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.     

The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent

This study aimed to determine the effect of insulin-like growth factor-I (IGF-I) on early antral bovine follicular development, and the expression of insulin-like growth factor-binding protein-2 (IGFBP-2). Antral follicles separated into three different size groups were cultured for 6 days in medium supplemented with either a low (10 ng/ml) or high (1 microg/ml) dose of human recombinant IGF-I. Oestradiol production by follicles in all size ranges, cultured in the presence of the high concentration of IGF-I, significantly increased by day 6 (P < 0.05). Follicles in the smallest size range, 165-215 microm, cultured in a high dose of IGF-I, were found to be significantly increased in size (P < 0.01). Oocyte health of the largest follicles (281-380 microm) was significantly improved by the addition of IGF-I to the culture medium. mRNA expression of IGFBP-2 was decreased in the granulosa cells of follicles, size range 216-280 microm, cultured with a high dose of IGF-I (P < 0.05). Granulosa cells (P < 0.05) and oocytes (P < 0.01) of the largest follicles (281-380 microm) showed a decrease in IGFBP-2 expression (protein) when cultured in the control and low-IGF-I treatment groups. Therefore, the response of a bovine follicle to IGF-I is both dose and stage dependent. This work supports a role for IGF-I in modulating somatic and germ-cell maturation and development in early antral follicles. Furthermore, the inverse relationship between the level of IGF-I stimulation and IGFBP-2 expression suggests a local regulatory system modulating IGF-I availability.     

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.     

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.     

Fas and Fas ligand protein and mRNA in normal and atretic mouse ovarian follicles

Apoptosis is the underlying mechanism of follicular atresia in the mammalian ovary. However, the apoptotic pathways governing this ovarian process are not completely elucidated. In the present study, expression of Fas and Fas ligand, the proximal members of the death receptor pathway, was evaluated in mouse ovarian follicles using immunofluorescence and in situ hybridization. Normal or atretic follicles were obtained from immature female Swiss mice after administration of 10 iu equine chorionic gonadotrophin for 48 or 72 h, respectively. Expression of both Fas and Fas ligand mRNA and protein was observed in granulosa cells of normal and atretic follicles. Although the oocytes of normal follicles failed to show any staining, those of atretic follicles stained intensely for Fas, indicating that the presence of Fas in the oocyte determines the fate of the follicle.     

Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus-oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 microM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1-0.5 microM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.     

Geraniin-mediated apoptosis by cleavage of focal adhesion kinase through up-regulation of Fas ligand expression in human melanoma cells

Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis; however, cell death characteristics for geraniin have not yet been elucidated. Here, we investigated the mechanism of geraniin-induced apoptosis in human melanoma cells and demonstrated that geraniin was able to induce cell apoptosis in a concentration- and time-dependent manner. We also examined the signaling pathway related to geraniin-induced apoptosis. To clarify the relationship between focal adhesion kinase (FAK) and geraniin-induced apoptosis, we treated human melanoma cells with geraniin and found that this resulted dose- and time-dependent degradation in FAK. However, FAK cleavage was significantly inhibited when cells were pretreated with a selective inhibitor of caspase-3 (Ac-Asp-Glu-Val-Asp-CHO). Here, we demonstrated for the first time that geraniin triggered cell death by caspase-3-mediated cleavage of FAK. There were two possible mechanisms for activating caspase-3, mitochondria-mediated and receptor-mediated apoptosis. To confirm the geraniin-relevant signaling pathway, using immunoblot analysis we found that geraniin-induced apoptosis was associated with the up-regulation of Fas ligand expression, the activation of caspase-8, the cleavage of Bid, and the induction of cytochrome c release from mitochondria to the cytosol. Treatment with geraniin caused induction of caspase-3 activity in a dose- and time-dependent manner followed by proteolytic cleavage of poly-(ADP-ribose) polymerase, and DNA fragmentation factor 45. The geraniin-induced apoptosis may provide a pivotal mechanism for its cancer-chemopreventive action.     

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.     

Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17beta (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10, 100 ng/ml) or FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of 100 ng/ml and ALK-5 mRNA expression at 10 ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.     

Effects of the persistent dominant follicle on the ability of follicle stimulating hormone to induce follicle development and ovulatory responses

