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变化及卵泡闭锁规律研究

旨在对不同日龄猪卵巢组织结构以及卵巢发育过程中卵泡闭锁规律进行研究。取3、40、50、60、72、86、95及165日龄猪卵巢各3例,采用常规石蜡切片、HE染色和TUNEL技术检测卵巢组织结构及卵泡闭锁规律,结果表明,猪卵巢发育从组织学上可分为卵原细胞增殖期、卵泡缓慢生长期及卵泡快速生长期3个阶段。卵原细胞增殖期主要以卵原细胞的增殖分裂为特点;在卵泡缓慢生长期,原始卵泡的数量随日龄增加逐渐减少,初级卵泡数量及体积则明显增加;至卵泡快速生长期,生长卵泡的数量及体积继续增大,其数量在86日龄达到最大值;72日龄卵巢中出现三级卵泡,至95日龄卵巢中出现近成熟卵泡。在各时期的卵巢组织,均可观察到卵原细胞及卵泡的闭锁现象,以原始卵泡的退化最为显著。TUNEL检测表明,在卵原细胞增殖期,可见大量原始卵泡及初级卵泡的闭锁,其闭锁主要源自卵泡卵母细胞的凋亡。在卵泡缓慢生长期,各级生长卵泡均可出现闭锁,初级卵泡的闭锁主要由卵母细胞的凋亡引起,也有部分是同时伴有卵母细胞和卵泡细胞凋亡的;次级卵泡的闭锁则主要由于颗粒层细胞的凋亡所致。在卵泡快速生长期,随着卵泡的快速生长,各级卵泡的闭锁也变得更加明显,次级卵泡及三级卵泡的闭锁主要是由颗粒细胞凋亡引起。     

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。     

不同年龄哈萨克羊卵巢卵泡分布及组织学特征

哈萨克羊作为中国三大粗毛羊品种之一,是我国宝贵的畜禽遗传资源之一,研究不同年龄哈萨克羊卵巢中卵泡的分布及各级卵泡形态学特征,可为进一步开展其卵巢基础生理研究和品种遗传资源的开发与利用提供参考依据。试验采用组织学方法,分别对胎儿、羔羊、青年母羊、老龄母羊卵巢切片进行分析,结果表明不同年龄哈萨克羊卵泡分布区域相异,胎儿卵巢组织中以原始卵泡为主,主要分布于皮质最外层,内含较大的卵母细胞核。羔羊卵巢组织中发育卵泡较少;青年母羊发育卵泡则较多,主要分布于与髓质交界的区域。发育卵泡外周常环绕单层或多层立方状颗粒细胞。随着年龄增长,老龄母羊中逐渐以闭锁卵泡为主,哈萨克羊闭锁卵泡卵母细胞皱褶,核固缩或颗粒细胞分布杂乱与基底膜分离。     

双峰驼卵巢的组织结构

应用组织学方法对双峰驼卵巢的形态结构进行了显微与亚显微观察。结果显示 ,双峰驼卵巢表面被覆单层立方至单层扁平的生殖上皮 ,无排卵窝 ,上皮下为致密结缔组织白膜 ,卵巢实质由外周的皮质和中央的髓质构成。皮质中原始卵泡和初级卵胞数量稀少 ,其分布位置更靠近皮质中层 ,偶见同一卵泡内含 2个卵母细胞。体积大小和发育阶段不同的次级卵泡与三级卵泡闭锁时 ,所发生的形态变化不一致 ,可出现实心和囊状 2种闭锁卵泡。黄体中以粒性黄体细胞占优势 ,其细胞质中含大量脂质泡而呈海绵样。卵巢髓质中除含大量血管外 ,还见卵巢网分布     

早期猪胎儿卵巢的组织学观察

母猪怀孕后33-61d期间,是胎猪卵母细胞发生的关键阶段。为掌握这一时期卵巢上组织细胞的发育变化,试验采集妊娠33、40、46、54、61d的雌性胎猪卵巢进行苏木精-伊红染色。结果表明,妊娠33d胎猪卵巢表面上皮由1-2层细胞构成,实质见到卵原细胞和髓质索结构;妊娠40d时出现卵原细胞共质体结构和前细线期卵母细胞,髓质索状结构断裂;妊娠46d共质体样结构增多,卵母细胞进入前细线期和细线期。髓质索断裂成片段;妊娠54d时表面上皮由单层细胞构成,卵巢皮、髓可分,皮质中层见到合胞体样卵母细胞群,髓质相邻的皮质深层有一些原始卵泡正在形成;妊娠61d胎猪卵巢皮、髓界限清晰,皮质中层是大量合胞体样卵母细胞群,皮质深层出现了较多的原始卵泡,髓质中卵泡结构退化,卵母细胞发生核固缩。     

