The cytoprotective peptide humanin is induced and neutralizes Bax after pro‐apoptotic stress in the rat testis

• Published in: Andrology (Oxford) Vol. 1; no. 4; pp. 651 - 659
• Main Authors: Jia, Y., Lue, Y.‐H., Swerdloff, R., Lee, K.‐W., Cobb, L. J., Cohen, P., Wang, C.
• Format: Journal Article
• Language: English
• Published: Schaumburg, IL American Society of Andrology 01.07.2013; Wiley Subscription Services, Inc
• Subjects: Animals; apoptosis; Apoptosis - drug effects; BAX; bcl-2-Associated X Protein - metabolism; Biological and medical sciences; Cytoprotection; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; germ cells; Gynecology. Andrology. Obstetrics; humanin; Immunoprecipitation; Injections, Subcutaneous; Intracellular Signaling Peptides and Proteins - administration & dosage; Intracellular Signaling Peptides and Proteins - metabolism; Male; Male genital diseases; Mammalian male genital system; Medical sciences; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - pathology; Oligopeptides - toxicity; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombinant Proteins - administration & dosage; STAT3 Transcription Factor - metabolism; Testis - drug effects; Testis - metabolism; Testis - pathology; Time Factors; Up-Regulation; Vertebrates: reproduction; Peptides; Rat; Rodentia; Humanin; Germinal cell; Testicle; Male genital system; Stress; Vertebrata; Mammalia; Cell death; Animal; BAX; germ cells; Apoptosis
• ISSN: 2047-2919; 2047-2927
• DOI: 10.1111/j.2047-2927.2013.00091.x

Summary We have previously demonstrated that the mitochondria‐derived cytoprotective peptide humanin (HN), when administered intratesticularly to rats, rescues germ cells from apoptosis secondary to testicular stress of hormonal deprivation induced by gonadotropin‐releasing hormone antagonist (GnRH‐A). To decipher the cellular mechanisms of HN action in the amelioration of GnRH‐A‐induced germ cell apoptosis, adult male rats received the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of GnRH‐A on Day 1 and daily IT injection of saline; (iii) daily IT injection of synthetic HN; and (iv) GnRH‐A injection on Day 1 and daily IT injection of HN (GnRH‐A+HN). HN alone had no effect on germ cell apoptosis. GnRH‐A increased germ cell apoptosis and BAX in the testicular mitochondrial fractions. Synthetic HN decreased germ cell apoptosis induced by GnRH‐A and BAX in the mitochondria. We deduced that the cytoprotective action of synthetic HN on GnRH‐A‐induced germ cell apoptosis was mediated by attenuating p38 mitogen‐activated protein kinase activity and increasing STAT3 phosphorylation. The effect of synthetic HN on the expression of endogenous rat HN in the testis was studied using rat HN specific antibody. GnRH‐A treatment increased, but concomitant treatment with synthetic HN reduced endogenous rat HN expression in both cytosolic and mitochondrial fractions in testis. Co‐immunoprecipitation experiments demonstrated that the increased rat HN was physically associated with BAX in the cytosolic testicular fractions after GnRH‐A treatment. Double‐immunofluorescence staining confirmed the co‐localization of BAX and rat HN in the cytoplasm of Leydig cells and spermatocytes after GnRH‐A treatment. We conclude that the cytoprotective effect of exogenously administered synthetic HN is mediated by interactions of endogenous rat HN with BAX in the cytoplasm preventing the entry of BAX to the mitochondria to govern the fate of germ cell survival or death during pro‐apoptotic stress to the testis in rats.