Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.     

A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.     

Leptin directly controls secretory activity of human ovarian granulosa cells: possible inter-relationship with the IGF/IGFBP system

AIMS: The aim of our in vitro studies was to understand the role of leptin and the insulin-like growth factor I/insulin-like growth factor protein (IGF/IGFBP) system in controlling human ovarian function. METHODS: We studied the action of leptin (0, 1, 10, or 100 ng/ml) and immunoneutralization of IGF-I using specific antiserum (0.1%) on the release of progesterone (P), estradiol (E), oxytocin (OT), IGF-I, IGFBP-3, and prostaglandins F (PGF) by these cells using radioimmunoassay/immunoradiometric assay. RESULTS: It was found that leptin stimulated the secretion of OT, IGFBP-3, and PGF. It suppressed the secretion of E and IGF-I, but not P, into the medium. The addition of antiserum against IGF-I decreased IGF-I output, increased P, OT, IGFBP-3, and PGF secretion, and had no effect on E release. Immunoneutralization of IGF-I also prevented or reversed the effects of leptin on P, E, IGF-I, IGFBP-3, PGF, but not on OT. CONCLUSIONS: These observations (1) demonstrate that leptin directly controls the secretory activity of human ovarian cells, (2) confirm the involvement of IGF-I in the regulation of ovarian cells, and (3) suggest an inter-relationship between leptin and the IGF/IGFBP system in the control of these functions and the involvement of IGF/IGFBP system in mediating leptin action on the ovary.     

Subpopulations of granulosa cells within the human ovarian follicle

Human follicular cells were separated according to their isopycnic densities. Three populations were isolated and identified in terms of their secretion of progesterone and oestradiol. Cells in the least dense population secreted approximately 60% as much progesterone and 20% as much oestradiol per cell as did cells in the two denser bands. It is proposed that cumulus cells compose the least dense band and that another band may be made up of antral cells.     

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

The effect of obestatin on porcine ovarian granulosa cells

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P(4)), testosterone (T) and estradiol (E(2)) was measured by EIA. Addition of obestatin (1-100ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100ng/ml, obestatin promoted the secretion of P(4), but not T or E(2). Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.     

人卵巢颗粒细胞的体外培养方法探讨

目的建立人卵巢颗粒细胞分离纯化、体外培养的有效方法。方法收集体外受精—胚胎移植(IVF-ET)穿卵时的卵泡液,用胰蛋白酶消化法及密度梯度离心法分离纯化颗粒细胞并用不同培养基进行培养。结果用体积分数为50%的Percoll细胞分离液分离,DMEM/F12或McCoy’5a液体培养基进行培养,细胞纯度高,存活率高,后续生长良好。结论建立了人卵巢颗粒细胞体外培养的稳定模型,为颗粒细胞的体外研究奠定良好的基础。     

The distribution of actin in cultured ovarian granulosa cells

Ovarian granulosa cells grown on glass coverslips were split by a "sandwich" technique. Using this technique we describe a complex filamentous network in the cytoplasm of cultured granulosa cells that was composed of a branching and anastomosing lattice of filaments 20-40 nm in diameter. Since filament identification is impossible on the basis of size, split cells were decorated with S-1 fragments of rabbit skeletal muscle myosin. It was readily apparent that the major constituent of the filamentous lattice was actin. Actin was organized in large bundles in which individual filaments were longitudinally aligned. Actin was also observed organized in a loose network throughout the remainder of the cytoplasm. Actin appeared to be intimately associated with organelle and plasma membranes. Coated pits were also a site of actin-filament interaction. Filament polarity was generally away from the membrane with which filaments were associated.     

Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines

In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIP(S) and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia.     

