonadal steroids or tamoxifen. An increase in the plasma concentration of LH after castration was accompanied by a significant increase in the content of LHRH-I in the posterior hypothalamus (including the mediobasal hypothalamus and median eminence) which was reversed by oestradiol benzoate given on days 14 and 15 after castration. Under similar circumstances, testosterone propionate did not modify the hypothalamic content of LHRH-I, even though both steroids reduced the plasma concentrations of LH to levels below those of intact cockerels. Treatment of intact cockerels with oestradiol benzoate significantly increased the content of LHRH-I in the posterior hypothalamus, whilst testosterone propionate was again without effect. Tamoxifen significantly raised the plasma concentration of LH in intact cockerels and partially antagonized the suppressive effect of oestradiol benzoate and testosterone on LH secretion in castrated cockerels. However, an anti-oestrogenic effect of tamoxifen on the hypothalamic content of LHRH-I was not demonstrated. There was no evidence of any changes in the hypothalamic content of LHRH-II after castration, with or without gonadal steroid replacement. A change in the hypothalamic content of LHRH-I in response to manipulation of the steroid environment would imply an involvement of this peptide in the mechanism by which gonadal steroids regulate the release of LH. The absence of changes in the hypothalamic content of LHRH-II in the same circumstances suggest that it is not directly involved in the control of LH secretion by the gonadal steroid negative feedback loop.
Journal of Endocrinology (1990) 125, 139–146
P. G. Knight, F. J. Cunningham and R. T. Gladwell
The validity of using a radioimmunoassay employing an antiserum raised against synthetic luteinizing hormone releasing hormone (LH-RH) for the quantification of luteinizing hormone releasing factor (LH-RF) in birds was investigated. Extracts of avian hypothalamus yielded displacement curves which were parallel to that of the synthetic LH-RH standard and the immunoreactive potencies of a number of extracts assayed concurrently using two different anti-LH-RH sera were found to be similar. Moreover, after chromatography of cockerel hypothalamic extract on carboxymethyl–cellulose. immunoreactive and biologically active LH-RF were found in the same eluate fractions. Immunoreactive LH-RH was shown to be widely distributed in cockerel hypothalamus with the highest concentrations present in the mediobasal hypothalamus (MBH; 6·55± 1·86 pg/μg protein, n = 6) and medial preoptic region (POR; 0·95 ±0·07 pg/μg protein, n = 6). The postcastration rise in plasma LH in the cockerel was accompanied by significant (P <0·05) increases in the concentration of LH-RH in five hypothalamic areas including the POR; testosterone replacement therapy completely reversed these effects. Although castration raised the mean concentration of LH-RH in four other hypothalamic areas including the MBH, these differences were not significant. However, testosterone replacement therapy depressed LH-RH in all four regions to levels significantly (P <0·05) less than those in castrated cockerels. These findings constitute the first direct evidence that the negative feedback action of testosterone on LH secretion in the cockerel is mediated, at least in part, by an action on hypothalamic LH-RF-producing neurones.
P. G. KNIGHT, R. T. GLADWELL and F. J. CUNNINGHAM
Concentrations of dopamine, noradrenaline and adrenaline in discrete areas of the diencephalon in male and female domestic fowl were correlated with changes in the plasma concentrations of LH induced by gonadectomy. Gonadectomized birds of both sexes exhibited raised plasma concentrations of LH and in castrated cockerels the daily administration of testosterone propionate was completely effective in preventing the post-castration rise in LH. Although no significant alterations in the brain concentrations of noradrenaline or adrenaline were observed in cockerels, the concentration of dopamine in the paraventricular nucleus (PVM), dorsomedial thalamic nucleus and mediobasal hypothalamus (MBH) were significantly raised in castrated compared with sham-operated birds by 136, 182 and 52% respectively. In each case the increase was partially suppressed by testosterone replacement therapy. In pullets, ovariectomy resulted in significant increases in the concentrations of dopamine (83%) and noradrenaline (78%) in the MBH and noradrenaline (35%) and adrenaline (34%) in the PVM. These findings suggest that in the fowl at least part of the negative feedback effect of gonadal steroids on LH secretion may be mediated by catecholaminergic mechanisms at the level of the hypothalamus.
