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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate (cAMP)-regulated chloride and bicarbonate ion channel found in many human cells. Its unique biochemical characteristics and role as a member of the adenosine triphosphate (ATP)-binding cassette transporters superfamily are pivotal for the transport of several substrates across cellular membranes. CFTR is known to interact, physically and functionally, with several other cellular proteins. Hence, its properties are essential for moving various substances across cell membranes and ensuring correct cell functioning. Genetic mutations or environmental factors may disrupt CFTR's function resulting in different possible phenotypes due to gene variations that affect not only CFTR's function, localization, and processing within cells, but also those of its interactors. This has been reported as an underlying cause of various diseases, including cystic fibrosis. The severe clinical implications of cystic fibrosis have driven intense research into the role of CFTR in lung function but its significance to fertility, particularly in men, has been comparatively understudied. However, ongoing and more recent research into CFTR and its interacting proteins in the testis or specific testicular cells is beginning to shed light on this field. Herein, we provide a comprehensive and up-to-date overview of the CFTR, its interactome, and its crucial role in male reproduction, highlighting recent discoveries and advancements in understanding the molecular mechanisms involved. The comprehension of these complex interactions may pave the way for potential therapeutic approaches to improve fertility of men suffering from alterations in the function of CFTR., (© 2024 Wiley Periodicals LLC.)

Atrazine (ATZ), a widely used herbicide with potent endocrine-disrupting properties, has been implicated in hormonal disturbances and fertility issues. Sertoli cells (SCs) play a crucial role in providing mechanical and nutritional support of spermatogenesis. Herein, we aimed to study the effects of environmentally relevant ATZ concentrations on the nutritional support of spermatogenesis provided by SCs. For that, mouse SCs (TM4) were exposed to increasing ATZ concentrations (in μg/L: 0.3, 3, 30, 300, or 3000). After 24 h, cellular proliferation and metabolic activity were assessed. Mitochondrial activity and endogenous reactive oxygen species (ROS) production were evaluated using JC-1 and CM-H 2 DCFDA probes, respectively. We also analyzed protein levels of lactate dehydrogenase (LDH) using Western Blot and live cells glycolytic function through Seahorse XF Glycolysis Stress Test Kit. ATZ exposure decreased the activity of oxidoreductases in SCs, suggesting a decreased metabolic activity. Although ATZ is reported to induce oxidative stress, we did not observe alterations in mitochondrial membrane potential and ROS production across all tested concentrations. When we evaluated the glycolytic function of SCs, we observed that ATZ significantly impaired glycolysis and the glycolytic capacity at all tested concentrations. These results were supported by the decreased expression of LDH in SCs. Overall, our findings suggest that ATZ impairs the glycolytic function of SCs through LDH downregulation. Since lactate is the preferential energetic substrate for germ cells, exposure to ATZ may detrimentally impact the nutritional support crucial for spermatogenesis, hinting for a relationship between ATZ exposure and male infertility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

In Brief: Mitochondrial uncoupling proteins (UCPs) regulate mitochondrial activity and reactive oxygen species production through the transport of protons and metabolites. This study identified the expression of UCPs in human Sertoli cells, which proved to be modulators of their mitochondrial activity., Abstract: Mitochondrial uncoupling proteins (UCPs) are mitochondrial channels responsible for the transport of protons and small molecular substrates across the inner mitochondrial membrane. Altered UCP expression or function is commonly associated with mitochondrial dysfunction and increased oxidative stress, which are both known causes of male infertility. However, UCP expression and function in the human testis remain to be characterized. This study aimed to assess the UCP homologs (UCP1-6) expression and function in primary cultures of human Sertoli cells (hSCs). We identified the mRNA expression of all UCP homologs (UCP1-6) and protein expression of UCP1, UCP2, and UCP3 in hSCs. UCP inhibition by genipin for 24 h decreased hSCs proliferation without causing cytotoxicity (n = 6). Surprisingly, the prolonged UCP inhibition for 24 h decreased mitochondrial membrane potential, oxygen consumption rate (OCR), and endogenous reactive oxygen species (ROS) production. The metabolism of hSCs was also affected as UCP inhibition shifted their metabolism toward an increased pyruvate consumption. Taken together, these findings demonstrate that UCPs play a role as regulators of the mitochondrial function in hSCs, emphasizing their potential as targets in the study of male (in)fertility.