Secretion of oestrogen from murine-induced pluripotent stem cells co-cultured with ovarian granulosa cells in vitro

POF (premature ovarian failure) is a distressing condition that is a common cause of infertility. No effective treatment is available to overcome the loss of fertility. A method to derive oestrogen from miPSCs (mouse-induced pluripotent stem cells) was explored as a potential treatment for POF. In this study, C57BL/6 female mice were injected with PMSG (pregnant mare's serum gonadotropin) to obtain ovarian GCs (granulosa cells) and then co-cultured with miPSCs. The morphological changes in the miPSCs co-cultured with GCs were observed by light microscopy. The expression of FSHR (follicle-stimulating hormone receptor) was detected by immunocytochemistry and flow cytometry. Radioimmunoassay was used to analyse the level of E2 (oestradiol) in culture supernatants. The results showed that the proportion of GCs expressing FSHR in GCs was over 90%. The E2 concentration of the culture supernatant of the GC group was 62.4 pg/ml on day 1 and decreased in a time-dependent manner. The opposite situation was observed in the miPSCs-GC co-cultured group with an E2 concentration of 87.9 pg/ml on day 1 that increased in a time-dependent manner to reach a concentration of 328.4 pg/ml on day 7. The data indicate that GC-like cells were effectively induced from miPSCs through indirect cell-to-cell contact. Our method provides a novel in vitro system to study miPSC differentiation, particularly the interactions between miPSCs and GCs. The ultimate goal of this approach would be to provide a treatment for POF in the future.     

Androgen-induced changes in rat ovarian granulosa cells in vitro

Ovarian granulosa cells collected from small antral follicles from immature rats were cultured in McCoy's 5A medium, for 1-6 days in the presence of delta 4-androstenedione, testosterone, dihydrotestosterone, and dehydroepiandrosterone (10(-5) M and 10(-7) M). Granulosa cells examined by electron microscopy demonstrated many lipid droplets, mitochondria with tubular cristae and profiles of smooth endoplasmic reticulum, all suggestive of active metabolism in the cell. Cells cultured in androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone produced estrogen and progesterone as measured by radioimmunoassay. By day 4, cells cultured in androgen had almost completely degenerated. The control cells acquired none of the aforementioned characteristics and survived up to beyond 6 days, at which time the experiments were terminated. This study supports the hypothesis that high concentrations of androgens in cultured granulosa cells contribute to their degeneration through altered structure, which is associated with functional change.     

Morphological variations in gap junctions of ovarian granulosa cells.

Granulosa cells in ovarian follicles of rat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the intramembranous A face particles. In large gap junctions, ranging up to 6 mu in diameter, particles were packed in rectilinear arrays separated by a labyrinthine network of particle-free 'aisles'. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear intramembranous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical intracytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepreted as a means of disposal of junctions by interiorization.     

Apoptosis in ovarian cells in postmenopausal women

Apoptosis is a natural process which accompanies human ovary from the moment of birth until old age. While it is a well-known process at the reproductive age, it still needs to be thoroughly examined when referring to the postmenopausal age. The study involved 30 postmenopausal women who had their ovaries removed by laparotomy due to nonneoplastic diseases of the uterus. The women were divided into 3 groups depending on the time that had passed since the last menstruation. Group A consisted of women who had their last menstruation no more than 5 years earlier. In group B menopause occurred 5 to 10 years earlier. Group C was composed of patients who had the last menstruation over 10 years earlier. In all the patients concentrations of follitropin (FSH) and estradiol (E2) in blood plasma were measured. Ovarian tissue was obtained during surgery. For morphological studies, ovaries were fixed in Bouin's solution and 4% formalin and embedded in paraffin. Morphological analysis was carried out after hematoxylin-eosin (H-E) staining. For histochemical detection of apoptotic cells (in situ localization of fragment DNA), the TUNEL method was used. The expression of caspase-3 positive cells was determined immunohistochemically in paraffin-embedded specimens. Comparing to groups A and B, the ovaries in group C contained small number of corpora albicantia located in the medullary part as well as thinned blood vessels and few lymphatic vessels and nerves. In contrast to group A where the number of TUNEL-positive cells was high and caspase-3 expression was observed, no TUNEL-positive nuclei and caspase-3 expression were found in the examined ovaries of group C women.