AREG对绵羊小腔卵泡卵母细胞体外成熟的影响

旨在研究双调蛋白（AREG）对绵羊小腔卵泡卵母细胞体外成熟（IVM）的影响。本研究从屠宰场绵羊卵巢上采集小腔和中腔卵泡的卵母细胞进行试验，利用自发荧光检测卵母细胞的NAD （P） H和FAD⁺⁺水平，利用JC-1检测卵母细胞的线粒体膜电位；两种来源的卵母细胞经体外成熟后，比较其卵丘扩展指数（CEI）、第一极体排出率（MII%）；比较AREG、AREG+GDF9、AREG+BMP15、AREG+GDF9+BMP15、GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的CEI、MII%及卵母细胞线粒体膜电位的影响；检测了AREG+GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的NAD （P） H和FAD⁺⁺水平及其受精后发育能力的影响。结果表明，成熟前，小腔卵泡卵母细胞的线粒体膜电位和FAD⁺⁺水平均显著低于中腔卵泡卵母细胞（P<0.05）；体外成熟培养后，小腔卵泡卵母细胞的CEI和MII%均显著低于中腔卵泡卵母细胞（P<0.05）。与对照组相比，AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的CEI、MII%和线粒体膜电位（P<0.05），且与中腔卵泡卵母细胞组差异不显著（P>0.05）；另外，AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的NAD （P） H和FAD⁺⁺水平（P<0.05），且与中腔卵泡卵母细胞组差异不显著（P>0.05）。与对照组相比，在成熟液中添加AREG+GDF9+BMP15可以明显提高小腔卵泡卵母细胞体外受精后的卵裂率和囊胚率（分别为（43.79±3.69）%、（28.54±4.31）%和（78.99±1.12）%、（47.46±2.50）%，P<0.05），而且与中腔卵泡卵母细胞组无显著差异（P>0.05）。综上表明，绵羊小腔卵泡卵母细胞的代谢水平及IVM质量较低， AREG在GDF9和BMP15的协同作用下可以显著提高小腔卵泡卵母细胞的代谢水平及IVM质量，并进一步提高小腔卵泡卵母细胞体外受精后的发育能力。     

生长分化因子8在哺乳动物中的功能研究进展

转化生长因子β(TGF-β)超家族成员在卵母细胞成熟和卵丘细胞(CCs)扩增中起关键作用,其中研究确认比较早的有卵母细胞分泌的生长分化因子9 (GDF9)、骨形成蛋白15 (BMP15)等。而TGF-β超家族的新成员生长分化因子8 (growth differentiation factor 8,GDF8),又称肌肉生长抑制素(myostatin, MSTN),最早是作为肌肉生长调控因子被认识与研究的。但近年来该因子作为旁分泌因子在卵泡壁颗粒细胞中表达,并可以在血液内检测到。新的研究证实它影响着卵泡的发育,并参与卵母细胞成熟调控。已有研究表明,GDF8通过与Ⅱ型受体ActRIIa、ActRIIb结合发挥调节效应,诱导Ⅰ型受体ALK 4/5,进而激活TGF-β传感器SMAD2/3,进而调节下游靶点发挥作用。众所周知,GDF8是肌肉生长分化的负调节因子,但最近GDF8对生育能力的正调节作用被确定。论文依据近年来GDF8对生育调节作用的研究报道,综述GDF8在哺乳动物卵母细胞发育影响的研究进展,为相关研究提供资料参考。     

C型钠肽和生长分化因子9预处理对绵羊体外成熟卵母细胞质量和发育能力的影响

为探讨生长分化因子9(growth differentiation factor 9,GDF9)和骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)在协同C型钠肽(C-type natriuretic peptide, CNP)调节卵母细胞减数分裂进程中的作用，提高卵母细胞体外成熟的质量，试验基于CNP预处理的“两步法”体外成熟(in vitro maturation, IVM)体系，研究了预处理阶段添加GDF9和BMP15在调节绵羊卵母细胞减数分裂进程和胞质成熟事件中的作用，并探讨CNP和GDF9联合预处理后对卵母细胞体外成熟质量及发育能力的影响。结果显示，GDF9可显著提高卵丘细胞中CNP受体NPR2基因的表达，从而增加CNP阻滞绵羊卵母细胞减数分裂的效率，而BMP15无此作用。预处理阶段联合添加CNP和GDF9能改善卵丘细胞功能，并且在CNP的作用基础上进一步增加卵母细胞线粒体的活性和数量、GSH含量，并降低ROS水平，从而提高卵母细胞体外成熟后的质量以及体外受精后的发育效率，卵裂率和囊胚率得到显著增加。研究结果不仅表明GDF9能强化CN...     

