Activation of AMP-Activated Protein Kinase Regulates Proliferation of Rat Sertoli Cells.

RIERA M FERNANDA; GALARDO MARÍA N; AGNELLO ANA C; PELLIZZARI ELIANA H; MERONI SILVINA B; CIGORRAGA SELVA B

Washington DC, USA

Congreso; 91st Annual Meeting Endo 2009; 2009

The Endocrine Society

Sertoli cells (SC) provide the structural and nutritional support for germ cell development. Considering that each SC is able to support a limited number of germ cells, the final number of SC reached during the proliferative periods determines sperm production capacity in adulthood. Hence, the importance of clarifying the molecular mechanisms involved in cessation of SC proliferation. The protein kinase activated by AMP (AMPK) is a serine/threonine protein kinase that has been involved in sensing cell energy levels and in down-regulating cell proliferation. In this context, it has been observed that activation of AMPK by 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) inhibits proliferation of various cell lines. The aim of this work was to analyze the participation of AMPK in the regulation of SC proliferation. Mitotically active SC were isolated from 8-day-old rats and cultured in chemically defined medium. Cultures were stimulated with AICAR (1mM) in the absence or presence of FSH (100ng/ml) for variable periods of time. AMPK activation by Western blot using a specific antibody against phosphorylated AMPK (P-AMPK), the number of SC by a cell proliferation assay (MTS) and the mRNA levels of the cell cycle inhibitor p19INK4d by Northern blot, were evaluated. As expected, AICAR treatment increased the levels of P-AMPK under basal and FSH-stimulated conditions. While AICAR did not modify cell viability evaluated by Trypan blue exclusion, it produced a decrease in FSH-stimulated SC mitosis. AICAR also promoted a 2.4±0.6-fold increment (mean±SD, n=3, p<0.05) in p19INK4 mRNA levels in FSH-treated cultures. Considering that triiodothyronine (T3) has been involved in the cessation of SC proliferation stimulated by FSH (1), that it activates AMPK in some cell lines (2) and that this hormone regulates the expression of some cell cycle inhibitors in SC (3), we decided to analyze whether T3 had effects on AMPK activation and on the expression of p19INK4d in Sertoli cells. Western blot analysis showed that T3 (500nM) treatment increased the levels of P-AMPK. Also, T3 produced a 2.1±0.3-fold increase (mean±SD, n=3, p<0.05) in p19INK4d mRNA levels and a decrease in SC mitosis in the presence of FSH. Altogether, these results suggest that activation of AMPK through the regulation of p19INK4d expression may be involved in the cessation of SC proliferation and that this mechanism may partially account for the reported effects of T3 on the final SC number. (1) Holsberger DR et al., Cell Tissue Res. 2005; 322:133(2) Yamauchi M,et al., Mol Endocrinol. 2008; 22:893(3) Buzzard JJ et al., Endocrinology. 2003; 144:3722Supported by CONICET Grant 5479; ANPCyT Grant 25365.