Distribution of binding sites for human chorionic gonadotropin in the preovulatory follicle of the rat.

The distribution of binding sites for human chorionic gonadotropin (hCG) in the preovulatory follicle was studied by autoradiography. An ovulatory dose (10 IU/rat) of [125I]hCG (1.4 muCi/IU) was administered intravenously, and large Graafian follicles were isolated 3 h later by microdissection. Injection of excess unlabeled hCG (500 IU/rat) prevented uptake of radioactivity by the follicle, indicating that binding of iodinated hormone was confined to specific and saturable receptor sites. The density of bound hormone molecules was highest in the theca interna and in three to four layers of mural granulosa cells adjacent to the basement membrane; labeling was chiefly associated with the cell borders. No significant binding could be detected either on the oocyte or on the cumulus cells surrounding the oocyte. We therefore suggest that the induction of ovum maturation does not require attachment of the hormone to the oocyte itself or to follicle cells in its immediate vicinity.

Immunocytochemical localization of progesterone receptor in the chick ovary.

The distribution of progesterone receptor (PR) in the chick ovary was studied using light- and electron-microscopic immunocytochemical methods. With the light microscopic technique, PR was observed in the germinal epithelium cells of estrogen-treated and estrogen-untreated immature chicks. With the preembedding immunocytochemical technique, which proved to be more sensitive than immunohistochemistry using paraffin sections, the germinal epithelium and also part of the thecal and stromal cells were stained when immature chicks were not treated with estrogen. After estrogen treatment, the number of PR-positive stromal and thecal cells increased, as did the immunostaining intensity in their nuclei. The granulosa cells were PR-positive only after estrogen treatment. In the ovary of laying hens, the most intense staining for PR was localized in the germinal epithelium. PR was also present in the thecal, stromal, and granulosa cells. At the subcellular level, PR was detected only in the cell nuclei, indicating that ovarian PR is intranuclear independent of the presence of progesterone. In conclusion, immunocytochemical methods proved to be suitable for studying steroid hormone receptors in steroid-producing tissues (e.g. the ovary), because excess endogenous hormones do not affect detection of the receptor with the antibody as they do detection with labeled ligands. Immunocytochemically, the germinal epithelium, stromal, thecal, and granulosa cells of the laying hen ovary were shown to be target cells for progesterone. The inducibility of PR by estrogen in the thecal, stromal, and granulosa cells suggests that these cell types are also sensitive to estrogen.

Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period.

Plasminogen activators (PAs) are believed to be involved in ovulation. Because both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are secreted by cultured rat granulosa cells, we have examined the activities of these proteins in ovarian homogenates as well as granulosa and theca-interstitial (TI) cells during gonadotropin-induced ovulation. Immature rats were injected with 20 IU pregnant mare serum gonadotropin (PMSG) to initiate follicle development, followed by treatment with 10 IU hCG 48 h later to induce ovulation. Ovarian proteins were separated by SDS-PAGE and PA activity determined by fibrin overlay. The activity of tPA, but not uPA, was stimulated following PMSG treatment in ovarian homogenates. Subsequent hCG injection further increased the tPA activity in a time-dependent manner, reaching a maximum (12 h after hCG treatment) immediately prior to ovulation and declined thereafter. Similar preovulatory increases in tPA activity were detected in isolated granulosa cells. Although both tPA and uPA activities were increased in TI cells after PMSG administration, no further increases were detected after hCG treatment. To estimate enzyme secretion, ovarian cells obtained at various preovulatory periods were incubated for 24 h in vitro. The ability of granulosa cells to secrete tPA, but not uPA, increased following in vivo PMSG and hCG treatment in a time-dependent manner, reaching a maximum immediately prior to ovulation. During the preovulatory period, an abrupt increase in tPA secretion by TI cells was also detected. Using immunohistochemical staining for tPA, it was found that ovarian sections from preovulatory rats at 12 h after hCG injection stained positively in granulosa, theca interna, and interstitial gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive beta-endorphin in human ovaries.

Beta-endorphin (beta-EP) immunostainable cells were demonstrated in human ovarian tissue using a non-cross-reacting anti-beta-EP serum and the avidin-biotin-peroxidase detection technique. In ovaries from ovulating and premenopausal women, beta-EP immunoreactivity was localized in the luteinized cells of theca interna of maturing follicles with almost negligible staining in granulosa cells; cells of primary follicles did not stain. In corpora lutea, luteinized cells in both theca interna and granulosa, layers were equally positive. In postmenopausal ovaries, staining was detectable only in scattered luteinized stromal cells. This is the first report on the presence of immunoreactive beta-EP in human ovaries, in which beta-EP seems to be produced by the same sex cord cells engaged in active steroidogenesis and may be under gonadotropin central regulation. The significance of this finding is discussed.

