Nuclei Of Granulosa Cells Research Articles

Ultrastructural localisation of calcium deposits in the mouse ovary.

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate-pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development.

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.

Stage-specific expression of Smad2 and Smad3 during folliculogenesis.

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.

Androgen receptor and follicle-stimulating hormone receptor in the pig ovary during the follicular phase of the estrous cycle.

Follicle-stimulating hormone (FSH) is an important regulator of follicular development. Some effects of FSH on ovarian follicles might be enhanced by androgens. The main objectives of the present study were to examine expression of the androgen receptor (AR) and FSH receptor (FSHR) in late developing follicles in pigs. Ovaries were collected from gilts on days 13, 15, 17, and 19 of the estrous cycle (day 0 = first day of estrus, n = 4 gilts/day), a period coincident with the follicular phase. One ovary was processed for immunohistochemistry (IHC) of AR. Samples of surface wall from the largest follicles (4-5 per gilt) were dissected from the other ovary, pooled and processed for determination of AR and FSHR mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Intense AR immunostaining was present in nuclei of granulosa cells of preantral and antral follicles. AR immunoreactivity was also present in the nuclei of oocytes. Weak staining for AR was observed in cells of the theca interna, ovarian surface epithelium, and in most cells of the ovarian stroma. Relative amounts of immunoreactive AR in granulosa cells of late developing follicles, or small antral follicles (< 2 mm), did not differ between days 13, 15, 17, and 19. However, amounts of AR in granulosa cells of small antral follicles was greater (P < 0.05) than in the largest follicles present in the same ovary. The relative amounts of AR mRNA in tissue from the largest follicles on days 13, 15, 17, and 19 did not differ; however, amounts of FSHR mRNA in the same follicles were not different between days 13, 15, and 17, but decreased (P < 0.05) by day 19. Results indicate that during the follicular phase in gilts, the AR protein is mainly present in granulosa cells. Relative amounts of AR protein in granulosa cells and mRNA in walls of late developing follicles did not significantly change from day 13 to 19; however, amounts of FSHR mRNA decreased in preovulatory follicles by day 19 of the estrous cycle.

Possible direct cytoxicity effects of cyclophosphamide on cultured human follicles: an electron microscopy study.

To evaluate the direct effect of cyclophosphamide on cultured human ovarian follicles. Human ovarian cortical slices from premenopausal women were incubated with medium containing cyclophosphamide (0.0005-0.5 mg/mL) for 2-48 h and assessed by transmission electron microscopy. Noncultured specimens and samples cultured without cyclophosphamide were used as controls. There were significantly more damaged granulosa cell nuclei after incubation with 0.5 mg/mL cyclophosphamide for at least 4 h. There were also more changes in the basement membrane after incubation with cyclophosphamide at concentrations of 0.05 and 0.5 mg/mL. Although the cyclophosphamide dose that caused damage to the granulosa cell nuclei was above the pharmacological level, our results suggest that cyclophosphamide, and not only its active metabolite phospharamide mustard, might have a destructive effect on human follicles, as it remains in the circulation longer. This effect could be mediated by damage to the granulosa cells and perhaps the basement membrane.

Effect of adrenal and ovarian androgens on type 4 follicles unresponsive to FSH in immature mice.

The present study investigates the physiological significance of dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, androstenedione (Delta(4)), dihydrotestosterone (DHT), estrone (E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4 follicles obtained from immature mice. Type 4 follicles of a diameter of 100-120 microm with one or two granulosa cell layers around the oocyte and an intact basal lamina with theca cells were isolated from the ovaries of 11-d-old BDF-1 mice and cultured with medium alone (control) or with dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, Delta(4), DHT, E1, or E2 at concentrations ranging from 1 x 10(-11) to 1 x 10(-7) M for 4 d. We examined the mean diameters of type 4 follicles, levels of immunoreactive (IR)-inhibin, and E2 and progesterone in the culture media on day 4. In addition, we evaluated follicular cell proliferation by immunofluorescence staining with 5-bromo-2'-deoxyuridine. All tested androgens significantly increased the diameter of type 4 follicles in a dose-dependent manner without the production of IR-inhibin and E2. The nuclei of granulosa cells in type 4 follicles cultured with all tested androgens exhibited intense 5-bromo-2'-deoxyuridine-positive staining, compared with those of controls. In contrast, neither E1 nor E2 had any stimulatory effects. The stimulatory effects of T, Delta(4), or DHT were inhibited by an AR antagonist in a dose-related fashion but not by an aromatase inhibitor. Furthermore, all tested androgens had a synergistic effect with rhFSH on follicular growth and the production of IR-inhibin and E2. These results demonstrated that neither adrenal nor ovarian androgens are arteriogenic but that they stimulate type 4 follicles unresponsive to rhFSH and augment the responsiveness of these follicles to rhFSH.

