Changes in aromatase activity in small ovarian follicles of the domestic fowl (Gallus domesticus) during growth and atresia

The tritium release assay for measuring aromatase activity was adapted to measure oestrogen production in intact small ovarian follicles of the domestic fowl. The activity was measured in three types of follicle classified as normal, atretic or grossly atretic. Normal follicles were distinguished from atretic by the presence of small haemorrhages on the surface of the atretic follicles. Grossly atretic follicles were identified by their deformed shape. The aromatase activity in normal and atretic follicles was related to follicular weight, the activity in atretic follicles being less than that in normal follicles. The aromatase activity in grossly atretic follicles was independent of follicular weight. The enzyme activity in this group of follicles was significantly less than that in either normal or atretic follicles. The significance of these results is discussed in relation to the induction of atresia within the small ovarian follicles of the domestic fowl.     

Haemorrhage and adrenocortical activity in the fowl (Gallus domesticus)

Plasma aldosterone concentrations were markedly elevated in chickens by the removal of 10-30% of their total blood volume. This aldosterone response to haemorrhage was delayed and of reduced magnitude in birds pretreated for 48 hr with dexamethasone (100 micrograms/kg every 12 hr), in which the basal aldosterone level was also suppressed. Corticosterone concentrations were elevated in both haemorrhaged and nonhaemorrhaged birds, although these responses were completely abolished by dexamethasone pretreatment, which also reduced the basal corticosterone concentration. Prolactin concentrations were unaffected by haemorrhage. These results demonstrate that haemorrhage specifically affects adrenocortical function by increasing aldosterone release and suggests that factors other than the pituitary-adrenal axis are responsible for this stimulation of secretion.     

Thyroid hormones inhibit growth hormone secretion in domestic fowl (Gallus domesticus)

Daily blood samples from four mares were assayed for cortisol through a total of eight ovulatory cycles. Mean cortisol concentrations on days -14, -13, -10, -9 and -8 before ovulation (dioestrus) were greater than on days -5 to -1 (oestrus). The highest mean (+/- S.E.M) value of cortisol occurred on day -10 (260 +/-28 nmol/l) and the lowest on day -2 (142 +/- 14 nmol/l). A single episode on a day in late dioestrus characterized the maximum cortisol value per cycle for five of eight cycles. Extraction of plasma samples with petroleum ether or chromatography before assay, to eliminate interference from progesterone and its metabolites, did not alter the pattern of high dioestrous and low oestrous cortisol concentrations. Maximum follicular diameter at ovulation was negatively correlated with mean cortisol concentration for that cycle. These results indicate that in the mare the adrenals secrete cortisol more actively during dioestrus than during oestrus and suggest that a decline in cortisol values at oestrus may favour full follicular growth and ovulation.     

Diurnal variation in plasma luteinising hormone levels in the domestic fowl (Gallus domesticus)

Variations in the concentration of luteinising hormone (LH) in the blood were measured over 24 hr in sexually immature chickens maintained on long daily photoperiods of 18L:6D, 16L:8D, or 14L:10D. A significant increase in the concentration of LH occurred shortly after the onset of the dark period in both sexes. The possible significance of this observation in relation to neural control of the ovulatory cycle is discussed.     

3 beta-hydroxy-delta 5-steroid dehydrogenase activity in the rapidly growing ovarian follicles of the domestic fowl (Gallus domesticus)

The total and specific activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, isocitrate dehydrogenase (NADP+) and glucose-6-phosphate dehydrogenase were measured in ovarian follicles from the domestic fowl. The enzymes were assayed in the five largest yolk-filled follicles which were sampled twice during the ovulatory cycle, at 1 h and 16 h before an expected ovulation. The total and specific activity of granulosa enzymes increased throughout the hierarchy and reached a maximum in the largest follicle. The relative increase in 3 beta-hydroxy-delta 5-steroid dehydrogenase activity was greater than that of the other two enzymes examined. The total thecal 3 beta-hydroxy-delta 5-steroid dehydrogenase activity reached a maximum in the third and fourth largest follicles. Thereafter its activity decreased up to the time of ovulation. The activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase and glucose-6-phosphate dehydrogenase in follicles collected 1 h before and ovulation were significantly less than the activity in corresponding follicles collected 16 h before an ovulation.     