AREG对绵羊小腔卵泡卵母细胞体外成熟的影响

旨在研究双调蛋白(AREG)对绵羊小腔卵泡卵母细胞体外成熟(IVM)的影响。本研究从屠宰场绵羊卵巢上采集小腔和中腔卵泡的卵母细胞进行试验,利用自发荧光检测卵母细胞的NAD(P)H和FAD~(++)水平,利用JC-1检测卵母细胞的线粒体膜电位;两种来源的卵母细胞经体外成熟后,比较其卵丘扩展指数(CEI)、第一极体排出率(MII%);比较AREG、AREG+GDF9、AREG+BMP15、AREG+GDF9+BMP15、GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的CEI、MII%及卵母细胞线粒体膜电位的影响;检测了AREG+GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的NAD(P)H和FAD~(++)水平及其受精后发育能力的影响。结果表明,成熟前,小腔卵泡卵母细胞的线粒体膜电位和FAD~(++)水平均显著低于中腔卵泡卵母细胞(P0.05);体外成熟培养后,小腔卵泡卵母细胞的CEI和MII%均显著低于中腔卵泡卵母细胞(P0.05)。与对照组相比,AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的CEI、MII%和线粒体膜电位(P0.05),且与中腔卵泡卵母细胞组差异不显著(P0.05);另外,AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的NAD(P)H和FAD~(++)水平(P0.05),且与中腔卵泡卵母细胞组差异不显著(P0.05)。与对照组相比,在成熟液中添加AREG+GDF9+BMP15可以明显提高小腔卵泡卵母细胞体外受精后的卵裂率和囊胚率(分别为(43.79±3.69)%、(28.54±4.31)%和(78.99±1.12)%、(47.46±2.50)%,P0.05),而且与中腔卵泡卵母细胞组无显著差异(P0.05)。综上表明,绵羊小腔卵泡卵母细胞的代谢水平及IVM质量较低, AREG在GDF9和BMP15的协同作用下可以显著提高小腔卵泡卵母细胞的代谢水平及IVM质量,并进一步提高小腔卵泡卵母细胞体外受精后的发育能力。     

体外成熟培养前后山羊卵泡发育相关基因及其受体表达特性的研究

为研究山羊卵母细胞体外成熟培养前后部分转移生长因子β基因及其受体表达特性,本试验以6~8周龄晋岚绒山羊母羔超数排卵获取的卵丘细胞-卵母细胞复合体为研究对象,应用Real time PCR技术检测成熟培养前后BMP15、GDF9、BMP6及其受体基因BMPRⅡ、TGFβRI和BMPRIB的表达差异。结果显示:山羊卵母细胞体外成熟培养后,卵泡发育相关的基因及受体基因中,BMP6 mRNA表达量显著增加(P0.05),BMPRⅡ、TGFβRI和BMPRIB的mRNA表达量极显著上调(P0.01),Ptgs2基因的表达量极显著下调(P0.01),BMP15和GDF9基因差异不显著(P0.05)。     

BMP15促进牛颗粒细胞FSH受体表达并调控其激素分泌

<正>1前言骨形态发生蛋白-15(BMP-15)是转化生长因子-β(TGF-β)超家族成员。在卵巢中,BMP15是卵母细胞特异的衍生因子,可以与卵巢产生的GDF9共同作用,以自分泌或旁分泌的形式影响优势卵泡的发育及卵母细胞的生长,是对于雌     

三倍体虹鳟卵巢发育中细胞凋亡与发育相关基因的表达

为了研究三倍体雌性虹鳟在生长过程中出现的性腺发育停滞、卵原细胞发育异常的原因,试验采集180~300 dpf时期二倍体和三倍体虹鳟的性腺组织,利用光镜、电镜切片和TUNEL染色进行组织与细胞学对比研究,利用实时荧光定量PCR分析对卵巢发育和配子发生过程发育具有调节作用的生长分化因子9(GDF9)、骨形态发生蛋白4(BMP4)和骨形态发生蛋白7(BMP7)在二、三倍体相同时期的表达差异。结果表明:180~300 dpf虹鳟三倍体卵巢中的卵原细胞发生凋亡,除240 dpf三倍体虹鳟BMP4表达量显著高于二倍体外,三倍体虹鳟中GDF9、BMP4和BMP7表达量显著低于同时期二倍体。说明三倍体虹鳟卵原细胞发育异常,最终发生凋亡,可能与GDF9、BMP4和BMP7的低表达有关,BMP4在调节卵母细胞发育的同时可能有抑制细胞凋亡的作用。     

