Global analysis of differential gene expression related to long-term sperm storage in oviduct of Chinese Soft-Shelled Turtle Pelodiscus sinensis

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.

Molecular and Cellular Mechanisms of Apoptosis during Dissociated Spermatogenesis

“…The standard Illumina protocol was followed to develop RNA-Seq libraries. Three cDNA libraries of P. sinensis named MT_1, MT_2, and MT_3 were constructed separately from the tissues in April, July, and October, and sequenced according to a previously reported method (Liu et al, 2016b). The Illumina HiSeq 2500 platform was used for sequencing, and 150-bp paired-end reads were generated.…”

“…Moreover, biochemical, and molecular genetic studies in female P. sinensis have discovered a number of apoptosis-related genes in which some critical genes were also demonstrated to be involved in spermatogenesis (Zhang et al, 2013; Liu et al, 2016b). Recently, the RNA sequencing (RNA-Seq) technique serves as an accurate and appropriate approach when combined with digital gene expression (DGE) tag profiling, and it has been successfully used for the expression analysis of transcriptional changes in some reptile species, including brown treesnake ( Boiga irregularis ), anolis lizard ( Anolis allogus ), alligator ( Alligator mississippiensis ), and western painted turtle ( Chrysemys picta bellii ; Mcgivern et al, 2014; Akashi et al, 2016; Jiang et al, 2016; Yatsu et al, 2016).…”

Genome-wide identification and comparison of differentially expressed profiles of miRNAs and lncRNAs with associated ceRNA networks in the gonads of Chinese soft-shelled turtle, Pelodiscus sinensis

“…The turtle has a sex-dependent growth pattern, with males showing a signi cantly larger weight and size, thicker and wider calipash, and lower levels of fat than females [3]. Similar to other reptiles and mammals, the soft-shelled turtle has the ability to store sperm in the ovary [4]. Spermatogenesis, copulation, and ovulation are seasonal and segregational in turtles [4,5].…”

“…Similar to other reptiles and mammals, the soft-shelled turtle has the ability to store sperm in the ovary [4]. Spermatogenesis, copulation, and ovulation are seasonal and segregational in turtles [4,5]. Many previous studies have focused on sex determination and differentiation in the turtle.…”

Genome-wide identification and comparison of differentially expressed profiles of miRNAs and lncRNAs with associated ceRNA networks in the gonads of Chinese soft-shelled turtle, Pelodiscus sinensis

“…The turtle has a sex-dependent growth pattern, with males showing a significantly larger weight and size, thicker and wider calipash, and lower levels of fat than females [3]. Similar to other reptiles and mammals, the soft-shelled turtle has the ability to store sperm in the ovary [4]. Spermatogenesis, copulation, and ovulation are seasonal and segregational in turtles [4,5].…”