猪人工授精和繁殖疾患的防治(续一)

第二讲母猪的生殖生理第一节母猪的内生殖器官母猪的生殖器官主要有内生殖器官(卵巢和生殖道—输卵管、子宫和阴道)和外生殖器官(包括尿生殖前庭、阴唇、阴蒂),还有副性腺体(位于子宫颈以及阴道的一些腺体)共同构成母猪的生殖器官。一、卵巢在卵巢表面皮质部有一层生殖上皮,其中发育着卵泡。卵泡由原始卵泡开始经初级卵泡发育为次级卵泡,再继续发育形成卵泡细胞,再和卵丘上的卵泡壁相连,发育为三级卵泡,最后发育为成熟卵泡。发情期卵泡体积增大,卵泡中通常只有一个卵母细胞。未成熟的卵泡退化为闭锁卵泡。发情前夕,卵泡不断增长,卵泡液增多,…     

牛卵巢内卵泡及卵母细胞生长发育的组织学研究

随机摘取牛离体卵巢18枚,常规石蜡切片,光镜下共观察卵泡5 818个,并测得卵泡、卵母细胞直径和透明带厚度（平均值）。结果:在整个卵泡发育过程中,卵泡与卵母细胞发育是完全显著正相关（P<0.01,R=0.9906）,其中腔前期P<0.01,R=0.9917,有腔期P<0.01,R=0.9951。腔前卵泡时期,卵泡和其卵母细胞直径增长幅度大体相等,而从有腔卵泡始,卵泡生长速度远远大于其卵母细胞。透明带与卵母细胞发育呈不显著正相关（P>0.05,R=0.9521）。     

梅花鹿卵巢颗粒细胞的体外培养与鉴定

卵巢颗粒细胞在卵母细胞生长和卵泡发育过程中起着重要作用,为卵母细胞提供营养,分泌孕酮和雌二醇,与膜细胞一起调控卵泡的增殖和分化。卵巢颗粒细胞也具有干细胞特性,可以作为核移植的替代物,用卵泡刺激素受体(FSHR)抗体能很快地鉴定颗粒细胞。本研究通过对梅花鹿卵巢颗粒细胞进行体外培养,旨在探索梅花鹿颗粒细胞长期体外培养的条件,描述颗粒细胞的形态和表型特征,为进一步研究颗粒细胞的功能奠定基础。试验结果表明,梅花鹿卵巢颗粒细胞呈多角形或梭形,并伸出伪足,呈放射状生长,FSHR抗体鉴定结果为颗粒细胞纯度>90%。用含15%胎牛血清和1%双抗的DMEM/F12培养液培养,可获得生长活性好、细胞纯度高的梅花鹿卵巢颗粒细胞,HE染色和FSHR免疫荧光染色可以简便快速地鉴定梅花鹿卵巢颗粒细胞。     

水牛胎儿卵泡细胞和卵母细胞凋亡的特征

选择5月龄胎儿水牛的卵巢，采用石蜡切片、HE染色的光镜观察和DNA末端原位标记技术（TUNEL）进行了研究。结果表明：5月龄胎儿卵巢上分布着大量的凋亡细胞；在皮质部存在的大量腔前卵泡中，均匀分布有单个凋亡的卵泡细胞和卵母细胞，凋亡细胞的主要组织学变化是细胞缩小，核浓缩；DNA末端标记变化主要显示出TUNEL阳性。上述结果提示：水牛卵泡细胞和卵母细胞凋亡在胎儿时期已经存在，细胞凋亡是启动水牛腔前卵泡闭锁的潜在机理。     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

