| Telocytes,as a peculiar type of interstitial cells named by Popescu in 2010,have been identified in various organs among various species.The most typical feature of telocytes in comparison with other cells are their extending cellular prolongation named telopodes,which is extremely long and thin.Telopodes consists of alternately podomeres and podoms,establishing moniliform features.Numerous studies have shown telocytes’ potential roles in different organizations,such as mechanical support,signal transduction,tissue regeneration and repair,immune surveillance,etc.Currently as reported,telocytes have been identified in various organs,such as cardio vascular system,respiratory system,digestive system,urinary system,gland,skin,skeletal muscle and other tissues,but as for the male reproductive system,especially in the testes,the research of telocytes is still in its infancy.In testis,current research on the local regulation mechanism of spermatozoa in testis mainly focused on the cellular interaction between Sertoli cells,peritubular myoid cells,Leydig cells and germ cells,based on the unclear research status on the cellular components of peritubular tissues in testis.Thus in this study we focused on rat testis as the research object,aimed to explore the existence of telocytes in rat testis.We investigated the distribution characteristics and ultrastructural features of telocytes through transmission electron microscopy,and revealed the relationship between telocytes and their adjacent cells.Through immunohistochemistry and double-immunofluorescence,we used alpha-SMA which is the marker of peritubular myoid cells and the commonly used telocytes’ marker CD34,to explored the cellular components of peritubular tissue in rat testes.Through double-immunofluorescence of PDGFR-alpha/alpha-SMA and CD34/vimentin,we explored the immune phenotype and distribution characteristics of telocytes in rat testis.This study aimed to reveal the distribution characteristics and immunolabling of telocytes in rat testis from the view of morphology,and provide a new perspective of the cellular elements of peritubular tissue in rat testis,thus provide morphological evidence that support the hypothesis that telocytes may participate in testicular regulation mechanism of spermatozoa.Experiment Ⅰ Study on the distribution characteristics and ultrastructure features of telocytes in rat testisThis experiment adopted the transmission electron microscopy method,to observe the ultrastructure features and distribution characteristics of telocytes in rat testis.According to the results,telocytes protrude around the seminiferous tubule and cover the peritubular myoid cells,as a separate layer of cells that surround the whole seminiferous tubules.Further observation found that these telocytes show different features in ultrastructural morphology.Some of the cells show typical features as small cell body and extremely long processes with alteration of podomers and podoms,while some of the other cells show diferent morphology.One of the cells form large and irregular nuclei and prolongation with plague attachment,another kind of cells show long and thin smooth nuclei.All of the cells share some characteristics of telocytes again,such as slim prolongation contained mitochondria and a large number of vesicles.It can be observed in the same field of vision that the neighbouring diiferent forms of telocytes were close to each other.It is speculated that these different features of telocytes were in differrent stages,and they surround the seminiferous tubule together.At the same time,it was observed that various kinds of connections were established by telopodes between these cells.Also,telocytes were observed in close contact with adjacent peritubular myoid cells,and extablishing close connection with Leydig cells through direct contact,at the same time surround blood vessels,as well as release vesicles,thus form a three-dimensional network structure in the testicular interstitial.Based on above evidence,the experiment provide an evidence that telocytes exist in the rat testis and may possess some kind of plasticity.Also,we speculate that telocytes play an important role in the communication between sertoli cells,peritubular myoid cells,leydig cells and spermatogenic cells in testis.Experiment Ⅱ Study on the immune markers of telocytesin rat testisIn this experiment,we adopted the markers CD34 and alpha-SMA in immunohistochemistry and double-immunofluorescence of CD34/alpha-SMA,PDGFR-alpha/alpha-SMA and CD34/vimentin,to explore the cellular components of peritubular tissue and the immune markers of telocytes in rat testis.The results showed that in rat testis,the peritubular tissue mainly consists of two layers of cells,inner layer of cells is peritubular myoid cells marked by alpha-SMA and the outer layer of cells is telocytes marked by CD34.The wall of blood vessels was marked by alpha-SMA and CD34 positive cells were observed locating around blood vessels and the endothelial cells.It is speculated that CD34 marked also on telocytes except for the vascular endothelium.Additional,double-immunofluorescence showed telocytes also show PDGFR-alpha positive/vimentin negative.The CD34 positive cells were widely distributed around seminiferous tubule and also surround the blood vessels and string Leydig cells,thus form a network structure in the stroma of rat testis,which is inaccord with our ultrastructural observation under the transmission electron microscopy.From another hand,all of the cells in the outer layer show the same CD34 positivity,which confirmed that these differrent forms of cells observed under transmission electron microscope can all be regarded as telocytes.This experiment revealed the component of peritubular tissue and searched out a reliable marker of telocytes in rat testis--CD34.Based on the results that telocytes establish close connection with neighbouring cells and form a network in the stroma,it can be hypothesized that telocytes may play a potential functions in sertoli cells,peritubular myoid cells,leydig cells and spermatogenic cells interaction.Test Ⅲ The preparation procedures of transmission electron microscope samples for rat testisDue to the prolongation of telocytes possess extremely long and thin features which can reach a length of hundreds of microns,it is more difficult to observe the complete images of telocytes and telopodes in the field of a mesh in a 400-mesh copper net under transmission electron microscopy.Based on the unique morphological characteristics of telocytes,the transmission electron microscope samples prepared in this experiment were supported by copper net with 200-mesh which provides a wider field of vision.However,smaller number of meshes reduces the contact area between the copper net and the sample,which leads to the insufficient bearing capacity of the copper net to bear the sample and the sample is vulnerable to damage.Therefore,higher requirements for sample preparation are put forward.In order to solve the problem of sample vulnerability,thicker sections(70 nm)were adopted in this experiment,and the embedding process in sample preparation was optimized,that is,the embedding agent concentration was gradually increased.The results showed that the use of 200-mesh copper net enlarged the field of view under the transmission electron microscope observation area and was propitious to observe the overall perspective of telocytes in one mesh.The sections prepared by this method were relatively complete,and the ultrastructure of seminiferous tubules of testis could be clearly observed under transmission electron microscopy. |
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