Peripheral ghrelin reduces food intake and respiratory quotient in chicken.

Ghrelin injection, either centrally or peripherally strongly stimulates feeding in human and rodents. In contrast, centrally injected ghrelin inhibits food intake in neonatal chickens. No information is available about the mechanism and its relationship with energy homeostasis in chicken. Since ghrelin is predominantly produced in the stomach, we investigated the effect of peripherally injected ghrelin (1 nmol/100g body weight) on food intake and energy expenditure as measured in respiratory cells by indirect calorimetry for 24h in one-week-old chickens. Plasma glucose, triglycerides, free fatty acids, total protein and T(3) were measured in a separate experiment until 60 min after injection. Food intake decreased until at least 1h after intravenous ghrelin administration. The respiratory quotient (RQ) in ghrelin-injected chickens was reduced until 14 h after administration whereas plasma glucose and triglycerides concentrations were not altered. Free fatty acids and total protein levels also remained unchanged. Ghrelin did not influence heat production and this was supported by the absence of changes in plasma T(3) levels when compared to the control values. In conclusion, peripheral ghrelin reduces food intake as well as RQ and might influence the type of substrate (macronutrient) that is used as metabolic fuel.

The release of growth hormone (GH): relation to the thyrotropic- and corticotropic axis in the chicken.

In the chicken and other avian species, the secretion of GH is under a dual stimulatory and inhibitory control of hypothalamic hypophysiotropic factors. Additionally, the thyrotropin-releasing hormone (TRH), contrary to the mammalian situation, is also somatotropic and equally important in releasing GH in chick embryos and juvenile chicks compared to the (mammalian) growth hormone-releasing hormone (GHRH) itself. Consequently, the negative feedback loop for GH release not only involves the insulin-like growth factor IGF-I but also thyroid hormones. In adult chickens, TRH does no longer have a clear thyrotropic activity, whereas its somatotropic activity depends on the feeding status of the animal. In addition, as in mammals, the secretion of GH and glucocorticoids is stimulated by ghrelin, a novel peptide predominantly synthesized in the gastrointestinal tract. Two chicken isoforms of the ghrelin receptor have been identified, both of which are highly expressed in the hypothalamus and pituitary, suggesting that a stimulatory effect may be directed at these levels. GH and glucocorticoids control the peripheral thyroid hormone function by down-regulating the hepatic type III deiodinating enzyme (D3) in embryos (GH and glucocorticoids) and in juvenile and adult chickens (GH). Moreover, glucocorticoids help to regulate T3-homeostasis in the brain during embryogenesis by stimulating the type II deiodinase (D2) expression. This way not only a multifactorial release mechanism exists for GH but also a functional entanglement of activities between the somatotropic-, thyrotropic- and corticotropic axis.

Distribution and regulation of chicken growth hormone secretagogue receptor isoforms.

Chicken ghrelin has recently been isolated as a hormone which stimulates growth hormone and corticosterone secretion in chicken. Ghrelin mediates these actions in mammals by binding to the growth hormone secretagogue receptor (GHS-R). In this study, we describe the partial cloning of two chicken GHS-R (cGHS-R) isoforms: cGHS-R1a and cGHS-R1c. cGHS-R1a and cGHS-R1c cDNA show, respectively, 81 and 78% homology with the corresponding parts of the human GHS-R1a cDNA. In contrast to the human GHS-R1b isoform, which is truncated after transmembrane domain 5 (TM-5), the chicken GHS-R1c isoform lacks 16 amino acids in TM-6 suggesting that this isoform is not active in ghrelin signal transduction. The cystein residues, N-linked glycosylation sites and potential phosphorylation sites, found in the human GHS-R1a, were also conserved in both chicken isoforms. RT-PCR analysis demonstrated cGHS-R1a and cGHS-R1c mRNA expression in all tissues tested, except liver and pancreas, with highest levels in the pituitary and the hypothalamus. Intermediate levels of expression were detected, in descending order, in the ovary, telencephalon, heart, adrenal gland, cerebellum, and optic lobes whereas low expression was detected in the brainstem, lung, kidney, proventriculus, duodenum, and colon. Very low expression was found in skin, stomach, and muscle. cGHS-R1c was expressed in lower amounts than cGHS-R1a in all analysed tissues. Administration of 1 microM chicken ghrelin to pituitaries in vitro resulted in a down-regulation of both cGHS-R isoforms within 15 min, whereas after 1h levels returned to control values. Growth hormone and corticosterone down-regulated cGHS-R1a and cGHS-R1c mRNA expression within 60 min of exposure, whereas growth hormone-releasing factor 1-29 (1 microM) only reduced cGHS-R1a mRNA expression after 60min. Thyrotropin-releasing hormone (1 microM) did not alter cGHS-R expression.

