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1.
To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.  相似文献   

2.
Because IGFBP inhibit IGF-stimulated cellular proliferation and differentiation, it is hypothesized that variations among IGFBP in individual follicles might contribute to the regulation of recruitment, selection, dominance, and turnover of ovarian follicles. Sources of IGFBP in fluid of bovine follicles are not well established; thus, objectives of this study were to determine levels of IGFBP binding activities and messenger RNA (mRNA) in granulosa and theca interna cells at different stages of follicular development (small [< 6 mm], medium [6 to < 8 mm], and large [> or = 8 mm]) and to characterize associations of these levels measured in the cells with levels of IGFBP and steroids in follicular fluid. Thecal and granulosa cells from large healthy follicles contained two- to twentyfold less (P < 0.05) IGFBP-2, -3, and -5 than cells from small, medium, and large atretic follicles. Thecal cells from small, medium, and large atretic follicles contained more (P < 0.05) IGFBP-3 and -4 than granulosa cells from these follicles, whereas granulosa cells from these follicles contained more IGFBP-2 activity than thecal cells. Differences in IGF binding activity were paralleled by differences in levels of mRNA for the respective IGFBP. Developmental differences in IGFBP activity in follicular fluid were positively associated with activity in granulosa and/or thecal cells, with the exception of IGFBP-4, which was low in fluid from large healthy follicles but markedly increased (mRNA and binding activity) in granulosa cells from these follicles. It is concluded that developmental changes in follicular fluid IGFBP-2 and -5 binding activities seem to be controlled in part by alterations in synthesis of these IGFBP by granulosa and thecal cells, whereas diminished IGFBP-4 in fluid from large healthy follicles occurs concomitantly with increased levels of IGFBP-4 mRNA and activity in granulosa cells, implicating posttranslational regulation by specific proteases.  相似文献   

3.
Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.  相似文献   

4.
In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.  相似文献   

5.
Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5 mm) and large (LG) (8–22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3–500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P > 0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P > 0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P < 0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.  相似文献   

6.
The first wave of follicular development following ovulation in cattle is characterized by selection and growth of a large, estrogenic dominant follicle. After the follicle becomes morphologically dominant, concentrations of estradiol in its follicular fluid decrease abruptly. The purpose of this study was to determine whether this decrease in estrogen production is caused by an insufficient supply of androgen from theca interna or decreased aromatization of androgen precursor by granulosa cells. Dominant follicles were collected from Holstein heifers on d 4, 6, or 8 of the first follicular wave (n = 5/d). Amounts of 17alpha-hydroxylase mRNA in theca interna were sevenfold higher (P < 0.01) on d 4 than on d 8. After 3 h in culture, secretion of androstenedione by theca interna collected on d 4 (236 +/- 44 pg/microg of protein) tended to be lower (P = 0.055) compared with d 6 (517 +/- 162 pg/microg protein) and was lower (P < 0.05) compared with d 8 (387 +/- 51 pg/microg of protein). In granulosa cells, amounts of aromatase mRNA decreased (P < 0.05) on d 8 compared with d 6 but not d 4. In vitro secretion of estradiol was higher in granulosa cells collected on d 4 (3.5 +/- 0.8 ng/[10(5) cells x 3 h]) compared with d 6 (1.8 +/- 0.6 ng/[10(5) cells x 3 h]; P < 0.05) and tended to be higher on d 4 than on d 8 (2.2 +/- 0.2 ng/[10(5) cells x 3 h]; P = 0.058). We conclude that the decrease in estradiol production observed during atresia of the dominant follicle is not due to lack of androgen substrate for aromatization or downregulated expression of the aromatase gene, but may be the direct result of decreased activity of the aromatase enzyme within granulosa cells.  相似文献   

7.
A decrease in insulin-like growth factor (IGF) binding protein (BP) amount occurs within the follicular fluid of dominant ovarian follicles. At the same time, concentrations of follicular fluid IGF-I do not change. The mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 in dominant and subordinate follicles were measured to determine if changes in IGF or IGFBP gene expression are associated with follicular dominance. Heifers were ovariectomized during a follicular wave, either during early-dominance (emerging dominant follicle, 9 mm diameter) or mid-dominance (established dominant follicle, 14–16 mm diameter). Follicles were classified as either dominant (DF), subordinate (SF), or not-recruited (NRF; small antral follicles). mRNA was localized by in situ hybridization and measured by image analyses. The IGF-I mRNA (granulosa cells) was greatest in DF and increased in DF, SF, and NRF from early- to mid-dominance. Likewise, IGF-II mRNA (theca cells) was greatest in DF compared with SF or NRF. The IGFBP-2 mRNA (granulosa cells), however, was nearly undetectable in DF, whereas adjacent SF expressed abundant IGFBP-2 mRNA. The NRF were not uniform in their IGFBP-2 expression because only 5 of 13 NRF had IGFBP-2 mRNA. The IGFBP-3 mRNA (granulosa cells) was found only in two NRF, suggesting that local synthesis is not a predominant source of follicular fluid IGFBP-3. These data show that changes in gene expression for IGFBP-2 are opposite to those for IGF-I or IGF-II. Increased IGF-I and IGF-II mRNA and decreased IGFBP-2 mRNA within the DF may be one mechanism leading to follicular dominance. The opposite pattern of IGFBP-2 gene expression in SF and some NRF may lead to follicular atresia.  相似文献   