能量水平和来源对后备母猪卵母细胞质量及相关基因表达的影响

选择初始体质量（59&#177;4．2）kg的长白&#215;大白杂交母猪54头,采用3&#215;2试验设计,研究日粮能量水平和来源对后备母猪卵泡发育和卵母细胞体外成熟以及排卵前卵丘-卵母细胞复合物（COCs）中生长分化因子-9（GDF-9）、骨形态发生蛋白-15（BMP-15）、促黄体素受体（LHR）、促卵泡素受体（FSHR）mRNA表达的影响。3个能量水平分别为NRC推荐能量需要的87．5％、100％和112．5％;每个能量水平分别以淀粉和油脂为主要能量来源。试验母猪在第2个发情期的第19天屠宰。结果表明：随能量水平升高,大卵泡数增多（P〈0．05）,能量来源对卵泡数量没有显著影响（P〉0．05）;高能量水平下卵丘扩散程度（P〈0．05）和卵母细胞成熟的比例最高（P〈0．01）,能量来源对卵丘扩散程度影响差异不显著（P〉0．05）,淀粉组卵母细胞成熟的比例高于油脂组（P〈0．05）;基因分析表明,饲喂低能量水平组的GDF-9和BMP-15mRNA的表达量最高（P〈0．05）,淀粉组上调GDF-9mRNA的表达（P〈0．05）,能量来源对BMP-15mRNA表达影响差异不显著;各处理小卵泡的卵丘-卵母细胞复合物中GDF-9和BMP-15mRNA表达量高于大卵泡（P〈0．01）;高能量水平组的LHR和FSHRmRNA表达量最高（P〈0．05）,能量来源对大卵泡卵丘-卵母细胞复合物中FSHRmRNA表达无显著影响,淀粉组上调LHRmRNA表达（P〈0．05）。研究表明,提高日粮能量水平增加母猪排卵前大卵泡数量和卵母细胞成熟的比例,以淀粉为能量来源对卵泡数量没有显著影响,但提高了卵母细胞成熟的比例;能量影响卵泡数量和卵母细胞成熟可能与GDF-9、BMP-15和LHR、FSHRmRNA表达有关。     

抑制素、激活素与雌性动物繁殖生理

抑制素-激活素-卵泡抑素轴系统在动物繁殖生理中的作用日益突出,它参与调节下丘脑-垂体-卵巢轴系统,调节腺垂体FSH的分泌和合成;并可作为自分泌或旁分泌因子调节卵泡的发育和成熟.本综述着重阐述INH、ACT在雌性动物生殖调控、卵泡发育中的作用及其在生产中的应用.     

BMP15-FAT1对猪卵丘细胞凋亡的影响

猪卵丘细胞位于卵母细胞外周,作为一种特殊的颗粒细胞,参与卵母细胞发育、卵泡发育与闭锁以及胚胎的生存和发育等重要生殖生理过程。为探求BMP15通过调控卵丘细胞增殖与凋亡,进而影响卵母细胞生长及成熟的作用机制,本研究通过体外添加BMP15,利用RNA干扰技术、实时荧光定量PCR、流式细胞术等方法,分别检测了添加BMP15后,猪卵丘细胞凋亡情况及凋亡相关通路基因的m RNA表达变化。结果表明:猪BMP15由卵母细胞特异性分泌,可抑制猪卵丘细胞凋亡,同时也可促进FAT1基因的表达;沉默FAT1基因后,BCL2/BAX比率下降,进而影响猪卵丘细胞的凋亡。     

Role of oocyte‐derived paracrine factors in follicular development

Mammalian oocytes secrete transforming growth factor β (TGF‐β) superfamily proteins, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 6 (BMP6) and BMP15, and fibroblast growth factors (FGFs). These oocyte‐derived paracrine factors (ODPFs) play essential roles in regulating the differentiation and function of somatic granulosa cells as well as the development of ovarian follicles. In addition to the importance of individual ODPFs, emerging evidence suggests that the interaction of ODPF signals with other intra‐follicular signals, such as estrogen, is critical for folliculogenesis. In this review, we will discuss the current understanding of the role of ODPFs in follicular development with an emphasis on their interaction with estrogen signaling in regulation of the differentiation and function of granulosa cells.     

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor‐β (TGF‐β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c‐mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ–GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c‐mos [a regulatory subunit of mitogen‐activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M‐phase‐promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)‐test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.     

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.     

Bone morphogenetic protein 15 supplementation enhances cumulus expansion,nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.     