Involvement of thyrotropin-releasing hormone receptor, somatostatin receptor subtype 2 and corticotropin-releasing hormone receptor type 1 in the control of chicken thyrotropin secretion.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

A beta-turn endocytic code is required for optimal internalization of the growth hormone receptor but not for alpha-adaptin association.

Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of Pro335-Asp335, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that alpha-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with alpha-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of alpha-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not alpha-adaptin but an uncharacterized factor, presumably interacting with the identified beta-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate.

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.

Evidence of a thyrotropin-releasing activity of ovine corticotropin-releasing factor in the domestic fowl (Gallus domesticus).

Ovine corticotropin-releasing factor (oCRF) administered to 19-day-old chicken embryos (E19) increased plasma concentration of pituitary glycoprotein alpha-subunit concentrations within 15 min for at least 4 hr. Follicle stimulating hormone levels were unchanged, while plasma luteinizing hormone concentrations only began to increase 1 hr after the oCRF treatment. Calculation of circulating thyrotropin (TSH) indicator values revealed a rapid elevation in TSH plasma levels following oCRF. Concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), and corticosterone increased from 1 hr postinjection. Hypothalamic outer ring deiodinating type II increased and hepatic inner ring deiodinating type III fell after 2 and 4 hr, explaining at least in part the plasma T3 increase at the end of the experiment. In a second experiment, using E18 chicks, a comparison was made between the effects of a single injection of 2 micrograms oCRF and 20 mlU bovine TSH. Both hormones increased T4, T3, and rT3 plasma concentrations, supporting the hypothesis of a TSH-releasing activity for oCRF in the embryonic chicken. The proposed TSH-mediated effect of CRF on thyroid function was further confirmed in two in vitro experiments in which oCRF did not directly influence the thyroidal T4 secretion but, when applied to pituitaries, clearly increased the alpha-subunit release. In chickens CRF is concluded to not only control the adrenal axis, but also to participate in the coordination of avian TSH release.

A radioimmunoassay for African catfish growth hormone: validation and effects of substances modulating the release of growth hormone.

A highly sensitive radioimmunoassay has been developed for measuring plasma growth hormone (GH) concentrations in the African catfish (Clarias gariepinus). The lower detection limit of the assay was 0.1 ng/ml and the standard curve had an ED50 value of 0.5 ng/ml. The validity of the assay was established and the effects of several neurotransmitters on the release of GH were examined. In vitro experiments, using a static culture system for dispersed pituitary cells, demonstrated that the GH release in African catfish was affected by growth hormone-releasing hormone and somatostatin. Single intraperitoneal injections with a dopamine agonist, apomorphine, produced significant and dose-dependent increases in plasma GH levels. Unlike carp, goldfish, and tilapia, a super-active analogue of salmon gonadotrophin-releasing hormone did not alter plasma GH levels in African catfish.

Consequences of food restriction on short-term growth variation and on plasma circulating hormones in Oreochromis niloticus in relation to sex.

In tilapia, there is a sex-related growth difference between males and females. This study tried to detect any correlation between the somatic growth and the plasma endocrine status. For this, individually marked (Floytags) male and female tilapia (BW 82 +/- 10 g) were either starved or fed on different daily food rations (1, 2, or 3% of the biomass) during 15 days. We have found that specific growth rates (SGR) were positively and significantly related to feeding levels. Growth hormone (GH) plasma levels tended to increase with the decrease in food levels, and thus with the decrease in growth rate. No significant correlation was found between GH levels and SGR. Triiodothyronine (T3) levels in well-fed fish were higher than those in restricted fish (0 and 1%), but no differences in thyroxine (T4) levels were observed. No significant relationship was found between plasma levels of steroid hormones and feeding ration, even though 11-ketotestosterone (11-KT) levels tended to increase with the ration in fed males. SGR were not significantly different between males and females at the same feeding level, but taken as a whole, they were significantly different in favor of males (P < 0.05). There was no important difference in GH levels between the two sexes. Steroid hormones were, in general, higher in males for 11-KT and in females for 17 beta-estradiol (17 beta-E2). Males and females exhibited significant differences in T3 levels (respectively 4.25 +/- 0.18 and 2.71 +/- 0.09 pmol/ml), whatever the food ration, but no significant differences in T4 levels were observed except in the high-ration group. The correlation between T3 levels and SGR was low but stronger in males (r2 = 0.21; n = 90) than in females (r2 = 0.10; n = 105). The slope of the log-log regression of T3 levels with body weight was much lower in females (b = 0.87) than in males (b = 1.31). This relationship suggests the involvement of T3 in tilapia growth and probably in the differential growth between males and females. In both males and females, a significant but low correlation was observed between T3 and 11-KT levels (respectively r2 = 0.12; n = 82 and r2 = 0.08; n = 89), while no correlation was found between the levels of T3 and 17 beta-E2. T3 plasma levels were found to be the most different parameter between males and females. This hormone seemed to be involved in the control of somatic growth, and could explain the differential growth rate between males and females.