8.
Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.  相似文献   

9.
旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...  相似文献   

10.
Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.  相似文献   

11.
To determine the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P>0.05) on estradiol production by granulosa cells from both small (1–5 mm) and large (≥8 mm) follicles. However, in the presence of ≥3 ng/mL of insulin, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED50) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7–14 ng/mL in the presence of FSH. Similarly, in the presence of ≥3 ng/mL of IGF-I, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the ED50 of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5–6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and were not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin-stimulated estradiol production by 52% on day 1 and 19% on day 2 of treatment. Insulin, IGF-I and FSH also increased (P<0.05) granulosa cell numbers and progesterone production but their maximal effects were less (i.e., <4-fold increase) than their effects on estradiol production. In conclusion, insulin and IGF-I synergize with FSH to directly regulate ovarian follicular function in cattle, particularly granulosa cell aromatase activity.  相似文献   

12.
Involvement of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied during the last decade. In all mammalian species, IGF-I stimulates granulosa cell proliferation and steroidogenesis. The concentrations of IGF-I and -II do not vary during terminal follicular growth and atresia. In contrast, the levels of IGFBP-2 and -4, as well as IGFBP-5 in ruminants, dramatically decrease and increase during terminal follicular growth and atresia, respectively. These changes are responsible for an increase and a decrease in IGF bioavailability during follicular growth and atresia, respectively. They are partly explained by changes in ovarian expression. In particular, expression of IGFBP-2 mRNA decreases during follicular growth in ovine, bovine and porcine ovaries, and expression of IGFBP-5 mRNA dramatically increases in granulosa cells of bovine and ovine atretic follicles. Changes in IGFBP-2 and -4 levels are also due to changes in intrafollicular levels of specific proteases. Recently, we have shown that the pregnancy-associated plasma protein-A (PAPP-A) is responsible for the degradation of IGFBP-4 in preovulatory follicles of domestic animals. Expression of PAPP-A mRNA is restricted to the granulosa cell compartment, and is positively correlated to expression of aromatase and LH receptor. From recent evidence, the bone morphogenetic protein (BMP) family would also play a key role in ovarian physiology of domestic animals. In particular, we and others have recently shown that a non-conservative substitution (Q249R) in the bone morphogenetic protein-receptor type IB (BMPR-IB) coding sequence is fully associated with the hyperprolific phenotype of FecB(B)/FecB(B) Booroola ewes. BMP-4 and GDF-5, natural ligands of BMPR-IB, strongly inhibit secretion of progesterone by ovine granulosa cells in vitro, but granulosa cells from FecB(B)/FecB(B) ewes are less responsive than those from FecB(+)/FecB(+) to the action of these peptides. It is suggested that in FecB(B)/FecB(B) ewes, Q249R substitution would impair the function of BMPR-IB, leading to a precocious differentiation of granulosa cells and of follicular maturation. Interestingly, recent findings have described mutations in BMP-15 gene associated with hyperprolific phenotypes in Inverdale and Hanna ewes, suggesting that the BMP pathway plays a crucial role in the control of ovulation rate.  相似文献   

13.
To determine whether the hormonal regulation of IGF-I production differs between granulosa and thecal cells in cattle, granulosa and thecal cells from bovine follicles were collected, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 24 h in serum-free medium with various hormones. In Exp. 1, granulosa cells were treated with 0 or 100 ng/mL of insulin and(or) 50 ng/mL of follicle-stimulating hormone (FSH), insulin plus 10 ng/mL of epidermal growth factor, or insulin plus 10 ng/mL of basic fibroblast growth factor. In Exp. 2, thecal cells were treated as described in Exp. 1 except that 100 ng/mL of luteinizing hormone (LH) was used instead of 50 ng/mL of FSH. In Exp. 3, granulosa and thecal cells were treated with 0 or 30 ng/mL of cortisol with or without 100 ng/mL of insulin, 300 pg/mL of glucagon, or glucagon plus insulin. In Exp. 4, granulosa and thecal cells were treated with 0 or 300 ng/mL of estradiol with or without 100 ng/mL of insulin and(or) 100 ng/mL of LH. At the end of treatment, medium was collected, concentrated with Centricon-3 concentrators, and assayed for IGF-I by radioimmunoassay. Cell numbers were determined by Coulter counting at the end of culture. Thecal cells produced low amounts of IGFI (0.48 +/- 0.04, 0.63 +/- 0.03, and 0.82 +/- 0.03 ng per 100,000 cells per 24 h in Exp. 2, 3, and 4, respectively), and this production was not influenced (P > 0.05) by the various treatments. In contrast, IGF-I production by granulosa cells (2.0 to 6.2 ng per 100,000 cells per 24 h) was influenced by treatment in Exp. 1, 3, and 4 and was greater than IGF-I production by thecal cells (Exp. 2, 3, and 4). Alone, insulin, FSH, LH, and cortisol (but not estradiol) each decreased (P < 0.05) granulosa-cell IGF-I production by 20 to 57%; combined treatments of insulin plus FSH or insulin plus cortisol decreased IGF-I production to levels seen with insulin alone. Glucagon had no effect (P > 0.10) on IGF-I production in the absence or presence of insulin. In the presence of insulin, epidermal growth factor, basic fibroblast growth factor, and estradiol decreased (P < 0.05) IGF-I production below that observed for insulin alone. These results indicate that, during follicular development in cattle, changes in intrafollicular levels of IGF-I may be due to hormonally-induced changes in granulosa-cell, but not thecal-cell, IGF-I production.  相似文献   

