The enzymes in cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism in human corpora lutea: dependence on luteal phase, cellular and subcellular distribution

Eicosanoids synthesized within corpus luteum are presumed to regulate luteal function in women. However, the potential cellular source(s) of the eicosanoids, whether small and large luteal cells differ in eicosanoid synthesis and whether eicosanoids other than prostaglandin (PG)E2, PGF2 alpha and 6-keto-PGI1 alpha can be synthesized, have not been investigated. The present immunocytochemical studies were undertaken to answer these questions using mono and polyclonal antibodies to several enzymes in arachidonic acid metabolism by cyclooxygenase and lipoxygenase pathways. Human corpora lutea from early (n = 5), mid (n = 6) and late (n = 3) luteal phases were specifically immunostained for all the enzymes. All the enzymes were present in small and large luteal cells as well as in non luteal cells. However, small luteal cells contained more immunoreactive 5-lipoxygenase, PGD2 and PGF2 alpha synthases; large luteal cells contained more TXA2 synthase and 12-lipoxygenase; small and large luteal cells contained similar amounts of cyclooxygenase and PGI2 synthase. In all the cells, immunoreactive PGD2, PGI2 and TXA2 synthases increased from early to mid luteal phase and then declined in late luteal phase. Cyclooxygenase, 5- and 12-lipoxygenases and PGF2 alpha synthase, on the other hand, increased from early to mid and mid to late luteal phases. Immunoreactive cyclooxygenase and 5- and 12-lipoxygenases were present primarily in rough endoplasmic reticulum (ER) and/or smooth ER and cytoplasm. Quite unexpectedly, all three enzymes were also found in nuclear membranes, condensed chromatin and especially at the perimeter of condensed chromatin. Dispersed chromatin contained very little or no immunoreactive enzyme. These results indicate that regulation of human luteal function by eicosanoids synthesized within the corpus luteum is complex involving perhaps a) small and large luteal as well as non luteal cells, b) eicosanoids which have not been previously considered to play a role in luteal function and c) coordinate regulation of more than one enzyme in the pathways of arachidonic acid metabolism.     

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.     

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period.

The hormonal and second messenger regulation of plasminogen activator (PA) activities in avian granulosa and theca cells has been documented. However, the physiological role(s) of PAs in the avian ovary remains poorly understood. The present studies were designed to evaluate PA activity in hen granulosa cells collected from the most mature (F1) preovulatory follicle at three discrete time points relative to a spontaneous ovulation and from follicles collected at various stages of follicular development. Levels of PA activity in the granulosa layer of the F1 follicle declined by greater than 90% as follicles were collected closer to their anticipated time of ovulation (e.g., from 17-16 h to 0.75-0.15 h; p less than 0.05). Timing of tissue collection was confirmed by evaluation of serum progesterone levels, which peaked as expected at the 6-5-h time point. During follicular development, PA activity was several times greater in rapidly growing follicles (6-12 mm, 1-3 wk prior to ovulation) than in slowly growing (1-5 mm) or preovulatory (F3 and F1) follicles (p less than 0.05). Granulosa cells of these rapidly growing follicles also incorporated significantly higher levels of 3H-thymidine than did granulosa cells of mature follicles (p less than 0.05), suggesting a higher level of DNA synthesis. Similarly, granulosa cells of the mitotically active germinal disc region of the F1 granulosa layer were found to possess at least 3-fold higher (p less than 0.05) levels of PA activity and a 2-fold greater level of 3H-thymidine incorporation than the more mature granulosa cells isolated from the remaining F1 granulosa layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levels of expression of lipoxygenases and cyclooxygenase-2 in human breast cancer

Lipoxygenases and cyclooxygenase are key mediators of arachidonic acid metabolism. The eicosanoids metabolites from these oxygynases have been shown to regulate the growth and death of cancer cells. This study determined the level of expression of 5-, 12-, 15-lipoxygenase and cyclooxygenase-2 expression in a cohort of breast cancer patients and their correlation with clinical outcomes. Compared with normal breast tissues, tumour tissues exhibited a significantly higher levels of 12-lipoxygenase and cyclooxygenase-2 (P<0.05), and significantly lower level of 15-lipoxygenase (P=0.05). Lobular carcinomas had a higher level of cyclooxygenase-2 and lower level of 15-lipoxygenase than ductal carcinomas. The lowest level of 15-lipoxygenase was seen in TNM3 and TNM4 tumours and from patients who died of breast cancer. Levels of 12- and 5-lipoxygenases were also particularly high in tumours from patients who died of breast cancer. This study shows that human breast tumours aberrantly express lipoxygenases and cyclooxygenase-2 and that decreased level of 15-lipoxygenase and raised level of cyclooxygenase-2 and 12-lipoxygenase has prognostic value in patients with breast cancer.     