Ghrelin: a hypothalamic GH-releasing factor in domestic fowl (Gallus domesticus).

Ghrelin, a recently discovered peptide in the mammalian hypothalamus and gastrointestinal tract is thought to be the endogenous ligand for the GH secretagogue (GHS) receptor and it stimulates GH release in rats and humans. The possibility that ghrelin is present in birds was therefore assessed, since a GHS receptor is present in the chicken pituitary gland. Although immunoreactive ghrelin is readily detectable in the rat stomach and ileum, ghrelin immunoreactivity could not be detected in the chicken proventriculus, stomach, ileum or colon, whereas somatostatin immunoreactivity, in contrast and as expected, was readily detectable in the chicken gastrointestinal tract. Ghrelin immunoreactivity was, however, present in the chicken hypothalamus, although not in the arcuate (infundibular) nucleus, as in rats. Discrete parvocellular cells and neuronal fibers with ghrelin immunoreactivity were present in the anterior medial hypothalamus. This immunoreactivity was specific and completely abolished following the preabsorption of the antibody with an excess of human ghrelin. Ghrelin immunoreactivity was also present in clusters of large ovoid magnocellular cells in the nucleus magnocellularis preopticus pars medialis, nucleus magnocellularis preopticus supraopticus and in the chiasmaopticus. Immunoreactivity for ghrelin was restricted to the cytoplasm of the perikarya and their axonal sprouts. Immunoreactivity for ghrelin was not seen in any other hypothalamic nuclei. In a preliminary experiment, circulating GH concentrations in conscious immature chicks were promptly increased following bolus i.v. administration of human ghrelin. The increase in GH concentration (approximately three times that in the controls) was comparable with that induced by the same dose (10 microg/kg) of human GH-releasing hormone, although less than that (approximately sixfold) induced by thyrotropin-releasing hormone. These results demonstrate the presence of a ghrelin-like protein in the chicken hypothalamus and suggest that it participates in the regulation of GH secretion in birds.     

Ontogeny of growth hormone and prolactin secretion in the domestic fowl (Gallus domesticus).

Concentration of growth hormone (GH) and prolactin were measured in plasma from embryonic and neonatal chicks. GH was not detectable (<4 ng/ml) until after 17 days of incubation (6 ng/ml). The concentration increased progressively to 16 ng/ml on the day after hatching and then sharply to 50 ng/ml in 3-day-old chicks. Plasma concentrations of prolactin were low (<6 ng/ml) in 9-, 11-, and 13-day-old embryos, increased to 78 ng/ml after 17 days of incubation, then decreased to 37 ng/ml in 19-day-old embryos. On the day after hatching plasma prolactin concentration was 140 ng/ml but it fell progressively to 49 ng/ml in 5-day-old chicks.     

Calcium participation in thyroid function in fowl (Gallus domesticus)

The effects of the calcium antagonists ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid (EGTA), cobalt chloride (CoCl2), and magnesium chloride (MgCl2) on the concentrations of plasma thyroxine (T4) and triiodothyronine (T3) and on the basal and stimulated release of T4 from incubated thyroid glands have been determined in the domestic fowl. Plasma T4 levels were consistently reduced 2 hr after the administration of each calcium antagonist, although only EGTA and CoCl2 lowered the concentration of plasma total calcium. Concentrations of plasma T3 were increased following MgCl2 treatment but reduced after CoCl2 administration. The basal release of T4 by incubated thyroid glands was reduced following in vivo EGTA and CoCl2 treatment, but increased after MgCl2 injection. The addition of bovine thyroid stimulating hormone (TSH, 200 mU) to the incubation consistently stimulated in vitro T4 release, although the magnitude of the stimulation was increased following in vivo EGTA or CoCl2 treatment and reduced after MgCl2 administration. These results demonstrate the involvement of calcium dependent mechanisms in the control of T4 release in fowl.     