Effect of Adrenal and Ovarian Androgens on Type 4 Follicles Unresponsive to FSH in Immature Mice

The present study investigates the physiological significance of dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, androstenedione (Δ4), dihydrotestosterone (DHT), estrone (E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4 follicles obtained from immature mice. Type 4 follicles of a diameter of 100–120 μm with one or two granulosa cell layers around the oocyte and an intact basal lamina with theca cells were isolated from the ovaries of 11-d-old BDF-1 mice and cultured with medium alone (control) or with dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, Δ4, DHT, E1, or E2 at concentrations ranging from 1 × 10−11 to 1 × 10−7m for 4 d. We examined the mean diameters of type 4 follicles, levels of immunoreactive (IR)-inhibin, and E2 and progesterone in the culture media on day 4. In addition, we evaluated follicular cell proliferation by immunofluorescence staining with 5-bromo-2′-deoxyuridine. All tested androgens significantly increased the diameter of type 4 follicles in a dose-dependent manner without the production of IR-inhibin and E2. The nuclei of granulosa cells in type 4 follicles cultured with all tested androgens exhibited intense 5-bromo-2′-deoxyuridine-positive staining, compared with those of controls. In contrast, neither E1 nor E2 had any stimulatory effects. The stimulatory effects of T, Δ4, or DHT were inhibited by an AR antagonist in a dose-related fashion but not by an aromatase inhibitor. Furthermore, all tested androgens had a synergistic effect with rhFSH on follicular growth and the production of IR-inhibin and E2. These results demonstrated that neither adrenal nor ovarian androgens are arteriogenic but that they stimulate type 4 follicles unresponsive to rhFSH and augment the responsiveness of these follicles to rhFSH.

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.

Expression of ER α and ER β in human granulosa-lutein cells.

Expression of ER α and ER β in human granulosa-lutein cells.

Immunocytochemical localization of estrogen receptors alpha and beta in the human reproductive organs.

To better define the role of estrogens in reproductive functions, we have proceeded to the immunocytochemical localization of the estrogen receptor (ER) subtypes, ERalpha and the recently discovered ERbeta+, in human reproductive tissues. In the ovary, ERbeta+ immunoreactivity was found in nuclei of granulosa cells of growing follicles at all stages from primary to mature follicles, interstitial gland, and germinal epithelium cells. Nuclear staining for ERalpha occurred in thecal, interstitial gland, and germinal epithelium cells. In the uterus, strong ERalpha immunoreactivity was detected in nuclei of epithelial, stromal, and muscle cells. Similar localization was obtained for ERbeta+, although the staining was much weaker. In the vagina, only ERalpha could be detected; a nuclear reaction was observed in deep layers of the stratified epithelium as well as in stromal and muscle cells. In the mammary gland, both ER subtypes were observed in epithelial and stromal cells. In the testis, ERbeta+ was detected in nuclei of Sertoli and Leydig cells, whereas ERalpha immunoreactivity was only observed in Leydig cells, with no tubular labeling. In the efferent ducts, only ERbeta+ could be detected, whereas neither ERbeta+ nor ERalpha could be found in the epididymis. In the prostate, ERbeta+ nuclear immunolabeling was observed in both basal and secretory cells in alveoli as well as in stromal cells, whereas ERalpha could not be detected. The present results demonstrate that there is a cell-specific localization for each of the ER subtypes in the majority of the reproductive organs studied. Moreover, they contribute to establish the exact sites of action of estrogens in male and female human reproductive systems.

Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat

Atresia, a degenerative process through which many follicles are removed from the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in-vivo rat model was used which allowed the study of atresia of pre-ovulatory follicles in a synchronized and chronological order. By blocking the pre-ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, after which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles. Atresia involves apoptosis of granulosa cells, and therefore internucleosomal DNA fragmentation was examined. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulatory follicles was detectable 24 h after GnRH antagonist treatment. In order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of proliferation. Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulosa cells.

Culture of bovine oocytes from small antral follicles in meiosis-inhibiting medium with butyrolactone I: RNA synthesis, nucleolar morphology and meiotic competence

Bovine oocytes originated from follicles of two different size categories (medium (M), 3–6mm and small (S), 1–2mm) were cultured for 24 and 70h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100μM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (++++, +++) of the GV. (2) When the COC were labeled after 24 and 70h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (+++, ++) after 24h and negligible labeling after 70h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48h. The subsequent 24h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.

Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells.

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs.

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.

Immunolocalisation of oestrogen receptor beta in human tissues.

Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.

Effects of Gonadotropin-Releasing Hormone Agonist on the Incidence of Apoptosis in Porcine and Human Granulosa Cells

Gonadotropin-releasing hormone (GnRH) has been found to be expressed in ovaries of various species and to modulate cell differentiation in ovarian cells. GnRH agonists (GnRHa) are widely used in the stimulation protocols of assisted reproduction. In the present study, the direct effects of a GnRHa on the incidence of apoptosis and sterodogenesis in porcine in human granulosa cells were investigated. Cells were obtained from 6-month-old swine and in vitro fertilization patients, and cultured in minimum essential medium (MEM) supplemented with 5% fetal bovine serum for 24 h. The GnRHa (burserelin acetate) was added to the MEM at various concentrations (0, 5, 50, 500 pg/ml, and 5 ng/ml). The nuclei of granulosa cells were examined by fluorescence microscopy after Hoechst 33258 staining, and the incidence of apoptotic cells was tabulated. The GnRHa directly increased the incidence of apoptosis in both types of granulosa cells. The concentration of GnRHa in clinical use is much higher than the concentration which produced apoptosis in granulosa cells here. The clinical dosage of GnRHa should be reconsidered with regard to its apoptosis-inducing effect.

