【作者】 张莉；

【导师】 陈秋生；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2008， 博士

【摘要】 繁殖是动物生活史中最为重要的一个环节,能否正常、顺利地进行繁殖,关系到物种的延续及物种的多样性。由于爬行动物处于动物进化过程中的特殊地位,是脊椎动物最先登陆的类群,能否在陆上繁殖就成为进一步发展的主要矛盾。因此,对此环节进行深入研究显得尤为重要。然而,有关中华鳖生殖特性方面的研究鲜有报道。中华鳖(Pelodiscus sinensis)是龟鳖目鳖科的代表动物之一,是我国重要的特种水产经济动物,具有重要的营养价值和药用价值。在生殖周期中,由于中华鳖具有典型的冬眠现象和精子储存的生理学特征,是研究爬行动物生殖生物学的理想模式动物。本文首先对中华鳖精子的超微结构及其变态发生过程进行了观测,进而探究了生精上皮细胞显微结构的年周期变化以及在这个过程中细胞增殖和凋亡动态。在此基础上探讨了激素受体在相关生殖器官(由于雌性的输卵管具有精子储存功能,所以也在此一并研究)的分布规律。研究结果将从细胞和分子水平阐明雄性中华鳖生殖生物学特性,并为中华鳖的繁殖、保护及资源利用等应用研究奠定基础。试验Ⅰ中华鳖精子的超微结构研究应用透射电镜和扫描电镜技术观察了中华鳖精子的超微结构。成熟的精子由头部、连接段、中段、主段和末段组成。头部呈细长的锥体状,细胞核前1/3为核前突,表面被顶体复合体覆盖。顶体下锥直接包绕核前突,顶体帽与顶体下锥之间为顶体下间隙。3～5条纵行的核内小管与顶体下锥头端的杆状物相延续。连接段位于核后窝内,由纵行短的节柱组成。中段由中央的中心粒和周围的线粒体鞘组成。线粒体鞘由7～8圈线粒体环形排列而成,每圈约有5个线粒体,上、下层线粒体错位1/2排列。线粒体形态为8～10层排列的同心圆状,其平行的板层实为长而弯曲分布的线粒体嵴。这种线粒体嵴和细胞内大量的糖元颗粒,与中华鳖成熟精子长时间贮存并保持活力有关。线粒体鞘末端的楔形终环明显。近-远端中心粒相互垂直排列。近端中心粒的结构模式是9×3+0,管壁三联微管呈典型的风车状。远端中心粒的结构模式为9×3+2,中央微管和外周三联微管周围均与致密纤维重合排列,前部的外周致密纤维相互融合成连续的一圈,向后各致密纤维逐渐分离。主段具有发达的环行纤维鞘,前厚(数层)后薄(一层)。中央的轴丝复合体呈9×2+2,在与第3和第8号二联微管相对的部位可见两条与纤维鞘融合的纵行纤维。横切面上,主段二联微管A管上的动力臂明显,两条中央微管间有短的横桥连接,中央微管通过放射状辐与二联微管A管相连。末段仅由轴丝复合体和表面的细胞膜组成,其中的微管逐渐分散成单个排列。中华鳖精子的结构在一些细节和数量上与龟鳖目的海龟和乌龟存在一定差异,更与其它纲动物和龟鳖目之外的其它爬行动物差异明显。试验Ⅱ中华鳖精子的形成应用透射电镜技术观察了中华鳖精子的形成过程。精子的形成过程包括细胞核的拉长、染色质的浓缩、顶体和鞭毛的形成、细胞质的排除。第1期,前顶体泡形成并移向细胞核一侧,同侧核膜凹陷成浅窝。前顶体泡底部中央出现小的顶体颗粒,纤维物质层位于核前端与前顶体泡底壁之间,其核膜一侧的中央形成更小的顶体下颗粒,将与核内小管的形成有关。细胞核开始端移和变形。第Ⅱ期,前顶体泡变成扁囊状覆盖于隆突的核顶端,顶体颗粒弥散成中等电子致密物分布于顶体帽中,纤维物质层发育为顶体下锥。环形核套微管在顶体后端的核周围逐渐形成,核内染色质开始浓缩成圆形颗粒,伴随着细胞核的偏移,细胞质开始发生重排。第Ⅲ期,拉长的细胞核前端凸出,表面有顶体复合体覆盖,变态的精子头部扎在支持细胞的凹陷中,核后端最宽并出现植入窝,染色质进一步浓缩。线粒体向后拖移位于细胞核的后端,许多微管开始组装成鞭毛的轴丝复合体。第Ⅳ期,染色质浓缩成致密均质物,环形核套微管先后改建为斜行和纵行核套微管,支持细胞突起形成“袖领”包绕顶体。多余的细胞质后拖,形成与头部和中段相连的胞质叶,它逐渐的脱落排除。第Ⅴ期,精子的头部开始成熟,核套微管解聚而消失,顶体周围的“袖领”也消失。线粒体开始排列在近端中心粒和远端中心粒周围,在线粒体的后端,纤维鞘环绕在鞭毛轴丝复合体的周围。试验Ⅲ中华鳖睾丸和附睾形态结构的年周期变化应用组织学方法,对性成熟雄性中华鳖睾丸、附睾在年周期中的形态结构变化进行了较系统的观察和分析,以揭示其生殖的年周期变化规律。结果表明:中华鳖睾丸的年周期活动由7个特征明显的时期构成。4月份为第Ⅰ期,为精原细胞的增殖期;5～6月份是精原细胞复苏、增殖、分化形成精母细胞时期,为第Ⅱ期;7～8月份为第Ⅲ期,是精母细胞分裂成为精细胞时期;9～10月份是精子形成期,大量的精子细胞变态成为成熟的精子,此期为Ⅳ期;11月份为第Ⅴ期,生精上皮主要由精原细胞和大量的精子组成,精母细胞基本上已消耗完;12月至次年1月份为第Ⅵ期,精子发生渐趋停滞,只有残留的少量精子。2月份为第Ⅶ期,精子发生已经停滞。睾丸系数和附睾系数的季节性变化反应了雄性中华鳖的生殖年周期规律,而且与组织学观察结果相一致。中华鳖精子产生限定在一年内的特定阶段,雄性产生精子与雌性的排卵及受精间是异相的。因此,中华鳖的生精类型属于分离型模式,而且精子发生的模式是时间性的发育模式,明显与鸟类和哺乳类不同。试验Ⅳ中华鳖繁殖周期中生精细胞的增殖和凋亡应用电镜技术、增殖细胞核抗原(PCNA)以及脱氧核糖核酸末端转移酶介导的缺口末端标记法(TUNEL),研究了繁殖期和非繁殖期生精上皮细胞的增殖和凋亡情况。PCNA免疫组织化学反应显示:在7月、9月(繁殖期)精原细胞和初级精母细胞的细胞核都呈阳性反应;12月、1月(非繁殖期)大部分的精原细胞和少量的初级精母细胞呈阳性,但繁殖期初级精母细胞的阳性率比非繁殖期的高很多。TUNEL法标记的大量凋亡细胞出现在12月、1月份的曲细精管中,其中大量的初级精母细胞和少量的精子细胞发生凋亡,并且这种阳性细胞在整个生精停滞期(非繁殖期)都存在,但生精期几乎未见任何的凋亡细胞。初级精母细胞是主要的凋亡细胞类型。电镜下所见的凋亡细胞同样发生在非繁殖期,核染色质固缩边聚,呈半月形、环形、球形或不规则形高电子密度的浓缩核,有的凋亡细胞出现凋亡小体。而繁殖期各级生精上皮细胞器结构完整,核呈均质状,胞质无浓缩现象。制作超薄切片时保留半薄切片,甲苯胺兰染色,光镜观察在非繁殖期也看到凋亡现象。基于以上的结果,我们认为初级精母细胞和精子细胞的凋亡是非繁殖期生精上皮损失的主要原因,并且正常的季节改变对中华鳖生精上皮细胞具有时间和空间特殊性。试验Ⅴ雄激素受体和雌激素受体α在中华鳖生殖器官中的分布首次用雄激素受体(Androgen receptor,AR)和雌激素受体α(Estrogen receptorα,ERα)的多克隆抗体在中华鳖睾丸、附睾和输卵管中进行免疫组织化学定位研究。结果表明,特异性AR免疫阳性反应见于睾丸的间质细胞、肌样细胞、支持细胞、精原细胞、精母细胞和精子细胞。雄激素受体在中华鳖生精细胞中的阶段性表达表明,它与相应的激素结合后,调节精子发生,可能与精原细胞的增殖、精母细胞的成熟分裂及精子形成密切相关。雌激素受体α主要表达于睾丸的间质细胞,生精细胞只在生精活跃期呈弱阳性反应,表明雌激素主要通过体细胞起调节作用,而它在生精细胞中阶段性分布表明它也可以直接调节生精细胞。由于雌性中华鳖的输卵管具有精子储存功能,在此与雄性生殖器官一并进行了研究,发现这两种类固醇激素受体在中华鳖输卵管内均有阳性分布,主要表达于上皮细胞腔面、腺细胞和基质细胞,表明它们可能均与精子的储存及输卵管的腺体分泌有关。试验Ⅵ中华鳖睾丸雄激素受体mRNA的表达采用原位杂交技术(In situhybridization,ISH)研究雄激素受体mRNA在中华鳖睾丸的存在及细胞定位。取10只健康成年中华鳖的睾丸,进行冰冻切片。用地高辛标记的寡核苷酸探针对组织切片进行原位杂交,检测雄激素受体mRNA在睾丸组织细胞内的表达定位。结果显示:大部分雄激素受体mRNA阳性细胞呈圆形或椭圆形,数量少且分布在间质细胞中,杂交阳性信号物质只分布在细胞核中,曲细精管内没有阳性反应。本研究从基因水平证明中华鳖睾丸组织中有雄激素受体表达,为在基因水平上阐明中华鳖睾丸中雄激素的生理调节过程提供理论依据。更多还原

