Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead‐like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft‐shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft‐shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.

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.

Telocytes (TCs) are novel interstitial cells that have been found in various organs, but the existence of TCs in the testes has not yet been reported. The present ultrastructural and immunohistochemical study revealed the existence of TCs and differentiate these cells from the peritubular cells (Pc) in contact with the surrounding structures in the testes. Firstly, our results confirmed the existence of two cell types surrounding seminiferous tubules; these were Pc (smooth muscle like characteristics) and TCs (as an outer layer around Pc). Telocytes and their long thin prolongations called telopodes (Tps) were detected as alternations of thin segments (podomers) and thick bead‐like portions (podoms), the latter of which accommodate the mitochondria and vesicles. The spindle and irregularly shaped cell bodies were observed with small amounts of cytoplasm around them. In contrast, the processes of Pc contained abundant actin filaments with focal densities, irregular spine‐like outgrowths and nuclei that exhibited irregularities similar to those of smooth muscle cells. The TCs connected with each other via homocellular and heterocellular junctions with Pc, Leydig cells and blood vessels. The Tps of the vascular TCs had bands and shed more vesicles than the other TCs. Immunohistochemistry (CD34) revealed strong positive expression within the TC cell bodies and Tps. Our data confirmed the existence and the contact of TCs with their surroundings in the testes of the Chinese soft‐shelled turtle Pelodiscus sinensis, which may offer new insights for understanding the function of the testes and preventing and treating testicular disorders.

Steroidogenesis 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. In 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). The 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. In 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.

Sperm storage in vivo extends the time window for fertilisation in several animal species, from a few days to several years. The underlying storage mechanisms, however, are largely unknown. In this study, spermatozoa from the epididymis and oviduct of Chinese soft-shelled turtles were investigated to identify potentially relevant morphological features and transformations at different stages of sperm storage. Large cytoplasmic droplets (CDs) containing lipid droplets (LDs) were attached to the midpiece of most spermatozoa in the epididymis, without migrating down the sperm tail. However, they were absent from the oviductal spermatozoa, suggesting that CDs with LDs may be a source of endogenous energy for epididymal spermatozoa. The onion-like mitochondria recovered their double-membrane morphology, with typical cristae, within the oviduct at a later stage of storage, thus implying that mitochondrial metabolism undergoes alterations during storage. Furthermore, a well developed fibrous sheath on the long principal piece was the integrating ultrastructure for glycolytic enzymes and substrates. These novel morphological characteristics may allow turtle spermatozoa to use diverse energy metabolism pathways at different stages of storage.