骨形态发生蛋白基因mRNA在牛卵巢组织中的表达研究

骨形态发生蛋白(BMP15)基因主要在哺乳动物卵巢中表达,对卵泡的发育和分化起重要作用.研究根据其他物种BMP15基因的保守序列设计特异性引物,采用RT-PCR技术,从牛卵巢中提取总RNA,扩增出BMP15 cDNA序列;将此片段克隆到pGM-T载体中,经PCR鉴定和DNA序列测定分析验证,符合BMP家族基因结构特征,然后根据此序列构建cRNA探针,利用原位杂交技术检测牛卵巢BMP15基因 mRNA的表达情况.原位杂交结果显示,牛BMP15基因,在初级卵泡和次级卵泡早期表达,在初级和次级卵泡的颗粒细胞中也表达,在次级卵泡晚期也有表达,同时BMP15基因在透明带周围表达,这可能是透明带周围细胞中的BMP15基因渗透到透明带中.     

骨形态发生蛋白15基因mRNA在牛不同组织中的表达分析

由于BMP15基因对卵泡发育、优势卵泡的选择及排卵等雌性生殖过程的许多环节都起着关键作用,因此该研究应用RT-PCR技术检测了BMP15基因mRNA在蒙古牛卵巢、子宫、输卵管和肾脏组织中的表达情况.结果表明,BMP15基因在这4种组织中均有表达,只是在不同组织中的表达丰度不同,蒙古牛BMP15基因在卵巢组织中的mRNA水平极显著高于子宫组织(P＜0.01),显著高于肾脏组织(P＜0.05);说明BMP15基因在卵巢和输卵管中高度表达,在子宫和肾脏组织中表达丰度较低.     

湘西黄牛超数排卵和冲胚效果与卵巢Figla基因表达水平之间的关系

利用real-time PCR和原位杂交技术分析了Figla(factor in the germline alpha)基因在湘西黄牛卵巢中的相对表达量与定位表达情况。结果显示:能冲出较多优质胚胎的湘西黄牛个体,卵巢组织中Figla基因mRNA水平的表达量要显著高于对照组(P0.05)。Figla基因mRNA在牛卵巢组织中的初级卵泡、次级卵泡和成熟的有腔卵泡都有表达。而且Figla基因在有腔卵泡的卵母细胞中的胞质中表达,但在细胞核中没有表达。结果说明:湘西黄牛超数排卵和冲胚效果与卵巢Figla基因表达水平之间存在一定的关系,Figla基因可能是卵泡和早期胚胎发育的双重关键基因。     

牦牛卵巢组织学结构的观察

本试验对牦牛的卵巢结构进行了研究,主要是通过大体解剖和显微结构的观察。通过研究,把牦牛的卵泡分为了原始卵泡、初级卵泡、次级卵泡和囊状卵泡。研究发现,牦牛的卵巢中数量较多的是原始卵泡,它主要分布在皮质的浅表层。原始卵泡是由卵母细胞以及周围单层的扁平卵泡细胞组成。初级卵泡体积增大,透明带开始形成。次级卵泡体积迅速增大,透明带厚度明显增加,卵泡膜增厚。囊状卵泡腔体大,卵母细胞位于卵泡一侧,形成放射冠状的卵丘。     

休情期银黑狐卵巢形态和卵泡发育的显微结构观察

研究休情期银黑狐卵巢形态和卵泡的显微结构,以揭示银黑狐卵巢发育的一般规律。本试验于2012年12月份采集5只健康一岁龄银黑狐卵巢10枚,用游标卡尺测量其长、宽、厚,用电子天平测量其重量,并对其表面可见卵泡数量进行统计,然后利用光学显微镜对各级卵泡分别观察1~3个,共计原始卵泡30个,初级卵泡20个,次级卵泡15个,三级卵泡12个,成熟卵泡10个,并进行拍照。结果表明:随银黑狐卵巢体积不断增大,其中80%的卵巢重量也随之增大;可见卵泡数量与卵巢体积及重量没有相关性;卵巢由被膜、皮质和髓质构成,髓质位于卵巢内层,分布着较多血管,皮质位于卵巢外层,内有不同发育阶段的卵泡;原始卵泡由卵母细胞和颗粒细胞构成,初级卵泡开始出现透明带物质,到次级卵泡阶段发育完整,三级卵泡出现卵泡腔,卵泡及卵母细胞直径在有腔卵泡阶段比腔前卵泡阶段增长速度快,成熟卵泡的直径及透明带厚度达到最大,各级卵泡均有闭锁现象。     

