In this study, we investigated the localization, morphological features and cellular interactions of telocytes in the rat testicular interstitium. Transmission electron microscopy (TEM) and immunohistochemical and immunofluorescence analyses of the rat testicular interstitium showed a distinct layer of telocytes surround the seminiferous tubules along with inner layer of peritubular myoid cells. The majority of the telocytes were made up of a small cell body and moniliform prolongations that contained mitochondria and secretory vesicles. Some other telocytes were observed possessing large cell bodies. Within the testicular interstitium, the telocytes formed a network connecting peritubular myoid cells, Leydig cells as well as blood vessels. Immunohistochemical and double immunofluorescence analyses showed that rat testicular telocytes express CD34 and PDGFRα, but are negative for vimentin and α-SMA. Our findings demonstrate the presence of telocytes in the rat testicular interstitium. These cells interact with peritubular myoid cells, seminiferous tubules, Leydig cells and blood vessels via long telopode extensions, which suggests their vital role in the intercellular communication between different cell types within the rat testis.

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.

Telocytes (TCs) have been identified as a distinct type of interstitial cells, but have not yet been reported in the gastrointestinal tract (GIT) of ruminants. In this study, we used transmission electron microscopy (TEM) and double-labelling immunofluorescence (IF) (antibodies: CD34, vimentin and PGP9.5) to seek TCs and investigate their potential functions in the muscle layers of the goat rumen. TCs were distributed widely in the myenteric plexus (TC-MYs) between the circular and longitudinal muscle layers, within circular muscle layers (TC-CMs) as well as in longitudinal muscle layers (TC-LMs). Ultrastructurally, TCs displayed small cell bodies with several long prolongations—telopodes—harboring alternate thin segments (podomers) and dilated segments (podoms). The podoms contained mitochondria, rough endoplasmic reticulum, and caveolae. Telopodes frequently established close physical interactions with near telopodes, collagen fibers (CFs), nerve fibers (NFs), smooth muscle cells (SMCs), nerve tracts, and smooth muscle bundles, as well as with blood vessels (BVs). Furthermore, both homo- and heterotypic connections were observed. In addition, telopodes were capable of releasing extracellular vesicles (EVs). IF analyses proved that TCs were reliably labeled as CD34+/vimentin+ cells, displaying spindle- or triangle-shaped bodies with long prolongations, consistent with TEM results. Specifically, podoms were visible as obvious bright spots. These positive cells covered entire muscular layers, surrounding ganglions, intermuscular BVs as well as entire smooth muscle bundles, forming a network. TC-MYs were distributed as clusters in the external ganglion, encompassing the entire ganglion and spreading to the muscle layers where TC-CMs and TC-LMs seemingly surround whole smooth muscle bundles. TC-MYs were also scattered within the interior of the ganglion, surrounding each ganglionic neuron, following the glial cells layer. We speculate that TCs support the muscle layer structure of the goat rumen and facilitate intercellular signaling directly or indirectly via the TC network.

Autophagy is a unique catabolic pathway that is linked to several physiological processes. However, its role in the process of spermiogenesis is largely unknown. The aim of the current study was to determine the in vivo role of autophagy and the origin of autophagosome membrane biogenesis within male haploid cells. Our immunohistochemistry results demonstrated that LC3 and ATG7 localization were increased dramatically in round to elongated spermatids (haploid cells) towards the lumen of seminiferous tubules, however, poorly expressed in the early stages of germ cells near the basal membrane. Moreover, transmission electron microscopy revealed that the numbers of lysosomes and autophagosomes increased in the elongated spermatids as spermiogenesis progressed. However, no evidence was found for the presence of autophagosomes in the Sertoli cells, spermatogonia or early primary spermatocytes (diploid cells). Furthermore, TEM showed that many endoplasmic reticula were transformed into a “chrysanthemum flower center,” from which a double-layered isolation membrane appeared to develop into an autophagosome. This study provides novel evidence about the formation of autophagosomes through the chrysanthemum flower center from the endoplasmic reticulum, and suggests that autophagy may have an important role in the removal of extra cytoplasm within male haploid cells during spermiogenesis.