The testicular morphology of the Chinese soft-shelled turtle was similar to that previously reported by our research group and in other reptiles [
9,
28‐
30]. The dissociated reproductive pattern in the soft-shelled turtle indicates a seasonal activity in the tubular and interstitial compartments. The active reproductive phase was from May to Oct, and the quiescent or hibernation phase was from Nov to April [
31]. During the quiescent phase, the seminiferous tubules regressed due to a single cohort releasing of spermatozoa into the epididymis. A parallel pattern was also found in
Trionyx sinensis, which suggested the recrudescence of spermatogenesis [
32]. Contrarily, mammals (rodents, monkeys, and human) produce sperm stored in the epididymis until ejaculation [
33,
34]. After ejaculation, the principle activity of the Sertoli cell is removing the residual spermatids by apoptosis in mammals, while in reptiles, this occurs by entosis [
29,
35,
36]. The interstitium cell’s fibroblasts and telocytes showed a communication via vesicles with the LC, suggesting cell to cell vesicles communication, hence these cells influenced the Leydig cell (LC) shape and testosterone production [
37‐
39]. Hereafter, in past few decades the role of vesicles in cell-to-cell communication are of great interest in shaping their local environment by releasing factors that either effect adjacent cells or manipulate the biochemical properties of extracellular milieu [
40]. The seasonal variation in the LC population is the indicative of interstitial precursor cells, that divide, then differentiate and replenish the LC numbers during testicular seasonal cycle [
41]. Whereas the LC area related with steroidogenic activity based organelles such as SER that associated with androgen synthesis [
42]. In contrast with present study results, In adults’ rats, the significant increased numbers and area of Leydig cell treated with hCG (human chorionic gonadotrophin) and LH (Luteinizing hormone) was reported [
43]. While in the seasonal Golden hamster the LCs area was 30% reduced during inactive phase after photoinhibition [
44] whereas in bat, the LCs became hypertrophied on the renewal of spermatogenesis [
45]. During hibernation, the LC showed 3β-HSD activity and abundant tubular endoplasmic reticulum (ER) by IHC and TEM, which suggested an increased area in
Chrysemys picta and
Chelydra serpentine [
30,
46]. These findings are also consistent with temperate-zone reptiles such as the lizard during different phases of the reproductive cycle [
47]. The LC in turtles have the potential to become steroidogenic at any time of the year [
48] and are known to contain tubular or SER at all times [
46]. During reproductive activity, a similar consistency was found in present study by the weak expression of 3β-HSD and the existence of tubule-vesicular ER by TEM in the LC, which indicated a persistent steroidogenic activity, whereas in the LC of mammals (hamster, rat, rabbit, dog, guinea pig), the amount of SER was related to the testosterone secretion [
49]. Moreover, we observed numerous lipid droplets (LD) within the LC by ORO staining during hibernation. The strong immunopositive reaction of vimentin (intermediate filament) and their appearance around the LD by 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]. In mammals, a similar constant of vimentin positivity was reported [
51] and an in-vitro analyses presumed the facilitation of LD for the mitochondria [
52]. As a result, the direct contact of the mitochondria with the LD facilitates its expansion in mammals [
53], which was morphologically (TEM) observed in the current study.
Our study, the first for a non-mammalian vertebrate, illustrates the lipophagy within the Leydig cell of the Chinese soft-shelled turtle, which has previously been well studied in mammals by using the traditional methods of electron microscopy [
26,
27] and autophagic markers (ATG7, LC3 and p62). We observed that, phagophore appeared close to the LDs during hibernation, which is the initial step in autophagosome formation [
54] through the expansion of the endoplasmic reticulum [
16]. The autophagosome then fused the with lysosome to develop the autolysosome [
16]. In an In-vitro study of the LC, this phenomenon was associated with steroid biosynthesis by providing cholesterol to the LD and was known as lipophagy [
14]. This phenomenon included lysosomal lipolysis to release fatty acids to meet functional demands [
55,
56]. Hence, the current findings provided clear evidence of an autophagic to autolysosome (Fig.
8). Fascinatingly, we found lysosomes attached to LDs forming the autophagic tube and engulfing LDs (Fig.
7c and d, and
8). In yeast, micro-autophagy by autophagic tube engulfment has been described, but this has not yet been well characterized in other eukaryotes [
17]. An in-vitro study using HeLa cells, concluded that LAMP1 and LAMP2 bind with cholesterol and are involved in cholesterol export [
25]. Thus, for the TEM and immunofluorescence results, we suggest that macro-autophagy along with micro-autophagy were involved in the lipid metabolism. However, the lysosome is the endpoint of numerous vesicle trafficking pathways including for endocytosis, phagocytosis and autophagy [
57]. To associate the function and activity of different autophagy types and abundant mitochondria, the lysosome and their attachment to LDs were observed during hibernation. This findings reflects that lipid metabolism releases the endogenous energy for steroidogenesis by different ways because the LDs are reserved as energy stock [
58]. Furthermore, our findings demonstrate that the expression of specific autophagic markers are strongly expressed in the LC during hibernation compared to the reproductive phase. It has been suggested that the autophagy level was higher than other non-steroid-producing cells in mice and rats [
27,
59] because of the increased cellular demand for autophagy imposed by the high turnover rate of steroid producing apparatus components [
15]. Therefore, our autophagy marker and TEM findings are in line with the alteration of autophagy activity in turtle LC during the annual reproductive cycle.