猪卵巢发育的组织学特点及糖组织化学

运用组织学常规石蜡切片-HE染色和过碘酸-雪夫染色(PAS)方法,分别对3日龄及165日龄育成猪卵巢组织结构、各级卵泡的发育变化特点及其黏多糖分布情况进行了研究。结果显示,3日龄的猪卵巢皮质、髓质界限模糊,皮质部分由外向内依次分布着密集的卵原细胞和卵原细胞巢、共质体样的卵原细胞群,皮质深层与髓质相邻处分布着合胞体样的卵母细胞簇状结构和卵泡。育成猪卵巢中,皮、髓质界限明显,能观察到原始卵泡、初级卵泡、次级卵泡及近成熟卵泡,但是很少能见到黄体的结构。PAS染色结果表明,3日龄的猪卵巢中PAS阳性反应主要分布于卵原细胞周围的基质、卵原细胞巢和合胞体的外膜上,以及原始卵泡周围的基质、初级卵泡的卵泡膜及刚形成的透明带上,此外卵巢的白膜、髓质的结缔组织、血管壁及卵巢网周围也可见到广泛的阳性着色。育成猪卵巢中,PAS阳性反应主要分布于基质、生长卵泡的卵泡膜、卵泡的透明带、次级卵泡的卵泡液、黄体被膜及髓质的结缔组织和血管壁。     

猪卵巢发育的组织学特点及糖组织化学

运用组织学常规石蜡切片-HE染色和过碘酸-雪夫染色（PAs）方法,分别对3日龄及165日龄育成猪卵巢组织结构、各级卵泡的发育变化特点及其黏多糖分布情况进行了研究。结果显示,3日龄的猪卵巢皮质、髓质界限模糊,皮质部分由外向内依次分布着密集的卵原细胞和卵原细胞巢、共质体样的卵原细胞群,皮质深层与髓质相邻处分布着合胞体样的卵母细胞簇状结构和卵泡。育成猪卵巢中,皮、髓质界限明显,能观察到原始卵泡、初级卵泡、次级卵泡及近成熟卵泡,但是很少能见到黄体的结构。PAS染色结果表明,3日龄的猪卵巢中PAS阳性反应主要分布于卵原细胞周围的基质、卵原细胞巢和合胞体的外膜上,以及原始卵泡周围的基质、初级卵泡的卵泡膜及刚形成的透明带上,此外卵巢的白膜、髓质的结缔组织、血管壁及卵巢网周围也可见到广泛的阳性着色。育成猪卵巢中,PAS阳性反应主要分布于基质、生长卵泡的卵泡膜、卵泡的透明带、次级卵泡的卵泡液、黄体被膜及髓质的结缔组织和血管壁。     

雷州黑鸭早期卵巢卵泡发育规律及减数分裂基因表达研究

为了揭示雷州黑鸭早期卵巢发育规律及开产期早的特性,试验采用H.E.染色法对雷州黑鸭0～120日龄卵巢组织形态学进行观察,并通过荧光定量PCR对0～28日龄卵巢组织中减数分裂相关基因Str8、Sycp3、Spo11、Dmc1的表达量进行测定分析。结果表明:雷州黑鸭0～28日龄卵巢发育与前人对鸡卵巢研究结果基本一致,但在雷州黑鸭21日龄时有卵泡闭锁现象,推测14～21日龄由初级卵泡向次级卵泡发育中存在卵泡选择过程。Sycp3、Dmc1和Spo11基因在0日龄表达量最高且在0～28日龄呈下降趋势;Str8基因表达呈现一个波动,在7日龄时表达量最高,推测该基因参与诱导原始卵泡的发育和启动初级卵泡的减数分裂。说明雷州黑鸭开产期早的原因可能是在14～21日龄的卵泡选择过程中使28日龄后的次级卵泡发育速度快于其他品种;此外,Str8可能通过调控原始卵泡和初级卵泡的分裂时间影响雷州黑鸭开产期。     