14.
A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.  相似文献   

15.
Ovarian paracrine mediation by components of the wingless-type mouse mammary tumor virus integration site ligands (WNT1 to 11) and their receptors, frizzled family members (FZD1 to 10), has been proposed. Secreted truncated forms of FZD proteins (e.g., secreted frizzled-related protein 4 [SFRP4]) block the action of WNT ligands. Dickkopf-1 (DKK1) is another WNT antagonist, and R-spondin-1 (RSPO1) is one of a group of four secreted proteins that enhance WNT/β-catenin signaling. Our hypothesis was that granulosa cells signal theca cells (TCs) via SFRP4, DKK1, RSPO1, and WNT secretion to regulate TC differentiation and proliferation. Therefore, in vitro experiments were conducted to study the effects of WNT family member 3A (WNT3A), WNT5A, RSPO1, DKK1, insulin-like growth factor 1 (IGF1), bone morphogenetic protein 7 (BMP7), Indian hedgehog (IHH), and fibroblast growth factor 9 (FGF9) on bovine TC proliferation and steroidogenesis. TCs of large (8 to 20 mm) and small (3 to 6 mm) follicles were collected from bovine ovaries; TC monolayers were established in vitro and treated with various doses of recombinant human WNT3A, WNT5A, RSPO1, DKK1, IGF1, FGF9, BMP7, IHH, and/or ovine luteinizing hormone (LH) in serum-free medium for 48 h. In experiment 1, using LH-treated TC, IGF1, IHH, and WNT3A increased (P < 0.05) cell numbers and androstenedione production, whereas WNT3A and BMP7 inhibited (P < 0.05) progesterone production. In experiment 2, FGF9 blocked (P < 0.05) the WNT3A-induced increase in androstenedione production in LH plus IGF1-treated TC. In experiment 3, RSPO1 further increased (P < 0.05) LH plus IGF1-induced progesterone and androstenedione production. In experiment 4, SFRP4 and DKK1 alone had no significant effect on TC proliferation or progesterone production of large-follicle TC but both blocked the inhibitory effect of WNT5A on androstenedione production. In contrast, DKK1 alone inhibited (P < 0.05) small-follicle TC androstenedione production whereas SFRP4 was without effect. We conclude that the ovarian TC WNT system is functional in cattle, with WNT3A increasing proliferation and androstenedione production of TC.  相似文献   

16.
The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.  相似文献   

17.
Angiogenic factors are associated with angiogenesis during follicular development in the mammalian ovary. The aim of the present study was to determine the relationships between the vascular network and mRNA expressions of angiopoietins (Ang)-1, Ang-2 and hepatocyte growth factor (HGF), and their receptors in follicles at different developmental stages during follicular development. Ovaries in gilts were collected 72 h after equine chorionic gonadotropin (eCG, 1250 IU) treatment for histological observation of the capillary network. Granulosa cells and thecal tissues in small (<4 mm), medium (4-5 mm) or large (>5 mm) individual follicles were collected for detection of mRNA expression of HGF, Ang-1 and Ang-2 in granulosa cells, and HGF receptor (HGF-R) and Tie-2 in the theca cells by semi-quantitative RT-PCR. The number of capillaries in the thecal cell layer increased significantly in healthy follicles at all developmental stages in the eCG group compared with those in controls. The expression of Ang-1 mRNA declined in granulosa cells of medium and large follicles and the level of Ang-2 mRNA increased in granulosa cells of small follicles after eCG treatment. The ratio of Ang-2/Ang-1 increased in small, medium and large follicles from ovaries after eCG treatment, but Tie-2 mRNA expression in the theca cells did not change. The level of HGF mRNA increased in granulosa cells of small follicles after eCG treatment but HGF-R in theca cells was not increased by eCG. These data suggested that the angiopoietins might be associated with thecal angiogenesis during follicular development in eCG-treated gilts.  相似文献   

18.
Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.  相似文献   

19.
The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.  相似文献   

20.
Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.  相似文献   

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