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  • Author: Ryosuke Nakano x
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Kenichi Furukawa, Mareo Yamoto, Nobuyoshi Kokawa and Ryosuke Nakano

Furukawa K, Yamoto M, Kokawa N, Nakano, R. Purification of high-molecular-weight folliclestimulating hormone binding inhibitor in porcine follicular fluids. Eur J Endocrinol 1994;130:625–33. ISSN 0804–4643

We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 ± 24.6, 176.7 ± 3.0 and 141.3 ± 3.6U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 ± 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.

K Furukawa, Department of Obstetrics and Gynecology, Wakayama Medical College, Shichibancho 27, Wakayama 640, Japan

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Nobuyoshi Kokawa, Mareo Yamoto, Kenichi Furukawa and Ryosuke Nakano

We performed partial purification of low molecular weight luteinizing hormone binding inhibitor from porcine follicular fluids and examined its biological activities. Following ultrafiltration, gel filtration and anion exchange of the pooled porcine follicular fluids, low molecular weight fractions (500–10,000 MW) inhibited [125I]hLH binding to porcine granulosa cells in a dose-dependent manner. The binding inhibition kinetics study revealed that the luteinizing hormone binding inhibitor may indicate a non-competitive inhibition with [125I]hLH binding. In vitro bioassay using adult mouse testicular interstitial cells revealed that the partially purified luteinizing hormone binding inhibitor reduced ovine LH-stimulated testosterone and cAMP production in a dose-dependent manner, whereas the luteinizing hormone binding inhibitor did not affect basal production of testosterone and cAMP. The inhibitory activity was heat stable and did not disappear with activated charcoal adsorption. The results of the present study suggest that the luteinizing hormone binding inhibitor may play an important role as an ovarian non-steroidal regulator modulating the receptor binding of LH and LH-mediated steroidogenesis.

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Hisako Otani, Mareo Yamoto, Hiroyuki Fujinaga and Ryosuke Nakano

Otani H, Yamoto M. Fujinaga H, Nakano R. Presence and localization of endothelin receptor in the rat ovary and its regulation by pituitary gonadotropins. Eur J Endocrinol 1996:135:449–54. ISSN 0804–4643

In the present study we examined the regulation of receptors for endothelin 1 (ET-1) in rat granulosa cells. We examined the localization and regulation of ET receptors in immature rat ovary and the effects of ET-1 on steroidogenesis in cultured rat granulosa cells. The ovaries used in autoradiography were derived from pregnant mare serum gonadotropin and human chorionic gonadotropin-treated immature rats. Granulosa cells were obtained from diethylstilbestrol-treated immature rats and incubated with 125I-ET-1. Granulosa cells were cultured with ET-1 in the presence or absence of ovine follicle-stimulating hormone. The concentrations of sex steroid hormones in conditioned media were measured by radioimmunoassay. The binding site for ET-1 was localized in the granulosa cells, but not in thecal and luteal cells. Follicle-stimulating hormone (FSH) induced a dose-dependent increase in specific binding for ET-1 to cultured rat granulosa cells. In contrast. luteinizing hormone (LH) induced a dose-dependent decrease in specific binding for ET-1 to cultured rat granulosa cells. Conversely, treatment with prolactin and several sex steroid hormones had no effects on the specific binding of ET-1. Treatment with ET-1 inhibited FSH-stimulated accumulation of progesterone and estradiol in cultured rat granulosa cells. The results indicate that both FSH and LH influence the expression of ET-1 receptor, and that ET-1 may play a regulatory role in the ontogeny of the granulosa cell.

Ryosuke Nakano, Department of Obstetrics and Gynecology. Wakayama Medical College, 27 Shichibancho, Wakayama, 640, Japan

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Mitsuko Kobayashi, Mareo Yamoto, Sawako Minami, Miyako Imai and Ryosuke Nakano

Kobayashi M, Yamoto M, Minami S, Imai M, Nakano R. Immunohistochemical localization of inhibin α- and βA-subunits in the ovary of immature female rats. Eur J Endocrinol 1995;132:97–102. ISSN 0804–4643.

Immunohistochemical localization of inhibin α- and βA-subunits was examined in the ovaries of immature female rats. The granulosa cells in various sized ovarian follicles obtained from rats that were 10–24 days old exhibited positive staining for inhibin α- and βA-subunits. The relative intensities of immunostaining for α- and βA-subunits increased during follicular growth and maturation. Ova and internal thecal cells did not show any immunostaining for inhibin α- and/or βA-subunits. These results suggest that granulosa cells of immature rat ovaries may produce inhibin from the 10th day after birth, and that an increase in the number of mature ovarian follicles results in an increase in inhibin production in the immature rat ovary during prepubertal development.

Mitsuko Kobayashi, Department of Obstetrics and Gynaecology, Wakayama Medical College, 1 shichibancho, Wakayama 640, Japan

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Hiroko Hamada, Shiroh Kishioka, Mareo Yamoto and Ryosuke Nakano

Hamada H, Kishioka S, Yamoto M, Nakano R. [3H]Naloxone binding sites in porcine ovarian follicles and corpora lutea during the ovarian cycle. Eur J Endocrinol 1995;132:622–6. ISSN 0804–4643

We demonstrated the presence of opioid receptors in the porcine ovary using [3 H]naloxone. We also examined the change in the number of opioid receptors during follicular maturation. In addition, we found specific binding of [3H]naloxone in the porcine ovary using naloxone, β-endorphin, methionine-enkephalin and dynorphin. The binding of [3H]naloxone to porcine granulosa cells and the 2000-g subcellular fraction of corpora lutea was examined to demonstrate the presence of specific [3H]naloxone binding in the porcine ovary. Binding of [3H]naloxone to porcine granulosa cells was displaced by cold naloxone and β-endorphin but not by dynorphin and methionine-enkephalin. A similar phenomenon was also demonstrated in the 2000 g subcellular fraction of porcine corpora lutea. However, Scatchard analyses revealed a single class of high-affinity (Kd = 28.5 × 10−9 mol/l) and low-capacity binding sites (Bmax = 30.5 fmol/5 × 106 cells) in porcine granulosa cells. Similar binding parameters were obtained in the 2000-g subcellular fraction of porcine luteal tissue (Kd = 28.3 × 10−9 mol/l, Bmax = 59.3 nmol/kg protein). [3H]Naloxone binding sites in the porcine ovary showed binding characteristics similar to those of opioid receptors in other organs like brain, uterus and placenta. Furthermore, we demonstrated that the specific binding sites of [3H]naloxone in porcine granulosa cells decreased during follicular maturation. Opioid receptors have been detected in the uterus, placenta and Sertoli cell cultures in some species. However, there is no detailed study on opioid receptors in granulosa cells and luteal tissues in any species. Our data suggest a relationship between folliculogenesis and ovarian opioid peptides. The opioid system may participate in the regulation of follicular maturation.

Hiroko Hamada, Department of Obstetrics and Gynecology, Shichibancho 27, Wakayama 640, Japan