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Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.  相似文献   

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The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.  相似文献   

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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.  相似文献   

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Satisfactory development of bovine follicles in vitro remains elusive. This study used a serum-free system to evaluate the effects of insulin-like growth factor-1 (IGF-1) on bovine preantral follicles in culture and to identify the activity of gelatinase matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in vitro to assess their potential as markers of development. Preantral follicles were cultured for 6 days in serum-free medium containing insulin and IGF-1 (10 ng/ml). No difference was observed in follicular growth, health, or antrum formation between IGF-1-treated follicles and controls. However, IGF-1 had a negative effect (P < 0.01) on oocyte size and granulosa cell proliferation. When MMP-9 was secreted, the probability of follicles having healthy granulosa or theca cells at the end of the culture period was 0.85 and 0.60, respectively. If TIMP-1 was released, the probability of follicles having healthy somatic cells was 0.79. When TIMP-2 was detected, the probability of granulosa and theca cell health was 0. 78 and 0.67, respectively. These results demonstrate no positive effects of IGF-1 on bovine follicles in this system. Furthermore, MMP-9 and TIMPs are related to follicular health and, therefore, can be used as markers of follicular development.  相似文献   

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Paracrine regulations between the oocyte and granulosa cells are likely to be key regulators of early folliculogenesis. Evidence obtained from genetic mutants as well as in vivo experiments suggest that Kit and Kit Ligand (KL) may regulate early follicular morphogenesis and function. In this study, we used in vitro culture of intact mouse follicles to confirm and extend these findings. Two concentrations of Kit Ligand (20 and 50 ng/ml) or an antibody blocking the Kit-Kit Ligand interactions (SC1494) were added to preantral follicles grown individually for 12 days and which were finally triggered to ovulate. Effects on follicle and oocyte survival, granulosa cell function (antrum formation, cell numbers, steroidogenesis), and oocyte function (growth, survival, nuclear and/or cytoplasmic maturation) were then analyzed. In optimal culture conditions (presence of 5% fetal calf serum), 50 ng/ml of KL significantly improved cytoplasmic maturation of the oocyte and increased follicular testosterone output, but other parameters were not altered. In serum-free culture conditions, KL was mitogenic for granulosa cells at 50 ng/ml, but could not induce antrum formation and no differences were observed between control and treated groups for steroidogenesis or oocyte growth. Blockade of Kit-Kit Ligand interactions by addition of a blocking antibody decreased oocyte survival 6-9 days after addition of the antibody, but did not upset granulosa cell proliferation. Antrum formation was, however, strongly inhibited. In addition, the blocking antibody markedly reduced aromatase activity of granulosa cells. We conclude that Kit/KL interactions are important for antrum formation and follicular steroidogenesis and regulate survival and cytoplasmic maturation of the oocyte.  相似文献   

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Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.  相似文献   

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Choi J  Lee B  Lee E  Yoon BK  Bae D  Choi D 《Cryobiology》2008,56(1):36-42
Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.  相似文献   

13.
Characterization of integrin expression in the mouse ovary   总被引:7,自引:0,他引:7  
Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.  相似文献   

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This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.  相似文献   

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

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While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.  相似文献   

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Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.  相似文献   

18.
In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
Within the ovary, Estrogen Receptor β (ERβ) is localized to the granulosa cells of growing follicles. 17β-estradiol (E2) acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERβ-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b) that ERβ regulates expression of ECM components in the mouse ovary.  相似文献   

20.
The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.  相似文献   

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