荣昌猪BMP15基因通过TGF-β RⅡ激活SMAD4信号分子机制研究

本研究旨在阐明荣昌猪BMP15基因在荣昌猪性成熟前不同发育期的表达特性及其与SMADs信号相关基因的表达关系。采集1月龄、3月龄及5月龄荣昌猪卵巢组织,利用qRT-PCR及石蜡切片免疫荧光染色法分析荣昌猪不同月龄卵巢组织内BMP15基因表达及细胞定位特征;利用5月龄卵巢活组织添加重组人BMP15蛋白及TGF-β受体抑制剂(LY215799和LY2109761),应用qRT-PCR及Western blot方法分析BMP15及SMADs信号通路相关基因SMAD2、SMAD3、SMAD4、SMAD7、TGF-β1、TGF-β2、TGF-β3、TGF-βRⅠ和TGF-βRⅡ表达特征及BMP15/SMADs信号通路。结果表明:从1月龄至5月龄,随着荣昌猪生长发育,卵巢组织内BMP15基因mRNA表达量呈上调表达(P0.05);石蜡切片免疫荧光试验表明,5月龄卵巢组织卵母细胞周围颗粒细胞内存在BMP15蛋白荧光信号;从3月龄至5月龄的卵巢组织内BMP15、SMAD4、TGF-β1及TGF-βRⅡ基因在mRNA水平呈上调表达(P0.05),而SMAD2、TGF-β2及TGF-βRⅠ呈下调表达(P0.05);通过5月龄卵巢活组织添加重组人BMP15蛋白、TGF-βRⅠ/Ⅱ及TGF-βRⅠ受体抑制剂培养,发现TGF-βRⅠ/Ⅱ抑制剂(LY2109761)明显抑制TGF-βRⅡ受体蛋白的表达,TGF-βRⅠ抑制剂(LY2157299)不能抑制TGF-βRⅡ受体蛋白的表达。上述结果表明,荣昌猪BMP15基因在荣昌猪性成熟前的卵巢组织内呈上调表达,SMAD4、TGF-β1及TGF-β RⅡ也呈上调表达。本试验研究表明BMP15基因通过TGF-βRⅡ介导SMAD4信号分子调控下游基因的表达,为进一步研究荣昌猪BMP15基因在卵泡发育中发挥的作用提供理论依据。     

FOXL2基因与蛋鸡卵巢发育的相关性研究

试验采集120、160、220、330、480日龄海兰褐蛋鸡的卵巢,通过石蜡切片观察卵泡发育情况,采用荧光定量PCR检测FOXL2基因在卵巢的表达情况。结果显示:120~480日龄,卵巢重量呈现先增加后减少的趋势,220日龄达到最大;卵巢的初级卵泡数逐渐减少,而次级卵泡数先增加后减少,160日龄次级卵泡数最多;蛋鸡卵泡萎缩率增加,闭锁卵泡数逐渐增多;FOXL2基因的m RNA水平在120~480日龄呈下降趋势,暗示其可能与蛋鸡卵巢和卵泡的发育存在相关性。     

β-catenin和BMP2基因在幼龄水貂皮肤中表达的研究

本试验旨在探讨β-catenin和BMP2基因在幼龄水貂皮肤中定量表达的规律及意义。试验选用0～6周龄的幼龄公水貂21只,每周龄各3只,采集背中部的皮肤样品,运用实时荧光定量PCR技术相对定量方法, 检测不同周龄的幼龄水貂皮肤中β-catenin和BMP2基因相对表达量的变化。结果显示,随着周龄的增加β-catenin mRNA丰度呈上调趋势,在4周龄时显著增大(P<0.05),5周龄时达最大(P<0.01),6周龄时β-catenin相对表达量与5周龄相比极显著下降(P<0.01);而BMP2 mRNA的表达保持稳定(P>0.05)。β-catenin mRNA的丰度与次级毛囊发育变化趋势一致,与前人相关报道一致,提示其可能参与水貂次级毛囊发育;而BMP2 mRNA的丰度在毛囊发育阶段变化不明显。     

Differential expression of bone morphogenetic protein 4-6 (BMP-4, -5, and -6) and growth differentiation factor-9 (GDF-9) during ovarian development in neonatal pigs

