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.     

Apoptosis of bovine ovarian surface epithelial cells by Fas antigen/Fas ligand signaling

Ovarian surface epithelial cells (OSEs), a single layer of cells that cover the surface of the ovary, undergo turnover at the site of follicular rupture at ovulation. Greater than 90% of ovarian cancers arise from the OSEs. The objective of this study was to determine whether OSEs have the capacity to regulate their own demise through expression of Fas antigen (Fas) and Fas ligand (FasL) and activation of Fas-mediated apoptosis. In initial experiments, primary cultures of bovine OSEs responded to treatment with recombinant FasL by undergoing apoptosis. The percentage of cell death was not affected by the presence or absence of serum in the media or by co-treatment with interferon-gamma, a treatment shown to potentiate Fas-mediated apoptosis in a number of cell types. Subsequent experiments tested the ability of stress-inducing drugs, anisomycin and daunorubicin, to promote apoptosis by stimulating an endogenous Fas-FasL pathway in OSEs. Treatment with FasL, anisomycin or daunorubicin induced cell death and this was suppressed by co-treatment with a peptide inhibitor of caspases, ZVAD. Treatment with anisomycin or daunorubicin in the presence of ZVAD increased expression of FasL mRNA and protein but did not alter expression of Fas mRNA or protein. Treatment of OSEs with a recombinant protein that blocks interaction of FasL with Fas (Fas:Fc) reduced apoptosis in response to anisomycin and daunorubicin, indicating that drug-induced apoptosis was mediated at least partially through endogenous Fas-FasL interactions. In summary, OSEs undergo apoptosis in response to stress-inducing drugs through activation of an endogenous Fas pathway.     

Changes in the localization of MHC class II positive cells in hen ovarian follicles during the processes of follicular growth, postovulatory regression and atresia

The aim of this study was to determine the changes in the population of major histocompatibility complex class II positive (MHC-II(+)) cells in ovarian follicles during the processes of follicular growth, postovulatory regression and follicular atresia in hens. Cryostat sections of ovarian stroma containing cortical follicles, small white follicles, the largest (F(1)) and third largest (F(3)) preovulatory follicles, postovulatory and atretic follicles of laying hens were prepared. The sections were immunostained for MHC-II molecules using mouse anti-chicken MHC-II monoclonal antibody and positive cells were counted using a computer-assisted image analyser under a light microscope. MHC-II(+) cells were localized in the theca layer of normally growing follicles including cortical follicles, small white follicles and F(3) and F(1) preovulatory follicles, whereas they were found in both the theca and granulosa layers in postovulatory and atretic follicles. The frequency of MHC-II(+) cells in the theca layer was significantly increased during follicular growth from cortical follicles to F(3) preovulatory follicles. Although the population of MHC-II(+) cells did not differ between F(3) and F(1) preovulatory follicles, it increased significantly in postovulatory follicles (P < 0.01). The population of MHC-II(+) cells was significantly greater in the theca layer of atretic follicles than in non-atretic follicles (P < 0.01). These results indicate that the antigen-presenting function via MHC-II increases in association with follicular growth. A marked increase in MHC-II(+) cells indicates that these cells may be involved in regression of postovulatory and atretic follicular tissues.     

Effect of negative energy balance on the insulin-like growth factor system in pre-recruitment ovarian follicles of post partum dairy cows

Post partum negative energy balance (NEB) in dairy cattle is associated with a delayed return to ovarian cyclicity and reduced fertility. This study compared the IGF system of pre-recruitment ovarian follicles between cows in mild (n = 6) or severe (n = 6) NEB during early lactation. Ovaries were collected in the second week post partum, when circulating concentrations of IGF-I and glucose were lower (P < 0.01) in severe NEB cows. mRNA expression for IGF-II, type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1 to IGFBP-6 was determined by in situ hybridisation in individual follicles using radiolabelled oligonucleotide probes. Follicles were classified as very small (1-2.5 mm) or small (2.5-5 mm) and healthy or atretic. Relative mRNA concentrations were measured as optical density (OD) units using image analysis. Thecal IGF-II mRNA expression was highest in very small, healthy follicles (P < 0.05). Granulosa cell IGFBP-2 was the only component to change with EB status, with higher mRNA expression in mild compared with severe NEB cows (P < 0.05). IGFBP-1 and IGFBP-3 mRNA expression were undetectable. IGF-1R, IGFBP-4 and IGFBP-5 mRNA expression were not significantly altered by follicle size or health, but IGFBP-5 tended to increase in atretic follicles. The pattern of IGFBP-6 mRNA expression in theca paralleled that of IGF-II mRNA, with higher (P < 0.05) levels in healthy, very small follicles. In conclusion, the reduced expression of IGFBP-2 mRNA in severe NEB cows may alter the bioavailability of circulating IGF-I and locally produced IGF-II to modulate the pre-recruitment stages of follicles required to maintain normal post partum ovarian cyclicity.     

