Effects of Steroid Hormone in Avian Follicles.

The aim of the present study was to examine the effects of testosterone (T) and estradiol-17ß (E2) on the production of progesterone (P4) by granulosa cells, and of the E2 on the production of P4 and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest (F1) and third largest (F3) preovulatory follicle were incubated for 4 h in short-term culture system, P4 production by granulosa cells of both F1 and F3 was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or E2. In the second experiment, F1 and F3 granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and E2. Basal P4 production for 48 h during 48 to 96 h of the cultured was about nine fold greater by F1 granulosa cells than by F3 granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not E2, stimulated in a dose-dependent manner P4 production in both F1 and F3 granulosa cells. In addition, when the time course of P4 production by F1 granulosa cells in response to oLH, cVIP, T and E2 was examined for 48 h during 48 to 96 h of culture, although E2 had no effect on P4 production by granulosa cells of F1 during the period from 48 to 96 h of culture, P4 production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, P4 production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when F1 granulosa cells were precultured with E2 for various times before 4 h culture with oLH at 96 h of culture, the increase in P4 production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of F1, F2 and the largest third to fifth preovulatory follicles (F3-5) were incubated for 4 h in short-term culture system with increasing doses of E2. The production of P4 and T by theca internal cells were increased with the addition of E2 of 10(-6) M. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on P4 production, and on E2 in long-term action which may enhance the sensitivity to LH for P4 production, and thus, in theca internal cells, E2 in short term action may stimulate the production of P4 and T.

Expression of messenger RNA for gonadotropin receptor in the granulosa layer during the ovulatory cycle of hens.

The present experiments were conducted to evaluate the mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) in granulosa layers during the ovulatory cycle of hens, in relation to the release of LH and steroid hormones. After the release of LH, progesterone (P4) and estradiol-17beta (E2), found 4-5 h before ovulation, LHR and FSHR mRNA levels were observed to decrease in the granulosa layers of the largest (F1) and second largest (F2) preovulatory follicles, with the greatest in the LHR mRNA level of F1. P4 concentrations in the granulosa layers of F1 and F2 increased 4-5 h before ovulation, with greater in F1 than in F2. F2 concentrations in the theca layers were greater in F2 than in F1 throughout the ovulatory cycle. Also, the injection of ovine LH caused decreases in the mRNA levels of LHR and FSHR in the granulosa layers. However, these decreases were abolished by the injection of aminoglutethimide, an inhibitor of steroid synthesis. These results suggest that in hen granulosa cells, the mRNA levels of not only LHR but also FSHR are down-regulated by LH and the down-regulation may be mediated steroid hormones.

Chicken lutropin acts like follitropin in rat ovarian follitropin receptor: an isoelectric focusing study.

This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity.

Molecular cloning and functional expression of chicken luteinizing hormone receptor.

A complementary DNA for chicken luteinizing hormone (LH) receptor containing the entire coding region was isolated from chicken F1 granulosa cell cDNA library. Nucleotide sequence analysis revealed that there are characteristic GC-rich regions around the N-terminal part. Chicken LH receptor consists of a 19-residue signal peptide, a 366-residue extracellular domain, a 267-residue region containing seven transmembrane segments, and a 76-residue cytoplasmic C-terminal tail. The deduced amino acid sequence of the chicken LH receptor shares 67%, 69%, and 69% identity with the human, rat and porcine LH receptor sequences, respectively, and 51% with chicken FSH receptor. However, an insertion of about 30 amino acid residues is found in chicken LH receptor in the extracellular domain about 44 amino acid residues upstream of the first transmembrane segment. In addition, alternative splicing seems likely to occur at the point where the insertion starts (nucleotide position 933), resulting in the truncated forms of chicken LH receptor with only the extracellular domain. Northern blot analysis revealed the presence of multiple transcripts of LH receptor, a major 3.0-kb and minor 7-kb and 1.5-kb bands, in chicken F1 to F3 granulosa cells. The full length chicken LH receptor cDNA was transiently expressed in COS-7 cells and the transfected cells displayed a concentration-dependent increase in cAMP production when exposed to varying concentrations of chicken LH. This clearly indicates that the cloned cDNA encodes a functional chicken LH receptor protein.

Evaluation of a sensitive, in vitro bioassay for chicken thyroid stimulating hormone using FRTL-5 cells, a rat thyroid cell line.