In vitro release of triiodothyronine and thyroxine from thyroid glands of the domestic fowl (Gallus domesticus)

Basal and thyrotrophin (TSH)-stimulated release of iodothyronines (triiodothyronine, T3, and thyroxine, T4) from intact chicken thyroid glands was determined in vitro. In the absence of TSH, T3 and T4 were released in measurable amounts in the incubation media. The release of both iodothyronines was directly related to the media TSH concentrations and incubation period. Lineweaver-Burke analysis revealed that the Vmax for T3 was 99.4 pg/gland, with an apparent Km of 17.8 mU TSH, and that the Vmax for T4 was 323.35 ng/gland, with an apparent Km of 51.5 mU TSH, demonstrating that T4 is the major iodothyronine released by avian thyroid glands. The basal release of T4 was suppressed by the addition of a calcium chelator (ethyleneglycol-bis-(beta-aminoethylether)-N,N,N', N'-tetraacetic acid; EGTA), a calcium antagonist (cobalt chloride, CoCl2), or prostaglandin E1 (PGE1) to the incubation media. Basal T4 released was increased in the presence of a calcium agonist (lanthanum chloride, LaCl3), a calcium ionophore (A23187), dibutyryl cyclic adenosine 3'3'-monophosphate (dbcAMP), isobutylmethylxanthine (IBMX), indomethacin, magnesium chloride (MgCl2), and potassium iodide (KI). Thyrotrophin-stimulated T4 release was reduced by CoCl2, PGE1, and indomethacin but enhanced by LaCl3, MgCl2, and KI. These results demonstrate that it is possible to measure the release of thyroid hormones in an in vitro system in the chicken. Basal and stimulated iodothyronine release from the chicken thyroid gland appears to be mediated by calcium- and cAMP-dependent mechanisms.     

Diminution of pulsatile growth hormone secretion in the domestic fowl (Gallus domesticus): evidence of sexual dimorphism

Temporal plasma GH secretory patterns were measured in cannulated male and female meat-type chickens of two commercial strains at 17, 38 and 60 days of age. Pulse height, amplitude and baseline values were similar in both male and female chickens at 17 days of age, with high amplitude and low baseline values. However, by 38 days of age pulsatile GH secretion was not detectable in females, whereas males exhibited a continued pulsatile secretory pattern similar to that at 17 days of age. Pulsatile GH secretion was not evident in either males or females at 60 days of age. These results clearly demonstrate a sexually dimorphic ontogeny of GH secretion in meat-type chickens.     

The role of thyroid hormones in the growth hormone response to protein restriction in the domestic fowl (Gallus domesticus)

The possible role of thyroid hormones in the rise in plasma GH observed in protein-restricted chicks was examined. Increased sensitivity of protein-restricted chicks to secretagogue challenge (TRH or GH-releasing factor) appears to account, at least in part, for increased GH concentrations in protein-restricted chicks. Thyroid hormones administered acutely were able to suppress plasma GH concentrations in protein-restricted chicks. Further, chronic thyroid hormone supplementation to low protein diets normalized circulating thyroid hormone concentrations and also normalized the response to GH secretagogue challenge. This decreased sensitivity to TRH provocation occurred without an accompanying change in plasma concentrations of insulin-like growth factor-I, a reputed inhibitor of GH secretion in the chicken.     

Distinct features of dehydrocorticosterone reduction into corticosterone in the liver and duodenum of the domestic fowl (Gallus gallus domesticus)

The mammalian 11-beta hydroxysteroid dehydrogenase type 1 (11 betaHSD1) reduces glucocorticoids (GC) at C11 from the 11-keto-GC nonactive form to the 11-hydroxy-GC active form, an action essential for survival. Whereas GC metabolism at C11 and the role of 11 betaHSD1 are studied extensively in mammals, information about these in birds is scattered. Herein, we report the GC bidirectional metabolism in chickens. In hens' liver and duodenal mucosa, 11 betaHSD1-like mRNA expression was detected; and 11 betaHSD1-like immunoreactivity was found linked to membranes of hepatocytes and duodenal enterocytes. With either NADH or NADPH, the membranal fraction of liver and duodenal mucosa converted dehydrocorticosterone (A) into corticosterone (B) with K(m) (1.1-8.7 microM) and V(max) (10-40 pmol/mg protein/min) values similar to those reported for mammalian 11 betaHSD1. In the presence of NADP(+) or NAD(+), these membranal fractions oxidized B into A. With either NADPH or NADH, the cytosol of chicken liver and duodenal mucosa reduced A into B (K(m) of 1.1 - 2.3 microM and V(max) of 260-960 pmol/mg protein/min). These cytosolic fractions did not convert any amount of B into A when incubated with either NADP(+) or NAD(+). This may suggest that chicken liver and duodenal mucosa express 11 betaHSD1 that is a membrane-bound oxoreductase which uses both NADPH/NADP(+) and NADH/NAD(+) as cosubstrates. The substantial reduction of A into B (but no conversion of B into A) found in the cytosol is most likely executed by a unidirectional soluble reductase, different than 11 betaHSD1.     