【Abstract】 Breeding is the most important link in the animal life, whether they can normal and successful breed, is related to the continuation of species and species diversity. Because reptiles were in the special status during the evolution of animals, and was the first landing in vertebrate groups, whether they can successful breed in the land became the principal contradiction for further development. Therefore, a thorough study of this link is particularly important. However, they are few studies that have been reported the reproductive characteristics of soft-shelled turtle, Pelodiscus sinensis. Soft-shelled turtles, which are distributed widely in China, are one of the most representative classes of terrapin. This species is famous for its economical and pharmacological value, and hence they are subject to harvest pressure. Because the soft-shelled turtles have physiology characteristics which own typical hibernation and sperm storage in the reproductive periodicity, it is an ideal model for studying on reptile biology. In present study, we observed the characteristics of spermatozoon as well as spermiogenesis in the Pelodiscus sinensis by electron microscope; and examined the relationship between seasonal changes of testes histological structure, and the apoptosis and proliferation of germ cells in different season; then gave a further analysis of the distribution of the hormone receptor in the gonad tissues (Because the oviduct in the female soft-shelled turtle has a function for sperm storage, here we study this structure with the male reproductive organ). Those researches explain the reproductive characteristics of cell and molecule in the male Pelodiscus sinensis, and provide basic datum for the works of reproduction, protection and resource utilization of Chinese soft-shelled turtle.ExperimentⅠUltrastructure of spermatozoon in soft-shelled turtle, Pelodiscus sinensis Electron and scanning microscopies were undertaken to determine the ultrastructure of spermatozoon in soft-shelled turtle in present study. The mature spermatozoon is vermiform in shape, which consists of a head, connecting piece, middle piece, principal piece and end piece. In the head, acrosomal complex covers the anterior protrusion of the nucleus, where there is a narrow lucent space between the subacrosomal cone and the outer acrosomal cap. 3～5 intranuclear tubules go through the anterior tow-thirds of the nucleus and their central cores extend towards the perforatorial rods. The connecting piece is located in the concave implantation fossa in the caudal end of the nucleus. The mitochondrial sheath in the middle piece is composed of concentric mitochondria arranged in 7～38 circles each of which contains 5 mitochondrion. Concentric layers in the mitochondrion are double-membranes and they are cristae mitochondriales actually. The