Species differences in the ovarian distribution of 3beta-hydroxysteroid dehydrogenase/delta5-->4 isomerase (3beta-HSD) in two marsupials: the brushtail possum Trichosurus vulpecula and the grey,short-tailed opossum Monodelphis domestica

The ovarian distribution of the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase/delta(5-->4) isomerase (3beta-HSD) was investigated by immunocytochemistry in two marsupial species throughout the reproductive cycle, using a rabbit polyclonal antibody raised against human placental 3beta-HSD. In the polyoestrous and polyovular South American opossum Monodelphis domestica, immunostaining was positive for 3beta-HSD in the adrenal cortex, the ovarian interstitial tissue, the corpus luteum and the granulosa cells of antral and atretic follicles. The theca interna was weakly positive for 3beta-HSD, but only in late preantral to early antral stages of follicular development. The adrenal medulla and smaller preantral follicles were completely negative for 3beta-HSD. In contrast, in the polyoestrous and monovular Australian brushtail possum Trichosurus vulpecula, immunostaining showed a strong positive reaction for 3beta-HSD in the theca, whereas the granulosa layer remained predominantly negative for 3beta-HSD except in the largest follicles. The atretic follicles were completely negative for 3beta-HSD. The ovaries of pregnant animals contained grossly enlarged, persistent, antral follicles, which reacted positively for 3beta-HSD. The function of these follicles in T. vulpecula and the 3beta-HSD-positive atretic follicles in M. domestica has not been determined. The differences between the two marsupials represent species variations. The situation in M. domestica does not represent a marsupial-eutherian dichotomy as previously conjectured.     

Reduced recruitment and survival of primordial and growing follicles in GH receptor-deficient mice

GH influences female fertility. The goal of the present study was to obtain more insight into the effect of loss of GH signalling, as observed in humans suffering from Laron syndrome, on ovarian function. Therefore, serial paraffin sections of ovaries of untreated and IGF-I-treated female GH receptor knock-out (GHR/GHBP-KO) mice were examined to determine the follicular reserve and the percentage of follicular atresia in each ovary. Our observations demonstrate that the amount of primordial follicles was significantly elevated in GHR/GHBP-KO mice, while the numbers of primary, preantral and antral follicles were lower compared with wild-type values. The reduced number of healthy growing follicles in GHR/GHBP-KO mice was accompanied by a significant increase in the percentage of atretic follicles. IGF-I treatment of GHR/GHBP-KO mice for 14 days resulted in a reduced number of primordial follicles, an increased number of healthy antral follicles, and a decreased percentage of atretic follicles. The results of the present study suggest that GH may play a role, either directly or indirectly, via for instance IGF-I, in the recruitment of primordial follicles into the growing pool. Furthermore, GH seems to protect antral follicles, directly or indirectly from undergoing atresia.     

Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.     

Expression of Fas and Fas ligand protein and mRNA in mouse oocytes and embryos

During mammalian embryonic development, abnormal eggs and embryos are eliminated by apoptosis; however, the precise apoptotic pathways remain as yet unidentified. In the present study, expression of Fas and Fas ligand - the proximal members of the death receptor pathway, was evaluated in mouse preimplantation embryos by immunofluorescence and in situ hybridization techniques. Ovulated oocytes were collected from oviducts of cyclic mice on the day of oestrus (day 0), and one-cell, two-cell embryos and eight-cell morulae were collected from oviducts of mated animals on days 1, 2 and 3 of pregnancy, respectively. Blastocysts were flushed from the uterine horns on day 4. Expression of Fas and Fas L mRNAs and proteins was absent from embryos at days 0, 1 and 2. A marked increase in Fas and Fas L mRNA and protein expression was detected in all morphologically normal embryos on day 3 and day 4. In addition, embryos on days 3 and 4 were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining; however, absence of caspase 8 and 3 and intense localization of proliferating cell nuclear antigen confirmed the proliferative status of these embryos. Furthermore, TUNEL staining was absent in postimplantation embryonic sections obtained on day 6. The results of the present studies thus indicate an equilibrium between proliferation and apoptosis in the preimplantation embryo.     

