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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.)

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.)

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. Invitro 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.

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.

Multilamellar bodies (MLBs) are produced and secreted by many cell types. In this study, we report the existence and ultrastructure of MLBs that are produced by Leydig cells and identification of telocytes in the testicular interstitium of naked mole rat. This study was performed on both breeder and non-breeder male naked mole rats using light microscopy, transmission electron microscopy, and morphometric approaches. In the testicular interstitium, the most prominent cells were Leydig cells, which contained numerous lipid droplets (LDs) in the cytoplasm. We found that MLBs were associated with the LDs of Leydig cells and were secreted into the extracellular or interstitial environment via exocytosis. After their release from Leydig cells, MLBs localized to the space between Leydig cells near blood vessels and attached to telocytes. We also identified telocytes in the testicular interstitium, and their cellular extensions were distributed throughout the interstitium. MLBs were aligned along the cellular extensions of telocytes, and membrane-to-membrane contact was observed between the cellular extensions of telocytes and MLBs, suggesting that telocytes may play a role in the transport of MLBs within the interstitial space. No ultrastructural differences were found in Leydig cells, telocytes, or MLBs between breeder and non-breeder testes. However, morphometric analysis revealed a significant difference in the number of MLBs between the breeder and non-breeder animals. Furthermore, both selective autophagy of LDs and non-selective autophagy were observed in Leydig cells. Typical features of macrolipophagy were also observed, as a few LDs were entirely enclosed by a limiting membrane. Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. The appearance of MLBs in the testicular interstitium of naked mole rats could thus be related to lipid storage and trafficking. • This study reported the presence of MLBs and their association with telocytes in the testicular interstitial tissues of the naked mole rat. • We proposed that telocytes may facilitate the transport of multilamellar bodies to the target cells in the testicular interstitial space. • Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. [ABSTRACT FROM AUTHOR]

Sertoli cells (SCs) play their nursing role as structural and functional supporting cells during spermatogenesis to ensure the production of highly specialized mature spermatozoa. Besides that, the role of SCs in autophagy during active (adult) and inactive (young) spermatogenesis in the caprine testis is still largely unknown. In this study, we investigated autophagy in goat SCs by light microscopy, immunohistochemistry (IHC), double immunofluorescence (double-IF), and transmission electron microscopy. Light microscopy showed active seminiferous tubules with SCs and layers of developing germ cells in the adult goat testis. In young goats, layer of germ cells and SCs was viewed on the basal membrane in the seminiferous tubule. IHC of autophagy-related 7 (ATG7) showed moderate expressions in the cytoplasmic extensions of SCs during inactive spermatogenesis, and strong expression was observed during active spermatogenesis in the testis of goat. Co-immunolabeling of p62 or light chain 3 (LC3) with vimentin showed increasing expression from the basal to the luminal compartment of the seminiferous tubule and stronger expression during active than inactive spermatogenesis in the testis of goat. Ultrastructure assessment of the cytoplasm in SCs showed phagophores, generated from the endoplasmic reticulum during active spermatocytogenesis. Numerous autophagosomes and autolysosomes were noted in the SCs cytoplasm, which surrounds the spermatogenic cells in the basal compartment of the seminiferous tubules. At a later stage, SCs showed autophagosomes and autolysosomes, together with multivesicular bodies (MVB), during spermiogenesis at different phases of the acrosome formation. Numerous embedded elongated spermatozoa were found in the cytoplasm of SCs, surrounded by autophagic components and MVB. Under TEM, the mean diameter of autophagosomes was 952.35 nm and that of autolysosomes was 504.38 nm. Collectively, these results suggest that autophagy is active in SCs during caprine spermatogenesis and that the level of autophagy becomes more evident as spermatogenesis advances from the basal to the luminal compartment of SC. • Autophagy in Sertoli cell support different cellular events during caprine spermatogenesis. • Autophagy was more involved during active than inactive spermatogenesis. • Autophagy was associated with advancement of spermatogenesis. [ABSTRACT FROM AUTHOR]

Despite many studies being conducted over the past few decades, the origin of autophagosomal membranes remains unclear. The present study aimed to uncover the formation process of autophagosomal membranes in hepatocytes of zebrafish ( Danio rerio ), a model organism in medical science. Immunohistochemistry of zebrafish hepatocytes indicated that light chain 3-like protein 2 (LC3-II) is highly active in some hepatocytes, but poorly expressed in others. Under transmission electron microscopy, the amount of autophagosomes (APs) varied in different hepatocytes. When the endoplasmic reticulum (ER) is dispersed in the cytoplasm, few isolation membranes (IMs) and APs were observed. Subsequently, when the ER assembles into a particular “lamellar structure” (LS), IMs arise from it and extend to enwrap the mitochondria. With further aggregation of the ER, the LS developed into an over twenty-layered structure, and mitophagy was more obvious in the hepatocytes and cavities appeared in mitochondria. Finally, most ERs were assembled into several LSs. At this point, mitophagy was most active in the hepatocytes. Thereafter, glycogen and lipid droplet increased gradually, while the LS degenerated and ER scatter increased. Then, the glycogen and lipid droplets dominated the hepatocellular cytoplasm. After suppressing the formation of autophagosomes using 3-Methyladenine (3-MA), the LS could no longer be visualized in the hepatocellular cytoplasm, and mitophagy decreased drastically. Taken together, the results suggested that this LS in the hepatocytes of zebrafish, might be another manifestation of a pre-autophagosomal structure in zebrafish liver, analogous to the omegasome in yeast or the ER-IM complex in mammalian cell lines. Furthermore, selective mitophagy and consequent cyclic utilization of its products were probably relevant to dynamic cycle of the hepatocellular cytoplasm. [ABSTRACT FROM AUTHOR]