Localization of Humanin in Human, Rabbit and Mouse Sperm

Abstract

Humanin (HN) is a 24-amino acid peptide showing potent neuroprotective properties against damage associated with Alzheimer's disease (1). In neuronal cells, HN acts by inhibiting apoptosis via multiple mechanisms: it can suppress mitochondrially-mediated apoptosis by interfering with Bax activation (2), additionally it can bind to the Insulin Growth Factor-Binding Protein 3 (IGFBP-3) antagonizing its apoptotic actions (3). Ying et al. (4) demonstrated that HN inhibited beta-amyloid (Aβ) 42-induced neurotoxicity by binding to G-protein-coupled receptor FPRL-1. Moreover, neuronal protection by HN involves activation of tyrosine kinases, STAT-3 phosphorylation (5) and the binding to a complex or complexes involving CNTFR/WSX-1/gp180 (6). The homologues of HN in the rat is called Rattin (HNr) (7) and share, like HN, neuroprotective properties. HN and HNr have a broader spectrum of protective activity also in non-neuronal cells. Regarding the male reproductive system, studies reported the presence of HN both in human and rat, proposing that this peptide may play a role as a testicular anti-apoptotic factor (8-10). This study aimed to investigate the presence and to localize HN, mainly by immunocytochemistry (ICC), directly in human, mouse and rabbit sperm. MATERIAL AND METHODS Immunocytochemistry (ICC)Human semen samples were evaluated following World Health Organization guidelines (11) and processed for the ICC analyses. Ejaculated spermatozoa, washed in phosphate-buffered saline (PBS), were smeared on glass slides, air dried, rinsed in PBS and saturated for 20 min with PBS-bovine serum albumin (BSA) 1% containing 5% normal goat serum (NGS). The specimens were then incubated overnight at 4°C using two different rabbit polyclonal anti-HN antibodies (Sigma-Aldrich, USA and a self made antibody P04, kindly gift of Prof. Matzuoka) both diluted 1:50 in PBS/0.1% BSA/1% NGS. For the detection a goat antirabbit FITC conjugate antibody (Sigma-Aldrich), diluted 1:300, was used. The same ICC analysis was carried out also in sperm from rabbit and mouse. The fluorescence was observed with a Leitz Aristoplan light microscope equipped with fluorescence apparatus. Incubation in primary antibodies was omitted in control samples. Test for antibody specificityPreadsorption: An aliquot of rabbit polyclonal anti-HN antibody (Sigma-Aldrich) was pre-incubated 64