Tembusu virus induced apoptosis in vacuolate spermatogenic cells is mediated by Cytc-mediated mitochondrial apoptotic signaling pathway.

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that infects mainly poultry and has caused huge economic losses to the poultry farming industry in China. Also known as duck hemorrhagic ovarian disease, DTMUV principally destroys ovarian tissue in ducks, causing a dramatic drop in egg production. and can also invade the male reproductive system causing lesions. Currently, little research has been done to reveal the underlying mechanisms of reproductive dysfunction in ducks caused by DTMUV infection. In this study, histopathological analysis and electron microscopy of testes of ducks infected with DTMUV showed that DTMUV caused testicular atrophy and cytoplasmic vacuolation in ducks. Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling (TUNEL) staining and real-time quantitative PCR(RT-qPCR) results further indicated that DTMUV induced spermatogenic cells apoptosis. After DTMUV infection, a large amount of cytochrome c(Cytc) was released from the mitochondrial matrix into the cytoplasm, activating downstream target proteins and causing apoptosis. To sum up, DTMUV induces spermatogenic cell apoptosis through the Cytc-induced mitochondrial apoptosis pathway, our study provides evidence for DTMUV infection-induced male reproductive disorders.

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3ß-HSD 24 h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

Duck Tembusu virus infection causes testicular atrophy.

In recent years, Duck Tembusu virus (DTMUV) is becoming an important emerging and re-emerging pathogen that severely harms the poultry industry in China. The DTMUV disease was principally identified by a sharp decline in egg production, whereas few studies were focused on the virus-reproductive system interaction, especially male reproductive system. Herein, the present study was aimed at investigating the in vivo morphological changes in testis from DTMUV-infected adult Shaoxing ducks. After DTMUV infection, the gross observation indicated that the testis of DTMUV-infected ducks was significantly atrophied at 2 days post-infection (dpi), 4 dpi, and 8 dpi. At microscopic and ultrastructural level, morphological analysis revealed that DTMUV could lead to cytoplasmic vacuolation and exfoliation in seminiferous epithelium, decrease in the diameter of seminiferous tubule (ST), and even induce interstitial inflammation in duck testis. Ulteriorly, the spermatogenic cells, especially spermatocytes, are identified as the target cells of DTMUV infection in the testis of ducks through immunohistochemistry (IHC). And more notably, single virus particles and clustered virus particles were observed in the spermatogenic cells from infected ducks. In summary, our results comprehensively illustrated the effects of DTMUV infection on the testis, the morphological changes underlying testicular atrophy and identified the target cells of DTMUV infection in the testis of ducks.

Transformation of Mitochondrial Architecture and Dynamics in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis) During Hibernation.

Hibernation is a biological status during which hibernating animals acclimatize themselves to reduced energy consumption through extreme but governed decline in self-metabolism. The role of mitochondria (Mt) in metabolic suppression during hibernation has already been elaborated in different organs and species. Nonetheless, the concretely changing process of mitochondrial architecture and the mechanism underlying this transformation during hibernation remains unclear. Herein, the present study was aimed at clarifying the detailed alteration of mitochondrial morphology and its potential role in the Chinese soft-shelled turtle (Pelodiscus sinensis) during different stages of hibernation. Compared with the nonhibernation period, the mitochondrial architecture was changing from round to crescent, and lipid droplet (LD)/Mt interaction was enhanced during hibernation, as observed by transmission electron microscopy (TEM). Further ultrastructural analysis uncovered that mitochondrial fusion was promptly accelerated in the early stage of hibernation, followed by mitochondrial fission in the middle stage, and mitophagy was boosted in the late stage. Moreover, gene and protein expression related to mitochondrial fusion, fission, and mitophagy accorded closely with the mitochondrial ultrastructural changes in different stages of hibernation. Taken together, our results clarified that the transformation of mitochondrial architecture and mitochondrial dynamics are of vital importance in maintaining internal environment homeostasis of Pelodiscus sinensis.

Subcellular Evidence for Biogenesis of Autophagosomal Membrane during Spermiogenesis In vivo.

Although autophagosome formation has attracted substantial attention, the origin and the source of the autophagosomal membrane remains unresolved. The present study was designed to investigate in vivo subcellular evidence for the biogenesis of autophagosomal membrane during spermiogenesis using transmission-electron microscopy (TEM), Western blots and immunohistochemistry in samples from the Chinese soft-shelled turtle. The testis expressed LC3-II protein, which was located within spermatids at different stages of differentiation and indicated active autophagy. TEM showed that numerous autophagosomes were developed inside spermatids. Many endoplasmic reticulum (ER) were transferred into a special "Chrysanthemum flower center" (CFC) in which several double-layer isolation membranes (IM) were formed and extended. The elongated IM always engulfed some cytoplasm and various structures. Narrow tubules connected the ends of multiple ER and the CFC. The CFC was more developed in spermatids with compact nuclei than in spermatids with granular nuclei. An IM could also be transformed from a single ER. Sometimes an IM extended from a trans-Golgi network and wrapped different structures. The plasma membrane of the spermatid invaginated to form vesicles that were distributed among various endosomes around the CFC during spermiogenesis. All this cellular evidence suggests that, in vivo, IM was developed mainly by CFC produced from ER within differentiating spermatids during spermiogenesis. Vesicles from Golgi complexes, plasma membranes and endosomes might also be the sources of the autophagosome membrane.

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

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.

Cellular evidence for nano-scale exosome secretion and interactions with spermatozoa in the epididymis of the Chinese soft-shelled turtle, Pelodiscus sinensis.

The epididymis is the location of sperm maturation and sperm storage. Recent studies have shown that nano-scale exosomes play a vital role during these complicated processes. Our aim was to analyze the secretory properties of epididymal exosomes and their ultrastructural interaction with maturing spermatozoa in the Chinese soft-shelled turtle. The exosome marker CD63 was primarily localized to the apices of principal cells throughout the epididymal epithelium. Identification of nano-scale exosomes and their secretory processes were further investigated via transmission electron microscopy. The epithelium secreted epididymal exosomes (50~300 nm in diameter) through apocrine secretion and the multivesicular body (MVB) pathway. Spermatozoa absorbed epididymal exosomes through endocytosis or membrane fusion pathways. This study shows, for the first time, that nano-scale exosomes use two secretion and two absorption pathways in the reptile, which may be contribute to long-term sperm storage.

Androgen-related sperm storage in oviduct of Chinese Soft-Shelled Turtle in vivo during annual cycle.

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.