Transferrin and somatomedin C receptors in the human ovarian follicles.

Transferrin and somatomedin C receptors (TFr and SMCr) were studied in human ovaries by immunostaining using monoclonal antisera. The oocyte of evoluting follicles was intensely positive for both types of receptors, but this positivity decreases in large follicles. An evident positivity for both TFr and SMCr was shown in granulosa cells of evoluting follicles. However, not all of these cells were equally immunoreactive. An intense positivity was present in the developing thecal cells of early cavitary follicles as well as in thecal cells of follicles of medium and large size (greater than 6 mm) and in some cells of involuting follicles in initial atresia. These results seem to demonstrate that TFr and SMCr are present in different cellular components of the human developing and early involuting follicles.

Ovarian delta-5-3-beta-hydroxysteroid dehydrogenase in aging rats.

Ovaries of rats 1, 2, 4, and 18 months old revealed significant delta-5-3-beta-hydroxysteroid dehydrogenase (3beta-OHSD) activity when dehydroepiandrosterone (DHA), pregnenolone, and 16-dehydropregnenolone served as substrates. No enzyme activity was evident in ovaries 4 and 18 months of age when the substrates 17alpha-hydroxypregnenolone, pregnenolone sulfate, and dehydroepiandrosterone sulfate were used. A marked increase in enzyme activity occurred in the granulosae of mature vesicular follicles at 18 months of age. DHA, pregnenolone, and 16-dehydropregnenolone provided similar activities. On the other hand, interstitial enzyme activity exhibited an aging decline with DHA and 16-dehydropregnenolone but increased with pregnenolone. Corpora lutea were less evident in aging ovaries but did exhibit strong 3beta-OHSD activity when DHA was used. Pregnenolone use by corpora lutea was sharply increased in aging rats but no change occurred with 16-dehydropregnenolone. Thus aging was associated with changes in 3beta-OHSD distribution and use of steroid substrate; a change in function is suggested.

Histomorphological and histochemical studies on the female genitalia of aging goat. I. Lipid histochemistry of ovary.

Paraffin sections of ovaries from 24 goats stained by Acetone Sudan Black B method for bound lipids and Copper-Phthalocyanin method for phospholipids revealed at least two categories of interstitial cells containing sudanophilic lipids with phospholipids. First category were those which were assumed to be formed from theca interna cells of atretic follicles. Second category included those originating probably from the granulosa cells of primordial follicles and form a continuous zone under the tunica albuginea. Another zone of interstitial gland cells presumed to be originated from the germinal epithelium has also been recognized under the latter. Appearance of sudanophilic material in relation to atresia of follicles in goat ovary has been reported.

Developmental changes in the properties of gonadotropin receptors in the ovarian follicles of amago salmon (Oncorhynchus rhodurus) during oogenesis.

The presence of specific, saturable, high-affinity gonadotropin receptors was demonstrated in membrane preparations from preovulatory ovarian follicles of amago salmon (Oncorhynchus rhodurus), including intact follicles, isolated thecal layers, and isolated granulosa cells. Optimum conditions for the binding study using the amago salmon receptor system were similar to those previously reported for postovulatory ovaries of the same species (A. Kanamori, H. Kagawa, and Y. Nagahama, 1987, Gen. Comp. Endocrinol. 66, 210-217). Scatchard analysis of chum salmon gonadotropin (CSG-SII) binding to the membrane fraction suggested the presence of high-affinity binding sites in the intact follicles, isolated thecal layers, and isolated granulosa cells at all stages of development. The dissociation constant is consistent with those reported for gonadotropin receptors in several teleost gonads and in other vertebrate classes (about 0.1-1 nM). During oogenesis, the number of binding sites per follicle increased from about 20 to about 60 pg. Similarly an increase in binding sites was observed with granulosa cells and thecal layers during oogenesis. These findings show an increase in the number of gonadotropin receptors in the follicles and are in good temporal agreement with the developmental changes in follicular steroidogenesis in response to gonadotropin. The increase in gonadotropin receptors in the thecal layer was associated with the increased capacity for production of testosterone, whereas the increase in gonadotropin receptors in the granulosa cells was associated with an increase in gonadotropin sensitivity in terms of 20 beta-hydroxysteroid dehydrogenase activation.

