Abstract
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.
Highlights
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 Leydig cells in the testis of the Chinese soft-shelled turtle were characterized by a compact morphology during reproductive phase and a significantly increased area during hibernation (Fig. 1a and d)
The strong immunopositive reaction of vimentin and their appearance around the LD by transmission electron microscopy (TEM) reflected the integrative role of intermediate filaments within the LC for steroid biosynthesis by facilitating the LD, and these LDs are a source of cholesterol for steroidogenesis [50]
Summary
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 adult male testis has two significant roles, the production of spermatozoa and the secretion of sexual steroids [1]. Like other vertebrates, these two important functions in reptiles occur in the tubular and interstitial compartments of the testis, respectively. Structural changes in the male germinal epithelium have been associated with fluctuating reproductive patterns and variation in LC activity [7,8,9]. Our research group produced detailed cellular work on the germ cell, Sertoli cell and their novel role during the annual cycle [10, 11], but an ultrastructural analysis of the LC in the Chinese shelled-turtle has not been reported
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