An in vitro bioassay for mammalian thyroid stimulating hormone (TSH) based on TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production in FRTL-5 cells, a rat thyroid cell line, was used to measure chicken TSH. The addition of chicken pituitary homogenate equivalent to > or = 25% of a chicken pituitary gland to cultured FRTL-5 cells increased cAMP within these cells in a dose-dependent manner. The glycoprotein fraction derived from the pituitary homogenate was further fractionated by isoelectric focusing within a pH range of 5 to 11. Analysis of the focused fractions by the bioassay detected three major components with isoelectric points of 9.30, 7.12, and 3.82, in addition to several minor ones distributed over a wide range of pH, from alkaline to acidic. The isoelectric focusing profile obtained by the bioassay was clearly different from those obtained by radioimmunoassay for chicken LH and radioreceptor assay for chicken FSH, indicating that fractions contained chicken TSH. The homogenate of the cephalic portion of the chicken anterior pituitary gland was 4.46 times more active than that of the caudal portion in the bioassay, which is consistent with previous findings on localization of TSH in the chicken pituitary. We conclude that the bioassay using FRTL-5 rat thyroid cells is a sensitive, specific, and time-saving method of measuring chicken TSH.

Complementary DNA sequence of chicken thyroid-stimulating hormone (TSH) beta subunit.

Chicken TSH beta subunit cDNA was cloned and sequenced. The cDNA encodes a putative signal peptide and a mature protein consisting of 20 and 114 amino acids, respectively. The amino acid sequence of chicken TSH beta subunit is highly conserved (98.5%) between chicken and Japanese quail, whereas it has a low homology between chicken and mammals (67-69%), an amphibian (58%) and fish (40-49%). Structural characteristics of TSH beta subunits of avian species are discussed in comparison with those of non-avian vertebrates.

Mesotocin receptor binding of cortical and medullary kidney tissues of the hen.

[125I]Mesotocin (MT) binding of membrane fractions of the cortical tissue and the medullary tissue (medullary cone) of the kidney of nonlaying hens was measured by the use of radioligand binding assays to determine the distribution of two distinct MT receptors within the hen kidney. The binding to [125I]MT in the medullary tissue was found to be highly competitive with unlabeled MT. In the cortical tissue, the binding was competitive with both unlabeled MT and arginine vasotocin. Kinetic and Scatchard analyses of specific binding revealed that the binding affinity was higher in the cortical tissue than in the medullary tissue, but the binding capacity was less in the cortical tissue. The localization of two distinct MT receptor having different binding properties may be related to the biphasic action of MT within the tissue of the kidney of the hen.

Mesotocin binding to receptors in hen kidney plasma membranes.

Radioligand assays were performed to demonstrate the presence of a receptor for mesotocin (MT) in the membrane fractions of the kidney of the hen. Specific [125I]MT bindings were decreased by the presence of Mg2+ and Ca2+, increased by the presence of EDTA, increased during the first 4 h of incubation and then reached a plateau, and increased with the increase in the protein concentration from 2.5 to 20 micrograms. The membrane fraction showed binding specificity to [125I]MT. The Scatchard plot revealed a curvilinear profile that indicated the presence of two classes of binding sites: a high affinity site and a low affinity site. The equilibrium dissociation constant was 0.08 +/- 0.01 nM (mean +/- SEM; n = 5) in the high affinity site and 0.87 +/- 0.08 nM (n = 5) in the low affinity site. The maximum binding capacity of the high and low affinity sites was 42 +/- 4 and 129 +/- 6 fmol/mg protein, respectively. The results suggest the presence of two distinct MT receptors in the kidney of the hen.

Effects of progesterone, estrogen, and androgen on progesterone receptor binding in hen oviduct uterus (shell gland).

The specific [3H] progesterone binding per milligram of protein of the cytosolic fraction of the hen uterus was found to have decreased at 1 and 4 h after the injection of progesterone (P4), but increased at 4 and 8 h after the injection of estradiol-17 beta (E2), and at 1 and 4 h after the injection of testosterone (T). The equilibrium dissociation constant obtained by Scatchard analysis was not significantly different between the hens receiving steroid injections [P4], E2, T, and dihydrotestosterone (DHT)] and controls. The maximum binding capacity was lower in hens injected with P4 and greater in hens injected with E2, T, or DHT than in uninjected hens. The results suggest that the cytosolic progesterone receptor binding in the hen uterus may be modulated by the sex steroid hormones.

A vasoactive intestinal peptide binding component in hen granulosa cells.

In radioligand assays, the vasoactive intestinal peptide (VIP) binding component in the membrane fraction of granulosa cells of the ovary of the hen was shown to possess characteristic properties of a receptor, such as reversible binding, binding specificity, high-affinity, and limited capacity. The binding site was of a single class. The binding affinity was higher in the largest (F1) and the second largest follicle (F2) than in the third largest follicle (F3), and the binding capacity was greater in F2 and F3 than in F1. During the ovulatory cycle, changes in affinity and capacity were observed only in F1 shortly before ovulation. The results suggest the presence of VIP receptor in the hen granulosa cells and its binding is assumed to be related to the follicular growth and ovulation.