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.     

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.     

Capillary angiogenesis and degeneration in bovine ovarian antral follicles

Angiogenesis and capillary degeneration are both evident during ovarian follicle growth. However, the characteristics and distribution of thecal capillary proliferative and degenerative structures have not been fully defined. Indeed, the role of thecal microvasculature changes in follicular atresia is still a matter of debate. The present study examined the distribution of thecal capillary changes occurring during follicular growth and related the changes to capillary morphology (by scanning electron microscopy, SEM, on bovine ovarian corrosion casts) with the incidence of capillary apoptosis (TdT-mediated dUTP nick end-labelling, TUNEL) and follicular status (as confirmed by follicular fluid steroid concentrations). SEM demonstrated well-perfused vascular plexuses of small to large antral follicles with structural and functional changes to capillaries. Angiogenesis was evident mainly in the apical part of the inner capillary layer of medium follicles and the middle or basal part of the inner capillary layer of dominant follicles that exhibited high oestradiol:progesterone ratios. Degenerative capillaries were observed mainly in the outer vascular layers of small follicles, and in the inner and outer vascular layers of medium antral follicles. Although apoptotic structures were present only in the outer capillaries of the theca interna of morphologically healthy antral follicles, atretic follicles showed apoptotic structures in both the outer and inner thecal capillary layers. These results show that angiogenesis increases during bovine follicular growth and occurs unevenly in different inner theca regions of the follicles. The differential angiogenic and degenerative response of theca interna capillaries may reflect differences in the microenvironment of the follicles, which in turn determine the fate of the follicles (continued growth versus atresia).     

Short photoperiod-induced ovarian regression is mediated by apoptosis in Siberian hamsters (Phodopus sungorus)

Siberian hamster reproduction is mediated by photoperiod-induced changes in gonadal activity. However, little is known about how photoperiod induces cellular changes in ovarian function. We hypothesized that exposing female hamsters to short (inhibitory) as opposed to long (control) photoperiods would induce an apoptosis-mediated disruption of ovarian function. Ovaries and plasma from hamsters exposed to either long (LD, 16 h light:8 h darkness) or short (SD, 8 h light:16 h darkness) days were collected during diestrus II after 3, 6, 9 and 12 weeks and processed for histology or RIA respectively. Apoptosis was assessed by in situ TUNEL and active caspase-3 protein immunolabeling. No significant differences were observed among LD hamsters for any parameter; therefore, these control data were pooled. SD exposure induced a decline in preantral follicles (P < 0.05), early antral/antral follicles (P < 0.01) and corpora lutea (P < 0.01) by week 12 as compared with LD. Terminal atretic follicles appeared by SD week 9; by week 12, these had become the predominant ovarian structures. Estradiol concentrations decreased by weeks 9 and 12 SD when compared with both LD and week-3 SD hamsters (P < 0.05); however, no changes were observed for progesterone. TUNEL-positive follicles in SD ovaries increased at week 3 and subsequently declined by week 12 as compared with LD ovaries (P < 0.01). Active capsase-3 protein immunostaining peaked at SD week 3 as compared with all other groups (P < 0.01). TUNEL and capsase-3 immunolabeling were localized to granulosa cells of late-preantral and early-antral/antral follicles. These data indicate that SD exposure rapidly induces follicular apoptosis in Siberian hamsters, which ultimately disrupts both estradiol secretion and folliculogenesis, resulting in the seasonal loss of ovarian function.     

Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans

During mammalian follicle development, a fluid-filled antrum develops in the avascular centre of the follicle. We investigated the hypothesis that follicular fluid contains osmotically-active molecules, sufficiently large so as to not freely escape the follicular fluid. Such molecules could generate an osmotic differential and thus recruit fluid from the surrounding vascularised stroma into the antrum. Follicular fluid was collected from bovine follicles classified histologically as healthy (n = 4 pools) or atretic (n = 4 pools). Dialysis of the follicular fluid at 300 kDa or 500 kDa resulted in a reduction in colloid osmotic pressure of 35% and 60%, respectively, in fluid from healthy follicles and 29% and 80% from atretic follicles. Digestion of follicular fluid with Streptomyces hyaluronidase, chondroitinase ABC or DNase 1 followed by dialysis resulted in reductions in osmotic pressure of 43%, 53% and 43% respectively for fluids from healthy follicles and 34%, 20% and 31% for atretic follicles. Digestion with collagenase I, proteinase K, heparanase 1 or keratanase had no significant effect on the osmotic pressure of follicular fluid of healthy follicles. Ion exchange and size exclusion, Western blotting and ELISA identified the proteoglycans versican and inter-alpha trypsin inhibitor and the glycosaminoglycan hyaluronan in follicular fluid. We conclude that these molecules or aggregates of them are of sufficient size to contribute to the osmotic potential of follicular fluid and thus recruit fluid into the follicular antrum. DNA may also contribute but it is probably not a component that is regulated for this role.     

Expression of endothelial nitric oxide synthase in the ovine ovary throughout the estrous cycle

This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corpora lutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2) determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble beta3 (GUCY1B3), and (3) co-localize eNOS and vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment 1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries were collected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collected from superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, the expression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles. However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatory period, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expression of eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment 3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteins in the CL demonstrated that both were found in the blood vessels.     

Analysis of atresia in equine follicles using histology,fresh granulosa cell morphology and detection of DNA fragmentation

Follicular atresia has been examined previously by various biochemical and histological methods. The aim of this study was to compare, for the first time, detection of granulosa cell apoptosis by biochemical DNA analysis and microscopic examination of fresh granulosa cell morphology with the established method of detecting atresia by histology in equine follicles. DNA extracted from granulosa cells was examined by staining with ethidium bromide and end-labelling with [(32)P]dideoxy-ATP, which labels the free 3'-end of DNA fragments. In 25 of 26 follicles (96%) there was agreement between end-labelling and staining of DNA with ethidium bromide (P < 0.001). Granulosa cell apoptosis was distinguished more easily in the end-labelled samples than by staining with ethidium bromide. Histological atresia and apoptosis as detected by biochemical DNA analysis were significantly correlated (P < 0.02) with 20 of 22 follicles (91%) receiving corresponding classifications with the two methods. No follicles with granulosa cell apoptosis as detected by biochemical DNA analysis were histologically viable, but some of the histologically early atretic follicles did not display DNA laddering. Stereomicroscopic evaluation of morphology of the fresh granulosa cells was significantly correlated (P < 0.001) with the histological findings, with 29 of 33 follicles (88%) receiving corresponding classifications. There was a potential error in determining follicle health by biochemical DNA analysis only, as both histologically early and late atretic follicles in some cases did not show DNA laddering. Thus, if relying solely on biochemical detection of apoptosis, severely atretic follicles could wrongly be classified as healthy follicles.     

Conditional ablation of macrophages disrupts ovarian vasculature

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48 h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16 h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.     

The grape and wine constituent piceatannol inhibits proliferation of human bladder cancer cells via blocking cell cycle progression and inducing Fas/membrane bound Fas ligand-mediated apoptotic pathway

Piceatannol (3,3',4,5'-Tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine. Piceatannol is a protein kinase inhibitor that modifies multiple cellular targets exerting immunosuppressive, antileukemic, and antitumorigenic activities in several cell lines and animal models. In this study, the antiproliferative activity of piceatannol was investigated. The results showed that piceatannol inhibited the proliferation of T24 and HT1376 human bladder cancer cells by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. ELISA showed that the G0/G1 phase arrest is due to an increased in the expression of p21/WAF1. An enhancement in Fas/APO-1 and membrane-bound Fas ligand (mFasL) might be responsible for the apoptotic effect induced by piceatannol. Our study reports the novel finding, that the induction of p21/WAF1 and activity of the Fas/mFasL apoptotic system may participate in the antiproliferative activity of piceatannol in T24 and HT1376 cells.     

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.