Upregulated Autophagy in Sertoli Cells of Ethanol-Treated Rats Is Associated with Induction of Inducible Nitric Oxide Synthase (iNOS), Androgen Receptor Suppression and Germ Cell Apoptosis

Abstract

This study was conducted to investigate the autophagic response of Sertoli cells (SCs) to acute ethanol toxicity using in vivo and in vitro models. Adult Wistar rats were intraperitoneally injected with either 5 g/kg ethanol or phosphate-buffered saline (for the control group) and sacrificed 0, 3, 6 and 24 h after injection. Compared to the control group, enhanced germ cell apoptosis was observed in the ethanol-treated rats (ETRs) in association with upregulation of iNOS and reduced expression of androgen receptor protein levels in SCs, which were resistant to apoptosis. Meanwhile, autophagy was upregulated in ETR SCs (peaking at 24 h) compared to the control group, as evidenced by transcription factor EB (TFEB) nuclear translocation, enhanced expression of microtubule-associated protein 1 light chain3-II (LC3-II), lysosome-associated membrane protein-2 (LAMP-2), pan cathepsin protein levels and reduced expression of p62. This upregulation of SC autophagy was confirmed ultrastructurally by enhanced formation of autophagic vacuoles and by immunofluorescent double labelling of autophagosomal and lysosomal markers. Study of cultured SCs confirmed enhanced autophagic response to ethanol toxicity, which was cytoprotective based on decreased viability of SCs upon blocking autophagy with 3-methyladenine (3-MA). The results highlighted the molecular mechanisms of prosurvival autophagy in ETR SCs for the first time, and may have significant implications for male fertility.

Accelerating Onset of Puberty Through Modification of Early Life Nutrition Induces Modest but Persistent Changes in Bull Sperm DNA Methylation Profiles Post-puberty

“…In contrast, the induction of the meiosis gatekeeper STRA8 at the onset of meiosis is associated with a repression of autophagy and with ULK1 and TFEB downregulation ( Ferder et al, 2019 ). In Sertoli cells, TFEB mediates the initiation of autophagy upon exposure to toxins such as ethanol, in order to promote cell survival and sustain spermatogenesis ( Horibe et al, 2017 ). Interestingly, ULK1 directly relates autophagy with the mTOR pathway, which regulates many metabolic processes and essential cell functions in response to the nutrient and energy status of the cell ( Kim et al, 2011 ).…”

Ethanol-Induced Autophagy in Sertoli Cells Is Specifically Marked at Androgen-Dependent Stages of the Spermatogenic Cycle: Potential Mechanisms and Implications

“…Morphologically, autophagy is characterized by the formation of isolation membranes that engulf a region of the cell forming early AVs or autophagosomes, which are mediated by LC3 (a specific autophagosome marker). The latter then fuse with lysosomes to form late AVs or autolysosomes, and the contents are degraded by cathepsins [3,4]. Autophagy may be a selective process in the clearance of damaged mitochondria (mitophagy) or excessive lipid droplets (lipophagy) in hepatocytes in association with exposure to acute or chronic ethanol intake.…”

“…Fourth, germ cells and spermatozoa, in particular, depend on lactate production by SCs via autophagy and glycolysis for viability and motility under normal and stressed conditions [10,11]. Fifth, autophagy upregulation has been found to be a prosurvival mechanism for cultured SCs upon exposure to heat stress [11] or high dose of ethanol [4].…”

“…Excessive alcohol intake has been reported to enhance germ cell apoptosis, resulting in infertility problems via mechanisms related to oxidative stress, upregulation of inducible nitric oxide synthase (iNOS) and mitochondrial dysfunction [4,5,6]. The authors recently reported that acute ethanol exposure upregulated autophagy in SCs of adult rats, and that this was accompanied by elevated germ cell apoptosis, androgen receptor (AR) suppression in testicular somatic cells and enhanced expression of iNOS in SCs [4]. The rat spermatogenic cycle includes 14 stages, each involving specific morphological and functional criteria (simplified into four groups consisting of stages I–VI, VII–VIII, IX–XI and XII–XIV) [12].…”

Effect of ethanol on the changes in testicular protein expression in adult male rats

“…receptor expression in testis (Horibe et al, 2017;Horibe, Eid, Ito, Otsuki, & Kondo, 2019). It has been also demonstrated that testicular proteins such as steroidogenic acute regulatory (StAR) protein (Jana, Jana, De, & Guha, 2010;Radhakrishnakartha, Appu, & Indira, 2014) and cytochrome P450 side-chain cleavage enzyme (P450scc) are lower expressed in ethanol animals (Radhakrishnakartha et al, 2014).…”