Diuretic and antidiuretic effects of mesotocin as compared with the antidiuretic effect of arginine vasotocin in the hen.

Mesotocin and arginine vasotocin were injected intravenously once at various dosage levels (.0025 to 25 micrograms/kg body weight) immediately after an intravenous injection of 15 mL/kg body weight saline solution into hens in which an artificial anus had been surgically created, and the volume of urine excreted during a 5-h period, following the injection, was measured. Arginine vasotocin caused a monophasic, dose-dependent decrease in urine volume. By contrast, mesotocin was biphasic: at .025 and .25 micrograms/kg body weight it decreased urine volume with the same efficacy as arginine vasotocin, whereas it dramatically increased urine volume at the higher doses (2.5 and 25 micrograms/kg body weight).

An opiate receptor in the neurohypophysis of laying hens.

An opiate binding component in the membrane fraction of the neurohypophysis of the laying hen showed a high affinity, limited capacity, reversible binding, and binding specificity to [3H]diprenorphine. The binding sites were of a single class. The equilibrium dissociation constant was .30 +/- .03 nM (mean +/- SEM; n = 5) in Scatchard analysis and .31 +/- .02 nM (n = 5) in kinetic analysis. The maximum binding capacity determined by Scatchard analysis was 619 +/- 34 fmol/mg protein (n = 5). The association and dissociation rate constants determined by kinetic analysis were .088 +/- .002 nM-1 min-1 (n = 5) and .027 +/- .001 min-1 (n = 5), respectively. The results suggest that the binding component is regarded as being an opiate receptor.

Presence of vasoactive intestinal peptide receptor in the hen hypothalamus.

Radioligand assays of the membrane fraction of hen hypothalamic tissues involving the preoptic (HPOA) or median eminence (HMEA) areas revealed the presence of a specific binding component to chicken vasoactive intestinal peptide (cVIP) having properties of a receptor. The equilibrium dissociation constant (Kd) was 0.70 +/- 0.07 nM (Mean +/- SEM; N = 5) in HPOA and 1.02 +/- 0.15 nM (N = 5) in HMEA as estimated by Scatchard analysis of saturation studies, and was 0.91 +/- 0.11 nM (N = 3) (HPOA) and 1.25 +/- 0.09 nM (N = 3) (HMEA) as determined by a kinetic analysis. The maximum binding capacity (Bmax) obtained by Scatchard analysis was 167 +/- 19 fmol/mg protein (N = 5) (HPOA) and 133 +/- 17 fmol/mg protein (N = 5) (HMEA). The Kd and Bmax values obtained by Scatchard analysis were similar in the two areas of the hypothalamus and in both laying and nonlaying hens. Administration of cVIP in vivo caused a decrease in specific cVIP binding. These results suggest the presence of a VIP receptor in the hen hypothalamus.

Changes in progesterone receptor binding of preoptic hypothalamus during an ovulatory cycle of the hen.

By the use of radioligand assays, cytosolic and nuclear fractions of hypothalamic tissue involving preoptic area (HPOA) of the hen was found to contain a specific [17 alpha-methyl-3H]promegestone ([3H]-R5020; a synthetic progesterone) binding component having properties of receptor for progesterone. The equilibrium dissociation constant (Kd) was different neither between cytosolic and nuclear fractions nor between the fractions of laying and nonlaying hens. The maximum binding capacity (Bmax) per milligram of tissue of both fractions was greater in laying hens than in nonlaying hens. During the ovulatory cycle of laying hens, the specific [3H]-R5020 binding in cytosolic fraction showed a decrease, with an increase in the nuclear fraction from 21 to 18 h and from 6 to 3 h before ovulation. Such a change was not observed in nonlaying hens during a 24-h day. The results suggest that progesterone receptors are present in the preoptic hypothalamus and may be related to the incidence of ovulation in the hen.

Arginine vasotocin receptor binding in the hen uterus (shell gland) before and after oviposition.

The binding affinity and capacity of arginine vasotocin (AVT) receptor in the hen uterus changed during a period before and after oviposition. Three hours before oviposition, the binding capacity of the AVT receptor increased. An injection of prostaglandin (PG) F2 alpha caused an increase in the AVT receptor Bmax in the uterus, and indomethacin blocked the normal rise in the AVT receptor Bmax and PGF content prior to oviposition. However, just prior to oviposition, the binding affinity increased with a decrease in the binding capacity. A progesterone injection caused an increase in binding affinity and a decrease in binding capacity of the AVT receptor. The specific binding of the progesterone receptor in the uterus increased 2 h before oviposition and remained high until oviposition. Serum AVT levels increased at oviposition. An injection of AVT caused an increase in the affinity of the AVT receptor with a decrease in the capacity. The change in the affinity and capacity of AVT receptor at oviposition may result from the action of progesterone via increased progesterone receptor binding, and the action of AVT on its own receptors.

