Dihydrotestosterone induces a sexual dimorphism in estrogen uptake by specific anterior pituitary cell types in vivo

Summary Castrated male and female rats pretreated with dihydrotestosterone (DHT) were injected i.v. with ³H-estradiol (E₂). Nuclear uptake and retention of ³H-E₂ was measured in each of five cell types of the anterior pituitary gland using a combined quantitative autoradiographic and immunocytochemical procedure. In non-pretreated groups, each cell type bound a characteristic amount of ligand but no sex differences were apparent. DHT pretreatment, however, caused a significant decrease in ³H-E₂ retention by gonadotrophs in both males and females. The treatment also caused a decrease in binding by lactotrophs and somatotrophs, but only in the females. No other cell types were altered. Thus, androgen appears to modulate E₂ binding and retention by pituitary cells in both a cell-type and sexdependent manner. Our results also indicate that the inhibitory effects of androgens on E₂ binding by the pituitary gland is more complex than can be explained by simple competition for the estrogen receptor.     

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.     

Characterization by high performance liquid chromatography of nuclear metabolites of testosterone in the brains of male rhesus monkeys

Testosterone (T) restores the potency of castrated male rhesus monkeys, and our autoradiographic data have demonstrated that ³H-T or its metabolites concentrate in cell nuclei in the corticomedial amygdala, bed nucleus of stria terminalis, preoptic area, and hypothalamus. In rat, ³H-estradiol (³H-E₂) is a major nuclear metabolite of ³H-T in areas of the limbic system, but comparable data are lacking for the primate. We have therefore developed an improved technique using high performance liquid chromatography for investigating metabolites of ³H-T that accumulate in cell nuclei in small amounts of tissue obtained from the brain of the rhesus monkey. Two castrated male rhesus monkeys were injected with 5 mCi of ³H-T and were killed 30 min later. In amygdala, preoptic area-bed nucleus of stria terminalis, and hypothalamus, 48–70% of the nuclear radioactivity was in the form of ³H-E₂ (Type I tissues). In six other brain areas and in pituitary, 35–85% of the nuclear radioactivity was in the form of ³H-T (Type II tissues), whereas in genital tract tissues, 86–99% of the nuclear radioactivity was in the form of ³H-dihydrotestosterone (³H-DHT) (Type III tissues). In plasma and in supernatants from both Type I and Type II tissues, the proportions of ³H-T were high, and ³H-E₂ did not exceed 10% of the total extractable radioactivity. These data suggest that, as in rodents, some of the central actions of T in primates may be mediated by estrogen target neurons.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN1-15. Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors. Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutea of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutea in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of AN1-15, confirming the presence of androgen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early folliculogenesis in primate ovaries: testing the role of estrogen.

The purpose of this study was to examine the effect of an exogenous estrogen, diethylstilbestrol (DES), on follicle development in the ovary of a juvenile primate. The immature cynomolgus monkey (12-22 mo) was used as a model since ovaries at this age lack endogenous gonadotropin support but are capable of responding to exogenous hormonal stimulation. In addition, the pituitary gland receives virtually no GnRH stimulation and under these conditions lacks responsiveness to estrogen feedback. Two groups of three monkeys each received DES for 14 days. Members of the second group also were given GnRH antagonist to assure no GnRH action upon the gonadotropes. The left ovary of each monkey was removed just prior to Day 1 of DES treatment and served as the control. The right ovary was removed on Day 14 of treatment. Both ovaries from each monkey were prepared for evaluation by light microscopy. Results indicated that both the number of preantral follicles and the mean number of medium-sized (0.5-1 mm in diameter) developing antral follicles decreased significantly (p less than 0.05) in the DES-treated ovaries with no increase in early-atretic antral follicles. These data suggest that DES, at the amount administered, inhibits the growth of both preantral and medium-sized antral follicles in the primate. Whether these effects are manifest directly at the follicle level or are mediated by other mechanisms remains to be determined.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

Estrogen receptor protein and mRNA expression in the ovary of sheep

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep. Mol. Reprod. Dev. 48:53–62, 1997. © 1997 Wiley-Liss, Inc.     

Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans

Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERalpha] and beta [ERbeta]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERbeta all recognized recombinant human (h) ERbeta but did not bind to recombinant hERalpha. The ERbeta protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERbeta protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERalpha protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERbeta than ERalpha. In conclusion, three independent antibodies have demonstrated convincingly that ERbeta is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERbeta:beta dimers. In antral follicles, however, ERalpha is also expressed, and the formation of homo- or heterodimers containing ERalpha may influence the pattern of gene activation within these cells.     