Oocyte Morphology from Primordial to Early Tertiary Follicles of Yak

The objective of this study was to investigate the developmental morphology of yak oocytes from the primordial follicle to the tertiary follicle. Yak oocytes from resting primordial (n = 6), activated primordial (n = 12), primary (n = 9), secondary (n = 7) and early tertiary (n = 5) follicles were processed and analysed by light and transmission electron microscopy. The resting primordial follicular oocyte was characterized by relatively smooth surface on the oolemma, the accumulation of free and organelle‐related smooth (SER) and rough endoplasmic reticulum (RER), round or oval mitochondria, and polyribosomes on the surface of the RER and throughout the ooplasm. The activated primordial follicular oocyte was dominated by numerous coated pits and coated vesicles on the oolemma, and round mitochondria. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, polyribosome, Golgi complexes and mitochondria with distinct cristae. During the secondary follicular stage, formation of the zona pellucida, development of a desmosome‐like connection between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and elongated mitochondria in nearly all oocytes were seen. In the early tertiary follicular oocyte, the perivitelline space was present and a decrease in free SER and RER in the ooplasm occurred; finally, the nucleus migrated from an eccentric to a peripheral location. In conclusion, the growth of the yak oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte‐specific structures such as the zona pellucida, desmosome‐like connection and cortical granules.     

Histochemical detection of glycoconjugates in the inner perivitelline layer of Japanese quail (Coturnix japonica)

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. In this glycohistochemical study, a panel of fluorescein isothiocyanate (FITC)-labelled lectins was used to characterise and localise the oligosaccharide sequences of the IPVL glycoproteins at different stages of follicular development in the quail ovary. Deacetylation and sialidase digestion were also performed prior to lectin cytochemistry. Contrary to mammals, where the topographical distribution of these carbohydrates is not uniformly distributed throughout the zona pellucida, indicating the regionalisation of oligosaccharide chains, our results demonstrated a homogenous lectin staining of the comparatively thin IPVL. We also found variations in the presence and distribution of the carbohydrate residues in the IPVL during different stages of follicular growth. The IPVL of pre-vitelline follicles distinctly stains with WGA, sWGA and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α-D-GalNac in the glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc, VAA (Gal), DBA (α-D-GalNAc(1-3)-GalNAc and UEA-I (α-L-Fuc). With continuing follicular growth of the oocyte and the follicle, this staining pattern changed. LCA and PNA-staining in the IPVL became distinctly positive. As the IPVL of immature oocytes distinctly stains with WGA/sWGA-FITC, but spermatozoa do not bind to immature zona, it appears questionable that carbohydrate residues detected by WGA/sWGA play a major role in sperm-IPVL binding, as suggested in previous investigations.     

Ovarian Structure and Oogenesis of Catfish Pimelodella vittata (Lütken, 1874) (Siluriformes,Heptapteridae)

The morphology of the ovaries and oogenesis of Pimelodella vittata were studied using anatomical and histological techniques to provide information of its reproductive biology. Eighty adult females were captured trimonthly during the period November 2005 to October 2006. The ovaries are paired, saculiform organs, which are coated with tunica albuginea and contain ovigerous lamellae, where the oocytes develop before being released into the ovarian lumen and following the ovarian duct until reaching the genital papilla. Oogenesis was divided into stages based on the alterations to the nucleus, ooplasm and surrounding follicular layers. Oogonia form groups from the germinal epithelium have asynchronous development and differentiate into initial perinucleolar oocytes. The formation of the zona pellucida is initiated in the advanced perinucleolar oocytes reaching a thickness of 1.46 ± 0.58 μm in the vitellogenic oocytes. The follicular cells are squamous in perinucleolar oocytes, become cubical in the pre‐vitellogenic oocytes and prismatic in the vitellogenic oocytes with a height of 11.20 ± 4.74 μm. The histochemical reactions indicate that zona pellucida, cortical alveoli and yolk globules contain neutral glycoproteins and the follicular cells contain neutral glycoproteins in association with carboxylated and sulphated glycoconjugates. Statistical analyses showed significant differences in the diameter of the oocytes and follicular cells height as oocytes matured. This study represents the first data about the ovarian structure and oogenesis of this species.     

