Mammalian target of rapamycin (mTOR): a central regulator of male fertility?

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolic phenotype and is involved in virtually all aspects of cellular function. It integrates not only nutrient and energy-sensing pathways but also actin cytoskeleton organization, in response to environmental cues including growth factors and cellular energy levels. These events are pivotal for spermatogenesis and determine the reproductive potential of males. Yet, the molecular mechanisms by which mTOR signaling acts in male reproductive system remain a matter of debate. Here, we review the current knowledge on physiological and molecular events mediated by mTOR in testis and testicular cells. In recent years, mTOR inhibition has been explored as a prime strategy to develop novel therapeutic approaches to treat cancer, cardiovascular disease, autoimmunity, and metabolic disorders. However, the physiological consequences of mTOR dysregulation and inhibition to male reproductive potential are still not fully understood. Compelling evidence suggests that mTOR is an arising regulator of male fertility and better understanding of this atypical protein kinase coordinated action in testis will provide insightful information concerning its biological significance in other tissues/organs. We also discuss why a new generation of mTOR inhibitors aiming to be used in clinical practice may also need to include an integrative view on the effects in male reproductive system.

mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo.

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Signaling pathways regulating blood-tissue barriers - Lesson from the testis.

Signaling pathways that regulate blood-tissue barriers are important for studying the biology of various blood-tissue barriers. This information, if deciphered and better understood, will provide better therapeutic management of diseases particularly in organs that are sealed by the corresponding blood-tissue barriers from systemic circulation, such as the brain and the testis. These barriers block the access of antibiotics and/or chemotherapeutical agents across the corresponding barriers. Studies in the last decade using the blood-testis barrier (BTB) in rats have demonstrated the presence of several signaling pathways that are crucial to modulate BTB function. Herein, we critically evaluate these findings and provide hypothetical models regarding the underlying mechanisms by which these signaling molecules/pathways modulate BTB dynamics. This information should be carefully evaluated to examine their applicability in other tissue barriers which shall benefit future functional studies in the field. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Estrogenic regulation of bicarbonate transporters from SLC4 family in rat Sertoli cells.

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. Bicarbonate is essential not only to ionic homeostasis but also to pH maintenance along the male reproductive tract. Previous studies support an association of high 17ß-estradiol (E2) levels with modulation of specific ion transporters expression. Herein we determined the effect of E2 on the expression/functionality of SLC4 family bicarbonate transporters in rat Sertoli cells (SCs). All studied transporters [anion exchanger 2 (AE2), Na(+)-driven Cl(-)/HCO3 (-) exchanger (NDCBE), electrogenic Na(+)/HCO3 (-) co-transporters (NBCe1), and electroneutral Na(+)/HCO3 (-) co-transporters (NBCn1)] were identified in SCs, being AE2 and NBCn1 the most abundant. In E2-treated cells (100 nM), increases in AE2 and NBCn1 protein levels were observed, as well as altered transcellular transport. E2-treated SCs presented a significant perturbation of ATP-induced short-circuit current. This alteration was concurrent with augmented AE2 and NBCn1 levels. Overall, we report a relation between increased E2 levels and the expression/function of AE2 and NBCn1 in rat SCs, providing new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis, with direct influence on male reproductive potential.

Aquaporin-4 as a molecular partner of cystic fibrosis transmembrane conductance regulator in rat Sertoli cells.

Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively. The possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein. The co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB. The full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport.

Aquaporin-9 is expressed in rat Sertoli cells and interacts with the cystic fibrosis transmembrane conductance regulator.

Men with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are usually subfertile/infertile. Besides playing a role in Cl(-)/HCO3(-) transport, it has been proposed that CFTR interacts with water membrane transport systems, particularly aquaporins, to control seminiferous tubular secretion, which is regulated by the somatic Sertoli cells (SCs). As aquaporin-9 (AQP9) is highly expressed throughout the male reproductive tract, we hypothesized that it is also present in rat SCs and that it physically interacts with CFTR. To test this hypothesis, primary cultures of rat SCs were established, and expression of CFTR and AQP9 was assessed by RT-polymerase chain reactions (mRNA) and Western blot analysis (protein). A coimmunoprecipitation assay was used to evaluate the physical interaction between CFTR and AQP9. Our results show that CFTR and AQP9 are expressed in rat SCs. We were also able to detect a molecular interaction between CFTR and AQP9 in rat SCs. This is the first report describing the presence of AQP9, and its interaction with CFTR, in rat SCs. Moreover, our results provide evidence that CFTR is involved in water homeostasis of the seminiferous tubular secretion. These mechanisms may open new insights on therapeutic targets to counteract subfertility/infertility in men with cystic fibrosis and mutations in the CFTR gene.