Diabetic nephropathy (DN) and insulin resistance (IR) in kidney cells are considered main causes for end-stage renal failure. However, it is unclear how IR affects early stages of the disease. Here, we investigate the impact of mild (11 mM) and severe (22 mM) hyperglycemia, with and without induced IR, on cellular metabolism and mitochondrial bioenergetics in a human kidney cell line (HK-2). IR in HK-2 cells was induced with palmitic acid and cellular cytotoxicity was studied. We evaluated the impact of mild and severe hyperglycemia with and without IR on the metabolic secretome of the cells, their live-cell mitochondria function, mitochondrial membrane potential, and mitochondrial complex activities. Furthermore, we measured fatty acid oxidation and lipid accumulation. Cells cultured under mild hyperglycemic conditions exhibited increased mitochondrial bioenergetic parameters, such as basal respiration, ATP-linked production, maximal respiration capacity, and spare respiration capacity. However, these parameters decreased when cells were cultured under higher glucose concentrations when IR was induced. Our data suggests that progression from mild to severe hyperglycemia induces a metabolic shift, where gluconeogenic amino acids play a crucial role in supplying the energy requirements of HK-2. To our knowledge, this is the first study to evaluate the progression from mild to severe hyperglycemia allied to IR in human kidney cells. This work highlights that this progression leads to mitochondrial dysfunction and alters the metabolic profile of kidney cells. These results identify possible targets for early intervention in DN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Fluoroacetic acid (FAA) is a poison commonly used for the lethal control of invasive species in Australia and New Zealand. Despite its widespread use and long history as a pesticide, no effective treatment for accidental poisoning exists. Although it is known to inhibit the tricarboxylic acid (TCA) cycle, specific details of FAA toxicology have remained elusive, with hypocalcemia suggested to be involved in the neurological symptoms prior to death. Here, we study the effects of FAA on cell growth and mitochondrial function using the filamentous fungi Neurospora crassa as model organism. FAA toxicosis in N. crassa is characterized by an initial hyperpolarization and subsequent depolarization of the mitochondrial membranes, followed by a significant intracellular decrease in ATP and increase in Ca 2+ . The development of mycelium was markedly affected within 6 h, and growth impaired after 24 h of FAA exposure. Although the activity of mitochondrial complexes I, II and IV was impaired, the activity of citrate synthase was not affected. Supplementation with Ca 2+ exacerbated the effects of FAA in cell growth and membrane potential. Our findings suggest that an imbalance created in the ratio of ions within the mitochondria may lead to conformational changes in ATP synthase dimers due to mitochondrial Ca 2+ uptake, that ultimately result in the opening of the mitochondrial permeability transition pore (MPTP), a decrease in membrane potential, and cell death. Our findings suggest new approaches for the treatment research, as well as the possibility to use N. crassa as a high-throughput screening assay to evaluate a large number of FAA antidote candidates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Osmoregulation plays a vital role in sperm function, encompassing spermatogenesis, maturation, and fertilization. Aquaglyceroporins, a subclass of aquaporins (AQPs), facilitate the transport of water and glycerol across the sperm membrane, with glycerol serving as an important substrate for sperm bioenergetics. This study aimed to elucidate the significance of AQP-mediated glycerol permeability in sperm motility. The presence and localization of AQP3 and AQP7 in human sperm were assessed using immunofluorescence. Subsequently, the glycerol permeability of spermatozoa obtained from normozoospermic individuals ( n = 30) was measured, using stopped-flow light scattering, after incubation with specific aquaporin inhibitors targeting AQP3 (DFP00173), AQP7 (Z433927330), or general aquaglyceroporin (phloretin). Sperm from asthenozoospermic men ( n = 30) were utilized to evaluate the AQP7-mediated glycerol permeability, and to compare it with that of normozoospermic men. Furthermore, hypermotile capacitated sperm cells were examined, to determine the AQP7 expression and membrane glycerol permeability. AQP3 was predominantly observed in the tail region, while AQP7 was present in the head, midpiece, and tail of human sperm. Our findings indicate that AQP7 plays a key role in glycerol permeability, as the inhibition of AQP7 resulted in a 55% decrease in glycerol diffusion across the sperm membrane. Importantly, this glycerol permeability impairment was evident in spermatozoa from asthenozoospermic individuals, suggesting the dysregulation of AQP7-mediated glycerol transport, despite similar AQP7 levels. Conversely, the AQP7 expression increased in capacitated sperm, compared to non-capacitated sperm. Hence, AQP7-mediated permeability may serve as a valuable indicator of sperm motility, and be crucial in sperm function.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7 α -acetoxy-6 β -benzoyloxy-12- O -benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53 -induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

