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.     

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries.     

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.     

Immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase in the ovary of pregnant pigs and fetal gonads

The study was designed to localize P450 17alpha-hydroxylase/c17-20 lyase (P450c17) in the ovaries of pregnant pigs and fetal gonads. Immunoexpression of P450c17 was investigated in the porcine ovaries (follicles and corpora lutea; CL) collected on days 10, 18, 32, 50, 70 and 90 post coitum (p.c.), and fetal gonads (testes and ovaries) on days 50, 70 and 90 p.c. The presence of P450c17 in ovarian follicles was demonstrated on all examined days of pregnancy but was restricted to theca interna cells. In CL, P450c17 was detected on all examined days of pregnancy but only in small luteal cells. In the female porcine fetuses, P450c17 immunostaining was found in oocyte nests and granulosa cells of primary ovarian follicles, while in the male fetuses in fetal Leydig cells. In conclusion, the immunolocalization of P450c17, detected in the ovaries of pregnant pigs and fetal porcine gonads, indicates the potential sites of androgen synthesis. We suggest that androgens may play a role in the maintenance of pregnancy and in the development of prenatal gonads in pigs.     

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.     

Gonadotropin releasing hormone treatment of dairy cows with ovarian cysts: II. Histology of ovarian cyst walls

Dairy cows known to have ovarian cysts were assigned to receive either sterile water or 100 μg GnRH (5 cows/group). The ovaries were removed 9 to 13 days post-treatment and prepared for histologic study. The cyst walls of ovaries removed from cows following GnRH treatment were 2 to 15 times thicker than those from cows treated with water due to apparent luteinization of cells of theca interna. Following GnRH treatment, the cells in the outermost portion of the theca interna exhibited the greatest morphologic evidence of secretory activity whereas the innermost zone was hyalinized due to poor blood supply. The luteal cells in ovarian cystic cows treated with GnRH were similar to the small luteal cells in normal corpora lutea. In summary, GnRH treatment of cows with ovarian cysts appeared to induce luteinization of the cells in the outermost portion of the theca interna.     

Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis

The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.     

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.     

Localization of some steroidogenic enzymes in the ovary of the rabbit

The distribution of delta 5-3 beta-HSD, peroxidase and cytochrome oxidase in immature, sexually mature and pregnant rabbit ovary has been studied histochemically. Corpora lutea are found only in pregnant rabbits. delta 5-3 beta-HSD is present in the theca interna of mature follicles, corpora lutea and interstitial gland cells but is absent in the granulosa cells of both developing and mature follicles. The granulosa cells of mature and developing follicles, hypertrophied theca interna and the luteal cells all show intense cytochrome oxidase activity. Peroxidase is present in the corpora lutea only. It is suggested that delta 5-3 beta-HSD in the theca interna and interstitial gland cells is the enzyme responsible for steroid synthesis in the ovaries of immature as well as sexually mature rabbits, while peroxidase and delta 5-3 beta-HSD present in the corpora lutea together regulate luteal steroidogenesis during pregnancy. The intense cytochrome oxidase activity together with peroxidase and delta 5-3 beta-HSD confirms the observations that this tissue is a site of intense oxidative activity.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary

Immunohistochemical localization of cholesterol side-chain-cleavage, 17 alpha-hydroxylase and aromtase cytochromes P-450 was performed in 35 morphologically normal human premenopausal ovaries by using specific antibodies against the enzymes. In well-developed ovarian follicles in the late stages of follicular growth, immunoreactivity of P-450AROM was only seen in granulosa cells while P-450(17 alpha) and P-450SCC activity was confined to theca interna cells, confirming that follicular oestrogen is produced in granulosa cells by the aromatization of androgens derived from the theca interna cells. In the corpus luteum, this functional differentiation is maintained, since immunoreactivity of P-450AROM was exclusively present in luteinized granulosa cells while that of P-450(17 alpha) was present in luteinized theca calls. Immunoreactivity of P-450SCC was present in both types of cells in the corpus luteum.     

Biochemical assessment of limits to estrogen synthesis in porcine follicles

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.     

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.     

Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture.