concentric cristae mitochondriales and lots of glycogens show the relationship to the vitality maintaining during long-time storage of the spermatozoon in the turtle. There is a wedge-shaped annulus around the end of the mitochondriall sheath. The structural modal of proximal centriole is 9×3+0, in which the typical nine triplet microtubules arrange in a pinwheel fashion and are surrounded by a dense material. By comparison, the structural modal of distal centriole is 9×3+2, and the central microtubules and outer triplet microtubules are embed in dense fibers respectively. The distal centriole stands vertically to the proximal one. The axonemal complex of the principal piece is surrounded by several layers of circumferential fibers, which obviously produces a fibrous sheath reducing backward gradually in thickness. In the axonemal complex that posses a structural model of 9x2+2, there are two thick fibers corresponding to No.3 and No.8 doublet microtubules respectively, which merge with the fibrous sheath. On the transection of principal piece, two dynein arms attached to a microtubule of the doublet which connected with the central microtubules through an obvious radial spoke. Two central microtubules are linked by a bridging substance. The end piece only contains an axonemal complex surrounded by cell membrane, the microtubules in which are dispersed gradually. The spermatozoon of Pelodiscus sinensis are unique in several structures and numbers to that of the turtles, Chrysemys picta and Chimemys reevesii. The structural characteristics above were different evidently from other class animals or other class reptile.ExperimentⅡSpermiogenesis in the soft-shelled turtle, Pelodiscus sinensis Spermiogenesis in the Pelodiscus sinensis, was examined by transmission electron microscopy. The process includes nuclear elongation, chromatin condensation, acrosomal and flagellar development, and elimination of excess cytoplasm. In stageⅠ, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. A smaller subacrosomal granule in the middle of the fibrous layer is related to the development of intranuclear tubules. The nucleus begins to move eccentrically. In stageⅡ, the round proacrosomal vesicle is flattened by protrusion of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle, as the fibrous layer forms the subacrosomal cone. Circular manchettes develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. In stageⅢ, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; the spermatids come to lie within deep recesses of the Sertoli cell cytoplasm; the caudal end of the nucleus is wide and now lodged into a depression; coarse granules replace the small ones within the nucleus. At the posterior pole of the head, mitochondria move backwards. Numerous microtubules begin to assemble the axoneme of flagellum. In stageⅣ, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the cytoplasm move to the posterior pole of the spermatid. The residual bodies are derived from cytoplasmic lobes of elongated spermatids, which are eliminated gradually. In stageⅤ, the sperm head matures. Following dissolution of the longitudinal