Mitochondrial-derived humanin peptides: New cytoprotective factors for ovarian cells?

GOTTARDO, MARÍA FLORENCIA; ROMANOWSKI VICTOR; MARVALDI CAROLINA; PIDRE MATIAS; SEILICOVICH ADRIANA; MARTIN DANIEL; IMSEN MERCEDES; JAITA GABRIELA

Washington DC

Congreso; SSR 2017 Annual Meeting; 2017

Along folliculogenesis, the fate of each follicle is different and controlled by endocrine and also by diverse paracrine factors. Humanin (HN) and Rattin (rHN, rat homologous peptide) have cytoprotective action in several cell types such as neurons, spermatogonias and Leydig cells. Also, it was reported that HN could be secreted from cells. In the ovary, it was showed that HN mRNA is present in luteal cells. However, it has not been described the protein expression and humanin peptides action in the ovary. We aimed to explore the expression and function of HN peptides in the ovary from prepuberal rats, cycling adult rats and in a human granulosa-like tumor cell line (KGN). We investigated the expression of rHN in ovarian sections from untreated prepuberal rats (rich in preantral follicles), or treated with DES (rich in early antral follicles) or PMSG (rich in preovulatory follicles). Immunohistochemical (IHC) staining showed rHN expression in granulose and theca cells, and also in oocytes. In PMSG-treated rats, rHN was mainly expressed in theca cells. In ovarian sections from cycling rats the pattern of rHN expression was similar to that of treated prepuberal rats and rHN was also expressed in luteal cells. In addition, KGN cells expressed HN. To study the role of HNr, we performed the TUNEL assay together with IHC for HNr in ovarian sections. HNr positive cells were TUNEL-negative in PMSG-treated rats. Also, we analyzed the effect of HN on viability of KGN cells by MTT assay. HN increased the viability of KGN cells (C: 0.24 ± 0.02, HN 0.25 µM: 0.40 ± 0.02, HN 0.5 µM: 0.36 ± 0.02, HN 1µM: 0.41 ± 0.01, p < 0.01 vs control. Data are the means ± SEM of quintuplicate samples from one experiment representative of two. ANOVA). Also, we study the effect of HN inhibition with a shRNA plasmid on the apoptosis of KGN cells. We observed an increase of percentage of apoptotic cells in the presence of shRNA plasmid (plcontrol: 41% ± 11; plshRNA: 81% ± 7. p < 0.01, data are the percentage of apoptotic cells ± confidence intervals of total cells. n=1, 2). Our results show that HNr is present in all follicular cells, including oocytes, and also show strong intensity in luteal cells. Considering that HN inhibition increases the apoptosis and exogenous HN increases the viability of KGN cells, our results suggest that HN peptides may play a cytoprotective role in ovarian cells. This research was supported by Agencia Nacional de Investigaciones Científicas y Tecnológicas, CONICET and Universidad de Buenos Aires, Argentina.