Expression of SCC in ovarian granulosa cells and cultured cells, induced rapid structural changes in mitochondria.

In this study, the relation of P450scc expression and mitochondrial ultrastructure was examined in rat granulosa cells at the time of ovulation, and in the NIH/3T3 cells. Before ovulation in the ovary, granulosa cells of Graafian follicle which expressed only mRNA of P450scc had elongated mitochonria with lamellar cristae. After ovulation, granulosa lutein cells which expressed both P450scc mRNA and protein had oval and round mitochondria with tubular or vesicular cristae. Two different cytochrome P450scc cDNA fragments in length were subcloned into pEGFPN vector, transfected into NIH/3T3 cells, and the mitochondrial structure was examined under fluorescent microscope with GFP and by electron microscopy. 5'end of cytochrome P450scc contained mitochondrial localization signal, and was composed of about 40 amino acids. NIH/3T3 cells had filamentous and elongated mitochondria with lamellar cristae, free ribosomes, and rER. After the transfection of short fragment of SCC(scc-s:200bp), mitochondria remained filamentous and their cristae also remained lamellar. On the other hand, when almost full length of SCC fragment(scc-f:1.1kb) was transfected, globular and round mitochondria were labeled with GFP, and round or oval mitochondria with vesicular or tubular cristae could be examined by electron microscope. Our study suggests that cytochrome P450scc located in mitochondrial inner membrane plays an important role to determine the mitochondrial morphology in vivo and in vitro.

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. In the cisternae of the endoplasmic reticula, we found intermembrane bridges linking opposite membranes of the cisternae. Membranes of adjoining rough endoplasmic reticulum cisternae were linked by intermembrane bridges crossing a narrow cytoplasmic gap between cisternae. Intermembrane bridges were also found in and between the Golgi cisternae and in nuclear envelopes. Three kinds of intermembrane bridges were found within mitochondria: one linking between outer and inner mitochondrial membranes and the other two spanning the intracristal space and intercristal matrix space. The presence of intermembrane bridges within membrane organelles, except for those between rough endoplasmic reticulum cisternae, was seen in all cell types examined. Intermembrane bridges within membrane organelles provide a structural basis for the membrane organization of the organelles and thus may contribute to the functional integrity of the organelles.

Steroidogenic activity and ultrastructural observation of atretic follicles in the cycling hamster ovary.

In mature hamster ovaries, many follicles undergo atresia and this atretic change occurs in any estrous day. In this study, the localization of enzymes involved in estrogen biosynthesis, Bromodeoxyuridine (BrdU) incorporation, and Proliferating Cell Nuclear Antigen (PCNA) were immunohistochemically examined in the atretic follicles always provided with an atrum variable in size. Moreover, the granulosa cells from the atretic follicle in various but parallel stages of development were examined by scanning and transmission electron microscopes. Very early morphological signs of atresia in these follicles was the pyknotic change of a few granulosa cells lining the antral cavity. In this kind of follicles, the number of BrdU incorporating granulosa cell was decreased and the immunoreactivity of PCNA and aromatase was gradually decreased. Even in the early stage of atresia, some granulosa cells showed a remarkable morphological change characteristic of apoptosis as revealed by scanning and transmission electron microscopy.

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 17 alpha-hydroxylase/C17-C20 lyase (17 alpha-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 17 alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic immunocytochemistry on the colocalization of ATP synthase and cytochrome P-450 of side chain cleavage enzymes in mitochondria of rat and bovine adrenal cortical cells.

To clarify whether ATP synthase and side chain cleavage enzymes (SCC) can coexist in a mitochondrion of the adrenal cortical cell, and whether the functional heterogeneity of mitochondria can occur in an adrenal cortical cell, the immunostainability of these two enzymes in mitochondria was compared by electron microscope using pre- and post-embedding methods. In the pre-embedding method, the inner membrane of most mitochondria in the adrenal cortical cell was positively stained for SCC, 11 beta-hydroxylase, and ATP synthase, but among these immunopositive mitochondria we often observed negatively immunoreacted ones in the same adrenal cortical cell. In the positively stained mitochondria, immunoreaction products were evenly localized along the inner mitochondrial membrane. We therefore think that these differences in the immunostainability are caused by technical artifacts, namely the inadequate penetration of the antibody. In the post-embedding immunocytochemistry, gold particles showing the presence of the enzymes were observed on all mitochondria. Two different antibodies, anti-ATP synthase antibody and anti-cytochrome P-450scc antibody, which were labelled with gold particles of varying diameter (5 nm, 10 nm each) could be observed on all the mitochondria of the bovine adrenal cortical cells. No heterogeneities as to the stainability for both ATP synthase and SCC were detected in mitochondria of the rat and bovine adrenal cortical cells. These results indicate that in the rat and bovine adrenal cortical cells, both ATP synthase and SCC are evenly and simultaneously present on the inner membrane of all mitochondria. Hence, mitochondria in the adrenal cortical cell seem to be homogeneous in their steroid and ATP synthesizing abilities.

Immunohistochemical studies on the localization of aromatase and 17 alpha-hydroxylase/C17-20 lyase (17 alpha-lyase) in estrous cycling and pregnant hamster ovaries.

In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00 am) of Day 1 to the evening (5:00 pm) of Day 4; in the night (9:00 pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction. As for 17 alpha-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00 am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00 pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00 pm) of Day 4 or morning (10:00 am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17 alpha-lyase in the theca interna cells of the ovary. On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17 alpha-lyase antibody. Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17 alpha-lyase show ultrastructural features typical of steroid secretory cells.

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

Immunocytochemical 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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

The localization of cytochrome P-450 of 17 alpha-hydroxylase/C17-C20 lyase (P-450(17 alpha, 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-450(17 alpha, 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-450(17 alpha, 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-450(17 alpha, lyase) again. The 17 alpha-hydroxylating activity of P-450(17 alpha, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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

The 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.

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

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-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 3 beta-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 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and its relation to the ultrastructure of steroidogenic cells in immature and mature rat ovaries.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its relation to the ultrastructure of steroidogenic cells were examined in mature and immature rat ovaries. In mature (8-10 weeks old) rat ovaries, the theca interna cells of secondary as well as Graafian follicles, and the interstitial gland cells were all strongly stained with anti-17 beta-HSD antibody. However, granulosa cells, corpus luteum cells, oocytes and peritoneal epithelial cells were negative against this staining. In the ovaries of 1-week-old rats, all these cells were negative to immunostaining for 17 beta-HSD. In the ovaries of 2-week-old rats, the theca interna cells of secondary follicles and the interstitial gland cells showed a positive reaction for the 17 beta-HSD activity. Electron microscopic examination demonstrated the presence of characteristic structures for steroid secretory cells such as many lipid droplets, well developed smooth endoplasmic reticulum, and oval mitochondria with tubular cristae in the theca interna cell of secondary as well as Graafian follicles and in the interstitial gland cell of mature rat ovaries. In the ovaries of 1-week-old rats, all the theca cells of the primary and secondary follicles were fibroblast-like in their shape and fine structure, and typical interstitial cells were not recognized. In the 2-week-old rats, some of the theca interna cells and interstitial cells were well differentiated in ultrastructure, showing characteristic features for steroid secretory cells. These findings indicate that by 2 weeks after birth, theca interna cells and interstitial gland cells acquire the ability for testosterone production as seen in mature rat ovaries.