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

Abstract

In a recent study, we reported that acute ethanol exposure enhanced autophagy in Sertoli cells (SCs) of adult rats. However, further research is needed to clarify the specific spermatogenic stage exhibiting the highest autophagic response, the mechanisms behind such specificity, and the related relevance to sperm. This brief report provides results indicating that stages VII–VIII (androgen-dependent or spermiation stages) of the spermatogenic cycle exhibited more marked autophagic response in acute-ethanol treated rats (ETRs) than other stages based on suppression of androgen receptor (AR), analysis of microtubule-associated protein 1 light chain 3 (LC3) (an autophagosomal marker) immunostaining in SCs, double labeling of LC3 and lysosomal proteins and electron microscopy. Ultrastructural observations and TUNEL method revealed a notable presence of phagocytosed apoptotic germ cells and retained sperm in SCs of ETRs at these specific stages—a finding rarely observed in control testes. In addition, PTEN-induced putative kinase 1 ( PINK1) (a sensor of mitochondrial damage and mitophagy) and giant lipid droplets were found to have accumulated in SCs of ETRs at same stages. Our data show novel findings indicating that stages VII–VIII of the spermatogenic cycle exhibit high levels of autophagy, specifically under stress conditions, as expressed by the term autophagic stages. This stage-specific upregulation of autophagy in SCs may be related to AR suppression, mitochondrial damage, lipid accumulation, and phagocytosis of apoptotic cells. The phenomenon may be an essential part of ensuring the viability of SCs and supporting germ cells in toxic environments.

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).…”

Ethanol-Induced Mitochondrial Damage in Sertoli Cells is Associated with Parkin Overexpression and Activation of Mitophagy

“…The immunohistochemical labeling methods were performed according to the manufacturer’s recommendations and our recent studies [6,18,21,22]. Paraffin-embedded sections (4-µm thickness) underwent a deparaffinization process, antigen retrieval, blocking of endogenous peroxidase activity, and non-specific antigen binding.…”

“…Using Adobe Photoshop, the tubules were captured and saved for computer analysis using Image J (National Institutes of Health, Bethesda, MA, USA). The intensity of protein expression in SCs was quantified as recently reported [22].…”

“…For double labeling of LC3 with either cytochrome c (a mitochondrial marker) or p62, we used a simultaneous application of two primary antibodies followed by Alexa Fluor 594 and 488-conjugated secondary antibodies [6,23,24]. For double labeling of pan-cathepsin (lysosomal marker) with either Parkin or LC3, we used a sequential method as previously reported [6,16,21,22]. In brief, following incubation with primary antibodies for 1 hour, Alexa Fluor 594 and VectaFluor™ R.T.U.…”

Autophagy in male reproduction

“…Recent research has shown that lipophagy is involved in the testosterone biosynthesis and likely supplies endogenous energy for the development of germ cells (Ahmed et al 2016;Gao et al 2018;Ma et al 2018;Tarique et al 2019). In addition, mitophagy may have an antiapoptotic role in the ethanol-treated rat Sertoli cells (Eid et al 2012;Horibe et al 2019), and it possibly participates in the elimination of paternal mitochondria after fertilization among different species (Sato and Sato 2011;Al Rawi et al 2012;Luo et al 2013;Politi et al 2014;Rojansky et al 2016;Song et al 2016). Herein we review relevant literature concerning the role of autophagy in the male spermatogenesis and testicular endocrinology, as well as in the existence of endocrine-disrupting chemicals.…”