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.

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.

Molecular Mechanisms and Signaling Pathways Involved in Sertoli Cell Proliferation.

Sertoli cells are somatic cells present in seminiferous tubules which have essential roles in regulating spermatogenesis. Considering that each Sertoli cell is able to support a limited number of germ cells, the final number of Sertoli cells reached during the proliferative period determines sperm production capacity. Only immature Sertoli cells, which have not established the blood-testis barrier, proliferate. A number of hormonal cues regulate Sertoli cell proliferation. Among them, FSH, the insulin family of growth factors, activin, and cytokines action must be highlighted. It has been demonstrated that cAMP/PKA, ERK1/2, PI3K/Akt, and mTORC1/p70SK6 pathways are the main signal transduction pathways involved in Sertoli cell proliferation. Additionally, c-Myc and hypoxia inducible factor are transcription factors which participate in the induction by FSH of various genes of relevance in cell cycle progression. Cessation of proliferation is a pre-requisite to Sertoli cell maturation accompanied by the establishment of the blood-testis barrier. With respect to this barrier, the participation of androgens, estrogens, thyroid hormones, retinoic acid and opioids has been reported. Additionally, two central enzymes that are involved in sensing cell energy status have been associated with the suppression of Sertoli cell proliferation, namely AMPK and Sirtuin 1 (SIRT1). Among the molecular mechanisms involved in the cessation of proliferation and in the maturation of Sertoli cells, it is worth mentioning the up-regulation of the cell cycle inhibitors p21Cip1, p27Kip, and p19INK4, and of the gap junction protein connexin 43. A decrease in Sertoli cell proliferation due to administration of certain therapeutic drugs and exposure to xenobiotic agents before puberty has been experimentally demonstrated. This review focuses on the hormones, locally produced factors, signal transduction pathways, and molecular mechanisms controlling Sertoli cell proliferation and maturation. The comprehension of how the final number of Sertoli cells in adulthood is established constitutes a pre-requisite to understand the underlying causes responsible for the progressive decrease in sperm production that has been observed during the last 50 years in humans.

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.

HIF involvement in the regulation of rat Sertoli cell proliferation by FSH.

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH increases the rate of proliferation of Sertoli cells; however, little is known about the transcription factors that are activated by the hormone in order to regulate Sertoli cell proliferation. On the other hand, Hypoxia Inducible Factors (HIFs) are master regulators of cell growth. HIFs are dimers of HIF-ß and HIF-α subunits. Considering that HIF-ß is constitutively expressed, HIF transcriptional activity is regulated through the abundance of HIF-α subunits. To date, three HIF-α isoforms have been described. The association of the different HIF-α subunits with HIF-ß subunit constitutes three active transcription factors -HIF-1, HIF-2 and HIF-3- which interact with consensus hypoxia-response elements in the promoter region of target genes. Hypoxia has been classically considered the main stimulus that increases HIF transcriptional activity, however, regulation by hormones under normoxic conditions was also demonstrated. The aim of this work has been to investigate whether HIFs participate in the regulation of rat Sertoli cell proliferation by FSH. Sertoli cells obtained from 8-day old rats were cultured in the absence or presence of FSH. It has been observed that FSH increases HIF transcriptional activity and HIF-2α mRNA levels without modifying either HIF-1α or HIF-3α expression. Incubations with FSH have been also performed in the absence or presence of a pharmacological agent that promotes HIF-α subunit degradation, LW6. It has been observed that LW6 inhibits the FSH effect on proliferation, CCND1 expression and c-Myc transcriptional activity. Altogether, these results suggest that HIFs might be involved in the regulation of Sertoli cell proliferation by FSH.

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.

Molecular Mechanisms and Signaling Pathways Involved in the Nutritional Support of Spermatogenesis by Sertoli Cells.

Sertoli cells play a central role in spermatogenesis. They maintain the blood-testis barrier, an essential feature of seminiferous tubules which creates the proper environment for the occurrence of the spermatogenesis. However, this confinement renders germ cells almost exclusively dependent on Sertoli cells' nursing function and support. Throughout spermatogenesis, differentiating sperm cells become more specialized, and their biochemical machinery is insufficient to meet their metabolic demands. Although the needs are not the same at all differentiation stages, Sertoli cells are able to satisfy their needs. In order to maintain the seminiferous tubule energetic homeostasis, Sertoli cells react in response to several metabolic stimuli, through signaling cascades. The AMP-activated kinase, sensitive to the global energetic status; the hypoxia-inducible factors, sensitive to oxygen concentration; and the peroxisome proliferator-activated receptors, sensitive to fatty acid availability, are pathways already described in Sertoli cells. These cells' metabolism also reflects the whole-body metabolic dynamics. Metabolic diseases, including obesity and type II diabetes mellitus, induce changes that, both directly and indirectly, affect Sertoli cell function and, ultimately, (dys)function in male reproductive health. Insulin resistance, increased estrogen synthesis, vascular disease, and pubic fat accumulation are examples of metabolic-related conditions that affect male fertility potential. On the other hand, malnutrition can also induce negative effects on male sexual function. In this chapter, we review the molecular mechanisms associated with the nutritional state and male sexual (dys)function and the central role played by the Sertoli cells.

Participation of HIFs in the regulation of Sertoli cell lactate production.

Hypoxia Inducible Factors (HIFs) are master regulators of glycolytic metabolism. HIFs consist of a constitutive HIFbeta (HIFß) subunit and a HIFalpha (HIFα) subunit, whose half-life depends on prolyl-hydroxylases activity. Inhibition of prolyl-hydroxylases by hypoxia or transition metals, or augmentation of HIFα subunit levels by hormonal stimuli lead to a higher HIF transcriptional activity. On the other hand, it is well known that lactate produced by Sertoli cells is delivered to and used by germ cells as an energy substrate. The aim of this work was to investigate whether HIFs participate in the regulation of lactate production in rat Sertoli cells and whether they are involved in the FSH mechanism of action. In order to reach a higher HIF transcriptional activity, Sertoli cells were treated with CoCl2. We observed that a higher HIF transcriptional activity leads to an augmentation of: lactate production, glucose uptake and LDH activity. Besides, an increase in Glut1, Pkm2 and Ldha mRNA levels was observed. These findings suggested that HIFs may participate in the modulation of Sertoli cell nutritional function. As FSH regulates lactate production, we evaluated whether HIFs were involved in FSH action. Sertoli cells were stimulated with FSH in the absence or presence of LW6, a drug which promotes HIFα subunit degradation. On the one hand, we observed that FSH increases HIF1α protein, Hif1α and Hif2α mRNA levels and, on the other hand, that LW6 inhibits FSH-stimulated lactate production, glucose uptake, Glut1, Pkm2 and Ldha expression. It is proposed that HIFs are key components of the intricate pathways utilized by FSH to regulate the provision of lactate for germ cells. Considering that FSH is the master endocrine regulator of Sertoli cells, it is not surprising that this hormone may employ several regulatory mechanisms to fulfill the nourishing functions of this cell type.