Search

Spermiogenesis is a highly organized process of the metamorphosis of round spermatids into spermatozoa in the testes. Autophagy is involved in the physiological process of spermiogenesis and its crucial role in germ-plasm clearance conserved across kingdoms. However, the fate of by-products generated through autophagy during spermiogenesis is still largely unknown. In the present study, we showed that the autophagy enhanced lipid droplets (LDs) formation during spermiogenesis in Chinese soft-shelled turtle, Pelodiscus sinensis. TEM and Oil Red O staining results found that the number and size of LDs within spermatid increased considerably during the process of spermiogenesis. RNA-Seq analysis revealed that autophagy was highly activated via the PI3K pathway during spermatogenesis. Inhibiting autophagy with 3-methyladenine (3-MA) significantly decreased testicular triglycerides (TGs) and fatty acid (FAs) content. In comparison with the control group, the number and size of LD within elongating spermatids was reduced significantly in the 3-MA group. Moreover, DGAT1, a diacylglycerol acyltransferase, which normally localize to the endoplasmic reticulum, was found to co-localize with LDs. Taken together, our results showed that FAs released through the autophagic degradation of germ-plasm was replenished LDs of spermatid, increasing LD number and size, during the process of spermiogenesis. These LDs facilitate long-term sperm storage in the epididymis of Chinese soft-shelled turtle., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Spermatogenesis in reptiles is a seasonally dependent physiological process that is not temporally associated with male mating behavior. Characteristics of seasonal spermatogenesis in reptiles, however, remain largely unknown. In this review, there is a coverage of the characteristics of soft-shelled turtle, Pelodiscus sinensis, during seasonal spermatogenesis that provides insights into spermatogenesis of testudines. The seminiferous epithelium of P. sinensis are undergoing spermatogenesis during the summer and fall, but are quiescent throughout the rest of the year; germ cells progress through spermatogenic stages in a temporal rather than a spatial pattern. While apoptotic germ cells mainly appear in the non-spermatogenic phase, these are seldom present during active spermatogenesis. It is inferred that apoptosis may be one of the reasons for germ cell loss during the resting phase of spermatogenesis. During the period when spermatogenesis is occurring, Sertoli cells become very narrow and are in contact with several round/elongated spermatids. Many residual spermatozoa can be internalized and degraded within Sertoli cells by entosis during the non-spermatogenic phase, which precedes the next reproductive cycle in P. sinensis. In the late spermatogenic phase, round-shaped mitochondria of spermatids become elongated and swollen, subsequently forming a crescent-like shape and develop into "onion-like" shaped mitochondria. As spermiogenesis progresses, the endoplasmic reticulum of spermatids is transferred into a specialized structure called the "Chrysanthemum flower center", which may be a source of autophagosomal membranes. The information provided in this review will help improve understanding of characteristics of seasonal spermatogenesis, which will hopefully promote interest in the study of reptilian species., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

The present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many "heterogeneous" MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome-MVB-exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.