manchette and the collar of the Sertoli cell process shrinks, the mitochondria arrange themselves around the proximal and distal centrioles. Caudal to the mitochondrial mass, a fibrous sheath surrounds the proximal portion of the flagellum.ExperimentⅢAnnual variation in testis and epididymis of male Pelodiscus sinensis The morphology and microstructure of testis, epididymis in male adult soft-shelled turtle, Pelodiscus sinensis were observed and analyzed relatively systemically using histological methods during the annual reproduction cycle, in an attempt to investigate its reproductive rule. The spermatogenetic cycle of soft-shelled turtle comprises seven stages with significant features: stageⅠin April, the testis was at the stage of spermatogonic proliferation; StageⅡfrom May to June, most spermatogonia proliferated and differentiated into the spermatocytes. StageⅢfrom July to August, spermatocytes were in meiotic division state and divided to form spermatides. StageⅣfrom September to October, spermiogenesis was well underway, and spermatids were transformed to spermatozoa. StageⅤin November, seminiferous tubules became filled with spermatogonia and lots of spermatozoa. StageⅥfrom December to January, the spermatogenesis was largely complete in this stage. Some residual spermatozoa scattered in the lumen. StageⅦin February, the testes were in the regression. The changes of testis index and epididymis index during the annual cycle were response the rule of the reproductive activity, and to accord with the results of histology. Spermatogenesis in the Pelodiscus sinensis was limited in some stages during one year, and spermatogenesis and ovulation were out if phase with each other. Therefore, the testicular cycle of soft-shelled turtle follows a disassociated pattern. This germ cell development strategy is temporal in the spermatogenesis. This strategy is different from bird and mammalia.ExperimentⅣSeasonal effects on apoptosis and proliferation of germ cells in the testes of the soft-shelled turtle, Pelodiscus sinensis To elucidate the proliferation and apoptosis of germ cells in the testes of the soft-shelled turtle during the spermatogenically active phase and nonspermatogenic phase, we used electron microscopy, the proliferating-cell nuclear antigen (PCNA), TdT-mediated dUTP nick end labeling (TUNEL) assay. The PCNA was expressed in nuclei of spermatogonia and primary spermatocytes during the spermatogenically active phase (July and September). During the regressive phase (December and January), PCNA-positive cells also included spermatogonia and spermatocytes, but the number of positive spermatocytes was less than that during the spermatogenically active phase. The TUNEL method detected few apoptotic cells in spermatogenic testis, with much larger numbers during the spermatogenically quiescent phase. Spermatocytes were the most common germ cell types labeled by the TUNEL assay and a few spermatides were also labeled. Using the electron microscope, apoptotic spermatocytes had membrane blebbing and chromatin condensation being mostly half-moon or horseshoe shaped during the resting phase, but not during