Synergistic effect of gonadotropin releasing hormone on LH-stimulated progesterone production in granulosa cells of the domestic fowl (Gallus domesticus)

A direct gonadal action of gonadotropin releasing hormone (GnRH) has been demonstrated in several mammalian species. The effect of mammalian GnRH on steroidogenesis in avian granulosa cells (GC) was investigated. When such cells from mature follicles were preincubated with GnRH, the subsequent production of progesterone in response to oLH was significantly enhanced. In GC from medium or small follicles (F₃ and F₅), no effect of GnRH was observed. Similarly, when steroidogenesis was stimulated with dibutyryl cyclic AMP preincubation with GnRH failed to alter progesterone responses. On the other hand, oFSH-stimulated steroidogenesis in GC from small follicles (F₄ and F₅) was moderately but consistently and significantly attenuated by GnRH. Further studies with homologous GnRH may be necessary for the elucidation of the possible ovarian action of this hormone.     

Effect of luteinizing hormone on progesterone production by the follicular granulosa in the ovarian hierarchy of the domestic fowl (Gallus domesticus)

A LH function test was used to assess the effect of a previously administered dose of ovine LH on the granulosa from the four largest preovulatory follicles, F1-F4, of laying hens. At intervals of 1, 3, and 6 hr after intravenous ovine LH (40 micrograms; a dose previously shown to cause atresia) or carrier solution (1% bovine serum albumin solution, BSA), a second dose of LH or carrier was injected. The progesterone contained in the dissected granulosa of the F1-F4 follicles 45 min after the second injection was measured by radioimmunoassay. With birds injected with BSA, the mean basal levels ranged from 17 to 59 ng progesterone per granulosa, irrespective of the follicular type. In short-term experiments, i.e., 45 min after LH, the mean progesterone contents of F1 (262 ng) and F2 (137 ng) were significantly higher than their controls, whereas those of F3 (59 ng) and F4 (21 ng) did not change. The response per cell was greater in the F1 granulosa than in that of F2-F4; other evidence is presented to support a hypothesis that the F1 granulosa contains a larger LH receptor population. The effect of LH over a longer period (6 hr) was shown by a decline in the steroidogenic response of the F1 granulosa only. The kinetics of this decline (estimated t1/2 13.7 hr) resembled that previously reported for adenylate cyclase activity in granulosa cells from postovulatory follicles (POF) (t1/2 about 14.4 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of aldosterone on water and electrolyte transport in the colon and coprodeum of the domestic fowl (Gallus domesticus) in vivo

White Leghorn laying hens were maintained on commercial poultry food (medium-Na+ diet) and fresh water. High-Na+ diet birds received, in addition, 10 ml 9% (w/v) NaCl/kg by stomach load for 2 days before perfusion experiments. The lumen of the coprodeum and colon of anaesthetized birds was perfused with a hyperosmotic solution resembling ureteral urine. Transmural solute and water fluxes and potential difference (p.d.) values were measured for 2.5 h before and for 8 h after i.v. injection of aldosterone (120 microgram/kg). After administration of aldosterone, the lag, increasing and stabilized plateau phases were identified for Na+, K+ and Cl- fluxes (which together formed an electroneutral ion-exchange system); plateau flux values were significantly greater than preinjection values and were comparable to values for birds maintained on low-Na+ diets in parallel experiments. Ammonium, phosphate and water fluxes were unresponsive to aldosterone and p.d. values showed a transient increase in medium-Na+ diet birds only. In parallel experiments on birds on low sodium diets the ammonium flux and p.d. increased but the osmotic flow and phosphate transfer did not respond. Therefore acute injection of aldosterone reproduced some but not all of the responses to dietary Na+ restriction in fowls.     

