Tomoko Yoshinaga-Hirabayashi

Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice.

SummaryThe precise localization of estrogen biosynthesis in the ovary of rats and mice were immunocytochemically studied using new antisera against aromatase cytochrome P-450. The positive reaction for aromatase was detected mainly on the granulosa cells of large, apparently preovulatory follicles. In addition, the cells of some corpora lutea showed very weak positive reaction but most corpora lutea were negative to the staining. Those cells such as the granulosa cells of smaller follicles, the theca interna cells, the interstitial gland cells, oocytes, peritoneal epithelial cells were entirely negative. These results indicate that in the ovary of rats and mice, the granulosa cells of preovulatory follicles are the main site for synthesis of estrogen from androgen which is provided by the theca interna cell and the interstitial gland cell.

Intermembrane bridges within membrane organelles revealed by quick‐freeze deep‐etch electron microscopy

Intracellular membrane–bounded organelles, such as the endoplasmic reticulum, Golgi apparatus, and mitochondrion, possess and maintain their shape and intrinsic relationship due to the nature of their membrane organization. To reveal the membranous attachments that support these shapes and relationships, we examined various kinds of cells by quick‐freeze deep‐etch electron microscopy.

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

SummaryImmunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

SummaryThe localization of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3β-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3β-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3β-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

Immunocytochemical and biochemical studies on the localization and changes of 17 α-hydroxylase/C17–C20 lyase activity in immature rat ovary treated with PMSG and hCG

SummaryThe localization of cytochrome P-450 of 17 α-hydroxylase/C17–C20 lyase (P-45017 α, lyase) and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-45017 α, lyase was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-45017 α, lyase was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-45017 α, lyase again. The 17 α-hydroxylating activity of P-45017 α, lyase was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG+hCG(3 h)-treated and PMSG+hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes. These immunocytochemical and biochemical findings suggest that 1) P-45017 α, lyase is localized in both the theca interna cell and interstitial gland cell, and these cells are the main site of the androstenedione production in the ovary, and that 2) the decreased production of estrogen occurring just before ovulation is not brought about by the decreased activity of P-45017 α, lyase, but done by the loss of the enzyme.

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17α-hydroxylase/C17–C20 lyase (17α-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17α-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles. Even in the early stage of atresia, the granulosa cells showed remarkable morphological change characteristic of apoptosis, as visualized by scanning and transmission electron microscopy. Cessation of granulosa cell proliferation may occur earlier than apoptotic change and the degeneration of the granulosa cells becomes rapid once atresia starts.