【作者】 王琦；

【导师】 杨平；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2022， 硕士

【摘要】 波尔山羊是优良的肉羊品种,具有极高的经济和科学价值。睾丸是雄性生殖系统中的重要器官,其发育状况将直接影响性成熟时间、精液品质,从而决定山羊的繁殖性能。充分了解山羊睾丸发育的生理特性,对山羊的选育、生精能力的调控具有重要意义。本试验从功能形态学角度出发,以幼龄组(1月龄,2月龄)和成年组(12月龄)山羊睾丸为研究对象,通过Telocytes(TCs)、管周肌样细胞(Peritubular myoid cells,PMCs)、支持细胞(Sertoli cells,SCs)、睾丸间质细胞(Leydig cells,LCs)的结构鉴定和变化特点,探究生精小管(Seminiferous tubule,ST)、固有层(Lamina propria,LP)的发育变化过程,为深入研究山羊雄性生殖系统疾病的发生、治疗提供理论基础。目前,对睾丸的研究主要集中在生精细胞、支持细胞和睾丸间质细胞,其他成分研究较少,本试验聚焦于睾丸间质固有层,证明了山羊睾丸中存在新型间质细胞TCs。试验借助了HE及特殊染色、透射电子显微镜(Transmission Electron Microscopy,TEM)、形态学计量统计、免疫荧光双标(Double-labelling Immunofluorescence,DIF)技术。结果显示:与幼龄山羊睾丸相比,成年山羊睾丸生精小管、固有层、血-睾屏障(Blood-testis barrier,BTB)发育完善。随着年龄增长,管周细胞(Peritubular cells,PCs)分化成了两种类型,内层是具有平滑肌特征的管周肌样细胞,外层是突起细长的长梭形细胞。试验还证明了山羊睾丸中存在TCs,观察了TCs的超微结构、分布特点、免疫表型,揭示了TCs与其他细胞的空间位置关系,光镜下TCs的特征与在TEM下观察到的相吻合,验证了CD34/Vimentin是山羊睾丸TCs的最佳标记物,证明了山羊睾丸管周细胞外层长梭形细胞的实质是TCs。本试验为了解生精小管、固有层的发育变化过程,证明管周细胞具有异质性以及TCs能够维持睾丸内环境稳态提供了理论依据。试验Ⅰ山羊睾丸生精小管和固有层的组织学特征本试验采用HE和特殊染色、TEM,结合形态学计量统计分析,通过显微结构和超微结构观察,探究了性成熟前后山羊睾丸生精小管、固有层的发育变化过程。结果显示:与幼龄山羊睾丸相比,成年山羊睾丸生精小管出现管腔、管径增大,生精细胞种类、数量增多,各生精细胞分层排布。固有层基膜变薄,支持细胞发育成熟、体积增大、胞质广泛,支持细胞之间的紧密连接在1月龄已经形成,成年山羊睾丸中紧密连接明显增多。睾丸间质细胞逐渐成熟,成群密集分布。胶原纤维广泛分布于固有层基膜和间质中,固有层、血-睾屏障发育完善。固有层中管周细胞层数明显增多,胞核和突起显著增长,值得注意的是,与幼龄组相比,成年组管周细胞分化为两种,内层是具有平滑肌特性的管周肌样细胞,外层是细长突起的长梭形细胞,外层细胞具有TCs的典型特征,但幼龄睾丸管周细胞分化不显著。本试验探究了生精小管、固有层的发育变化过程,证明了管周细胞的异质性,为深入研究睾丸的生理功能提供了理论依据。试验Ⅱ山羊睾丸Telocytes的鉴定和功能形态学研究本试验利用TEM、DIF的方法探究固有层中TCs的存在、分布和变化。结果显示:从超微结构来看,TCs在生精小管管周广泛存在,突起(Telopodes,TP)呈细段(Podomers,Pm)和膨大部(Podoms,Pd)相互交替的串珠状。TP弯曲折叠、相互缠绕,镶嵌分布于胶原纤维束之间、管周肌样细胞外侧、血管周围,并释放囊泡(Vesicle,V)。TCs与周围结构,如:血管(Blood Vessels,BV)、胶原纤维(Collagen Fiber,CF)、管周肌样细胞、睾丸间质细胞等形成异型细胞连接,构成复杂的三维网络,推测其可能在信息传递中发挥作用。从DIF结果来看,管周细胞具有异质性,幼龄组中管周细胞呈CD34~+/α-SMA~+,也存在CD34~+/Vimentin~+管周细胞,突起较短,结合DIF结果判断CD34~+/Vimentin~+管周细胞为TCs。成年睾丸CD34~+细胞和α-SMA~+细胞为完全不同的两层,生精小管周围存在大量CD34~+/α-SMA~-细胞,CD34~+/Vimentin~+管周细胞为TCs,具有细长的突起,呈多层分布,与TEM结果吻合。CD34和Vimentin可作为山羊睾丸TCs的可靠标记物,尽管PDGFR-α可作为TCs的常用标记物,但本试验证明其不能标记山羊睾丸TCs。本试验为山羊睾丸TCs参与睾丸微环境稳态提供了理论依据。更多还原

