The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of tumor and hematopoietic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, we demonstrated that human granulosa/luteal cells express the Fas antigen. An anti-human Fas antigen monoclonal antibody (Fas mAb; clone CH-11), which induces apoptosis in other cell types by binding to the Fas antigen, induced significant cell death (30%) in cultures pretreated with interferon gamma (IFN gamma). This agrees with studies on tumor cell lines showing that IFN gamma enhances cytotoxic effects of Fas mAb. Granulosa/luteal cells exhibited morphological characteristics typical of apoptosis, including membrane blebbing and condensed chromatin. DNA fragmentation into oligonucleosomal units of approximately 180 bp, typical of apoptosis, was detected at elevated levels in Fas mAb-treated cultures via 3' end-labeling and gel electrophoresis. Examination of cultured cells in situ for apoptotic DNA cleavage by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) indicated that more apoptotic death occurred in Fas mAb-treated cultures than in control cultures. Effects of hCG-induced luteinization of cultures on Fas mAb-induced cytotoxicity was examined: combined pretreatment with IFN gamma and hCG induced a synergistic increase in Fas mAb-induced cytotoxicity (40%) over that obtained with IFN gamma-pretreatment alone (15%). In summary, granulosa/luteal cells express the Fas antigen and are sensitive to Fas mAb-induced apoptosis. Human CG synergized with IFN gamma to increase Fas mAb-induced death.(ABSTRACT TRUNCATED AT 250 WORDS)
Growth factors and steroids play an important role in the regulation of ovarian follicular development. In cattle, two of the earliest detectable differences between the healthy dominant follicle selected for development to the ovulatory stage and subordinate follicles destined to undergo atresia are the greater availability of IGF and the greater capacity to produce estradiol in the dominant follicle. We have shown that IGF-I and estradiol stimulate the proliferation of bovine granulosa cells in vitro and promote granulosa cell survival by increasing resistance to apoptosis. Furthermore, the ability of IGF-I and estradiol to increase resistance to apoptosis is tied to their ability to promote progression through the cell cycle. Blocking the cell cycle at the transition between the first gap phase and the DNA synthesis phase using a specific inhibitor prevented the protective effects of IGF-I and estradiol against apoptosis. Further experiments showed that the protective effect of IGF-I against apoptosis is mediated by the stimulation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B/Akt. Constitutive activation of Akt by the infection of granulosa cells with a recombinant Akt adenovirus protected against apoptosis, and this effect also depended on cell cycle progression. These experiments show that the protective effect of estradiol and IGF-I against apoptosis depends on unperturbed progression through the cell cycle. Once follicles have developed to the preovulatory stage, the LH surge induces terminal differentiation of granulosa cells and withdrawal from the cell cycle. Bovine granulosa cells withdraw from the cell cycle by 12 h after the LH surge and become resistant to apoptosis, even in the absence of growth factors. Treatment with a progesterone receptor antagonist in vitro caused reentry of granulosa cells into the cell cycle and susceptibility to apoptosis, suggesting that induction of progesterone receptor expression by the LH surge is required for cell cycle withdrawal and resistance to apoptosis. In summary, the susceptibility of granulosa cells to apoptosis depends on the cell cycle. Proliferating granulosa cells in growing follicles depend on growth factors for survival, whereas cells that have terminally differentiated in response to the LH surge are resistant to apoptosis and relatively independent of growth factors for survival.
To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2(tm3(cre)Bhr) strain to target deletion of Wnt4 to granulosa cells. Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis.
The hedgehog (HH) signaling pathway plays an essential role in the Drosophila ovary, regulating cell proliferation and differentiation, but a role in the mammalian ovary has not been defined. Expression of components of the HH pathway in the mouse ovary and effects of altering HH signaling in vitro were determined. RT-PCR analyses show developmentally regulated expression of sonic (Shh), indian (Ihh) and desert (Dhh) HH in the ovary. Expression is detected in whole ovary, granulosa cells, and corpora lutea. The mRNAs for the two receptors, patched homolog 1 and 2 (Ptch1, Ptch2), and the signal transducer, smoothened (Smo), are also expressed. Immunohistochemistry using an antibody that detects all three HH ligands demonstrated HH protein primarily in granulosa cells of follicles from primary to antral stages of development. Follicles also stained for PTCH1 and SMO in both granulosa and theca cells. Treatment of cultured preantral follicles and granulosa cells with recombinant SHH increased growth and proliferation while treatment with the HH pathway inhibitor, cyclopamine, had no effect. Therefore, activation of HH signaling can increase cell proliferation and follicle growth but is not essential for these processes in vitro. Treatment of granulosa cells with SHH increased levels of mRNA for Gli1, a transcriptional target of HH signaling, while cyclopamine decreased expression. SHH had no effect on production of progesterone by cultured granulosa cells, while cyclopamine increased progesterone production. The results demonstrate a functional HH pathway in the follicle and identify granulosa cells as at least one of the potential targets of HH signaling.
