Michiharu Kamiyoshi

Nuclear estrogen receptor bindings in granulosa cells and estradiol-17β contents in follicular membranes of the ovary of the hen during the ovulatory cycle

Estrogen receptors were found in the nuclear fractions of granulosa cells of the largest (F1) and the second largest (F2) preovulatory follicle in the ovary of the hen. The maximum number of binding sites (NBSmax) of the nuclear estrogen receptors was greater in F2 than in F1, while the equilibrium dissociation constant (Kd) was not different. During the ovulatory cycle, the NBSmax of the estrogen receptors showed a change parallel to the change in the estradiol-17 beta content in the follicular membranes. The results suggest that estradiol-17 beta produced in the follicular membrane may exert a direct action on the granulosa cells in the hen ovary.

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.

Effects of progesterone on pituitary cells of the hen (Gallus domesticus) during the ovulatory cycle for production and release of LH and FSH

Abstract Dispersed pituitary cells of laying White Leghorn hens 8, 14, and 18 hr before ovulation were incubated in media containing progesterone for 2, 4, and 8 hr at 41°, and the amount of LH and FSH in the cells and media was measured by homologous RIA. When the pituitary cells of 8 hr before ovulation were incubated, an increase in the cellular LH and total LH (sum of cellular and medium LH) and in the medium FSH and total FSH was observed after 4-hr incubation. When the pituitary cells of 14 hr before ovulation were incubated, no change in the amount of LH and FSH in the cells and media was observed in any of the incubations. When the pituitary cells of 18 hr before ovulation were incubated, the cellular LH and the total LH were increased following the 4-hr incubation, and the medium FSH and the total FSH were increased following the 2-hr incubation. When LH-RH was added at 4 hr of the incubation of the pituitary cells of 18 hr before ovulation, the medium LH was increased following an additional 4-hr incubation. These results suggest that one of the actions of progesterone at the pituitary level is to stimulate the production of LH (which may not always result in the release) and FSH (which may result in the release), and that the effect differs at different times during the ovulatory cycle in the hen.

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.

A Vasoactive Intestinal Peptide Binding Component in Hen Granulosa Cells

Abstract 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.

Effect of serotonin and β-endorphin on the release of luteinizing hormone in the hen (Gallus domesticus)

Serotonin (5-hydroxytryptamine) and beta-endorphin administered into the third ventricle of the hen blocked normal and progesterone-induced ovulation, and suppressed the release of LH in normal and progesterone-injected hens. p-Chlorophenylalanine, an inhibitor of serotonin synthesis, caused the release of LH and diminished the effect of beta-endorphin. Naloxone, an antagonist of opiate peptides, diminished the effect of beta-endorphin but not the effect of serotonin. The results suggest that both serotonin and beta-endorphin are involved in the control of LH release in the hen as an inhibitory agent, and serotonin is predominant while beta-endorphin is subsidiary to the inhibition of the LH release.

Properties of Cytoplasmic and Nuclear Estrogen Receptors in the Hen Oviduct Uterus (Shell gland)

白色レグホーン種産卵鶏の卵管子宮部のCytosolおよび細胞核画分のいずれにおいてもエストロジェンに対する選択的結合性(結合特異性)と結合飽和性とが認められた.結合物質の解離定数(Kd)は Cytosolおよび細胞核画分のいずれにおいても10-10Mのオーダーの値であり,NBS max(最大結合部位数)は Cytosolにおいては蛋白質1mg当り10-14～10-15molesであり,細胞核画分においてはDNA 1μg当り10-16～10-15molesであった.この結合物質のKd値は産卵鶏と休産鶏とで差がなく,NBS maxは産卵鶏の方が大であった.休産鶏にエストラジオール•17βを投与すると Cytosolおよび細胞核画分のKd値には顕著な変化は認められないが,NBS max値はCytosolにおいては減少し,細胞核画分においては増加した.上記の結果より,鶏の卵管子宮部の Cytosolにはエストロジェン•レセプターが存在し,細胞核にはレセプター•エストロジエン複合体が存在にるものとみなされ,この組織に対にるエストロジェンの作用は血液中のエストロジェンが細胞質中のレセプターと結合し,この複合体が細胞核へ移行するという機序によるものと思われる.

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.

Enhancement of the response of hen granulosa cells to LH with norepinephrine in vitro.

1. The effect of norepinephrine (NE) on progesterone production in hen granulosa cells was examined. After 4 days of the culture, the progesterone production was stimulated by chicken LH but not by NE during a 4 hr incubation. 2. The LH-stimulation progesterone production was not affected by the presence of NE when given together with LH, but was significantly increased when the cells were precultured with NE for 2 days. 3. The effect of NE was inhibited by phentolamine, but not by propranolol. 4. The results indicate that NE may enhance the responsiveness of the hen granulosa cell to LH through alpha-adrenergic receptors.

Expression of messenger RNAs of luteinizing hormone and follicle-stimulating hormone receptors in the granulosa layer during the ovulatory cycle of the hen

inhibin or activin in the presence of 5 ng/ml IGF-I (maximally effective dose being 10 ng/ml IGF-I). At the end of the culture period, the medium was collected. Monolayer cells were thereafter incubated with luteinizing hormone (10 ng/ml) for 4 h and medium was again collected. All media were stored at –20°C until assayed for progesterone by specific RIA. In a separate experiment, the effects of inhibin or activin on granulosa cell proliferation were determined over a 48 h culture period. Cells were lysed in DNA assay buffer (10 mM phosphate buffer, 2 M NaCl) containing trypsin/EDTA and total DNA was determined by the method of Labarca and Paigen (1980). The results showed that inhibin had no effect on either progesterone production or cell proliferation. However, activin at low doses up to 1 ng/ml tended to increase basal, LH and IGF-I-stimulated progesterone production but higher doses significantly inhibited progesterone production. Basal and IGF-I stimulated DNA synthesis was inhibited by activin in a dose-dependent manner. The results suggest that activin but not inhibin may be involved in the regulation of steroid production and follicular growth in the adult chicken ovary.