Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.     

Relationship of Fas ligand expression and atresia during bovine follicle development

The Fas antigen (Fas) is a cell surface receptor that may be involved in the initiation and progression of follicle cell apoptosis during atresia. Fas initiates apoptosis in sensitive cells after binding Fas ligand (FasL). Other experiments have shown that expression of Fas mRNA and responsiveness to Fas-mediated apoptosis vary in bovine granulosa and theca cells during follicle development. In the present study, FasL mRNA content was measured and Fas and FasL protein expression was examined in bovine granulosa and theca cells of healthy dominant follicles and the two largest atretic subordinate follicles on day 5 of the oestrous cycle (day 0 = oestrus), and of dominant follicles from the first wave of follicle development after they had become atretic and showed no growth for 4 days. FasL mRNA content was higher in granulosa cells from atretic compared with healthy follicles. FasL mRNA content was also higher in theca cells from atretic subordinate compared with healthy dominant follicles on day 5, but did not differ between theca cells from healthy and atretic dominant follicles. Immunohistochemical staining for FasL was more intense in theca compared with granulosa cells and in atretic compared with healthy follicles. Immunohistochemical staining for Fas was more intense in granulosa compared with theca cells and in atretic subordinate compared with healthy dominant follicles on day 5. Immune cells, known to express Fas and FasL, were localized in the theca, but not the granulosa, cell layer of all follicles. Higher concentrations of Fas and FasL in cells from atretic follicles, together with the previous demonstration of increased responsiveness of granulosa cells from subordinate follicles to FasL-induced apoptosis, support a potential role for FasL-mediated apoptosis during ovarian follicle atresia.     

Transforming growth factor-beta: its role in ovarian follicle development

Ovarian follicular growth and differentiation in response to transforming growth factor-beta (TGFB) was investigated using postnatal and immature ovarian models. TGFB ligand and receptor mRNAs were present in the rat ovary 4-12 days after birth and at day 25. In order to assess the impact of TGFB1 on follicle growth and transition from the primordial through to the primary and preantral stages of development, we established organ cultures with 4-day-old rat ovaries. After 10 days in culture with FSH, TGFB1, or a combination of the two, ovarian follicle numbers were counted and an assessment of atresia was undertaken using TUNEL. Preantral follicle numbers declined significantly when treated with the combination of FSH and TGFB1, consistent with our morphological appraisal suggesting an increase in atretic primary and preantral follicles. To investigate the mechanisms behind the actions of TGFB1, we isolated granulosa cells and treated them with FSH and TGFB1. Markers of proliferative, steroidogenic, and apoptotic capacity were measured by real-time PCR. Cyclin D2 mRNA expression by granulosa cells was significantly increased in response to the combination of FSH and TGFB. The expression of forkhead homolog in rhabdomyosarcoma (Foxo1) mRNA by granulosa cells was significantly reduced in the presence of both FSH and TGFB1, individually and in combination regimes. By contrast, the expression of steroidogenic enzymes/proteins was largely unaffected by TGFB1. These data suggest an inhibitory role for TGFB1 (in the presence of FSH) in follicle development and progression.     

Hormonal regulation and differential expression of neuropilin (NRP)-1 and NRP-2 genes in bovine granulosa cells

Although much is known about the biology of vascular endothelial growth factor (VEGF) and its receptors, little is known about the role of the VEGF receptors neuropilin (NRP)-1 and NRP-2 in the process of bovine follicle development. The aim of the present study was to examine the hormonal regulation of NRP-1 and NRP-2 mRNAs by real-time PCR in follicles from the bovine ovary and in cultured granulosa cells. The NRP-1 gene was expressed in the granulosa and theca cells in the post-selection (POF) and pre-selection (PRF) follicles in the bovine ovary. In contrast, the NRP-2 gene was expressed only in the theca cells in the POF and the PRF. The level of NRP-1 mRNA was significantly increased by treatment of the cultured granulosa cells with 10 ng/ml estradiol (E2). In contrast, the addition of progesterone (P4) to the culture medium decreased the expression of the NRP-1 gene. The level of NRP-1 mRNA was increased by 10 ng/ml E2 with or without 1 ng/ml P4, but the level of NRP-1 mRNA was decreased if the P4 level was increased to 10 ng/ml, even when 1 ng/ml E2 was also added. Follicle-stimulating hormone did not stimulate the expression of the NRP-1 gene. These results are the first data showing that NRP-1, but not NRP-2, is expressed in the granulosa cells of bovine follicles and that NRP-1 gene expression is regulated by sex steroids. Our findings suggest the involvement of NRP-1 in follicle development in the cow.     

FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E(2)) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7-8?mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3?mm for 0, 0.1, and 1?μg/ml FGF10, respectively, at 72?h after treatment; P<0.05). In a third experiment, follicles were obtained 24?h after FGF10 (1?μg/ml) or PBS treatment through ovariectomy. In theca cells, FGF10 treatment did not affect mRNA encoding steroidogenic enzymes, LHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E(2) production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.     

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta ) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, BMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. In addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.     

Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.     

Differentially expressed plasma microRNAs in premature ovarian failure patients and the potential regulatory function of mir-23a in granulosa cell apoptosis

Recent studies implicate the regulatory function of microRNAs (miRNAs) in oocyte maturation and ovarian follicular development. Differentially expressed miRNAs are found in the plasma of premature ovarian failure (POF) patients and normal cycling women. In this study, miRNA-regulated signaling pathways and related genes were described using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The effect of mir-23a on granulosa cell apoptosis was also studied by examining the protein expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3, followed by subsequent counting of apoptotic cells after Hoechst 33258 staining. Both GO analysis and pathway analysis suggested that many signaling pathways, including the AKT signaling pathway, steroid hormone receptor signaling pathways, and others, were regulated by this group of differentially expressed miRNAs. A decrease in XIAP expression (mRNA and protein level) and caspase-3 protein levels and an increase in cleaved caspase-3 protein were observed in human ovarian granulosa cells transfected with pre-mir-23a, along with an increased occurrence of apoptosis. In conclusion, differentially expressed miRNAs in the plasma of POF patients may have regulatory effects on proliferation and apoptosis of granulosa cells by affecting different signaling pathways. Mir-23a may play important roles in regulating apoptosis via decreasing XIAP expression in human ovarian granulosa cells.     

Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation

During atresia of bovine follicles, granulosa cells are lost through the controlled form of cell death, apoptosis. The purpose of this study was to characterize the regulation of apoptotic death of granulosa cells in dominant bovine follicles during the first wave of follicular development. Dominant follicles were collected from Holstein heifers on days 4, 6 or 8 of the first follicular wave (n = 5/day). Regulation of apoptosis in granulosa cells was examined by annexin V and propidium iodide staining; measurement of relative levels of mRNA encoding Bcl-2, Bcl-xL and Bax; and activity of caspase-3, -8 and -9. Steady-state levels of mRNA encoding four oxidative stress-response proteins were determined. Compared with day 4, the incidence of apoptotic and nonviable granulosa cells tended to increase on day 6, and numbers of nonviable cells were higher on day 8. The ratios of relative levels of mRNA encoding Bcl-2 to Bax and Bcl-xL to Bax were higher on day 6 than days 4 and 8. Activity of caspases-3 and -9 in granulosa cells did not change among the 3 days, while caspase-8 activity decreased on day 8 compared with days 4 and 6. Amounts of GSHPx, MnSOD and Cu/ZnSOD mRNA in granulosa cells were higher on day 8 than day 6. In theca interna, amounts of Cu/ZnSOD mRNA decreased between days 4 and 6. From the decreased production of estradiol and increased numbers of apoptotic and nonviable granulosa cells, we conclude that atresia of the dominant follicle is initiated between days 4 and 6 of the first follicular wave. However, apoptosis of granulosa cells does not appear to be initiated by changes in expression of oxidative stress-response proteins.     

Implication of cortisol and 11beta-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts

This study investigated cortisol inactivation by 11beta-hydroxysteroid dehydrogenase (11beta HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP(+)-dependent 11beta-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11beta HSD, this studyalso investigated whether levels of 11beta HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11beta HSD1 activity in rat kidney homogenates decreased progressively from 50 +/- 8% inhibition by FF from small antral follicles (P < 0.001) to 23 +/- 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11beta HSD1 activity by 59 +/- 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35-48% inhibition, P < 0.05) and cyst fluid (45-75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11beta HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11beta HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.     

