TLR2/4 Expression In Chinese Soft Shelled Turtle Oviduct During Hibernation And Their Relationship With Sperm Storage

A variety of animals regard sperm storage as a reproductive strategy in order to achieve the fertilization.On the one hand,the structure of sperm own may benefit storage process in oviduct.On the other hand,the oviduct would provide nutrition materials from epithelial cells for sperms and take away the metabolism products.Meanwhile,oviduct would create suitable and secure microenvironment for sperm storage.It is visible that there is relationship between immunology system and sperm storage.TLR2/4,as two important members of TLR family in innate immunology system,act a principal role in resistance against infection and keeping the organism stable.Foreign sperms maintain a long time storage in oviduct.We suspect that TLR2/4 may play an important role in long sperm storage.To our knowledge,there are on reports about the TLRs expression on reptiles.In this paper we investigated microstructure and ultrastructure of Chinese soft shelled turtles oviduct and the TLR2/4 protein and mRNA expression and distribution in oviduct with sperm storage during hibernation.Then we discuss the relationship between the TLR2/4 expression and sperm storage in different storage stage.Experiment I:Microstructure and ultrastructure of Chinese soft shelled turtle oviduct with sperm storage The microstructure of different parts of oviduct and ultrastructure of epithelial cell and scretory glands were examined by microscopy and electron microscopy.The oviduct comprises the infundibulum,magnum,isthmus,uterus,and vagina.Apart from infundibulum,magnum,the rests are relative with sperm storage.The results by HE staining and electron microscopy reveal that each parts has similar structure with mucous layer,submucous layer,muscle layer,and mantle layer The epithelial cells comprise secretory cells and ciliated cell.Lamina propria contains many vascular and a mount of secretory glands.The glands contain many secretory particles inside and open to lumen.The particular structure provides a suitable environment for sperms survival.Experiment II:Expression and distribution of TLR2/4 in Chinese soft shelled turtle oviduct with sperm storage The initiation of innate immunology system could play an important role in the aspect of protection for sperms long term storage when the sperms got into oviduct of turtles and come into contact with epithelium.The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable.The objective of this study was to examine the gene and protein expression profiles in Chinese soft-shelled turtle during hibernation from November to April in the next year.The protein distribution of TLR2/4 was investigated in the magnum,isthmus,uterus and vagina of the turtle oviduct using immunohistochemistry,and the gene expression of TLR2/4 was analyzed using quantitative real-time PCR(qRT-PCR).The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct.TLR4 immunoreactivity was widely observed in almost every part of the oviduct,particularly in the epithelium and secretory gland membrane.Analysis of protein,mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus,uterus and vagina during hibernation.The protein and mRNA expression of TLR2 in the magnum,isthmus,uterus and vagina was decreased in April compared with that in November.TLR4 protein and mRNA expression in the magnum,isthmus,uterus and vagina was decreased in November compared with that in April.These results indicated that TLR2/4 expression might protect the sperm from microbial infections.In contrast to the function of TLR2,which protects sperm during the early stages of hibernation,TLR4 might play a role in later stages of storage.