Effect of estradiol-17 beta and progesterone on arginine vasotocin in nonlaying hens.

Effect of estradiol-17 beta (E2) and progesterone (P4) on serum arginine vasotocin (AVT) concentration was examined. Serum AVT concentration was lower in nonlaying hens than in laying hens. A single intramuscular injection of E2 or P4 into nonlaying hens caused an increase in serum AVT concentration, suggesting that estrogen and P4 may stimulate the release of AVT from the neurohypophysis.

Estrogen receptor binding in the hen hypothalamus and pituitary during the ovulatory cycle.

The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) of estrogen receptor in soluble (cytosolic) and insoluble (nuclear) fractions in a hypotonic buffer solution of hypothalamus containing preoptic (HPOA) and median eminence (HMEA) areas and of anterior pituitary (AP) of laying and nonlaying hens were examined by Scatchard analysis of specific [3H]estradiol-17 beta ([3H]-E2) binding. The Kd of receptor in all of the tissues was different neither between soluble and insoluble fractions, nor between laying and nonlaying hens. The Bmax in laying hens was greater in the insoluble fraction and lower in the soluble fraction than that in nonlaying hens, but the total binding capacity (sum of Bmax in the soluble and insoluble fractions) was not different between laying and nonlaying hens. In laying hens, the specific [3H]-E2 binding in the insoluble fraction of HPOA was found to increase at 21 h before ovulation and again at 8 to 6 h before ovulation, and of HMEA and AP at 18 to 11 h before ovulation. No change in the specific [3H]-E2 binding in the insoluble and soluble fractions was found in any of the tissues of nonlaying hens during a 24-h period. The results suggest that in laying hens, estrogen may act on the hypothalamus and pituitary at restricted hours during the ovulatory cycle.

Arginine vasotocin binding component in the uterus (shell gland) of the chicken.

The plasma membrane fraction of the uterus of the chicken was found to contain a component that shows specific binding to arginine vasotocin (AVT), arginine vasopressin (AVP) and oxytocin (OT). This binding component possesses a higher affinity to AVT than to AVP or OT, and the affinity to AVT was higher in laying hens than in non-laying hens and immature pullets, while the maximum number of binding sites per mg protein was less in the laying hens. Intramuscular injections of either estradiol-17 beta, progesterone or testosterone into the immature pullets for six consecutive days caused an increase in the affinity and number of binding sites. The results suggest that AVT receptors are present in the chicken uterus and that their binding properties are affected by ovarian steroid hormones.

Effect of progesterone in vitro on luteinizing hormone production in hen pituitary cells pretreated with estrogen.

Isolated cells from the pituitary gland of nonlaying hens were preincubated in vitro with or without estradiol-17 beta (E2), and then incubated with or without progesterone (P4), and luteinizing hormone (LH) in the cells and media was measured by a homologous radioimmunoassay. When the cells were preincubated with E2 for 2 h and then incubated with P4 for 4 h, cellular LH was increased, but LH in the medium remained unchanged. The increase in cellular LH in response to P4 was dose-dependent. Protein synthesis inhibitors (cycloheximide, actinomycin-D, and alpha-amanitin) blocked the response of the cells to P4. The results suggest that LH production in estrogen-primed pituitary cells of the hen was stimulated by P4 through a protein-synthesizing pathway.

Luteinizing hormone-releasing hormone receptor bindings of the hen pituitary: difference between laying and nonlaying hens, effects of ovarian steroid hormones in vivo, and changes during an ovulatory cycle.

The equilibrium dissociation constant (Kd) of the pituitary luteinizing hormone-releasing hormone (LHRH) receptors and the maximum binding capacity (Bmax) per milligram of membrane protein were greater in laying hens than in nonlaying hens. A single i.m. injection of progesterone, estradiol-17 beta, or 5 alpha-dihydrotestosterone into nonlaying hens caused an increase in Kd and Bmax values. During an ovulatory cycle of the laying hen, Kd and Bmax values decreased from 16 to 14 h and 8 to 6 h before ovulation. A decrease in the Bmax value was also found 24 to 21 h before ovulation. A single i.v. injection of chicken LHRH-I caused a decrease in the Kd and Bmax values within 2 min after the injection. The results suggest that the hen pituitary LHRH receptor bindings are affected by ovarian steroid hormones and that their changes during the ovulatory cycle may relate to the release of a gonadotropin from the pituitary.