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

This study was conducted to elucidate the involvement of the PINK1-Parkin pathway in ethanol-induced mitophagy among Sertoli cells (SCs). In the research, adult rats were given intraperitoneal injections of ethanol (5 gm/kg) and sacrificed at various time periods within 24 h. Transmission electron microscopy was applied to reveal enhanced mitochondrial damage in SCs of the ethanol-treated rats (ETRs) in association with a significant increase in numbers of mitophagic vacuoles (mitophagosomes and autolysosomes) in contrast to very low levels in a control group treated with phosphate-buffered saline (PBS). This enhancement was ultra-structurally verified via observation of trapped mitochondria within LC3-labeled membranes, upregulation of LC3 protein levels, colocalization of LC3 and cytochrome c, and reduced expression of mitochondrial proteins. Importantly, Parkin expression was found to be upregulated in ETR SCs, specifically in mitochondria and mitophagosomes in addition to colocalization with PINK1 and pan-cathepsin, indicating augmented mitophagy. Transcription factor EB (TFEB, a transcription factor for autophagy and mitophagy proteins) was also found to be upregulated in nuclei of ETR SCs and associated with enhanced expression of iNOS. Enhanced Parkin-related mitophagy in ETR SCs may be a protective mechanism with therapeutic implications. To the authors' knowledge, this is the first report demonstrating the ultrastructural characteristics and molecular mechanisms of Parkin-related mitophagy in ETR SCs.

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

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.

A Method for In Vivo Induction and Ultrastructural Detection of Mitophagy in Sertoli Cells.

An emerging body of evidences based on in vitro studies indicate that mitophagy (selective autophagic clearance of damaged mitochondria) is a prosurvival mechanism, specifically under exposure to various stressors. Sertoli cells (SCs) play essential roles in maintenance of spermatogenesis via paracrine interactions with germ cells and other somatic cells in the testis; however, studies investigating mitophagy in SCs are still very few. In this chapter, we give a brief review of mechanisms and detection methods of mitophagy in SCs based on our recent publications on animal models of ethanol toxicity and current literature. In addition, we provide a method for induction and ultrastructural identification of mitophagy in SCs of adult Wistar rats using a single intraperitoneal injection (5 g/kg) of ethanol. Proper understanding of mitophagy features and mechanisms in SCs may have therapeutic implications for infertility associated with alcoholism and other diseases characterized by mitochondrial dysfunction.

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.

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.

Ethanol-induced mitophagy in liver is associated with activation of the PINK1-Parkin pathway triggered by oxidative DNA damage.

Mitophagy is a cytoprotective mechanism against mitochondrial damaging agents. Studies demonstrating morphological evidence for the involvement of the PINK1-Parkin pathway in the hepatocyte mitophagic response to ethanol toxicity, and potential links to apoptosis and mitochondrial alterations such as spheroid formation are still lacking. We addressed these unresolved issues using a rat model of binge alcohol exposure. Adult rats were injected with ethanol (5g/kg) and liver samples were taken at 0, 3, 6, and 24 hours after ethanol administration and processed for light and electron microscopic studies. Ethanol induced a low level of hepatocyte apoptosis, peaking at 3 h and decreasing significantly by 24 h. In contrast, there was enhanced formation of mitophagic vacuoles in the majority of normal hepatocytes of ethanol-treated rats (ETRs), which peaked at 6 h and was maintained up to 24 h based on electron microscopy and TUNEL/LC3 double labelling. Moreover, enhanced mitophagy in ETR hepatocytes was confirmed by increased LC3 puncta formation, and co-localization of Parkin and LC3 with mitochondrial and lysosomal markers. Immunoelectron microscopy demonstrated the localization of PINK1 and Parkin to damaged mitochondria of ETR hepatocytes, which was consistent with co-localization of Parkin with 8-OHdG, a marker of oxidative mitochondrial DNA damage. Furthermore, electron microscopy showed enhanced formation of mitochondrial spheroids in ETR hepatocytes. These data are the first direct morphological evidence linking PINK1-Parkin pathway activation to the enhanced mitophagic response of hepatocytes to ethanol toxicity. Ethanol-induced hepatic mitophagy may be a prosurvival mechanism, which may have therapeutic implications.

A deletion in the endothelin-B receptor gene is responsible for the Waardenburg syndrome-like phenotypes of WS4 mice.

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomic sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.

Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2.

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(beta-D-mannuronate)lyases.