Despite the exploration of mitochondria-rich cells (MRCs) in different animal classes, very limited information has been documented about MRCs in reptiles. The present study was designed to investigate the effect of seasonal variation on the cell ultrastructure and ion transport protein activity of MRCs during hibernation and non-hibernation of Chinese soft-shelled turtle's intestine. Transmission electron microscopy revealed that, during hibernation the high-density cytoplasm of MRCs occupied large cross-sectional area and showed heterogeneous abundance of mitochondria and an expanded extensive tubular system as compared to non-hibernation. During hibernation the cytoplasm of MRCs exhibited more mitochondrial vacuolization, autophagosomes, phagophore formation and well-structured endoplasmic reticulum. During hibernation, MRCs connected with absorptive cells through wide interdigitation, and created tight junction and more desmosomes as compared to non-hibernation. Immunohistochemistry and immunofluorescence showed, the strong immunopositive reactions and immunosignaling of Na + /K + -ATPase (NKA) and Na + /K + /2Cl- cotransporter (NKCC) at basolateral region of mucosal surface of intestine during hibernation. However, weak immunopositive reactions and immunosignaling of NKA and NKCC during non-hibernation. The statistical analysis showed that the number and size of MRCs with NKA-associated immunoreactivity were significantly increased during hibernation. NKA and NKCC mRNA expression was significantly increased during hibernation via qPCR. Further confirmed, the intensity of NKA and NKCC proteins was more elevated during hibernation than non-hibernation shown by immunobloting. However, the concentrations of the plasma ions Na + and Cl- were significantly higher during hibernation; conversely, K + concentration was significantly higher during non-hibernation. The findings suggest that the potential role of MRCs is affected by seasonal fluctuations, during which intestinal homeostasis and hydromineral balance are essential for turtles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Autophagy is a subcellular process that is extensively involved in spermiogenesis. In this study, we observed ultrastructural malformation of acrosome and mitochondrial cristae during the spermiogenesis of Chinese soft-shelled turtle due to the inhibition of autophagy. Autophagy was blocked with 3-MA, and the inhibition of autophagy was confirmed through western blot analysis. The morphological abnormalities of acrosomes and mitochondria were observed under transmission electron microscopy (TEM). In the early spermiogenesis (Golgi and cap phases), damaged macrovesicle was observed, and its proper expansion over the nucleus failed to be form a normal acrosomal cap. As spermiogenesis proceeded, the malformation of the acrosome in spermatids became more severe. In the late spermiogenesis (acrosomal and maturation phases), defective acrosome with damaged acrosomal membrane that was detached from the nucleus was observed. Along with malformed acrosome, elongation failed nucleus having oval or round shaped morphology was also observed. Moreover, morphological damage to the mitochondrial cristae was observed. Lacuna formation, half and complete loss of cristae were observed in the mitochondria of developing spermatids. We proposed that autophagy is required for normal formation of the acrosome and mitochondrial cristae during turtle spermiogenesis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hibernation is an adaptive survival strategy in response to cold and foodless winter. To determine the underlying mechanisms of seasonal adaptions, transcriptome sequencing studies have been conducted in bears, ground squirrels and bats. Despite advances in identifying differentially expressed genes involved in metabolism, the precise mechanisms of these physiological adaptions remain unclear. In the present study, we examined liver of Chinese Soft-Shelled Turtle ( Pelodiscus sinensis ) and found that the contents of lipid droplet (LD) and triglyceride (TG) were significantly decreased during hibernation. Increases in mRNA expression levels of lipolysis-related genes and decreased levels of lipogenesis-related genes during hibernation indicated that LD hydrolysis was stimulated during hibernation. To continuously release fatty acids (FAs) from LD, adipose triglyceride lipase (ATGL) was recruited and accumulated on the surface of LDs via activation of Cyclic Adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling. Meanwhile, increased phosphorylation of the LD-associated protein, perilipin-5, activated the enzyme activity of ATGL via interaction between comparative gene identification-58 (CGI-58) and ATGL. Taken together, our results indicated that ATGL accumulation on the LD surface and its induced enzyme activity during hibernation promoted LD breakdown in the liver of Chinese Soft-Shelled Turtle ( Pelodiscus sinensis ), thereby enhancing mitochondrial β-oxidation to maintain energy hemostasis.

Background: 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., Method: 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)., Results: 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., Conclusion: 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.