Personalized Medicine in Medullary Thyroid Carcinoma: A Broad Review of Emerging Treatments.

Medullary thyroid carcinoma (MTC) arises from parafollicular cells in the thyroid gland, and although rare, it represents an aggressive type of thyroid cancer. MTC is recognized for its low mutational burden, with point mutations in RET or RAS genes being the most common oncogenic events. MTC can be resistant to cytotoxic chemotherapy, and multitarget kinase inhibitors (MKIs) have been considered a treatment option. They act by inhibiting the activities of specific tyrosine kinase receptors involved in tumor growth and angiogenesis. Several tyrosine kinase inhibitors are approved in the treatment of advanced MTC, including vandetanib and cabozantinib. However, due to the significant number of adverse events, debatable efficiency and resistance, there is a need for novel RET-specific TKIs. Newer RET-specific TKIs are expected to overcome previous limitations and improve patient outcomes. Herein, we aim to review MTC signaling pathways, the most recent options for treatment and the applications for personalized medicine.

TERTmonitor-qPCR Detection of TERTp Mutations in Glioma.

Telomerase promoter (TERTp) mutations are frequently observed in various types of tumours and commonly characterised by two specific hotspots located at positions -124 and -146 upstream of the start codon. They enhance TERTp activity, resulting in increased TERT expression. In central nervous system (CNS) tumours, they are integrated as biomarkers, aiding in the diagnosis and with a role in prognosis, where, in some settings, they are associated with aggressive behaviour. In this study, we evaluated the performance of TERTmonitor for TERTp genotyping in a series of 185 gliomas in comparison to the traditional method, Sanger sequencing. Against the gold-standard Sanger method, TERTmonitor performed with a 97.8% accuracy. Inaccuracy was mainly due to the over-detection of variants in negative cases (by Sanger) and the presence of variants that can modify the chemistry of the probe detection. The distribution of the mutations was comparable to other series, with the -124 being the most represented (38.92% for Sanger and TERTmonitor) and more prevalent in the higher-grade tumours, gliosarcoma (50.00%) and glioblastoma (52.6%). The non-matched cases are debatable, as we may be dealing with the reduced sensitivity of Sanger in detecting rare alleles, which strengthens the use of the TERTmonitor. With this study, we present a reliable and rapid potential tool for TERTp genotyping in gliomas.

Activation of an actin signaling pathway in pre-malignant mammary epithelial cells by P-cadherin is essential for transformation.

Alterations in the expression or function of cell adhesion molecules have been implicated in all steps of tumor progression. Among those, P-cadherin is highly enriched in basal-like breast carcinomas, playing a central role in cancer cell self-renewal, collective cell migration and invasion. To establish a clinically relevant platform for functional exploration of P-cadherin effectors in vivo, we generated a humanized P-cadherin Drosophila model. We report that actin nucleators, Mrtf and Srf, are main P-cadherin effectors in fly. We validated these findings in a human mammary epithelial cell line with conditional activation of the SRC oncogene. We show that, prior to promoting malignant phenotypes, SRC induces a transient increase in P-cadherin expression, which correlates with MRTF-A accumulation, its nuclear translocation and the upregulation of SRF target genes. Moreover, knocking down P-cadherin, or preventing F-actin polymerization, impairs SRF transcriptional activity. Furthermore, blocking MRTF-A nuclear translocation hampers proliferation, self-renewal and invasion. Thus, in addition to sustaining malignant phenotypes, P-cadherin can also play a major role in the early stages of breast carcinogenesis by promoting a transient boost of MRTF-A-SRF signaling through actin regulation.

Generation of an Obese Diabetic Mouse Model upon Conditional Atrx Disruption.