Three experiments were conducted to evaluate the effect of an induced first wave persistent dominant follicle on folliculogenesis and ovulatory responses induced by FSH. On d 6 of a synchronized estrous cycle (d 0 = estrus), cows were treated with a Syncromate-B implant and two injections of PGF2, (25 mg, 0700 h; 15 mg, 1900 h, i.m.). Cows in the control group retained a first-wave persistent dominant follicle, but in the aspirated group, the first-wave dominant follicle was removed via transvaginal aspiration on d 10 (d 0 = estrus). Beginning on d 12, cows received 32 mg of FSH-P i.m. in decreasing doses at 12-h intervals over a 4-d period. On d 15, the Syncromate-B implant was removed, and cows were ovariectomized (experiment 1, n = 8) or inseminated (experiment 2, n = 11) at 10 and 22 h after the onset of estrus. Cows in experiment 3 received a used controlled intravaginal drug releasing (CIDR) device and two injections of PGF2alpha (25 mg, 0700 h; 15 mg, 1900 h; i.m.) on d 6. On d 8, the first-wave dominant follicle was aspirated (n = 6) or left intact (n = 5), and FSH treatment was initiated (20 mg of Folltropin in decreasing doses at 12-h intervals over a 4-d period), and on d 10 the used CIDR device was removed from all cows. Ovarian follicle size and number were examined daily by ultrasonography from d 5 of the estrous cycle. The persistent dominant follicle increased in size from 10.7 mm on d 5 to 15.4 mm on d 10 (experiments 1 and 2), and from 9 mm on d 5 to 20.4 mm on d 11 (experiment 3). From d 11 to 14, the number of class 1 (2 to 5 mm) follicles was lower in the aspirated group than in the control group; the number of class 2 (6 to 9 mm) follicles was higher on d 12 and 13 for the aspirated group (experiments 1 and 2). The number of class 3 (> or =10 mm) follicles was higher in the aspirated group on d 14 to 16, but the same on d 17. Ovarian and embryo responses to superovulation did not differ between groups. In experiment 3, the numbers of class 1, 2, and 3 follicles, as well as ovarian and embryo responses following ovulation did not differ between groups. Initiation of exogenous FSH treatment appears to override any systemic inhibitory effect that a persistent dominant follicle may be exerting at the pituitary and possibly the ovary.     

Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses

Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF_2α metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF_2α 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d −16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n = 19), cows had their estrous cycle synchronized starting on study d −9 (GnRH and progesterone insert on d −9, and insert removal and PGF_2α on d −2). In experiment 2 (n = 25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d −4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2 mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2, estradiol and progesterone concentrations did not differ between treatments from d 0 to 16; however, the proportion of cows with a short luteal phase tended to increase in LP than HP (25 vs. 0%). Concentrations of PGFM were greater for LP than HP. Uterine biopsies had a greater abundance of progesterone receptor, and tended to have less estrogen receptor-α abundance on d 12 compared with d 16. An interaction between treatment and day of collection was detected for estrogen receptor-α because of an earlier increase in protein abundance on d 12. Reduced concentrations of progesterone during the development of the ovulatory follicle altered follicular dynamics and follicular fluid composition, increased basal LH concentrations, and prematurely increased estrogen receptor-α abundance and exacerbated PGF_2α release in the subsequent estrous cycle.     

Concentration of progesterone during the development of the ovulatory follicle: I. Ovarian and embryonic responses

Objectives were to evaluate the effects of differing progesterone concentrations during follicle development on follicular dynamics, fertilization, and embryo quality. Lactating Holstein cows (n = 154) were assigned randomly to 1 of 2 treatments. Cows underwent a presynchronization of the estrous cycle composed of an injection of GnRH concurrently with the placement of a progesterone insert, an injection of PGF_2α and insert removal 7 d later, and a second injection of GnRH 48 h later (study d −16). All cows were then submitted to a hormonal protocol identical to the presynchronization program starting on d 7 of the estrous cycle (study d −9). Cows enrolled in the high progesterone (HP) treatment received no further treatment. Cows in the low progesterone (LP) treatment received additional PGF_2α injections on study d −14, −13.5, and −13 and again on study d −9, −7, −6.5, and −6. Ovaries were evaluated by ultrasonography, and blood was sampled for concentrations of progesterone and estradiol throughout the study. Uteri were flushed 6 d after artificial insemination (AI) and recovered oocytes-embryos were evaluated. Concentrations of progesterone were less for LP cows from study d −7 to −2; concentrations of estradiol at PGF_2α and at the last GnRH of synchronization were greater for LP than HP. The proportion of cows in estrus at AI was greater for LP than for HP (38.0 vs. 5.3%). Ovulatory follicles of LP cows had larger diameters at the injections of PGF_2α (17.2 vs. 14.6 mm) and final GnRH (19.4 vs. 16.9%) of the synchronization, which resulted in a larger diameter of the corpus luteum 6 d after AI (24.3 vs. 22.6 mm). Double ovulation after the last GnRH of the synchronization was increased in LP (18.6%) compared with HP (4.5%). Fertilization rate was similar and averaged 82.7%. The proportion of embryos and oocytes-embryos classified as grades 1 and 2, proportion of degenerated embryos, and unfertilized-degenerated oocytes-embryos were not different between LP and HP. Number of blastomeres did not differ between LP and HP, but the proportion of live blastomeres tended to be less for LP than HP (94.2 vs. 98.7%). Reducing progesterone concentrations during the synchronization program altered concentrations of estradiol and follicular dynamics, but resulted in similar fertilization and only minor changes in embryo quality.