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization.

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.

Expression of the human inhibin alpha-subunit gene in preovulatory granulosa-theca cells.

Inhibin, a gonadal peptide that suppresses pituitary follicle-stimulating hormone, with lesser or no effect on luteinizing hormone, has recently been purified and the complementary deoxyribonucleic acid sequences cloned. Inhibin contains two subunits, labeled alpha-subunit and beta-subunit. Here we report for the first time the detection of human inhibin alpha-subunit gene expression in preovulatory granulosa-theca cells by Northern analysis. The transcript is the same size as previously reported for human placenta and corpus luteum, suggesting that the same gene is being expressed in all three tissues. These findings are consistent with previously reported Southern analysis of deoxyribonucleic acid, which showed only one copy of the alpha-inhibin gene in the human genome. Thus current data strongly suggest that there is only one copy of the inhibin alpha-subunit gene in the human genome, and this same gene is expressed in granulosa-theca cells, corpus luteum, and placenta.

[Description, cytochemistry, and electron microscopic autoradiography of nucleolar granular spheres and micronucleoli of lizard (Lacerta vivipara Jacquin) pyriform cells]. / Etude descriptive, cytochimique et autoradiographique en microscopie électronique sur les sphères granulaires nucléolaires et les micronucléoles des cellules piriformes chez le Lézard: Lacerta vivipara Jacquin

The granular spheres are constituted of granules of 300-350 A and show a higher contrast than the nucleolar part where they are localised. The micronucleoli consist of fine fibrils. These two structures contain RNP. A relation between the granular spheres and the degeneration of the pyriform cells is suggested. The RNP of the micronucleoli, probably synthesized on perinucleolar DNA, migrate into cytoplasm.

Histochemical observations on the ovaries of some Indian bats.

Ovarian follicles of the three species of bats, Pteropus giganteus giganteus, Taphozous longimanus and Megaderma lyra lyra were studied with regard to the distribution and nature of mucopolysaccharides. Oocyte contained only glycogen. Zona pellucida showed the presence of glycogen, neutral mucosubstances, sialo- and sulfomucins. Follicle cells showed the presence of glycogen and sialic acid except in megaderma lyra lyra where the follicle cells of the Graafian follicle showed the presence of sulfomucins in addition to these mucosubstances Cumulus cells contained glycogen., sulfomucin, sialomucins and hyaluronic acid. Liquor follicular showed the presence of sulfomucins, sialomucins and hyaluronic acid. Theca cells contained glycogen and sialic acid. The luteal cells contained glycogen only.

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles.

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.

Changes in catecholamine contents in ovarian follicular theca, progesterone contents in granulosa, and LH concentrations in peripheral plasma during the ovulatory cycle in the hen.

By the use of high-performance liquid chromatography, the content of norepinephrine (NE) and epinephrine (E) in the theca of the largest and the second largest preovulatory follicle of the ovary of the hen was found to increase from 15 h (NE) or 18 h (E) to reach a peak 9 h (NE) or 9-6 h (E) before ovulation of the largest follicle. The content of progesterone in the granulosa of these follicles and the concentration of LH in the peripheral plasma of the same hen, both of which were measured by radioimmunoassays, showed a peak 6 h before ovulation. The results suggest that the catecholamines are involved in some events occurring in both follicles some several hours before ovulation of the largest follicle during the ovulatory cycle.

Autoradiographic study of the distribution of LH(HCG) receptors in the ovary of untreated and gonadotrophin-primed immature rats.

The LH(HCG) receptors in the ovaries of immature rats which were either untreated, or primed with PMSG and HCG, have been studied with a histochemical method which has proved to be as effective as when earlier used in the rat testis. This method, which consists of the topical application of 125I-HCG to picric acid-formaldehyde (PAF) fixed frozen sections followed by autoradiography, is also suitable for quantitative studies on the distribution of receptors. In the ovary of the immature 26 days old rat, the LH(HCG) receptors are localized exclusively in the interstitial and thecal tissues. After PMSG treatment many receptors appear in the granulosa of the large antral follicles. These receptors are most numerous in the outer layers of cells and least numerous in the inner. At the same time there are fewer receptors in the thecal and interstitial cells which have undergone the process of luteinization. After PMSG and HCG treatment the newly formed corpora lutea have few receptors, but these become progressively more numerous on subsequent days. It is suggested that, in the rat, the luteinization of the ovarian LH-target cells is associated with an initial decrease in the number of their LH(HCG) receptors.

