SUN-214 Neonatal Metformin Administration Exerts a Suppressive Effect on Sertoli Cell Proliferation in Rats

Sertoli cell (SC) proliferation, a factor defining SC population size, occurs during a restricted period of time. In rats, SCs start proliferating during fetal development and undergo mitosis for up to 15 days after birth. In humans, SCs proliferate during fetal and neonatal periods for up to 6 months after birth and there is a second stage of proliferation at the onset of puberty. Metformin (MET), a first line anti-hyperglycemic agent, is used in pediatric population at ages when SCs are still proliferating. In addition to its strong anti-diabetic properties, numerous studies have demonstrated that MET has anti-proliferative activity (1). In this context, it has been demonstrated in SC cultures obtained from 8-day old rats that MET decreases FSH-stimulated SC proliferation and that this decrease is accompanied by a reduction in Cyclin D1, D2, E1 and E2 expression and an increase in cell cycle inhibitor p21Cip expression (2). The aim of this study was to analyze the potential effect of in vivo neonatal administration of Metformin on rat Sertoli cell proliferation and its impact on the spermatogenic capacity in adult animals. Male Sprague Dawley rat pups were randomly assigned at postnatal day 3 (PND3) to the following groups: MET (receiving daily 200 mg/kg MET i.p., from day 3 to 7 inclusive) and control (receiving daily sterile saline solution i.p). At PND8, SCs were isolated from a set of animals to analyze the expression of cell cycle regulators and of SC maturation markers by RT-qPCR. Another set of animals were injected with BrdU (50 mg/kg) 90 min before sacrifice to evaluate cell proliferation. Our results showed that MET group exhibited a significant decrease in BrdU incorporation in SCs compared to controls (p<0.05; n=7 per group). Consistent with decreased SC proliferation, MET group showed a reduction in Cyclin D1 and E2 and an increase in cell cycle inhibitor p21Cip mRNA levels in isolated SC (p<0.05; n=6). In addition, MET group showed a decrease in androgen receptor mRNA levels (p<0.05; n=6) without changes in AMH or FSH receptor mRNA levels compared to controls. Given that a single SC can support a limited number of germ cells, we studied the impact of neonatal treatment with MET on sperm parameters in adult animals. Animals were treated as mentioned before and epididymal sperm parameters were evaluated at PND90. No differences in sperm morphology, motility or number between groups were observed. Altogether these results suggest that neonatal MET treatment exerted a suppressive effect on Sertoli cell proliferation with no further impact on spermatogenesis and sperm epididymal maturation. Reference: (1) Cai et al., Oncol Rep. 2013 Nov;30(5):2449-57. (2) Rindone et al., Reproduction. 2018 Aug; 156(2):93-101. Sources of Research Support: ANPCyT Grants, PICT2015-0228 and PICT 2014/945.