Abstract
Important evolutionary and ecological consequences arise from the ability of female turtles to store viable spermatozoa for an extended period. Although previous morphological studies have observed the localization of spermatozoa in Pelodiscus sinensis oviduct, no systematic study on the identification of genes that are involved in long-term sperm storage has been performed. In this study, the oviduct of P. sinensis at different phases (reproductive and hibernation seasons) was prepared for RNA-Seq and gene expression profiling. In total, 2,662 differentially expressed genes (DEGs) including 1,224 up- and 1,438 down-regulated genes were identified from two cDNA libraries. Functional enrichment analysis indicated that many genes were predominantly involved in the immune response, apoptosis pathway and regulation of autophagy. RT-qPCR, ELISA, western blot and IHC analyses showed that the expression profiles of mRNA and protein in selected DEGs were in consistent with results from RNA-Seq analysis. Remarkably, TUNEL analysis revealed the reduced number of apoptotic cells during sperm storage. IHC and TEM analyses found that autophagy occurred in the oviduct epithelial cells, where the spermatozoa were closely attached. The outcomes of this study provide fundamental insights into the complex sperm storage regulatory process and facilitate elucidating the mechanism of sperm storage in P. sinensis.
Highlights
Sperm storage in the female reproductive tract is defined as the retention of viable spermatozoa for an extended period of time[1]
Considerable studies have suggested that the intimate contact between spermatozoa and the surface of oviduct epithelial cells is necessary to prolong spermatozoa survival, which is especially important for stimulating the synthesis of new proteins in the oviduct cells[7,8]
The differential expressions of several differentially expressed genes (DEGs) related to sperm storage were validated by RT-qPCR, and their protein levels were detected by Enzyme-linked immunosorbent assay (ELISA), western blot and IHC analysis
Summary
Sperm storage in the female reproductive tract is defined as the retention of viable spermatozoa for an extended period of time[1] It is typically used by a wide variety of animal species, including mammals, insects, fish, birds and reptiles, whose copulation is consistently asynchronous with ovulation[2]. The objectives of this study were to comprehensively identify the differentially expressed genes (DEGs) related to sperm storage and dissect the molecular mechanism underlying long-term sperm storage in P. sinensis. Morphological evidence further confirmed the storage of spermatozoa in the P. sinensis oviduct These results will enhance our understanding of sperm storage in female P. sinensis and help elucidate the underlying mechanism in turtles
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