Allometric Study on the Relationship between the Growth of Ovarian Follicles and Oocytes in Domestic Cats

The relationship between the growth of preantral and antral follicles and that of their oocytes in ovaries of domestic cats (Felis catus) was analyzed. Eight hundred and five pairs of follicles and oocytes from the ovaries of 51 female cats were collected, and only healthy and fresh follicles and oocytes with or without zona pellucida were used in this study. Immediately after collection, the diameters of follicles and their oocytes were measured. The relationship of the follicle diameter to the oocyte diameter was applied to four regression models and statistically analyzed. The best fitting model was found to be a hyperbolic regression (the coefficient of determination was 0.976 between the follicles and their oocytes with a zona pellucida, y=184x/(x+0.0738); the coefficient of determination was 0.983 between the follicles and their oocytes without a zona pellucida, y=122x/(x+0.0301)). The differentiated equations for the hyperbolic curves in the oocytes with or without a zona pellucida and the follicles were found to be y'=13.6/(x+0.0738)(2) and y'=3.67/(x+0.0301)(2), where y and x were the diameters of the oocytes (μm) and follicles (mm), respectively. When follicles grew to a size larger than 0.4 mm in diameter, the growth rates of their oocytes calculated by the differentiation equations showed an asymptotic depression around zero. Thus, it was suggested that when the follicles grew to a size larger than 0.4 mm in diameter, their oocytes reached full size and ceased to grow and that the zona pellucida stopped growing when the diameter of the follicles reached 0.3 mm in domestic cats.     

颗粒细胞凋亡影响家禽卵泡闭锁的研究进展

卵巢是家禽的重要繁殖器官,会产生大量卵泡,而卵泡在生长发育的各个阶段中都可能因为不同因素的调控而发生闭锁,最终导致繁殖性能衰退。颗粒细胞对卵泡的生长发育有重要调控作用,其凋亡会诱导卵泡发生闭锁。诱导颗粒细胞发生凋亡的因素较多,包括激素、细胞因子、氧化应激、线粒体及其他体外因素。颗粒细胞凋亡主要由线粒体途径导致,其涉及到半胱天冬酶（Caspase）家族参与,当线粒体裂解时会释放细胞色素C （Cyt-C）,随后形成凋亡小体激活Caspase-3和Caspase-8,最终激活Caspase-9导致颗粒细胞凋亡;当颗粒细胞发生凋亡,家禽体内卵泡丧失生物功能并且卵泡细胞之间的调控失衡,促使卵泡内卵母细胞和膜细胞凋亡,最终导致卵泡发生闭锁;颗粒细胞在存活状态下所分泌的生长因子、性腺类固醇、细胞因子能减少卵母细胞氧化损伤,防止细胞内活性氧（ROS）水平过高导致的线粒体DNA损伤,从而避免线粒体功能障碍而造成的颗粒细胞凋亡。作者从颗粒细胞凋亡及其影响因素、颗粒细胞凋亡和卵泡闭锁的关系、颗粒细胞凋亡对卵泡闭锁的影响3个方面进行阐述,以期为减少卵泡闭锁、提高家禽繁殖性能提供理论依据。     

Oocyte ultrastructural characteristics in camel (Camelus dromedarius) primordial to large antral follicles

This study was conducted to describe in detail the ultrastructural features and morphological characteristics of camel oocytes from preantral follicles in relation to the sequential stages of follicular development and also for oocytes from antral follicles in relation to their diameter. Camel oocytes from primordial, primary, secondary and also early to late antral follicles were processed and examined by light and transmission electron microscopy. Primordial follicular oocytes were characterized by a layer of flattened granulosa cells around and also eccentric nucleus and few cytoplasmic organelles in the peripheral region. Up to the secondary follicle stage, flat cells were replaced by cuboidal granulosa cells and their number increased and also an increase in the number of organelles such as vesicles, mitochondria and endoplasmic reticulum was observed. In the early antral stage, the formation of zona pellucida, appearance of microvilli and pleomorphic mitochondria was seen and the nucleus was dislocated to the peripheral region. During final growth phase, the extent of endoplasmic reticulum, vesicles and mitochondria increased, the number of lipid droplets decreased and cumulus cell process endings (CCPE) were observed. In conclusion, the growth of camel oocyte is associated with progressive increase in the number of mitochondria, endoplasmic reticulum, Golgi complexes and cytoplasmic vesicles as well as decrease in the number of lipid droplets and the nucleus migration from an eccentric in preantral to a peripheral location in antral follicles.