By means of histochemical technique the activities of delta53beta-, 17beta-, 20alpha-hydroxysteroid dehydrogenases and glucose-6-phosphate dehydrogenase, as well as alkaline and acid phosphatase were investigated in monolayer cultures of theca interna cells, isolated from preovulatory porcine ovarian follicles. It was found that theca interna cells exhibited high and constant activity of delta53beta-hydroxysteroid dehydrogenase and G6P-DH, whereas activities of both 17beta- and 20alpha-hydroxysteroid dehydrogenase were lower and showed some fluctuations during in vitro culture. Addition of LH to the medium brought about the increase of all studied dehydrogenases. FSH was less effective. Estradiol showed quite and inhibiting effect. All the hormones mentioned above caused the increase of alkaline and acid phosphatase activity in cultured porcine theca interna cells.     

Compartmentalization of steroidogen esis by the equine corpus luteum

The presence of cytochrome P450C17 within equine follicles and corpora lutea (CL) was detected by immunostaining. Two different antibodies were used which had previously been shown by immunoblotting to cross-react with equine P450C17. Strong positive immunostaining was present in the theca-derived cells of the CL during the estrous cycle and pregnancy. In the CL from mares after Day 40 of pregnancy there were also occasional bands of positively stained cells which resembled the polyhedral-shaped theca cells seen in preovulatory follicles. The pattern of immunostaining suggested compartmentalization of steroidogenesis within the equine CL with small cells possessing the potential to produce androgen which could then be aromatized to estrogen by the large luteal cells.     

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.     

Follicular development in the sheep after priming with PMSG

The administration of PMSG to sheep early in the oestrous cycle (Days 2 or 3) results in the formation of follicular cysts of varied morphology by Days 8 or 9. These may persist for up to 14 days after injection. If PMSG is given on Day 5 or later approximately 50% of such cysts ovulate. However, when PMSG is given at the beginning of the cycle (Day 2 or 3), the membrana granulosa is lost from the majority of the cysts and the theca interna luteinizes. The major hormone secreted by such luteinized follicles is progesterone. The structure of the steroid-secreting cells of the follicles is similar to that of large luteal cells of granulosa cell origin in cyclic corpora lutea. It is suggested that under suitable luteinizing conditions thecal cells may acquire many of the characteristics of granulosa cells.     

Ovarian cysts in high-yielding dairy cows

We examined the hormonal and morphologic changes associated with ovarian cyst formation in high-yielding dairy cows. Follicle fluid was aspirated from 90 cysts and 15 preovulatory and 18 subordinate follicles and used for hormonal determination. Pieces of cystic wall were subjected to morphologic and immunohistochemical evaluation. Cysts were characterized by low concentrations of insulin, insulin-like growth factor-I (IGF-I), and glucose and high activity of IGF binding proteins (IGFBPs). Insulin and IGF-I levels were (mean ± SEM) 205 ± 22 pg/mL and 146 ± 42 ng/mL in preovulatory follicles and 3 ± 1 pg/mL and 61 ± 6 ng/mL in cysts, respectively (P < 0.001). Insulin-like growth factor-binding proteins activity was about 10 times higher in cysts than in preovulatory follicles. Cysts were classified into three types according to their estradiol-to-progesterone (E/P) ratio. Type 1 cysts (n = 23) exhibited the highest E/P ratio (10.8 ± 2.3), partial loss of granulosa cells, and severe morphologic changes in the theca interna. Expression of P₄₅₀ side-chain cleavage and P₄₅₀ 17α-hydroxylase was noted in theca cells and expression of inhibin-α in granulosa cells. Type 2 cysts (n = 35) had a low E/P ratio (0.07 ± 0.02), and patches of luteal-like tissue in the cystic wall. Type 3 cysts (n = 32) had an E/P ratio of 0.91 ± 0.17, and no recognizable granulosa or theca cells. In summary, intrafollicular steroid levels as expressed by E/P ratio, together with IGF-I and insulin levels and morphologic changes in the follicular wall, may serve as accurate cyst-classification parameters. Because IGF-I and/or insulin play an essential role in the final stage of follicle development, it can be speculated that abnormal levels of these metabolic hormones might lead to follicle dysfunction, resulting in follicular regression or cyst formation.     

Histochemical changes in acid and alkaline phosphatase activities in the growing follicles and corpora lutea of the rat ovary

Changes in acid and alkaline phosphatase activities are studied histochemically in different components of the growing follicles and corpora lutea of different generations. Theca cells of the growing follicles showed strong acid phosphatase activity as compared to that observed in the oocyte and granulosa cells. Alkaline phosphatase activity is localized only in the theca interna cells of growing follicles. However, after ovulation with the formation of corpora lutea, the granulosa lutein cells also showed this activity. Changes in the histochemical distribution of the acid and alkaline phosphatases in the follicles and corpora lutea are discussed in relation to folliculogenesis and corpus luteum and regression.