The expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNA in the porcine ovary

The aim of this study was to visualize the expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNAs in various structures of porcine ovary. Porcine ovaries were frozen in liquid nitrogen, and 8 microm sections were prepared for in situ hybridization. In the small, medium and large antral follicles as well as in early, midluteal and regressing corpora lutea, mRNAs for androgen receptor, P450 aromatase and FSH receptor were detected. In small antral follicles high levels of mRNAs for androgen and FSH receptors were observed, mainly in the granulosa layer, while mRNA expression for P450 aromatase was negligible. As follicles grew, amount of mRNAs for androgen receptor and FSH receptor decreased, and that for P450 aromatase increased. Small amounts of androgen receptor mRNA were also present in corpora lutea at all examined stages. P450 aromatase mRNA was not detected in early and midluteal corpora lutea. However, regressing corpus luteum showed a weak expression of aromatase mRNA.     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luteal cells by prolactin

Regulation by PRL of aromatase (P450arom) mRNA and protein and estradiol (E) biosynthesis was examined in granulosa cells during early stages of luteinization in vitro and in vivo. PRL caused a dose-dependent (10-1000 ng/ml) decrease in P450arom mRNA and E biosynthesis (greater than 99%) in luteinized rat granulosa cells in vitro, even when the cells were cultured in the presence of insulin and hydrocortisone (hormones known to synergize with PRL to induce proteins in mammary tissue) or in the presence of forskolin (a nonhormonal stimulator of cAMP). PRL also prevented the marked increases in aromatase mRNA and E biosynthesis stimulated by FSH and forskolin in nonluteinized preovulatory granulosa cells in culture. These effects of PRL on granulosa cells in culture were specific for aromatase and were not observed for other proteins, such as cholesterol side-chain cleavage cytochrome P450 (P450scc) and alpha 2-macroglobulin. PRL also decreased P450arom mRNA and protein during the early stages of luteinization in vivo. PRL administered to rats beginning day 1 postovulation to mimic hormone release during pseudopregnancy reduced the progressive increase in P450arom mRNA occurring in corpora lutea on days 3-4 in ovulated rats not treated with PRL. CB 154, a dopamine agonist that inhibits pituitary release of PRL, caused P450arom mRNA and protein to decrease 50% if given to pregnant rats on days 8-10 of gestation, but increased P450arom mRNA and protein if given to pregnant rats on days 10-12 of gestation. These diverse effects of PRL in pregnancy suggest that placental factors may modify the response of luteal cells to PRL during gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Summary 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. 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.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.     

Immunocytochemical Localization of Aromatase in The Ovary of Superovulated Cattle,Pigs and Sheep

The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species. It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.     

Immunohistochemical localization of androgen receptor and aromatase in the ovary of the pregnant pig

The distribution of androgen receptor (AR) and cytochrome P450 aromatase was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32, 71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei of granulosa cells of follicles of various sizes. In addition, some follicles demonstrated staining in the nuclei of theca interna cells. Stroma cells also exhibited a positive immunostaining. At early and mid pregnancy (up to Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day 71 showed mainly cytoplasmic staining while on Day 90 almost all luteal cells showed staining exclusively in the cytoplasm. Immuno-staining for the presence of cytochrome P450 aromatase was very faint in all investigated ovarian structures. The results could suggest that the process of androgen aromatization plays a negligible role in the ovary of the pregnant pig.     

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.     

Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula)

The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome p450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta5,Delta4 isomerase (3betaHSD), cytochrome p45017alphahydroxylase (p45017alphaOH), and p450 aromatase (p450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain p450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed p450arom and 3betaHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, p450scc, 3betaHSD, and p45017alphaOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed p450arom, indicating that this tissue also has the potential to secrete estrogens in this species.     

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

Summary 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.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA.     

Differential regulation of aromatase and androgen receptor in granulosa cells

During follicular development, androgen acts in three distinct ways. During the early stage of follicular differentiation, androgen acts as an enhancer of FSH-stimulated follicular differentiation. As follicular differentiation progresses, this effect is decreased and androgen is mainly utilized as a substrate for estrogen synthesis under increasing stimulation of FSH and LH. These two events are mediated by androgen receptor (AR) and aromatase (P450arom), respectively. In the rat and marmoset monkey, AR and P450arom are predominantly expressed in granulosa cells, and both are developmentally regulated. The expression of AR is highest in preantral/early antral follicles and gradually decreases as follicles mature, whereas expression of P450arom is increased as follicular differentiation progresses. We propose that differential regulation of these two androgen-utilizing factors contributes to the smooth transition of developing follicles from the early stage of differentiation to the fully mature ovulatory status. A failure of this transition due to improper androgen stimulation might result in follicular atresia.     

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 localization and enzymatic distribution of rat ovarian aromatase

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.     

Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.     

Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro

We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450(arom)) mRNA abundance and 2) whether P450(arom) mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2-4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450(arom) mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450(scc)) mRNA abundance did not. FSH stimulated estradiol secretion and P450(arom) mRNA abundance; the effect was quadratic for both estradiol and P450(arom) mRNA. Estradiol secretion and P450(arom) mRNA levels were correlated. FSH stimulated progesterone secretion and P450(scc) mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450(arom) mRNA levels but not progesterone or P450(scc) mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450(arom) mRNA abundance responsiveness to FSH and insulin, whereas P450(scc) mRNA abundance and progesterone secretion were responsive to FSH but not insulin.