active spermatogenesis. Apoptotic cells were absent from the spermatogenically active seminiferous epithelium, which exhibited intact membranes and intact organelle morphology. Sections from the spermatogenically quiescent phase that were stained with toluidine blue for light microscopic examination also had apoptotic characteristics. We inferred that accelerated apoptosis of spermatocytes and spermatides partly accounted for germ cell loss during the nonspermatogenic phase. We concluded that seasonal variations in spermatogenesis in the soft-shelled turtle spermatogenesis were stage- and process-specific.ExprementⅤDistribution of androgen receptor and estrogen receptor a in the genital organs of Pelodiscus sinensis The localization of androgen receptor, estrogen receptor a in the testis, epididymis and oviduct were studied by immunocytochemistry in order to discuss their effects on the gonad. The specific AR immunostaining was observed in Leydig cells, peritubular myoid cells, Sertoli cells, spermatogonia, spermatocytes, spermatids. As a result, androgen receptor localized in spermatogeneous cells in phasic changing, which binding with androgen can exerts regulation role directly on spermatogenous cells. Therefore, there may be close relationship between androgen receptor and spermatogonia proliferation, spermatocytes meiosis and spermiogenesis. Estrogen receptor a positive cells were distributed mainly in the Ledyig cells. Spermatogenic cells only showed weak positive reaction in the spermatogenic active. Those results indicated that estrogen might be regulated through somatic cell mainly, and the stages of distribution in the spermatogenic cells showed that it might regulate directly on the spermatogenic cells. Because the oviduct in the female soft-shelled turtle has a function for sperm storage, here we study this structure with the male reproductive organ. Two sex steroid hormone receptors were localized in the oviduct and they were localized mainly in the luminal epithelial cell, gland cell and stromal cell, which indicated that these receptors may be all associated with sperm storage and gland secretion.ExperimentⅥExpression of androgen receptor mRNA in testis cells of Pelodiscus sinensis This study was to detect the expression and localization of androgen receptor mRNA in testis by means of the in situ hybridization histochemiatry (ISHH). Frozen sections of testis in ten healthy Pelodiscus Sinensis were made. In situ hybridization was conducted on the tissue sections by oligonucleotide probe marked by cardiox. The distribution of androgen receptor mRNA were detected in the cells of testis tissue. The results of in situ hybridization indicate that the AR positive cells distributing in the Leydig cells. The labeling cells are round or oval-shap in shape, and the signals are distributed in nuclei of all positive cells. The Sertoli cells and germ cells in the seminiferous tubules were negative for the reaction. This finding show that the AR mRNA are transcribed in the testis, and would provide further evidence that androgen might be regulation the function of the testis in the soft-shelled turtle.更多还原