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods after spermiation from the testes, but the molecular mechanisms underlying this storage are largely unknown. In this study, epididymal spermatozoa were investigated to determine the potential molecular mechanism for long-term sperm storage in P. sinensis. Transmission electron microscopy (TEM) and Oil red O staining indicated that unusually large cytoplasmic droplets containing lipid droplets (LDs) were attached to the epididymal spermatozoa. However, the content of LDs decreased gradually with the sperm storage. LDs were surrounded by autophagic vesicles and sequestered as degradative cargo within autophagosome. Immunofluorescence and western blotting demonstrated that autophagy in spermatozoa increased gradually with the storage time. In vitro studies found that spermatozoa obtained from soft-shelled turtles in January can survive more than 40 days at 4°C. Furthermore, immunofluorescence and TEM showed that autophagy was involved in the degradation of LDs with the extension of sperm incubation. Inhibition of autophagy with 3-methyladenine significantly suppressed LD degradation. Moreover, adipose triglyceride lipase was involved in the metabolism of LDs. These findings indicate that lipophagy was activated to maximise LD breakdown, which contributes to long-term sperm storage in the epididymis of P. sinensis. Epididymal spermatozoa of the Chinese soft-shelled turtle possess usually large cytoplasmic droplets with several lipid droplets (LDs), which serve as an important energy source. During long-term sperm storage in the epididymis, LDs were degraded by lipophagy to sustain sperm longevity. Understanding the mechanism underlying sperm storage would provide insights into the reproductive strategy of the Chinese soft-shelled turtle and contribute to further studies on sperm conservation in vitro. [ABSTRACT FROM AUTHOR]

Mitochondria are vital cellular organelles that have the ability to change their shape under different conditions, such as in response to stress, disease, changes in metabolic rate, energy requirements and apoptosis. In the present study, we observed remodelling of mitochondria during spermiogenesis and its relationship with mitochondria-associated granules (MAG). At the beginning of spermiogenesis, mitochondria are characterised by their round shape. As spermiogenesis progresses, the round-shaped mitochondria change into elongated and then swollen mitochondria, subsequently forming a crescent-like shape and finally developing into onion-like shaped mitochondria. We also noted changes in mitochondrial size, location and patterns of cristae at different stages of spermiogenesis. Significant differences (P < 0.0001) were found in the size of the different-shaped mitochondria. In early spermatids transitioning to the granular nucleus stage, the size of the mitochondria decreased, but increased subsequently during spermiogenesis. Changes in size and morphological variations were achieved through marked mitochondrial fusion. We also observed a non-membranous structure (MAG) closely associated with mitochondria that may stimulate or control fusion during mitochondrial remodelling. The end product of this sophisticated remodelling process in turtle spermatozoa is an onion-like mitochondrion. The acquisition of this kind of mitochondrial configuration is one strategy for long-term sperm storage in turtles. The sperm mitochondria of turtles possess a unique kind of morphology that is commonly known as 'onion-like mitochondria'. This is the end product of mitochondrial remodelling and was achieved after the mitochondria had passed through six different morphological types during spermiogenesis. Because mitochondria show great morphological diversity in response to physiological and pathological conditions, this normal transformation of mitochondria could be a good model in which to study different mitochondrial morphologies associated with disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Ductuli efferent is a conduit passage of spermatozoa from rete testis to the epididymis. After spermiation, various extracellular vesicles are released by epithelial cells to assist and transfer micro-molecules for spermatozoa maturation. To date, there is a lack of data in ductuli efferent. In this study, we investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their extracellular vesicles (EVs) in the epithelia of efferent ducts in a turtle by using light and transmission electron microscopy. Immunohistochemistry of CD63 (Cluster of differentiation 63) and ultrastructure demonstrates that ciliated and non-ciliated cells of ductuli efferent possess well developed exocytic systems. Ciliated cells and non-ciliated cells secrete extracellular vesicles via ciliary blebs and apical blebs, respectively. Results also showed numerous extracellular vesicles between these cells and indicate basolateral secretion. Moreover, these extracellular vesicles are associated with spermatozoa in the lumens of ductuli efferentes. Collectively, the present study provides cytological evidence that the ductuli efferentes (DE) epithelium secretes EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. Characterization and cellular distribution of these extracellular vesicles in the ductuli efferent of turtles may provide a study model to further investigate the transferring of micro-molecules via extracellular vesicles to the spermatozoa. The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa. [ABSTRACT FROM AUTHOR]