Oocyte-somatic cell-endocrine interactions in pigs

Oocyte-somatic cell communication is bi-directional and essential for both oocyte and follicular granulosa and theca cell function and development. We have shown that the oocyte secretes factors that stimulate porcine granulosa cell proliferation in serum-free culture, and suppress progesterone production, thereby preventing premature luteinisation. Possible candidates for mediating some of these effects are the bone morphogenetic proteins (BMPs) that belong to the transforming growth factor beta family. They are emerging as a family of proteins critical for fertility and ovulation rate in several mammals, and they are expressed in various cell types in the ovary. We have evidence for a functional BMP system in the porcine ovary and BMP receptors are present in the egg nests in the fetal ovary and in the granulosa cells, oocytes and occasional theca cells throughout subsequent development. In addition to paracrine interactions in the ovary, the porcine oocyte and its developmental potential can also be influenced by nutritional manipulation in vivo. We have demonstrated that feeding a high plane of nutrition to gilts for 19 days prior to ovulation increased oocyte quality compared to control animals fed a maintenance diet, as determined by oocyte maturation in vitro. This was associated with a number of changes in circulating reproductive and metabolic hormones and also in the follicular fluid in which the oocyte is nurtured. Further studies showed a similar increase in prenatal survival on Day 30 of gestation, demonstrating a direct link between oocyte quality/maturation and embryo survival. Collectively, these studies emphasise the importance of the interactions that occur between the oocyte and somatic cells and also with endocrine hormones for ovarian development, and ultimately for the production of oocytes with optimal developmental potential.     

Kit和KitL在卵泡发育过程中的作用

在卵泡内,卵母细胞及其周围的颗粒细胞之间的旁分泌作用对哺乳动物的卵子发生和卵泡发育是非常重要的。研究表明颗粒细胞衍生的KitL(Kitligand,stem cell factor,SCF)与卵母细胞和膜细胞产生的Kit的相互作用在PGCs迁移和增殖、卵泡发育过程中有重要的作用。本文重点阐述了由KitL通过Kit激活的PI3K(phosphatidylinositol 3-kinase,磷脂酰肌醇-3-激酶)通路,它对卵母细胞生长、凋亡和卵泡早期发育起重要的调节作用。     

哺乳动物卵母细胞发育机理的研究进展

卵母细胞的成熟过程是一个复杂的减数分裂过程，许多分子参与了这个过程的调控，本文就卵母细胞成熟抑制因子（OMI）、成熟促进因子（MPF）、生长分化因子（GDF）、微管辅助蛋白（MAP）、激素、Ca^2 及钙调素等对卵母细胞发育调控机理作一综述。     

Follicle selection in the avian ovary

Follicle development in the highly efficient laying hen is characterized by a well-organized follicular hierarchy. This is not the case in other chickens such as the broiler breeder hen that has excessive follicle development and lower reproductive efficiency. Although management practices can optimize egg production in less productive breeds of chickens, the factors that contribute to this difference are not known. Interactions between the oocyte and surrounding somatic cells are believed to be involved in promoting follicle selection. Anti-Müllerian hormone (AMH) has been shown to have a role in regulating rate of follicle development in mammals. In hens, the expression of AMH is restricted to the growing population of follicles and, similar to mammals, is markedly decreased at around the time of follicle selection. The oocyte factors, growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have been identified in the hen, and their expression pattern has been characterized. Anti-Müllerian hormone expression in hens is decreased by a protein factor from the oocyte (not GDF9) and is also decreased by vitamin D. Associated with the decrease in AMH expression by vitamin D, follicle-stimulating hormone receptor mRNA is increased. These data suggest that information about AMH regulation may enhance our understanding of follicle selection, particularly in birds with aberrant follicle development.     

绵羊发情周期中VEGF在卵巢中的表达

应用免疫组化方法结合计算机图像分析技术,分析同期发情后0、5、9、12、15d的绵羊卵巢中血管内皮生长因子（Vascular endothelial growth factor,VEGF）表达强度变化,以期了解VEGF在绵羊卵巢发情周期不同时期的表达规律。结果显示：VEGF阳性目标主要出现于卵泡膜与颗粒细胞。原始卵泡、初级卵泡、次级卵泡VEGF表达依次增强（P〈0．05）。发情周期0～5d,大窦腔卵泡（颗粒细胞4～8层）VEGF表达量骤然上升（P〈0．05）,而9d开始显著下降,与5d比较差异显著（P〈0．05）。12d继续下降（P〈0．05）且为最低值,15d又明显上升（P〈0．05）。VEGF在卵巢间质呈弱表达,各个时期之间差异不显著（P〉0．05）。结果表明,绵羊卵巢存在着血管周期性新生的变化特点,而VEGF在这种周期性血管新生过程中起着重要的调控作用。