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.

Long-term sperm storage in the female genital tract is essential for the appropriate timing of reproductive events in animals with asynchronous copulation and ovulation. However, the mechanism underlying the prolonged storage of spermatozoa is largely unexplored in turtles. In the present study, the role of androgen in sperm storage was investigated in the oviduct of the Chinese soft-shelled turtle, Pelodiscus sinensis. Morphological analysis revealed that spermatozoa were observed in the vagina, uterus and isthmus of the oviduct throughout the hibernation season. The increase of circulating testosterone and dihydrotestosterone levels were consistent with the arrangement of spermatozoa that had their head embedded among the cilia of the oviduct mucosal epithelium. Immunohistochemical analysis revealed that androgen receptor was distributed throughout the cytoplasm of gland cells and among the cilia of ciliated cells. Furthermore, marked variations in protein and mRNA levels of androgen receptor were validated through Western blot and qPCR analyses. The localization and the variation of androgen receptor demonstrated the crucial roles of androgens in sperm storage in the oviduct of P. sinensis. These results provide fundamental insights into the interaction of androgen and sperm storage and facilitate the elucidation of the mechanism of sperm storage in turtles.

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.

BackgroundSteroidogenesis is an indispensable process that is indirectly associated with spermatogenesis in the Leydig cell (LC) to utilize the lipid droplets (LDs) that are critical to maintaining normal testosterone synthesis. The regulation of LD mobilization, known as lipophagy, in the LC is still largely unknown.MethodIn the present study, the LC of the Chinese soft-shelled turtle was investigated to identify the steroidogenic activity and lipophagy during the annual reproductive cycle by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM).ResultsThe LC showed a dynamic steroidogenic function with strong activity of 3β-HSD, vimentin and tubular ER during hibernation by IHC and TEM. The tubulo-vesicular ER had a weak immunopositive reaction for 3β-HSD in the LC during reproductive phase, suggesting persistent steroidogenic activity. ORO staining and TEM demonstrated that a larger number of LDs had accumulated in the LC during hibernation than in the reproductive phase. These LDs existed in close association with mitochondria and lysosomes by being dynamically surrounded by intermediate filaments to facilitate LD utilization. Lysosomes were found directly attached to large LDs, forming an autophagic tube and engulfing LDs, suggesting that micro-lipophagy occurs during hibernation. Furthermore, the IHC of ATG7 (Autophagy Related Gene 7) and the IF of the LC3 (Microtubule-associated protein light chain 3), p62 (Sequestosome-1 (SQSTM1) and LAMP1(Lysosomal-associated membrane protein 1) results demonstrated strong expression, and further confirmation by TEM showed the existence of an autophagosome and an autolysosome and their fusion during the hibernation season.ConclusionIn conclusion, the present study provides clear evidence of LD consumption in the LC by lipophagy, lysosome and mitochondria during the hibernation period, which is a key aspect of steroidogenesis in the Chinese soft-shelled turtle.

Tembusu Virus (TMUV) is an emerging and re-emerging zoonotic pathogen that adversely affects poultry industry in recent years. TMUV disease is characterized by nonsuppurative encephalitis in ducklings. The duckling infection model was established to study the mechanism of TMUV crossing the blood-brain barrier (BBB) into the central nervous system (CNS). Here, we showed that no obvious clinical symptoms and enhancement of BBB permeability occurred at the early stage of infection (3∼5 dpi). While simultaneously virus particles were observed by transmission electron microscopy in the brain, inducing the accumulation of inflammatory cytokines. Neurological symptoms and disruption of BBB appeared at the intermediate stage of infection (7∼9 dpi). It was confirmed that TMUV could survive and propagate in brain microvascular endothelial cells (BMECs), but did not affect the permeability of BBB in vivo and in vitro at an early date. In conclusion, TMUV enters the CNS then causes encephalitis, and finally destruct the BBB, which may be due to the direct effect of TMUV on BMECs and the subsequent response of “inflammatory storm”. IMPORTANCE The TMUV disease has caused huge losses to the poultry industry in Asia, which is potentially harmful to public health. Neurological symptoms and their sequelae are the main characters of this disease. However, the mechanism of how this virus enters the brain and causes encephalitis is unclear. In this study, we confirmed that the virus entered the CNS and then massively destroyed BBB and the BBB damage was closely associated with the subsequent outbreak of inflammation. TMUV may enter the CNS through the transcellular and “Trojan horse” pathways. These findings can fill the knowledge gap in the pathogenesis of TMUV-infected poultry and be benefit for the treatment of TMUV disease. What’s more, TMUV is a representative to study the infection of avian flavivirus. Therefore, our studies have significances both for understanding of the full scope of mechanisms of TMUV and other flavivirus infection, and conceivably, for therapeutics.