Seasonal changes in plasma levels of steroid hormones in an asynchronous fish the gudgeon Gobio gobio L. (Teleostei, Cyprinidae).

Annual changes in plasma of estradiol-17 beta, testosterone, and 17,20 beta-dihydroxy-4-pregnen-3-one concentrations were measured, by radioimmunoassay, in female gudgeon Gobio gobio a fish which has an asynchronous-type ovary containing oocytes at various stages of development and spawns several times during the reproductive period. The gonadosomatic index and the relation between stages of maturity and steroid concentrations were also followed during the reproductive cycle. Plasma levels of estradiol-17 beta, testosterone, and 17,20 beta-dihydroxy-4-pregnen-3-one were low from October to April and increased rapidly in May to reach 0.61 +/- 0.31; 2.3 +/- 0.42; and 3.17 +/- 0.68 ng/ml, respectively. Elevated levels were maintained during spawning when vitellogenic oocytes are present alongside oocytes in final maturation. Histological analysis of the ovary indicated that an important number of spawnings has occurred since the proportion of oocytes in final maturation stage was very low (less than 1%). Fish in the regressive phase also presented high steroid levels. The vitellogenic oocytes in preovulatory atresia and the postovulatory follicles may be responsible for these events.

Effect of hypophysectomy and acute administration of growth hormone (GH) on GH-receptor binding in chick liver membranes.

The effects of hypophysectomy on GH binding to liver membranes of young chicks were studied 3 days and 1 week after surgery. Specific binding of 125I-labelled chicken GH (cGH) to MgCl2-treated liver microsomal fractions of hypophysectomized animals was two- to fivefold greater than to those of sham-operated or control (non-operated) birds. This effect was due to a rise in binding capacity rather than a change in binding affinity of the GH receptor. Two daily injections of cGH (20 micrograms/animal) returned the number of hepatic GH receptors from hypophysectomized chicks to the level of the sham-operated ones. Administration of GH to the latter group did not cause a significant lowering of specific binding or number of receptors. No positive correlation between GH binding and plasma concentrations of insulin-like growth factor-I (IGF-I) was observed; although GH binding increased, IGF-I levels were lower for the hypophysectomized group. Since the number of hepatic GH receptors and the plasma GH levels were inversely correlated, it was concluded that the GH receptors in the liver of the chicken can be down-regulated by GH. This possibly explains why GH binding is low in posthatch and young chicks, because circulating GH concentrations are high during this period.

Seasonal variations in prolactin, growth hormone and thyroid hormones and the prolactin surge at ovulation do not affect litter size of ewes during pregnancy in the oestrous or the anoestrous season.

Injection of bromocriptine from 5 days before until 5 days after mating clearly suppressed the periovulatory prolactin surge in ewes in the anoestrous and oestrous season but did not change the litter size significantly. Progesterone, GH, TSH or thyroid hormone concentrations were not influenced by the bromocriptine treatment. The progesterone concentrations were lower during the first weeks after mating in the anoestrous season compared to the oestrous season, while there was no difference between pregnant and non-pregnant ewes. During later gestation this seasonal difference was only observed in the non-pregnant ewes. At the same time there was a clear difference between pregnancy and non-pregnancy in both seasons. The prolactin, GH and thyroid hormone values also varied significantly during gestation. Since these patterns are identical in pregnant and non-pregnant ewes, the fluctuations are due to environmental factors and not to pregnancy or altered progesterone concentrations. In the anoestrous season prolactin, GH, T4 and T3 levels were higher than in the breeding season, while rT3 showed the opposite pattern. The TSH concentration did not differ between the two seasons. These results suggest that seasonal variations in prolactin, GH and thyroid hormones or the periovulatory prolactin surge do not affect litter size of ewes during pregnancy in the oestrous or the anoestrous season.

