Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.
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The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates. (+info)
Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells.
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Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles. (+info)
Activation of PKC delta in the rat corpus luteum during pregnancy. Potential role of prolactin signaling.
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Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC delta, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC delta in a luteinized granulosa cell model. We also assessed the activation status of PKC delta in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC delta was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC delta phosphorylated on serine 662. PKC delta activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC delta to phosphorylate the PKC delta-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC delta. Similarly, immunoreactivity with the phospho-epitope-specific PKC delta antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or following in vitro activation of recombinant PKC delta. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC delta in luteinized granulosa cells. PRL receptor agonists induced translocation PKC delta from the cytosolic to the Triton-soluble membrane fraction and increased PKC delta activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC delta antibody. Analysis of PKC delta activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC delta antibody revealed that PKC delta activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC delta in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC delta is increasingly expressed and active. (+info)
Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.
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RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT-PCR, follicle-derived macrophages showed a 100-1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle-derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea. (+info)
Histomorphometry of dominant and subordinate bovine ovarian follicles.
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The study was designed to quantitatively characterize the histomorphological attributes of dominant and subordinate follicles in relation to follicular wave dynamics. Heifers (n = 27) were examined daily using ultrasonography to record the growth of individual follicles from 2 days before ovulation until the day of ovariectomy to obtain growing (n = 7), early static (n = 6), late static (n = 6) and regressing (n = 5) phase anovulatory dominant follicles of Wave 1, as well as preovulatory (n = 6) and subordinate (n = 42) follicles. The wall thickness of Wave 1 dominant follicles decreased dramatically (P < 0.01) during the late-static (60.2 +/- 4. 3 microm) and regressing (41.8 +/- 4.3 microm) phases compared to earlier phases. Cells of the granulosa layer of the dominant follicle of Wave 1 became loose during the late-static phase, with an increase (P < 0.001) in number of degenerating cells. Dominant follicles of Wave 1 were lined by fibroblast-like flattened cells during the regressing phase. One day after wave emergence (i.e., before selection), the three largest follicles of the wave were histomorphologically indistinguishable. The wall of the preovulatory follicle close to the medulla of the ovary was thicker (P < 0.01) than the wall facing the ovarian surface. The wall of subordinate follicles was thinner (P < 0.01) and had a lower mitotic index (P < 0.01) than that of their dominant counterparts 3 days and 6 days after wave emergence. In summary, follicular status, ascribed by ultrasonography, was associated with quantitatively distinct histomorphological characteristics. Morphometric changes in the dominant follicle during immature, mature, and post-mature phases were similar to, but occurred later than, those of subordinate follicle. The dominant follicles of Wave 1 entered histological atresia at the time of emergence of Wave 2. (+info)
Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins.
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The relationship between microtubules and concanavalin A surface receptors during concanavalin A capping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 4 degree or 37 degree and then incubated at 37 degree for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization. (+info)
Expression and activity of the Fas antigen in bovine ovarian follicle cells.
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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. (+info)
Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia.
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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. (+info)