S. C. Wilson, F. J. Cunningham, R. A. Chairil and R. T. Gladwell
Treatment of chickens at different stages of sexual development with a single i.v. injection of synthetic chicken LHRH (cLHRH)-I or -II stimulated a rise in the plasma concentration of LH within 1 min. The activity of cLHRH-II was 1·3- to 2·7-fold greater than that of cLHRH-I in sexually immature cockerels and hens as determined by the changes in the plasma concentration of LH during the 5 or 10 min after injection. This could be attributed to both a greater effectiveness of cLHRH-II to stimulate LH release and to a more prolonged action. Thus, LH concentrations in plasma were maximal within 1–2 min of injection of all doses of cLHRH-I but within 2–5 min of injection at the higher doses of cLHRH-II. The responsiveness of the pituitary gland to cLHRH-I and -II was substantially greater in the sexually immature cockerel than in the hen and diminished during sexual development of the hen. Coincident with the onset of egg laying, the characteristics of the LH response to cLHRH-II changed to consist of an initial rise during the first 2 min, followed by a more sustained increase with LH concentrations still rising 10 min after injection. In contrast, after injection with cLHRH-I, plasma concentrations of LH rose to a peak at 2 min and thereafter declined gradually. Treatment of the sexually immature hen with oestradiol, progesterone or a combination of both steroids did not enable the expression of a laying hen-type response to the injection of cLHRH-II. It would appear, therefore, that unidentified events associated with the final stages of sexual maturation bring about changes in the mechanism of action of cLHRH-II which differ from those of cLHRH-I.
Journal of Endocrinology (1989) 123, 311–318
S. C. Wilson, R. A. Chairil, F. J. Cunningham and R. T. Gladwell
The contents of LHRH-I and -II in the anterior hypothalamus and posterior hypothalamus (including the mediobasal hypothalamus and median eminence) were measured at 90, 180 and 360 min after the i.m. injection of laying hens with progesterone. Whilst no changes were observed in the content of LHRH-I in the anterior hypothalamus, LHRH-I in the posterior hypothalamus tended to fall at 90 and 180 min after injection of progesterone in hens maintained on 16 h light:8 h darkness (16L:8D) and 8L:16D respectively. Pretreatment of laying hens with tamoxifen significantly increased the hypothalamic contents of LHRH-I and -II, raised the basal plasma concentration of LH and modified the LH response to progesterone injection. In hens in which tamoxifen prevented an increase in the plasma concentration of LH after progesterone injection, the content of LHRH-I in the posterior hypothalamus remained unchanged. In contrast, in hens in which progesterone stimulated a steep increase in LH within 90 min, there was a pronounced and significant fall in LHRH-I content of the posterior hypothalamus. No change in the hypothalamic content of LHRH-II was observed during the progesterone-induced surge of LH until plasma concentrations had attained maximal values or started to decline. Then, in hens maintained on 16L:8D, a significant fall in the content of LHRH-II in the anterior hypothalamus was found at both 180 and 360 min after injection with progesterone.
Tests in vitro and in vivo of the responsiveness of the pituitary gland to synthetic LHRH-I and -II revealed no change at 90 min after injection of laying hens with progesterone, when plasma concentrations of LH were increasing, but a pronounced reduction when plasma LH concentrations were maximal or falling.
These results suggest that LHRH-I mediates in the progesterone-induced increase in the plasma concentration of LH. Although the subsequent decline in plasma LH was associated with a reduced responsiveness of the pituitary gland to LHRH, a significant correlation between the contents of LHRH-I and -II in the anterior hypothalamus and a fall in the hypothalamic content of LHRH-II when plasma LH was maximal or declining allows the possibility of an involvement of this peptide in the neuroendocrine events preceding ovulation.
Journal of Endocrinology (1990) 127, 487–496
P. G. Knight, S. C. Wilson, R. T. Gladwell and F. J. Cunningham
The effects of various pharmacological treatments, designed to perturb central catecholaminergic neurotransmission, on the pattern of LH release during the preovulatory period in the domestic hen were studied. Treatment of hens with either l-dihydroxyphenylalanine or diethyldithiocarbamate which raised the concentration of dopamine in the hypothalamus by 42 and 110% respectively, or with apomorphine, attenuated the preovulatory surge of LH. In contrast, treatment with either α-methyl-p-tyrosine which produced a 65% decline in the concentration of dopamine in the hypothalamus without affecting the concentrations of noradrenaline or adrenaline or treatment with pimozide did not affect the LH surge. While treatment with propranolol was similarly ineffective, phenoxybenzamine attenuated the LH surge to a marked extent. These observations suggest that the preovulatory surge of LH in the hen is influenced by facilitatory α-adrenergic and inhibitory dopaminergic mechanisms. Evidence to corroborate these findings was sought by determining the steady-state concentrations of dopamine, noradrenaline and adrenaline in five discrete diencephalic regions of the hen throughout the ovulatory cycle.