Molecular and Cellular Mechanisms of Apoptosis during Dissociated Spermatogenesis

Abstract

Apoptosis is a tightly controlled process by which tissues eliminate unwanted cells. Spontaneous germ cell apoptosis in testis has been broadly investigated in mammals that have an associated spermatogenesis pattern. However, the mechanism of germ cell apoptosis in seasonally breeding reptiles following a dissociated spermatogenesis has remained enigmatic. In the present study, morphological evidence has clearly confirmed the dissociated spermatogenesis pattern in Pelodiscus sinensis. TUNEL and TEM analyses presented dynamic changes and ultrastructural characteristics of apoptotic germ cells during seasonal spermatogenesis, implying that apoptosis might be one of the key mechanisms to clear degraded germ cells. Furthermore, using RNA-Seq and digital gene expression (DGE) profiling, a large number of apoptosis-related differentially expressed genes (DEGs) at different phases of spermatogenesis were identified and characterized in the testis. DGE and RT-qPCR analysis revealed that the critical anti-apoptosis genes, such as Bcl-2, BAG1, and BAG5, showed up-regulated patterns during intermediate and late spermatogenesis. Moreover, the increases in mitochondrial transmembrane potential in July and October were detected by JC-1 staining. Notably, the low protein levels of pro-apoptotic cleaved caspase-3 and CytC in cytoplasm were detected by immunohistochemistry and western blot analyses, indicating that the CytC-Caspase model might be responsible for the effects of germ cell apoptosis on seasonal spermatogenesis. These results facilitate understanding the regulatory mechanisms of apoptosis during spermatogenesis and uncovering the biological process of the dissociated spermatogenesis system in reptiles.

Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

“…Seasonal spermatogenesis is characteristic of temperate and boreal reptilian species, including P. sinensis (Gribbins, 2011;Liu et al, 2017 (Chang & Karin, 2001;Lee et al, 2006;Taylor, Zheng, Liu, & Thompson, 2013). Apaf1, one of the important components of the p53 signaling pathway, can mediate apoptosis through its association with procaspase-9 (Zou, Li, Liu, & Wang, 1999).…”

“…Quantitative real-time PCR (qRT-PCR) analysis was performed according to previous reports (Liu et al, 2017;.…”

“…P. sinensis is an ectothermic aquaculture species with a specific evolutionary role linking ectothermic anamniotic animals (fishes and amphibians) and endothermic amniotic animals (birds and mammals) (Zimmerman, Vogel, & Bowden, 2010) and can thus be used as a potential animal model to study the evolution of critical genes or species (Liu, Chu, et al, 2016). Previous studies have revealed distinct seasonal apoptosis in the testis of P. sinensis on the basis of morphological and molecular evidence (Liu et al, 2017). Previous studies have revealed distinct seasonal apoptosis in the testis of P. sinensis on the basis of morphological and molecular evidence (Liu et al, 2017).…”

“…Previous studies have revealed distinct seasonal apoptosis in the testis of P. sinensis on the basis of morphological and molecular evidence (Liu et al, 2017). Fortunately, the public genomic sequences and RNA-seq data of P. sinensis (Liu et al, 2017;Wang et al,2013) can provide rich resources for the identification, phylogenetic analysis and functional exploration of PsHsp70/110 genes in P. sinensis. However, the relationship between apoptosis and stressrelated Hsp70/110 is poorly understood in P. sinensis.…”

“…Our previous studies examining the testis of P. sinensis demonstrated dynamic changes in apoptosis during spermatogenesis and showed that apoptosis was closely correlated with seasonal temperature variation: A mass of apoptotic cells was detected in April (spermatogenically quiescent phase), while decreased apoptosis was observed in July and October (intermediate and late spermatogenesis)(Liu et al, 2017). Our previous studies examining the testis of P. sinensis demonstrated dynamic changes in apoptosis during spermatogenesis and showed that apoptosis was closely correlated with seasonal temperature variation: A mass of apoptotic cells was detected in April (spermatogenically quiescent phase), while decreased apoptosis was observed in July and October (intermediate and late spermatogenesis)(Liu et al, 2017).…”