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs), comprise the largest subgroups of ligands in the TGF-β superfamily, and have been shown to be involved in follicle development in mammals. However, whether these factors are involved in folliculogenesis in pigs is still unknown. The present study was performed to determine the relationships between early folliculogenesis and the expression of GDF-9 and BMP (BMP-4, -5 and -6) mRNAs in neonatal pigs. Ovaries were removed at 5, 16, 28 and 39 days after birth to examine the follicular population (the right ovary of each animal) and to detect mRNA expression (the left ovary of each animal). Primordial follicles accounted for >80% of the ovarian follicles from 5 days until 39 days after birth. A marked increase in primary follicles and the appearance of secondary follicles were observed in the ovaries at 28 days after birth. BMP-4, -5, and -6 and GDF-9 mRNAs were expressed by ovaries at 5-, 16-, 28- and 39-day-old pigs. The peak expression of BMP-4, -5, and -6 and GDF-9 mRNAs was observed in the ovaries at 5, 39, 28 and 16 days, respectively, after birth. These data demonstrate that folliculogenesis in piglets might be controlled by the interaction with these factors. We conclude that BMPs and GDF-9 may have distinct functions in several stages of follicle development in neonatal pig ovaries.     

GDF9/BMP15对卵泡发育的调控

卵母细胞及其紧密连接的卵泡细胞之间的精细调节,促使卵母细胞成熟、受精和胚胎发育.在卵泡发育过程中,除了下丘脑-垂体-性腺轴间的内分泌调节外,卵母细胞源旁分泌或自分泌因子维持发育卵泡内微环境稳态,调节卵母细胞成熟和颗粒细胞增殖.目前发现,这些关键的调控因子主要是TGFβ超家族成员中的生长分化因子-9 （GDF9）和骨形态发生蛋白（BMP15）.GDF9/BMP15主要表达于卵母细胞,是卵泡发育必需的细胞因子.论文综述了GDF9/BMP15的结构特点、表达特性、信号通路及其在卵巢中的生物学作用等研究进展.     

Detection of Steroid Hormone Receptors in the Canine Circumanal Gland

The oestrogen receptor beta (ERß) is largely distributed in the ovary of many species but data for the bovine ovary are scare. Therefore, the expression of ERß mRNA in the different follicles of the bovine ovary was studied using in situ hybridization. Ovarian tissue sections of three cows with different plasma progesterone concentrations were used (cow 1: 3.50 ng/ml; cow 2: 1.00 ng/ml, cow 3: 0.35 ng/ml). A 602 bp fragment of ERß mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Subsequently, in situ hybridization was performed by incubating the sections with the DIG-labelled RNA anti-sense probe. For the semi-quantitative evaluation of ERß mRNA expression the ERß mRNA score (SER) was determined for the different follicular cell types using the formula: SER = 0.n0 + 1.n1+ 2.n2 + 3.n3 with n0, n1, n2, n3 indicating the percentage of cells exhibiting a staining intensity 0 (absent), 1 (weak), 2 (moderate) or 3 (strong), respectively. High ER mRNA levels were noticed in primordial and primary follicle cells, and suggest a role of ER mRNA in early folliculogenesis. A lower SER was observed in the granulosa cells of secondary and tertiary follicles. This significant difference in the SER of follicle cells during follicular growth may be associated with cell proliferation. In obliterative and cystic atretic follicles high SER were observed, although ERß mRNA levels in obliterative follicles showed much inter-individual variation. This is suggestive for ERß mediated oestrogen action in atretic follicles. In the corpora lutea moderate ERß mRNA levels were noticed. Our findings are in accordance with studies in the ewe in which corpora lutea cells synthesize estrogen. These preliminary findings will be further elaborated in a higher number of cows to examine the role of ERß in the ovary throughout the oestrus cycle.     

不同繁殖类型牦牛卵巢原始卵泡的观测

本文对不同繁殖类型牦牛卵巢原始卵泡进行了观测 ,结果发现 :原始卵泡数为青麻 (一年一胎者 ) >牙日玛 (两年一胎者 ) >干巴 (三年一胎者 ) (P <0 0 5 )     

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.     

Case of Pregnancy in Two Cows with Unicorn Horn of the Uterus either by Artificial Insemination at Ipsilateral or Embryo Transfer at Contralateral Corpus Luteum in the Ovary

Two Holstein heifers and a cow were diagnosed with White Heifer Disease by ultrasonography. Case 1 was a 14 month-old heifer with aplasia of both sides of the uterine horn. In case 2, a primiparous cow and case 3, an 18 month-old heifer, both showed aplasia of the right uterine horn. Case 2 became pregnant by artificial insemination at ipsilateral ovulatory follicle and corpus luteum in the left ovary, while case 3 became pregnant by embryo transfer at 7 days after oestrus with contralateral corpus luteum in the right ovary.