Effects of aging on ovarian fecundity in terms of the incidence of apoptotic granulosa cells.

The objective was to determine the effects of women's age on the ovarian fecundity as assessed by the incidence of apoptotic granulosa cells. Twenty-eight normo-ovulatory women underwent ovulation induction for standard IVF. The husbands of these women showed severe male infertility factors. The women were divided into four groups according to their ages. Women underwent follicle aspiration after the administration of human menopausal gonadotropin plus human chorionic gonadotropin. The nuclei of granulosa cells were examined by using fluorescence microscopy, and the incidence of apoptotic granulosa cells was tabulated. Granulosa cells in the older women revealed a significant increase in the number of apoptotic cells. The number of total oocytes and the number of mature oocytes obtained significantly decreased with age. However, endometrial thickness and follicular estradiol, progesterone, and free testosterone levels were not significantly different among four different age groups. Age increases apoptotic changes in granulosa cells and consequently decreases the ovarian fecundity.

P53, c-erbB, and Ki-67 expression in ovaries removed prophylactically from women with a family history of ovarian cancer.

Prophylactically removed ovaries from 64 women were compared with those from 30 women with no known family history of ovarian cancer for expression of the p53 tumor suppressor protein, the c-erbB-2 oncoprotein, and for the proliferation antigen Ki-67. All analyses were performed without knowledge of the family history. Ki-67 was expressed in rare nuclei in both the surface and cyst epithelial cells, whereas p53 was expressed in rare nuclei only in cyst epithelial cells. Neither the proportion of positive cases nor the intensity of staining differed between groups. c-erbB2 was not expressed in surface or cyst epithelium in any case from either group. Nuclei of granulosa and granulosa lutein cells expressed both Ki-67 and p53. In conclusion, increased expression of p53, c-erbB2, and Ki-67 was not found in the epithelium of prophylactically removed ovaries, suggesting that increased expression occurs later in the development of carcinoma or invasive tumor evolves too quickly to identify expression of these proteins in preinvasive epithelium.

Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin

The expression and function of estrogen receptor ERα/β subtypes and ERβ variants in granulosa cells have been determined using several integrated approaches: , Western blotting, indirect immunofluorescence, RT-PCR, and transient transfection assays. Each of these approaches has provided specific details concerning the dynamics of ER expression, ER functional activity, and estradiol (E) regulation of target genes in granulosa cells. Specifically, the studies presented herein document that messenger RNAs (mRNAs) encoding ERβ and its splice variants, as well as mRNA encoding ERα, are expressed in granulosa cells of immature rats before and during culture in serum-free medium. The results also provide the first documentation that functional (DNA binding and transcriptionally active) ER is present in cultured granulosa cells and that its ability to bind consensus estrogen response element (ERE) oligonucleotide and to transactivate an ERE promoter-reporter construct is associated with the level (type?) of receptor protein as well as the stage of granulosa cell differentiation. Using a labeled ERE consensus oligonucleotide and antibodies specific for ERβ and ERα, we show that ERβ but not ERα was detected (supershifted in electrophoretic mobility shift assays) in extracts of granulosa cells cultured overnight (0 h) in defined medium alone. When the cells were cultured with FSH and testosterone (T) to stimulate their differentiation, ERβ binding activity, as well as immunoreactive ERβ as determined by Western blot analyses, decreased progressively from 24 to 48 h and was undetectable by 72 h. ERβ mRNA was low, and ERβ binding activity was not observed in luteinized granulosa cells. ERα DNA binding activity was not observed in any of the granulosa cell cultures, although low levels of immunoreactive ERα were detected by Western blot analyses. Immunofluorescent analyses documented that ERβ, as well as ERα, were localized to granulosa cell nuclei and that the intensity of nuclear staining was related to agonist stimulation and differentiation: forskolin increased, whereas E decreased immunostaining for ERβ and ERα at 48 h. When an ERE-E1b-luciferase vector was transfected into granulosa cells of unprimed rats, basal luciferase activity was low but increased by forskolin (3–4×) and by E (2×), responses to both agonists being blocked by the ER antagonist, ICI. When the same vector was transfected into differentiated granulosa cells (cultured for 48 h with FSH/T), forskolin alone increased activity. Collectively, these results show that ERβ protein is preferentially expressed in immature granulosa cells, is functionally active (binds DNA), can transactivate (either as a homodimer or heterodimer with ERα) ERE-containing promoter constructs, and might be associated with increased expression of the endogenous gene encoding c-Jun.