Effect of Metformin on Sertoli Cell Fatty Acid Metabolism and Blood-Testis Barrier Formation.

Sertoli cells (SCs) are essential to maintaining germ cell development. Metformin, the main pharmacologic treatment for pediatric type 2 diabetes, is administered to children during SC maturation. The present study aimed to analyze whether metformin affects SC energy metabolism and blood-testis barrier (BTB) integrity. Primary SC cultures were used for the in vitro studies. In vivo effects were studied in Sprague-Dawley rats treated with 200 mg/kg metformin from Pnd14 to Pnd30. Metformin decreased fatty acid oxidation and increased 3-hydroxybutyrate production in vitro. Moreover, it decreased the transepithelial electrical resistance across the monolayer and induced ZO-1 redistribution, suggesting an alteration of cell junctions. In vivo, a mild but significant increase in BTB permeability and ZO-1 expression was observed in the metformin group, without changes in testicular histology and meiosis progression. Additionally, adult rats that received metformin treatment during the juvenile period showed no alteration in BTB permeability or daily sperm production. In conclusion, metformin exposure may affect BTB permeability in juvenile rats, but this seems not to influence spermatogenesis progression. Considering the results obtained in adult animals, it is possible to speculate that metformin treatment during the juvenile period does not affect testicular function in adulthood.

Low Doses of Glyphosate/Roundup Alter Blood-Testis Barrier Integrity in Juvenile Rats.

It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.

Beige adipocytes contribute to breast cancer progression.

Adipocytes are the main stromal cells in the mammary microenvironment, and crosstalk between adipocytes and breast cancer cells may play a critical and important role in cancer maintenance and progression. Tumor­induced differentiation to beige/brown adipose tissue is an important contribution to the hypermetabolic state of breast cancer. However, the effect of epithelial cell­beige adipocyte communication on tumor progression remains unclear. To contribute to the understanding of this phenomenon, we characterized components present in conditioned media (CM) from beige adipocytes (BAs) or white adipocytes (WAs), and evaluated the effects of BA­ and WA­CM on both adhesion and migration of tumor (LM3, 4T1 and MC4­L1) and non­tumor (NMuMG) mouse mammary epithelial cell lines. Additionally, we analyzed the expression of ObR, CD44, vimentin, MMP­9, MCT1 and LDH in tumor and non­tumor mouse mammary epithelial cell lines incubated with BA­CM, WA­CM or Ctrol­CM (control conditioned media). 3T3­L1 preadipocytes differentiated into beige adipocytes upon PPARγ activation (rosiglitazone) displaying characteristics that morphologically resembled brown/beige adipocytes. Levels of UCP1, CIDEA, GLUT4, leptin, MCT4 and FABP4 were increased, while adiponectin, caveolin 1 and perilipin 1 levels were decreased in BAs with respect to WAs. Tumor cell lines revealed lower cell adhesion and increased cell migration after incubation with BA­ and WA­CM vs. Ctrol­CM. ObR and MMP­9 in MC4­L1 cells were significantly increased after incubation with BA­CM vs. WA­ and Ctrol­CM. In addition, MC4­L1 and LM3 cells significantly increased their migration in the presence of BAs, suggesting that new signals originating from the crosstalk between BAs and tumor cells, could be responsible for this change. Our results indicate that beige adipocytes are able to regulate the behavior of both tumor and non­tumor mouse mammary epithelial cells, favoring tumor progression.

Postnatal metformin treatment alters rat Sertoli cell proliferation and daily sperm production.

BACKGROUND: The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. OBJECTIVE: The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. MATERIALS AND METHODS: Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200 mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. RESULTS: MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. DISCUSSION AND CONCLUSION: These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.

In vitro effects of glyphosate and Roundup on Sertoli cell physiology.

Roundup (R), a formulation that contains glyphosate (G) as the active ingredient, is a commonly used nonselective herbicide that has been proposed to affect male fertility. It is well known that an adequate Sertoli cell function is essential to maintain germ cell development. The aim of the present study was to analyze whether G and R are able to affect Sertoli cell functions, such as energy metabolism and blood-testis barrier (BTB) integrity. Sertoli cell cultures from 20-day-old rats were exposed to 10 and 100 ppm of G or R, doses which do not decrease cell viability. Neither G nor R caused impairment in lactate production or fatty acid oxidation. G and R decreased Transepithelial Electrical Resistance, which indicates the establishment of a Sertoli cell junction barrier. However, neither G nor R modified the expression of claudin11, ZO1 and occludin, proteins that constitute the BTB. Analysis of cellular distribution of claudin11 by immunofluorescence showed that G and R induced a delocalization of the signal from membrane to the cytoplasm. The results suggest that G and R could alter an important function of Sertoli cell such as BTB integrity and thus they could compromise the normal development of spermatogenesis.

Apoptotic germ cells regulate Sertoli cell lipid storage and fatty acid oxidation.

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.

Metformin counteracts the effects of FSH on rat Sertoli cell proliferation.

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 p21Cip expression. Altogether, these results suggest that MET can, at least in part, counteract the effect of FSH on postnatal Sertoli cell proliferation.

Germ cells regulate 3-hydroxybutyrate production in rat Sertoli cells.

Paracrine regulation of Sertoli cell function by germ cells is an outstanding characteristic of testicular physiology. It has been demonstrated that Sertoli cells produce ketone bodies and that germ cells may use them as energy source. The aim of the study was to analyze a possible regulation by germ cells of ketogenesis in Sertoli cells. Cultures of Sertoli cells (SC) obtained from 31-day-old rats were co-cultured with germ cells (GC). The results presented herein show that the presence of GC stimulated 3-hydroxybutyrate production and increased mRNA levels of two enzymes involved in ketogenesis-carnitine palmitoyltransferase 1a (CPT1a) and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (mHMGCoA) synthase- in SC. Additionally, GC increased monocarboxylate transporter 4 (Mct4) expression in SC, a transporter involved in ketone bodies exit. To evaluate if the observed effects might be mediated by soluble factors, SC cultures were incubated with germinal cell-conditioned medium (GCCM) or with two growth factors, bFGF and IGF1, which are known to be secreted by GC. We observed that GCCM and bFGF stimulated ketone bodies production but that IGF1 did not modify it. Also, we observed that GCCM and bFGF increased Cpt1a and Mct4 mRNA levels. In summary, results presented herein demonstrate that Sertoli cells are able to produce ketone bodies and that its production is regulated in a paracrine way by germ cells. This study adds new information about communication between Sertoli cells and developing germ cells.