miR-495-3p对山羊卵巢颗粒细胞功能的影响

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...     

VEGF modulates the effects of gonadotropins in granulosa cells

Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8 mm or 9 to 14 mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100 ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1 ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles (P = 0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P = 0.045); whereas higher doses of VEGF had no effect on proliferation (P = 0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (P = 0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles (P = 0.9). Granulosa cells from 9- to 14-mm follicles responded to 1 ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 (P = 0.003) but higher VEGF doses had no effect (P = 0.9). The PTGS2 response to 1 ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation (P = 0.003) and PTGS2 expression (P < 0.01) in granulosa cells from 9- to 14-mm follicles were abolished (P = 0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.     

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.     

lncRNA作为竞争性内源分子的作用机制及研究进展

竞争性内源RNA （competing endogenous RNA,ceRNA）假说提出具有相同短序列非编码微小（microRNA,miRNA）应答元件（microRNA response element,MRE）的转录物通过竞争的方式结合miRNA,从而影响转录物的表达水平。ceRNA假说颠覆了miRNA与靶基因单向调控的传统观念,在RNA调控网络中具有重要的生物学意义。在众多转录物中,长链非编码RNA （long non-coding RNA,lncRNA）是一类序列长度超过200个核苷酸的非编码RNA,对lncRNA的研究涉及到遗传、分子生物、基因调控、疾病（癌症、神经系统疾病等）等领域。miRNA与lncRNA形成了一个相互作用的调控网络,lncRNA可作为ceRNA抑制miRNA的功能,从而影响后续基因的表达。近年来,随着生物信息学技术的发展,科研人员发现ceRNA作用机制不仅涉及到人类癌症疾病,而且在各种复杂动物的肌细胞分化、脂肪细胞分化和颗粒细胞凋亡等生物过程中也发挥重要的调控作用。作者追溯了ceRNA调控机制,分析了ceRNA网络调控的影响因素,综述了ceRNA在不同动物中调控miRNA的研究进展,为进一步研究lncRNA与miRNA的调控网络提供参考,为畜牧业发展及复杂动物疾病治疗提供新的思路。     

FSH up-regulates angiogenic factors in luteal cells of buffaloes

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4–6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.     

水牛miR-302s真核表达载体的构建及生物信息学分析

试验旨在克隆并构建水牛miR-302s慢病毒真核表达载体（bbu-miR-302s）,对其进行生物信息学分析,并尝试将该载体应用于水牛体细胞重编程中。以水牛基因组DNA为模板扩增得到bbu-miR-302s前体序列,测序正确后将其连入pLVX-IRES-ZsGreen1构建重组慢病毒真核表达载体。重组的慢病毒真核表达载体经过酶切鉴定正确后,采用脂质体转染方法包装慢病毒颗粒,通过感染HEK-293T细胞及猪和水牛体细胞,检测重组慢病毒载体的有效性。bbu-miR-302s有效感染水牛胎儿成纤维细胞（BFF）后,经诱导培养,检测能否产生水牛诱导多能干细胞（iPSC）。采用CoGeMiR数据库查询法和SnapGene Viewer软件进行miR-302s家族在基因组中的定位分析;利用ClustalX 1.83软件分析miR-302s序列保守性;利用TargetScan和miRWalk软件预测bbu-miR-302s主要的靶基因,并运用DAVID程序对靶基因进行信号通路富集。测序结果显示,扩增获得的序列是水牛miR-302s家族,包装的重组慢病毒滴度为7.2×10⁶TU/mL,并可以有效感染3个物种的体细胞。bbu-miR-302s病毒感染BFF后,细胞经历形态变化并发生聚集形成克隆样,碱性磷酸酶检测阳性,但多能基因及表面抗原检测结果均为阴性,说明单因子miR-302s不足以完全重编程BFF为水牛iPSC,但促进了重编程进程。CoGeMiR数据库检索发现,miR-302s家族主要位于LARP7基因的内含子区;SnapGene Viewer软件进一步分析发现,bbu-miR-302s位于水牛7号染色体LARP7基因的内含子区。序列同源性分析表明,miR-302s家族成员间及miR-302s簇成员在不同物种间均高度保守。水牛miR-302s主要靶基因共255个,这些靶基因主要集中在33个信号通路中,其中PGK信号通路与胰高血糖素信号转导及调控干细胞信号通路最为显著。本研究结果为后续开展miR-302s簇在体细胞重编程中的作用奠定基础。     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Expression of Cyclins and Cyclin‐Dependent Kinase Inhibitors in Granulosa Cells from Bovine Ovary

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen‐inactive dominant follicles (EIDs), oestrogen‐active dominant follicles (EADs) and pre‐ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21^cip1) and CDKN1B (p27^kip1) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19^INK4d) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells

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.     

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte‐derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in‐vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin‐like growth factor I (IGF‐I) at 10 ng/ml for 48 hr after obtaining 75%–80%s confluency. Adiponectin at 10 µg/ml increased IGF‐I‐induced estradiol (E₂) and progesterone (P₄) secretion and FSH‐induced E₂ secretion from GC and also increased the abundance of factors involved in E₂ and P₄ production (cytochrome P45019A1 [CYP19A1] and 3‐beta‐hydroxysteroid dehydrogenase [3β‐HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.