The effect of hepatocyte growth factor on the initial stages of mouse follicle development

Interactions between theca and granulosa cells of the follicle are critical for the coordination of ovarian follicle development. The cell–cell interactions are mediated through the local production and actions of a variety of factors. The current study is designed to investigate the expression of Hgf and its receptor, c‐Met, in the mouse ovary during in vivo folliculogenesis. We found that Hgf and c‐Met mRNAs were already expressed in 2‐day‐old ovaries, and that, while c‐Met levels remained constant until 22‐day‐old, Hgf levels slightly but not significantly increased with age. The expression of Hgf mRNA in theca/interstitial cells was higher than in granulosa cells in 22‐day‐old ovaries. Immunohistochemistry analysis confirmed the expression pattern demonstrated by RT‐PCR. We investigated the role of hepatocyte growth factor (HGF) at the beginning of mouse folliculogenesis and its possible interaction with kit ligand (KL). Interestingly, both KL and HGF were able to increase the expression of each other, creating a positive feedback loop. In the presence of HGF, we observed an increase of granulosa cell proliferation and an increase in the number of pre‐antral and early antral follicles in ovary organ cultures. We also observed a significant increase in the diameters of follicles in individual follicle cultures. Moreover, HGF stimulated the expression of the FSH receptors, both in the whole ovary and in isolated pre‐antral follicle cultures. Based on the data presented, we concluded that HGF exerts multiple levels of control over follicular cell functions, which collectively enable the progression of follicular development. J. Cell. Physiol. 226: 520–529, 2011. © 2010 Wiley‐Liss, Inc.     

Estrogen receptor alpha and beta expression in the porcine ovary

In order to investigate the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the porcine ovary, in situ hybridization was applied. Specific ovine ERalpha and bovine ERbeta cDNA probes were labeled with [-32S]dCTP. In the porcine ovary, positive signals for ERbeta were found in both granulosa and theca cells of all types of antral follicles as well as in the corpora lutea at all stages of regression. ERalpha mRNA was limited exclusively to the granulosa cells of preovulatory follicles and was present in a few cells of early corpora lutea. Thus, we showed differential expression of ERalpha and ERbeta at the mRNA level. Large antral follicles and early corpora lutea are the site, where both forms of estrogen receptor are expressed.     

Localization of the renin-angiotensin system in the bovine ovary: cyclic variation of the angiotensin II receptor expression.

Angiotensin (Ang) II may modulate reproductive function in the bovine ovary. Therefore, expression and localization of a local ovarian renin-angiotensin system (RAS) were investigated by elucidating the influence of the estrus cycle, pregnancy, and the presence of follicular cysts. Receptor analysis and autoradiography were used to characterize and localize Ang II receptors. Cyclic variations in the density of ovarian Ang II receptors were found with a higher value in estrus than in diestrus. The density in ovaries with follicular cysts was in the same order of magnitude as in estrus. The Ang II receptor type 2 (AT(2)) dominated in all three groups. Autoradiography showed that the majority of antral follicles and follicular cysts had intense AT(2) receptor binding in the theca externa. Binding was less intense in the theca interna, whereas there was no binding in the granulosa layer. In the corpora lutea, the AT(2) receptor was dominant in the capsule and in connective tissue infoldings, whereas no binding was observed in the luteal tissue. The type 1 Ang II receptor (AT(1)) was dominant in the stroma and showed no cyclic changes. Angiotensin-converting enzyme (ACE) activity was detected in all aspirated follicular fluids and homogenates of ovarian tissue. Autoradiography showed that most of the ACE was localized on endothelial cells. Renin immunoreactivity was found in granulosa and thecal cells of antral follicles and in luteal cells. Furthermore, solitary cells in the stroma, presumably macrophages, displayed intense staining. Our finding of cyclic changes support the concept of an active and regulated RAS in the bovine ovary.     

A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary.

Estrogen receptor beta (ERbeta) is highly expressed, but ERalpha is not detectable in granulosa cells in the mouse ovary. In ERbeta knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERbeta there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.     

Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary

The present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well-characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20-day-old, 35-day-old, and adult rats, strong Cx43-immunore-activity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20- and 35-day-old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43-immunoreactivity, whereas Cx43-immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface. Shortly before delivery (GD 21), however, the staining pattern vanished and only few, presumably luteal cells remained immunoreactive. In Western blots (using homogenates of whole ovaries), the Cx43 antiserum recognized a major band of approximate Mr 43 × 10³, together with minor bands, which may reflect the presence of several differently phosphorylated Cx43 forms. This is indicated by treatment with alkaline phosphatase, which reduced the banding pattern to one single band. In summary, the gap junction molecule Cx43 is abundantly expressed in all endocrine compartments of the rat ovary. The staining pattern obtained in the present study indicates that Cx43 and presumably gap-junctional communication are associated with follicular development, atresia, and the development of the interstitial gland, as well as with the development and regression of the corpus luteum. The heterogeneous staining within the ovary furthermore hints to a contribution of the local intraovarian factors in the regulation of Cx43 expression. © 1995 Wiley-Liss, Inc.     

Characterization of estrogen and progesterone receptors in monkey endometrium: methodology and effects of estradiol and/or progesterone on endometrium of castrate monkeys.