【Abstract】 Boer goats are excellent meat goat breeds with high economic and scientific value.The testis is an important organ in the male reproductive system,and its development will directly affect the time of sexual maturity and semen quality,thus determining the reproductive performance of goats.A full understanding of the physiological characteristics of testicular development in goats is of great significance for the selection and breeding of goats and the regulation of spermatogenic ability.In this experiment,the testes of goats in the young(1 month old,2 months old)and adult(12 months old)groups were studied from the functional morphological point of view.Structural identification and characteristics of changes by Telocytes(TCs),Peritubular myoid cells(PMCs),Sertoli cells(SCs),and Leydig cells,(LCs).To investigate the developmental changes of Seminiferous tubule(ST)and Lamina propria(LP),and to provide a theoretical basis for in-depth research on the occurrence and treatment of male reproductive system diseases in goats.Currently,studies on testes have focused on spermatogenic cells,SCs and LCs,and other components are often neglected.This experiment focused on testicular mesenchyme and demonstrated the presence of novel mesenchymal cells TCs in goat testes.The experiment was performed with the help of special staining,Transmission electron microscopy(TEM),morphometric statistics,double-labelling Immunofluorescence(DIF)techniques.The results showed that the ST,LP,and Blood-testis barrier(BTB)were well developed in adult goat testes compared with those of young testes.With age,Peritubular cells(PCs)differentiated into two distinct types,the inner layer is PMCs with smooth muscle characteristics and the outer layer is a long spindle-shaped cell with elongated protrusions.The experiment also demonstrated the presence of TCs in goat testes,observed the ultrastructure,distribution characteristics,and immunophenotype of TCs,revealed the spatial location of TCs in relation to other cellular,and the characteristics of TCs under light microscopy are consistent with those observed under TEM,verifying that CD34/Vimentin was the best marker for TCs in adult goat testes.The parenchyma of the outer long spindle-shaped cells of goat testis PCs was demonstrated to be TCs.This experiment provides a theoretical basis for understanding the process of developmental changes in STs and LPs,demonstrating that PCs are heterogeneous and that TCs are able to maintain the homeostasis of the intra-testicular environment.ExperimentⅠHistological characteristics of seminiferous tubule and lamina propria in goat testisIn this experiment,we used special staining,TEM,combined with morphometric statistical analysis,and explored the developmental processes of ST and LP in goat testes before and after sexual maturation through microstructural and ultrastructural observations.The results showed that the adult testis ST showed an increase in the lumen and diameter of the ducts,an increase in the type and number of spermatogenic cells,and a stratified arrangement of each spermatogenic cell compared to the young testis.The LP basement membrane thinned,SCs were mature,enlarged and had extensive cytoplasm,tight junctions between SCs had formed by 1 month of age,and tight junctions were significantly increased in adult goat testes.LCs gradually matured and were densely distributed in clusters.Collagen fibers are widely distributed in the LP basement membrane and interstitium,and the LP and BTB are well developed.There was a significant increase in the number of PCs layers in LP and a significant increase in nuclei and cell protrusions.Notably,compared with young testes,adult testis PCs were divided into two distinct types,the inner layer is PMCs with smooth muscle characteristics and the outer layer is a long spindle-shaped cell with elongated protrusions,outer layer of cells with the typical characteristics of TCs,but young testis PCs were not significantly differentiated.This experiment explored the developmental change process of ST and LP,proved the heterogeneity of PCs,and provided a theoretical basis for an in-depth study of the physiological function of the testis.ExperimentⅡIdentification and functional morphology of Telocytes in goat testisIn this experiment,the presence,distribution and changes of TCs in LP were investigated by TEM and DIF.The results showed that from the ultrastructure,TCs were widely present in the peritubular area of ST,and the protrusions(Telopodes,TP)were in the form of a string of beads with alternating fine segments(Podomers,Pm)and expanded parts(Podoms,Pd).TP bends and folds,intertwines,and mosaics between collagen fiber bundles,outside of PMCs,around blood vessels,and releases vesicles(V).TCs form heterotypic cellular connections with surrounding structures,such as Blood Vessels(BV),Collagen Fiber(CF),PMCs,LCs,etc.,forming a complex three-dimensional network,presumably with a possible role in information transfer.From the DIF results,PCs were heterogeneous,and PCs in the young group were CD34~+/α-SMA~+and also CD34~+/Vimentin~+PCs with shorter protrusions,and combined with the DIF results,CD34~+/Vimentin~+PCs were judged to be TCs.Adult testicular CD34~+cells andα-SMA~+cells were two completely different layers,with a large number of CD34~+/α-SMA~-cells were present around the ST,and CD34~+/Vimentin~+PCs were TCs with elongated protrusions in a multilayered distribution,which was consistent with the TEM results.TCs were labeled as CD34~+/Vimentin~+cells with elongated protrusions and shuttle-shaped or triangular cytosol,which was consistent with the TEM results.CD34 and Vimentin can be used as reliable markers for TCs in goat testes,and although PDGFR-αcan be used as a common marker for TCs,this test demonstrated that it cannot label goat testicular TCs.This experiment provides a theoretical basis for the involvement of goat testicular TCs in testicular microenvironmental homeostasis.更多还原