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Mariana Regueira, Silvana Lucía Artagaveytia, María Noel Galardo, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga, Silvina Beatriz Meroni, and María Fernanda Riera

The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the physiological relevance of this mechanism. As for the regulation of PDC, analysis of pyruvate dehydrogenase kinase (Pdk) expression showed that FSH increases Pdk3 and decreases Pdk4 mRNA levels while bFGF increases the expression of all Pdks. In addition, we showed that bFGF increases phosphorylated PDC levels and that bFGF-stimulated lactate production is partially inhibited in the presence of a PDK inhibitor. Altogether, these results add new information regarding novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells. Considering that lactate is essential for the production of energy in spermatocytes and spermatids, these mechanisms might be relevant in maintaining spermatogenesis and male fertility.

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Gustavo Marcelo Rindone, Agostina Gorga, Mariana Regueira, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga, María Noel Galardo, Silvina Beatriz Meroni, and María Fernanda Riera

Metformin (MET) is one of the most widely used anti-hyperglycemic agents for treating patients with type 2 diabetes and it has started to be used in pediatric population at ages when Sertoli cells are still proliferating. It is well known that follicle-stimulating hormone (FSH) is the major Sertoli cell mitogen. The aim of the study is to investigate a possible effect of MET, which has been shown to have anti-proliferative properties, on FSH regulation of postnatal Sertoli cell proliferation and on the molecular mechanisms involved in this regulation. The present study was performed in eight-day-old rat Sertoli cell cultures. The results obtained show that MET in the presence of FSH increases phosphorylated acetyl-CoA carboxylase and decreases phosphorylated p70S6K levels. Moreover, we show that MET decreases FSH-stimulated Sertoli cell proliferation, and this decrease is accompanied by a reduction in FSH-stimulated Ccnd1 and Ccnd2 expression and an increase in cell cycle inhibitor p21 Cip expression. Altogether, these results suggest that MET can, at least in part, counteract the effect of FSH on postnatal Sertoli cell proliferation.

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Mariana Regueira, Agostina Gorga, Gustavo Marcelo Rindone, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga, María Noel Galardo, María Fernanda Riera, and Silvina Beatriz Meroni

The presence of lipid droplets (LD) and the utilization of fatty acids (FA) as a source of energy are Sertoli cell (SC) putative characteristics. It is well known that SCs can phagocyte and degrade apoptotic germ cells (AGC) resulting in increasing lipid content and ATP levels. A relationship between the regulation of lipid storage and of lipid oxidation in SC might be envisaged. The aim of this study was to analyze whether AGC and FA are able to simultaneously regulate molecular mechanisms involved in lipid storage and in FA oxidation in SC. The experimental model utilized in this study consisted in SC cultures obtained from 20-day-old rats that were co-cultured with AGC or treated with palmitic acid (PA, 500 μM) for 24 and 48 h. AGC and PA increase LD, triacylglycerol (TAG) content and mRNA levels of Plin1, Plin2, Plin3 (proteins involved in TAG storage). Simultaneously, AGC and PA rise the extent of FA oxidation and mRNA levels of Cpt1 and Lcad (proteins involved in FA degradation). Results also show that peroxisome proliferator-activated receptor (PPAR) transcriptional activity, transcription factor which participate in lipid metabolism regulation, increases by AGC and PA treatment in SC. Additionally, the presence of a PPARg antagonist decreases the upregulation of LD content and Plin1 expression. Similarly, the presence of a PPARb/d antagonist reduces the increase in FA oxidation and Cpt1 mRNA levels. Altogether these results suggest that AGC and FA, which probably generate PPAR ligands, regulate lipid storage and fatty acid utilization, contributing to the energy homeostasis in the seminiferous tubules.