Dietary protein restriction stress in the domestic fowl (Gallus gallus domesticus) alters adrenocorticotropin-transmembranous signaling and corticosterone negative feedback in adrenal steroidogenic cells

Previous work with growing chickens (Gallus gallus domesticus) indicates that transient dietary protein restriction induces long-term enhancement of adrenal steroidogenic function in response to adrenocorticotropin (ACTH). The present study investigated two possible cellular functions mediating this enhanced response: (a) ACTH signal transduction and dissemination and (b) short-loop feedback inhibition of ACTH-induced corticosterone production by exogenous corticosterone. Cockerels (2 weeks old) were fed isocaloric synthetic diets containing either 20% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of adrenal steroidogenic cells nearly devoid of chromaffin cells ( approximately 90% adrenal steroidogenic cells). Results of experiments to assess signal transduction and dissemination indicated that protein restriction selectively enhanced ACTH-induced corticosterone production mediated by the cyclic AMP (cAMP)-dependent pathway. In addition, protein restriction substantially counteracted exogenous corticosterone-dependent inhibition of acute ACTH-induced corticosterone production (by 40.7% vs control). The proximal portion of the cAMP pathway seemed most affected by this stressor. Protein-restricted cells exhibited enhanced homologous sensitization to ACTH (136% greater than that of control cells) which appeared to be localized at a step(s) prior to or at the formation to cAMP. Also, maximal ACTH-induced cAMP production and sensitivity to ACTH in terms of cAMP production by protein-restricted cells were, respectively, 2.2 and 15.8 times those of control cells. However, variable results were obtained from other experiments designed to pinpoint the altered early steps in ACTH-transmembranous signaling. For example, with intact cells, cAMP responses to cholera toxin (CT) and forskolin (FSK) did not corroborate the results suggesting an augmentation of ACTH-signal transduction induced by protein restriction. Furthermore, basal and stimulatable (by ACTH, CT, FSK, and NaF) adenylyl cyclase activities from membranes from protein-restricted cells were, respectively, 47.2 and 40.2% less than those from control cells (normalized to 10(7) cell equivalents of crude membranes). Collectively, these findings suggest that protein restriction stress potentiates ACTH-induced corticosterone secretion by chicken adrenal steroidogenic cells in at least two ways: (1) on the proximal end, by modulating unknown factors which enhance cellular sensitivity to ACTH, ACTH receptor-adenylyl cyclase coupling, and adenylyl cyclase activity, and (2) on the distal end, by suppressing end-product corticosterone negative feedback, thus facilitating an increase in net corticosterone secretion.     

Daily infusion of corticosterone and reproductive function in the domestic hen (Gallus domesticus)

The purpose of this study was to examine the effect of the daily infusion of corticosterone on reproductive function in the laying hen and to determine the relationship between the cyclic pattern of plasma concentrations of corticosterone on the open-period for the preovulatory release of LH. An exogenous rhythm of plasma levels of corticosterone was generated using an osmotic pump. Corticosterone was infused subcutaneously into laying hens at rates of 5, 10, 15 or 30 micrograms/hr for a duration of 10 hr beginning with the onset of darkness or at 15 micrograms/hr for 4 hr, or continuously at 30 micrograms/hr. Daily infusions greater than 15 micrograms/hr and the continuous infusion resulted in cessation of ovulation, ovarian and oviductal regression, hyperphagia, and elevated levels of plasma corticosterone compared to that observed in control hens. The hens which were infused with 5 or 10 micrograms/hr of corticosterone maintained normal reproductive function with plasma concentrations of corticosterone that were approximately the same as those in the control hens. The effect of infusing 10 micrograms/hr of corticosterone on the open-period for the preovulatory release of LH was determined under constant light. No significant changes were observed in the frequency distribution of the times of oviposition when hens were infused with 10 micrograms/hr of corticosterone for 12 hr from 9:00 to 21:00 hr or 21:00 to 9:00 hr each day.(ABSTRACT TRUNCATED AT 250 WORDS)