Hormonal regulation of apoptosis in rabbit granulosa cells in vitro: evaluation by flow cytometric detection of plasma membrane phosphatidylserine externalization

Annexin V and propidium iodide bivariate analysis and the TUNEL method were used to quantify hormonal regulation of apoptosis in rabbit granulosa cells from preovulatory follicles in vitro. The aim of this study was to analyse comparatively the effects of gonadotrophins and their second messenger in the regulation of granulosa cell apoptosis in (i) cultured isolated granulosa cells and (ii) granulosa cells scraped from cultured follicles. The results showed that increasing doses of FSH had no effect on apoptosis of cultured isolated cells but caused a decrease in the number of apoptotic granulosa cells from preovulatory follicles cultured in serum-free conditions. Unlike FSH, addition of hCG did not modify apoptosis of granulosa cells significantly. In contrast, dibutyryl cAMP had an apoptotic effect in the two cellular models in the presence of serum. Moreover, a biphasic effect of dibutyryl cAMP in isolated granulosa cells was observed with an increase in the incorporation of [(3)H]thymidine into DNA at the lowest dose and an increase in apoptotic cell death at the highest dose. It was concluded that, in rabbits: (i) FSH requires follicle integrity to exert its anti-apoptotic effect in granulosa cells; (ii) dibutyryl cAMP induces a dose-dependent apoptotic effect in granulosa cells cultured alone or obtained from cultured preovulatory follicles; and (iii) cAMP signals induce opposite effects on growth and apoptosis in granulosa cells.     

Developmental expression and cellular distribution of Mullerian inhibiting substance in the primate ovary

Ovarian follicle formation during development and follicle maturation in adulthood are crucial determinants of female fertility and disruptions in these processes may result in subfertility or infertility. Among the several factors that are involved in ovarian physiology, Müllerian inhibiting substance (MIS) also known as anti-Müllerian hormone has emerged as an important marker to predict the follicle reserve. However, the roles of MIS in human ovarian physiology are unknown. To gain an insight into the potential roles of MIS in human ovarian differentiation during development and its regulation in adulthood, the expression profiles of MIS mRNA in the developing and adult human and monkey ovaries was examined by in situ hybridization. The results revealed that in the fetal human ovaries, MIS is specifically expressed at low levels in the granulosa cells of the developing primordial follicles; a small subset (approximately 2-3%) of oocytes express high amounts of MIS. In the adult human and monkey ovary, MIS mRNA is expressed at low levels in the primordial follicles, maximally in the primary and secondary follicles, and the expression is downregulated in the antral and atetric follicles. MIS expression is extinguished in the granulosa cells only after ovulation. These observations strongly favor the regulatory roles of MIS in folliculogenesis. MIS in the primate ovary may exert its effect during the primordial follicle formation to the terminal granulosa cell differentiation. The presence of MIS in a small subset of oocytes in the fetal ovary further points towards its additional role during fetal oocyte development.     

Oestrogen receptor alpha and beta, androgen receptor and progesterone receptor mRNA and protein localisation within the developing ovary and in small growing follicles of sheep

A first step to elucidating the roles that steroids may play in the processes of ovarian development and early follicular growth is to identify the cell types that are likely to be receptive to steroids. Thus, cell types expressing receptors for oestrogen (alpha and beta form; ERalpha and ERbeta respectively), androgen (AR) and progesterone (PR) were determined by in situ hybridisation and immunohistochemistry in ovine ovarian tissues collected during ovarian development and follicular formation (days 26-75 of fetal life) as well as during the early stages of follicular growth. Expression of ERbeta was observed early during ovarian development and continued to be expressed throughout follicular formation and also during the early stages of follicular growth. ERbeta was identified in germ cells as well as in the granulosa cells. At the large preantral stage of follicular growth, expression of ERalpha was also consistently observed in granulosa cells. AR was first consistently observed at day 55 of fetal life in stroma cells throughout the ovary. Within the follicle, expression was observed in granulosa and thecal cells from the type-2 to -3 stage of follicular growth. PR mRNA did not appear to be expressed during ovarian development (days 26-75 of gestation). However, PR (mRNA and protein) was observed in the theca of type-3 (small preantral) and larger follicles, with mRNA -- but not protein -- observed in granulosa cells of some type-4 and 5 follicles. Expression of ERbeta, ERalpha and AR, as well as PR, was also observed in the surface epithelium and ovarian stroma of the fetal, neonatal and adult ovary. Thus, in sheep, steroid hormones have the potential to regulate the function of a number of different ovarian cell types during development, follicular formation and early follicular growth.     