Appropriate methods for repeated surgical collection of endometrial tissue from rhesus monkeys, and characterization of cytosol and nuclear estrogen (E₂) and progesterone (P) receptors (R) are described. Tissue collection was made in the mid-luteal phase at abdominal fundal hysterotomy. Functional status of the ovaries was determined by visual inspection and RIA of E₂ and P in serum. Receptor assay procedures were devised permitting the measurement of total cytosol and nuclear receptor concentration. Sucrose density gradients of labelled cytosol were made and a 4S saturable binding component for ³H P and for ³H E₂ were found. Equilibrium dissociation constants of ³H E₂ and ³H R5020 were 2.1×10^?10M and 3.6 × 10^?9M, respectively. These binding characteristics are similar to those found in human endometrium and suggest that these surrogate primates have extensive utility in investigation of factors influencing E₂R and PR concentrations in endometrial tissue during the menstrual cycle and implantation. Simulated menstrual cycle were produced in 20 castrate monkeys by sequential treatment with estradiol and progesterone in silastic capsules. RIA of E₂ and P, and gonadotropins in peripheral serum provided assuredness of the hormonal status of each monkey under treatment. Cytosol and nuclear receptors for E₂ and P were measured in the endometrium after different intervals of the treatment. E₂ receptor (E₂R) levels were not changed during the estrogen sequence, but were lowered by progesterone therapy in both cytosol and nuclear components. Progesterone receptor (PR) synthesis in cytosol was induced by exogenous estrogen. The total concentration of PR decreased with the uptake of P by the cell; meanwhile, the ratio of cytosol to nuclear P receptors declined. These data suggest that this sequential estrogen-progesterone regimen induces the changes in E₂R and PR patterns in the endometrium of ovariectomized monkeys which occurs due to ovarian cyclicity in the normal menstrual cycle.     

Expression of prostasin serine protease and protease nexin-1 (PN-1) in rhesus monkey ovary during menstrual cycle and early pregnancy.

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.     

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.     

Estrogenic and Antiestrogenic Down-regulation of Estrogen Receptor Levels: Evidence for two Different Mechanisms

Abstract

Preincubation of MCF-7 cells with estradiol (E₂) produces a decrease of H-E₂ binding capacity (“processing”); the strong antiestrogen methylhydroxytamoxifen (MHT) is also effective but with a ～ 100 fold lower efficiency. Parallel immunological measurement of estrogen receptor contents of the cells (ER-EIA from Abbott) revealed that the mechanisms by which these ligands operate are not of the same nature. Thus, while E₂ produced a loss of the ER peptide, MHT increased it; indicating an accumulation of a non-binding form of the receptor under its treatment. Measurement of the binding capacity of the cells for ³H-ORG 2058 showed a decrease of PgR concentration after preincubation with MHT which contrasted with the classical E₂-induced increase of the receptor. MHT at relatively low concentrations also antagonised the E₂-induced decrease of ³H₂ binding capacity; this property did not result from a difference in chemical structure between the ligands since bisphenol a weak estrogenic analogue of MHT failed to show a similar antagonistic activity. This property conferres to MHT the ability to reduce the efficiency of E₂ to induce PgR. Finally, actinomycin D a known antagonist of the E₂-induced processing was found to be totally ineffective towards the MHT processing. This clearly confirmed that the term “processing” covers at least two distinct mechanisms.     

Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine

A large number of oocytes are contained in the mammalian ovary. A very small number of these oocytes grow to the final size, mature, and are ovulated. In the ovary there are more early antral follicles than late antral or preovulatory follicles, offering a large pool of oocytes for IVM and IVF if appropriate culture conditions could be devised. In the present study, early antral follicles containing oocytes 90 to 99 microm in diameter were isolated from bovine ovaries. Cumulus-oocyte complexes (COC) with pieces of parietal granulosa (COCG) were then dissected from the follicles. The COCGs were embedded in collagen gels and cultured in Medium 199 with 10% fetal calf serum (FCS) for 8 d. In Experiment 1, the effect of hypoxanthine and FSH on the growth of bovine oocytes was examined. When hypoxanthine (2 and 4 mM) and FSH (10 ng/ml) were added to the culture medium, the number of granulosa cell-enclosed oocytes increased significantly (P < 0.05). All of the oocytes surrounded by granulosa cells showed a normal morphology and were at the germinal vesicle stage, while 75 to 94% of the denuded oocytes were degenerated and had resumed meiosis. The mean diameter of the oocytes showing normal morphology was significantly higher than that measured before culture (P < 0.05). In Experiment 2, the maturational competence of in vitro-grown bovine oocytes was examined. Oocytes which were 90 to 99 microm in diameter before culture did not have meiotic competence. After being in a growth culture of 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 7 or 11 d, granulosa cell-enclosed oocytes were recovered from the COCGs. No significant difference (P < 0.05) in the diameters of the oocytes was observed between 7 and 11 d of culture (7 d: 107.5 +/- 6.1 microm, n = 30; 11 d: 108.0 +/- 5.3 microm, n = 35). After a subsequent 24 h in a maturation free of hypoxanthine and FSH medium, only 17% of the oocytes cultured for 7 d underwent germinal vesicle breakdown. On the other hand, 89% of the oocytes cultured for 11 d underwent germinal vesicle breakdown, and 11% of the oocytes emitted the first polar body and reached metaphase II. These results demonstrate for the first time that bovine oocytes harvested from early antral follicles can grow, and acquire meiotic competence in vitro.