The autonomic innervation of the interstitial gland of the rat ovary during pregnancy.

The autonomic innervation of the interstitial gland of the rat ovary was studied on days 4, 6, 10, 14 and 18 of pregnancy with the acetylcholinesterase procedure, the Falck-Hillarp technique and electron microscopy after 5-hydroxydopamine treatment. Acetylcholinesterase-positive nerves were present as perivascular plexuses at all stages studied. Adrenergic nerves were present in the interstitial gland in all stages studied. The number and intensity of interstitial fluorescent adrenergic nerves increased as pregnancy progressed. Measurement of norepinephrine with the fluorometric procedure showed a highly significant (p less than 0.05) increase in the neurotransmitter in the ovary on days 14 and 18 as compared to day 4. Fine-structural studies after administration of the false transmitter, 5-hydroxydopamine, showed that the innervation of the steroidogenic cells of the interstitial gland is adrenergic.

Fluorescence histochemical demonstration of a relationship between adrenergic nerves and cells containing actin and myosin in the rat ovary, with special reference to the follicle wall.

Contractile proteins (actin and myosin detected by immunohistochemistry) were present in elongated cells forming concentric layers in the theca externa of Graafian follicles and around corpora lutea. Immunofluorescent cells were also found in the ovarian stroma. Study of adrenergic nerve fibres by the glyoxylic acid technique showed numerous branches in between and in close association with the contractile cells.

Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen.

The purpose of the present investigation was to measure the concentrations of progesterone (P4), testosterone (T) and estradiol-17 beta (E2) in isolated theca and granulosa layers of the five preovulatory follicles of the domestic hen. The largest follicle (F1), the second largest (F2), third largest (F3), fourth largest (F4) and fifth largest (F5) follicles were removed at 24, 18, 12, 6 and 2 h before the expected ovulation. Theca and granulosa layers were isolated and P4, T, E2 and protein concentrations determined. Protein concentrations of the granulosa and theca layers increased 5- and 15-fold, respectively, during the five ovulatory cycles prior to ovulation. As the follicle approached ovulation, there was a linear decrease in E2 concentration of the theca layer with the most significant decrease (P less than 0.001) occurring between 24 and 18 h of the ovulatory cycle. In the F3 and F4 theca layers, there was a significant increase (P less than 0.005) in E2 at 6 h of the ovulatory cycle. Fluctuations in T concentrations in theca and granulosa layers were similar. There was a significant increase (P less than 0.05) in T in both layers of the F2, F3 and F4 follicles at 6 h followed by a decrease (P less than 0.005) in the theca layers at 2 h of the ovulatory cycle. The P4 concentration of the granulosa layer increased gradually during follicular maturation, with the greatest increase occurring in the F2 and F1 granulosa layers between 18 and 12 h of the ovulatory cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased granulosa cell luteinizing hormone sensitivity and altered thecal estradiol concentration in the aged hen, Gallus domesticus.

Few studies have examined the effect of age on the ovulation cycle of the hen. Our aim was to determine if changes in the ovary account for the decrease in egg production with age. Young hens (28-38 wk of age) laying at least 20 eggs per sequence and old hens (53-63 wk of age) laying 3-6 eggs per sequence were used. We determined luteinizing hormone (LH) sensitivity of the ovary of young and old hens by measuring LH stimulable adenylyl cyclase (AC) activity of the granulosa layer. We also measured theca- and granulosa-layer weights and steroid concentrations of these layers and of the serum in young and old hens. Mean basal AC activity (pg/min/mg protein) for the largest (F1) and second largest (F2) follicles from young and old hens did not differ. A significant dose-response relationship to LH was present in all groups, and AC responsiveness to increasing doses of LH was greater in the F1 and F2 follicles of young hens than in the same follicles of old hens. The F4 and F5 follicles of young hens had a significantly greater estradiol (E2) concentration (pg/mg theca protein) compared to old hens, while the E2 concentration in the F2 follicle was greater in old hens. The theca layer of the F1 follicle of old hens weighed significantly more than that of young hens, whereas the theca layer of the F3, F4 and F5 follicles from young hens weighed more than those of old hens.(ABSTRACT TRUNCATED AT 250 WORDS)