Atrx loss was recently ascertained as insufficient to drive pancreatic neuroendocrine tumour (PanNET) formation in mice islets. We have identified a preponderant role of Atrx in the endocrine dysfunction in a Rip-Cre;AtrxKO genetically engineered mouse model (GEMM). To validate the impact of a different Cre-driver line, we used similar methodologies and characterised the Pdx1-Cre;AtrxKO (P.AtrxKO) GEMM to search for PanNET formation and endocrine fitness disruption for a period of up to 24 months. Male and female mice presented different phenotypes. Compared to P.AtrxWT, P.AtrxHOM males were heavier during the entire study period, hyperglycaemic between 3 and 12 mo., and glucose intolerant only from 6 mo.; in contrast, P.AtrxHOM females started exhibiting increased weight gains later (after 6 mo.), but diabetes or glucose intolerance was detected by 3 mo. Overall, all studied mice were overweight or obese from early ages, which challenged the histopathological evaluation of the pancreas and liver, especially after 12 mo. Noteworthily, losing Atrx predisposed mice to an increase in intrapancreatic fatty infiltration (FI), peripancreatic fat deposition, and macrovesicular steatosis. As expected, no animal developed PanNETs. An obese diabetic GEMM of disrupted Atrx is presented as potentially useful for metabolic studies and as a putative candidate for inserting additional tumourigenic genetic events.

Cell polarity, cell adhesion, and spermatogenesis: role of cytoskeletons.

In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP) is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2) in the testis. On the other hand, cell junctions at the Sertoli cell-spermatid (steps 8-19) interface are exclusively supported by adhesion protein complexes (for example, α6ß1-integrin-laminin-α3,ß3,γ3 and nectin-3-afadin) at the actin-rich apical ectoplasmic specialization (ES) since the apical ES is the only anchoring device in step 8-19 spermatids. For cell junctions at the Sertoli cell-cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-ß-catenin and nectin-2-afadin), tight junction (occludin-ZO-1 and claudin 11-ZO-1), and gap junction (connexin 43-plakophilin-2) and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2). In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both) and these polarity (or PCP) protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed.

Male fertility and obesity: are ghrelin, leptin and glucagon-like peptide-1 pharmacologically relevant?

Obesity is rising to unprecedented numbers, affecting a growing number of children, adolescents and young adult men. These individuals face innumerous health problems, including subfertility or even infertility. Overweight and obese men present severe alterations in their body composition and hormonal profile, particularly in ghrelin, leptin and glucagon-like peptide-1 (GLP-1) levels. It is well known that male reproductive health is under the control of the individual's nutritional status and also of a tight network of regulatory signals, particularly hormonal signaling. However, few studies have been focused on the effects of ghrelin, leptin and GLP-1 in male reproduction and how energy homeostasis and male reproductive function are linked. These hormones regulate body glucose homeostasis and several studies suggest that they can serve as targets for anti-obesity drugs. In recent years, our understanding of the mechanisms of action of these hormones has grown significantly. Curiously, their effect on male reproductive potential, that is highly dependent of the metabolic cooperation established between testicular cells, remains a matter of debate. Herein, we review general concepts of male fertility and obesity, with a special focus on the effects of ghrelin, leptin and GLP-1 on male reproductive health. We also discuss the possible pharmacological relevance of these hormones to counteract the fertility problems that overweight and obese men face.

Mammalian target of rapamycin controls glucose consumption and redox balance in human Sertoli cells.

OBJECTIVE: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress. DESIGN: Experimental study. SETTING: University research center and private assisted reproductive technology centers. PATIENT(S): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis. INTERVENTION(S): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of mTOR, for 24 hours. MAIN OUTCOME MEASURE(S): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied. RESULT(S): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated. CONCLUSION(S): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.

CFTR Regulation of Aquaporin-Mediated Water Transport: A Target in Male Fertility.

The formation of competent spermatozoa is associated with the movement of large quantities of water and electrolytes in the various tissues and luminal fluids of the male reproductive tract. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) and HCO3(-) membrane transporter. CFTR gene mutations cause cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Of note, one hallmark in CF is male infertility. Indeed, mutations of CFTR gene cause abnormal production of germ cells and a reduction in germ cell quality and number. Compelling evidence illustrates that CFTR is involved in several pivotal processes for male fertility, including spermatogenesis and sperm capacitation. Recent studies show that CFTR acts as a molecular partner of specific water channels, known as aquaporins, in somatic testicular cells. Aquaporins are water-selective channels that enable high permeability fluxes of water across plasma membranes. In the male reproductive tract, water movements and ion concentrations are determinants for the male reproductive function. Therefore, aquaporins expression and function play a key role in male fertility. Herein we present an overview of the expression and function of CFTR in the male reproductive tract, highlighting the reproductive outcomes in male carriers of CFTR mutations and CF couples. We also present an up-to-date discussion on the expression and role of aquaporins in the male reproductive tract. Finally, we discuss the regulation of aquaporin-mediated water transport by CFTR in the male reproductive tract and its implication for male fertility.