Ovarian follicle development is dependent on growth factors that stimulate cell proliferation and act as survival factors to prevent apoptosis of follicle cells. We examined the mechanism of the protective effect of IGF-I against Fas ligand-induced apoptosis of granulosa cells and its relationship to cell proliferation. IGF-I activated both the phosphoinositide 3'-OH kinase (PI3K) and the MAPK pathways. Experiments using specific inhibitors of these pathways showed that protection by IGF-I was mediated by the PI3K pathway and not the MAPK pathway. Recombinant adenoviruses were used to test whether the downstream target of PI3K activation, Akt kinase, was required for protection against apoptosis. Expression of dominant negative Akt prevented protection by IGF-I whereas expression of constitutively active Akt (myrAkt) mimicked the effect of IGF-I. Treatment with IGF-I, or expression of myrAkt, increased progression from G(0)/G(1) to S phase of the cell cycle whereas expression of dominant negative Akt inhibited G(0)/G(1) to S phase progression and prevented the stimulatory effect of IGF-I. We tested whether cell cycle progression was required for protection from apoptosis using the cyclin-dependent kinase-2 inhibitor roscovitine, which blocks cells at the G(1)/S transition. Roscovitine prevented the protective effect of IGF-I and myrAkt expression against apoptosis. Therefore, activation of Akt is not sufficient to protect granulosa cells from apoptosis in the absence of cell cycle progression. In summary, IGF-I protects granulosa cells from apoptosis by activation of the PI3K/Akt pathway. This protective effect can occur only when progression from G(1) to S phase of the cell cycle regulated by the PI3K/Akt pathway is unperturbed.
The role of the hedgehog (HH) signaling pathway in ovarian function was examined in transgenic mice in which expression of a dominant active allele of the signal transducer smoothened (SmoM2) was directed to the ovary and Müllerian duct by cre-mediated recombination (Amhr2(cre/+)SmoM2). Mutant mice were infertile and had ovarian and reproductive tract defects. Ovaries contained follicles of all sizes and corpora lutea (CL), but oocytes were rarely recovered from the oviducts of superovulated mice and remained trapped in preovulatory follicles. Measures of luteinization did not differ. Cumulus expansion appeared disorganized, and in vitro analyses confirmed a reduced expansion index. Microarray analysis indicated that expression levels of genes typical of smooth muscle were reduced in mutant mice, and RT-PCR showed that levels of expression of muscle genes were reduced in the nongranulosa, theca-interstitial cell-enriched fraction. Whereas a layer of cells in the outer theca was positively stained for smooth muscle actin in control ovaries, this staining was reduced or absent in mutant ovaries. Expression of a number of genes in granulosa cells that are known to be important for ovulation did not differ in mutants and controls. Expression of components of the HH pathway was observed in both granulosa cells and in the nongranulosa, residual ovarian tissue and changed in response to treatment with equine chorionic gonadotropin/human gonadotropin. The results show that appropriate signaling through the HH pathway is required for development of muscle cells within the theca and that impaired muscle development is associated with failure to release the oocyte at ovulation.
The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.
Experiments were conducted to test whether oestradiol (E2) protects granulosa cells from Fas ligand (FasL)-induced apoptosis and whether protection involves modulation of the cell cycle of proliferation. Treatment of cultured bovine granulosa cells with E2 decreased susceptibility to FasL-induced apoptosis. The effects of E2 were mediated through oestrogen receptor and were not mediated by stimulation of IGF production. E2 also increased the percentage of cells progressing from G1 to S phase of the cell cycle, and increased expression of cyclin D2 protein and the cell proliferation marker Ki67. Progression from G1 to S phase of the cell cycle was necessary for the protective effect of E2; blocking progression from G1 to S phase with the cdk2 inhibitor roscovitine, or blocking cells in S phase with hydroxyurea, prevented protection by E2. The stages of the cell cycle during which granulosa cells are susceptible to apoptosis were assessed. First, treatment with the G1 phase blocker, mimosine, protected cells from FasL-induced apoptosis, indicating that cells in G0 or early-to mid-G1 phase are relatively resistant to apoptosis. Secondly, examination of recent DNA synthesis by cells that became apoptotic indicated that apoptosis did not occur in S, G2 or M phases. Taken together, the experiments indicate that cells may be most susceptible to apoptosis at the transition from G1 to S phase. E2 stimulates transition from G1 to S phase and protects against apoptosis only when cell cycle progression is unperturbed.
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