Failure to relate thyroid hormones and in vitro 5'-monodeiodination activity to oocyte development and sex steroids in the giant swamp frog Dicroglossus occipitalis at the equator.

Females of the giant swamp frog Dicroglossus occipitalis were captured in Zaïre close to the equator in the course of 1 month. During this period, females with fully developed eggs were found, together with females of which the eggs were still in the first developmental stages. A close relationship was established between the maturation of the eggs and the studied gonadal factors: the gonadosomatic index, the oviduct weight, plasma estradiol-17 beta (E2) concentrations, plasma testosterone concentrations, and the total ovarian E2 concentrations. At the level of the thyroidal axis, the studied factors (plasma thyroxine (T4), plasma triiodothyronine (T3), plasma T3/T4 ratio, T4 and T3 concentrations, and the T3/T4 ratio in the thyroids and the 5'-monodeiodination activity (5'-D-activity) in the skin and kidney homogenates) did not show parallel changes with the maturation process of the eggs. These results indicate that no causal relation has to exist between the annual variation in thyroid hormones and the annual reproductive patterns as found in frogs from the tropical or temperate climatic region.

Estradiol-17 beta silastic implants in female Rana ridibunda depress thyroid hormone concentrations in plasma and the in vitro 5'-monodeiodination activity of kidney homogenates.

Estradiol-17 beta containing silastic tubings were implanted in female Rana ridibunda. Preliminary data, concerning in vitro incubations of such tubings in saline media, revealed that high concentrations of estradiol were released out of the tubings in the incubation medium. Compared to control-implanted frogs, the frogs that had the estradiol tubings implanted for 30 days showed a significant increase of the plasma estradiol concentration, the ovarian estradiol concentration, and the weight of the oviduct. Plasma triiodothyronine (T3) levels, plasma thyroxine levels, and the in vitro T3 production in the kidney homogenates were significantly decreased. These results indicate that high estradiol levels not only influence the gonadal axis, but also cause important effects on the thyroidal axis.

Relationship between the thyroidal and gonadal axes during the estrous cycle of ewes of different breeds and ages.

In order to define the patterns of TSH, T4, T3, rT3, GH and cortisol during the estrous cycle of sheep, pluriparous and primiparous ewes were synchronized with progestagen-impregnated pessaries (Veramix) at the start of the normal breeding season. After the pessaries were removed (day 0), daily blood sampling was carried out in cannulated ewes during the ovulatory cycle. Hormonal analyses of TSH, T4, T3, rT3, GH, cortisol, LH and progesterone (P) were performed by RIA. P and LH levels during the cycle were conform to the literature and were not different between the primiparous and pluriparous ewes of different breeds used in this study. Neither age nor breed influenced the hormone patterns. A significant negative correlation was found between TSH and P during the cycle, although the correlation between P and T4 was not significant; during the estrous period, low P levels were paralleled by high T4 levels, whereas the reverse was observed during the luteal phase. Higher T3 levels and T3/T4 ratios were observed during the luteal phase. No obvious pattern of rT3 and cortisol during the cycle was found. The GH concentration increased during the 17 days of the cycle. A positive correlation with P was calculated. During the estrous cycle obvious changes in thyroid hormones, GH and TSH occurred. However, this study shows no causal relationship between the thyroid and the gonadal axes.

Luteinizing hormone-releasing hormone induces thyroxine release together with testosterone in the neotenic axolotl Ambystoma mexicanum.

In male neotenic axolotls Ambystoma mexicanum plasma concentrations of thyroxine (T4) and testosterone were increased following intravenous injection of 10 micrograms luteinizing hormone-releasing hormone. A dose of 50 micrograms influenced only plasma T4 levels. This observation suggests for the first time that a hypothalamic hormone is capable of stimulating the thyroidal axis in the neotenic axolotl.

Luteinizing hormone-releasing hormone as a potent stimulator of the thyroidal axis in ranid frogs.