Incidence of apoptosis in granulosa cells from immature human follicles

The aim of this study was to investigate the incidence of apoptosis in granulosa cells from immature human follicles undergoing in vitro maturation (IVM) and to compare the incidence of apoptotic granulosa cells (i) between FSH-primed and unprimed normal ovaries and (ii) between polycystic and normal ovaries. Furthermore, the incidence of apoptosis was related to maturation and subsequent fertilization and cleavage of the oocytes from the corresponding ovary. Seventy women undergoing 70 IVM cycles were included. Group 1 consisted of patients with normal ovaries (n = 52) and group 2 consisted of patients with polycystic ovaries (n = 18). Patients in group 1 were subdivided into two groups according to priming with FSH before aspiration. In group 1a (n = 27 cycles) oocytes were obtained in unstimulated cycles. In group 1b (n = 25 cycles) oocytes were obtained after priming with recombinant FSH for 3 days initiated on day 3 after spontaneous menstruation. In group 2 all patients were primed with recombinant FSH for 3 days before aspiration. Aspiration was performed transvaginally and cumulus-enclosed oocytes were matured for 28-30 h before fertilization. Granulosa cells were collected from follicular aspirates. An APOPTAG detection kit was used to stain the granulosa cells and to detect apoptosis. The incidence of apoptosis in granulosa cells was decreased in follicles from FSH-primed normal ovaries compared with follicles from unprimed normal ovaries and FSH-primed polycystic ovaries. No difference was found between granulosa cells from FSH-primed polycystic ovaries and granulosa cells from unstimulated normal ovaries. No differences in maturation rate, fertilization rate, cleavage rate and implantation rate were observed when oocytes from a polycystic ovary were compared with oocytes from an unstimulated normal ovary. In unstimulated cycles, the ovaries were grouped according to the presence of a dominant follicle. The incidence of apoptosis was significantly higher in granulosa cells from an ovary without a dominant follicle compared with granulosa cells from an ovary with a dominant follicle. The rates of maturation, fertilization and cleavage did not differ between the two groups.     

Potential local regulatory functions of inhibins, activins and follistatin in the ovary

The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.     

Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 degrees C, 50% RH; heat stress: 35 degrees C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.     

灵芝总皂苷对结肠癌HCT116细胞的抑制作用

为探究灵芝总皂苷提取物对结肠癌HCT116细胞的抑制作用和相关机制，以灵芝子实体为原料，醇提并纯化总皂苷，通过CCK8法测定灵芝总皂苷提取物对结肠癌HCT116细胞的抑制效果，同时采用流式细胞术检测细胞凋亡，荧光分光光度计检测细胞内线粒体膜电位及细胞内活性氧含量，实时荧光定量PCR检测凋亡相关基因p53、noax、bax、bcl-2、caspase-9和caspase-3的表达。结果显示，与空白组相比，灵芝总皂苷质量浓度为40 mg/L以上时，可极显著地抑制HCT116细胞增殖，且抑制效果与时间呈正相关。经灵芝总皂苷干预后，细胞凋亡率显著增加，当加入灵芝总皂苷的质量浓度为150 mg/L时，其凋亡率达到50.2%，且灵芝总皂苷可显著降低细胞内线粒体膜电位，增加细胞内活性氧含量，增加促凋亡基因p53、noax、bax、caspase-9和caspase-3的表达，降低抗凋亡基因bcl-2的表达。综上，灵芝总皂苷对于结肠癌细胞具有显著抑制作用，且其抑制效果可通过线粒体凋亡途径实现，是值得进一步研究的抑结肠癌候选药物和食品添加剂。     

Interactive effects of granulosa cell apoptosis, follicle size, cumulus-oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus-oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8-16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73-84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.