Adenylyl cyclase system of the small preovulatory follicles of the domestic hen: responsiveness to follicle-stimulating hormone and luteinizing hormone

The purpose of this study was to determine if the granulosa cells of the small preovulatory follicles of the domestic hen are a target tissue for follicle-stimulating hormone (FSH). The third largest (F3), fourth largest (F4), and fifth largest (F5) follicles were removed from hens at 20, 12, 6 and 2 h before ovulation of the F1 follicle. Basal, FSH- and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activities were measured in the granulosa cells. Isolated granulosa cells of the F5 follicle, obtained 20 h before ovulation of the F1 follicle, were incubated with ovine (o) or turkey (t) FSH and progesterone (P4) was assayed in the medium. Basal AC activity was similar for F5, F4 and F3 granulosa cells except for an increase (P less than 0.01) in F3 follicles removed 2 h before ovulation of the F1 follicle. The FSH-stimulable AC activity of F5, F4 and F3 granulosa cells was elevated over basal (P less than 0.01). The greatest responsiveness was seen in the F5 follicle and the least in the F3 follicle. LH-stimulable AC activity was absent in the F5 follicle but present in the F4 and F3 follicles with the greater responsiveness in the F3 follicle. Isolated F5 granulosa cells secreted significant amounts of P4 in response to oFSH and tFSH. The data indicate that: 1) FSH stimulates the AC system of granulosa cells of the smaller preovulatory follicles (F5 greater than F4 greater than F3) while LH stimulates the AC system of granulosa cells of the larger follicles (F3 greater than F4), and 2) FSH promotes P4 production by granulosa cells of F5 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioreceptor and autoradiographic analysis of FSH, hCG and prolactin binding sites in primary to antral hamster follicles during the periovulatory period

As measured by radioreceptor assays, binding sites for FSH and prolactin were present at 09:00 h on the day of pro-oestrus in Stage 1-10 follicles (primary to antral) with prolactin receptors 3-6 times higher than FSH sites in Stages 1-3 (3 layers of granulosa cells). Specific binding sites for hCG were present in Stage 1 and 2 follicles (2 layers of granulosa cells) but thereafter their distribution was erratic and they were not consistently detectable until Stage 5, when thecal cells first appeared. Using topical autoradiography, specific binding for FSH was evident in Stage 1-4 follicles (4 layers granulosa cells) whereas specific hCG-binding was not. After the preovulatory gonadotrophin surges, by 21:00 h on pro-oestrus, FSH receptors declined in Stages 5-10, prolactin receptors fell in Stages 8 and 10 (small and large antral follicles) and hCG receptors were reduced in Stages 7 (start of antral cavity) to 10. On the morning of oestrus, for follicles from Stage 4 onwards, receptor numbers usually returned to levels found at 09:00 h on pro-oestrus. At oestrus, the few remaining Stage 10 follicles were all atretic and contained significantly reduced FSH and prolactin receptors but numbers of hCG binding sites comparable to those at 09:00 h of pro-oestrus. These results provide evidence of gonadotrophin receptors in small primary and secondary follicles which is consistent with increased DNA synthesis in small hamster follicles on the afternoon of pro-oestrus and on the morning and afternoon of oestrus. Periovulatory changes in gonadotrophin concentrations may therefore affect early stages of folliculogenesis.     

Quantitative analysis of in-vitro incorporation of [3H]thymidine into hamster follicles during the oestrous cycle

Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per microgram follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7-8 layers granulosa cells plus theca). When expressed as thymidine per follicle or microgram DNA, there was a significant increase in incorporation for stages 1-4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per microgram DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1-4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Granulosa cells as a source and target organ for tumor necrosis factor-alpha

Tumor necrosis factor (TNF-alpha), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF-alpha (5-10 units/300,000 cells per 15 h). This production was increased by addition of follicle-stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid-cycle follicles. TNF-alpha was found to increase prostaglandin F-2 alpha production by human granulosa cells (P less than 0.001). We conclude that granulosa cells are both a source and target organ for TNF-alpha.     

Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet forms of the enzyme

We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or disrupted tracheal epithelial cells metabolized arachidonic acid predominantly to (12S)-hydroxyeicosatetraenoic acid, and subcellular fractionation by differential centrifugation demonstrated that the 12-lipoxygenase activity was localized predominantly to the 100,000 x g supernatant (cytosol fraction). Analysis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-8.0), divalent cation effects (no dependence on cations), and kinetic characteristics (lag phase elimination by addition of hydroperoxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase reacted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an antibody (HPLO-3) against the platelet enzyme. Immunoaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bovine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa protein band. Activity assays of the purified enzymes demonstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently converted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipoxygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types.     

Demonstration of a 12-lipoxygenase activity in bovine polymorphonuclear leukocytes

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.     

Evidence for interaction between granulosa cells and theca in early progesterone synthesis

The temporal characteristics of steroidogenesis in vitro by hamster preovulatory follicles, were compared to granulosa cells and theca incubated separately. Gonadotropin-stimulated intact follicles or recombined granulosa cells and theca synthesized increased amounts of progesterone by 30-120 minutes of incubation. The granulosa cells and theca, when incubated separately, did not begin to accumulate progesterone until 4 to 6 hours. The relatively rapid rise in follicular progesterone synthesis after in vitro gonadotropin stimulation follows the same time course as the rapid rise in vivo of hamster and rat preovulatory progesterone after the gonadotropin surge. The sharp differences in the temporal characteristics of progesterone synthesis between follicles and separated follicular cell types suggest an interaction between granulosa cells and theca in at least one phase of progesterone synthesis.     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

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.