Plasma concentrations of T4, measured by radioimmunoassay, were raised significantly 2 and 4 hr after intravenous injection of synthetic luteinizing hormone-releasing hormone (LHRH) in Rana ridibunda (1 and 10 micrograms on 2 consecutive days) and in Rana esculenta (10 micrograms). A dose of 1 microgram LHRH was not so effective as 50 micrograms synthetic thyrotropin-releasing hormone (TRH) when injected in Rana ridibunda in November. However 10 micrograms LHRH was equipotent to 50 micrograms TRH. In February somewhat less than half of the Rana temporaria group was responsive to LHRH. There is no clear indication that fluctuating plasma T3 concentrations were caused by LHRH or TRH. Preinjection levels of T3 and T4 were higher during the breeding season (April) in R. esculenta (resp. 35.4 +/- 1.4 pg/ml; 744 +/- 134 pg/ml; n = 22) compared to the basal concentrations in the very closely related Rana ridibunda (November) (resp. 15.2 +/- 1.1; 162 +/- 24 pg/ml; n = 28). Four days after removal of the pars distalis plasma T4 concentrations were significantly decreased in Rana esculenta, whereas T3 could stay longer in circulation. T3 and T4 content of the thyroids was not altered by the short-term hypophysectomy. Injection of 10 micrograms LHRH had no influence on plasma T4 nor testosterone concentrations in these frogs, contrary to the sham-ectomized animals in which plasma testosterone remained elevated longer than T4. The results suggest that the stimulatory effect of intravenous injected LHRH on thyroid (and gonadal) activity in the frog is primarily mediated through the hypophysis. They also point to a possible correlation between the gonadal and thyroidal axis.

Thyroid hormones, testosterone, and estradiol-17 beta in plasma of Epomops franqueti (Tomes, 1860) (Chiroptera) in the rain forest of the Equator.

Using Japanese catching nets a total of 77 males and 37 females of the fruit eating bat Epomops franqueti (Megachyroptera) were captured during a period of 1 year around Kisangani (Zaïre). Adult males with white epaulets weighing 143 +/- 3.6 g (18) had their testes present in the scrotum and higher plasma concentrations of thyroxine (T4) and testosterone than adult males without epaulets and with abdominal testes (weight 107.5 +/- 2.0 g (39)) or juveniles (72.7 +/- 2.8 g (20)). Mean embryo weight of pregnant bats was 4.26 +/- 1.09 g (12) ranging from 1.8 to 14.8 g whereas body weight of mothers (+ embryos) was 117.0 +/- 3.6 g (12). Body weight of nonpregnant bats was 107.3 +/- 2.1 (10) and of female juveniles 74.6 +/- 4.3 (15). Plasma concentrations of triiodothyronine (T3) were lowest in nonpregnant adults and comparable in juveniles and pregnant bats. No difference in plasma T4 concentrations was found between all groups. Estradiol-17 beta concentrations were three times higher in pregnant bats compared to nonpregnant ones or juveniles. A possible relationship between these high figures and elevated T3 values has been discussed.

Glycosylated chicken growth hormone.

In an attempt to raise monoclonal antibodies to chicken pituitary glycoprotein hormones, mice were immunized with the concanavalin A-adsorbed components of a hypophyseal extract. Fusions of these spleen cells with myeloma cells repeatedly yielded hybridoma lines secreting antibodies that recognized specifically the pituitary caudal acidophils, known as the somatotropes. This paper reveals the existence of a glycosylated counterpart of the well-established holoprotein form of chicken growth hormone, similar to what has been established for human growth hormone and prolactin.

Effect of exogenous LH on plasma concentrations of progesterone and oestradiol in relation to the cessation of egg laying induced by different moulting methods.

Artificially induced cessation of egg laying caused regression of the reproductive tract in hens, as well as changes in circulating concentrations of sex steroids. Hens were bled at several stages during and after artificial moult induced by fasting or overfeeding a diet low in calcium or high in zinc. Hens received a single injection of 200 i.u. of horse LH at Day 0, 7, 21, 35 and 77 (Exp. 1) or Day 0, 8, 23, 35 and 71 (Exp. 2) after start of the treatment to induce moult. Blood samples were taken before and 20, 40 and 60 min (Exp. 1) or 15, 30 and 45 min (Exp. 2) after LH injection. Hens which were fasted or given the diet high in zinc had low plasma progesterone concentrations and the response to LH was reduced or delayed. In hens fed low calcium the reduction in plasma progesterone was less pronounced and the responsiveness to LH was more or less maintained. Conversely, there was no response of oestradiol to LH in laying hens. However, oestradiol concentrations increased in moulting hens after LH injection, due to the high oestradiol secretion from the small white follicles, since all yolky follicles were atretic.