Infertility is becoming a chronic and emerging problem in the world. There is a resistant stigma that this health condition is mostly due to the female, although the literature supports that the responsibility for the onset of infertility is equally shared between both sexes in more or less equal proportions. Nevertheless, male sex hormones, particularly testosterone (T), are key players in male-related infertility. Indeed, hypogonadism, which is also characterized by changes in T levels, is one of the most common causes of male infertility and its incidence has been interconnected to the increased prevalence of metabolic diseases. Recent data also highlight the role of aquaporin (AQP)-mediated water and solute diffusion and the metabolic homeostasis in testicular cells suggesting a strong correlation between AQPs function, metabolism of testicular cells, and infertility. Indeed, recent studies showed that both metabolic and sexual hormone concentrations can change the expression pattern and function of AQPs. Herein, we review up-to-date information on the involvement of AQP-mediated function and permeability in men with metabolic syndrome and testosterone deficit, highlighting the putative mechanisms that show an interaction between sex hormones, AQPs, and metabolic syndrome that may contribute to male infertility.

Obesity is associated with complex adipose tissue energy metabolism remodeling. Whether AT metabolic reprogramming differs according to body mass index (BMI) and across different obesity classes is unknown. This study’s purpose was to evaluate and compare bioenergetics and energy substrate preference of visceral adipose tissue (VAT) pertaining to individuals with obesity class 2 and class 3. VAT obtained from patients with obesity (n = 15) class 2 (n = 7; BMI 37.53 ± 0.58 kg/m2) or class 3 (n = 8; BMI 47.79 ± 1.52 kg/m2) was used to assess oxygen consumption rate (OCR) bioenergetics and mitochondrial substrate preferences. VAT of patients with obesity class 3 presented significantly higher non-mitochondrial oxygen consumption (p < 0.05). In VAT of patients with obesity class 2, inhibition of pyruvate and glutamine metabolism significantly decreased maximal respiration and spare respiratory capacity (p < 0.05), while pyruvate and fatty acid metabolism inhibition, which renders glutamine the only available substrate, increased the proton leak with a protective role against oxidative stress (p < 0.05). In conclusion, VAT bioenergetics of patients with obesity class 2 depicts a greater dependence on glucose/pyruvate and glutamine metabolism, suggesting that patients within this BMI range are more likely to be responsive to interventions based on energetic substrate modulation for obesity treatment.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that is crucial for fluid homeodynamics throughout the male reproductive tract. Previous evidence shed light on a potential molecular partnership between this channel and aquaporins (AQPs). Herein, we explore the role of CFTR on AQPs-mediated glycerol permeability in mouse Sertoli cells (mSCs). We were able to identify the expression of CFTR, AQP3, AQP7, and AQP9 in mSCs by RT-PCR, Western blot, and immunofluorescence techniques. Cells were then treated with CFTRinh-172, a specific CFTR inhibitor, and its glycerol permeability was evaluated by stopped-flow light scattering. We observed that CFTR inhibition decreased glycerol permeability in mSCs by 30.6% when compared to the control group. A DUOLINK proximity ligation assay was used to evaluate the endogenous protein-protein interactions between CFTR and the various aquaglyceroporins we identified. We positively detected that CFTR is in close proximity with AQP3, AQP7, and AQP9 and that, through a possible physical interaction, CFTR can modulate AQP-mediated glycerol permeability in mSCs. As glycerol is essential for the control of the blood-testis barrier and elevated concentration in testis results in the disruption of spermatogenesis, we suggest that the malfunction of CFTR and the consequent alteration in glycerol permeability is a potential link between male infertility and cystic fibrosis., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Stress, as a physiological response, is a major factor that affects several processes, including reproductive functions. The main hormonal players of stress are cortisol (humans) and corticosterone (rodents). Sertoli cells (SCs), as key contributors for the testicular homeostasis maintenance, are extensively challenged by different hormones, with glucocorticoid corticosterone being the signaling modulator that may impact these cells at different levels. We aimed to characterize how corticosterone modulates SCs energy balance, putting the mitochondrial performance and signaling output in perspective as the cells can disperse to the surroundings. TM4 mouse SCs were cultured in the absence and presence of corticosterone (in nM: 20, 200, and 2000). Cells were assessed for extracellular metabolic fluxes, mitochondrial performance (cell respirometry, mitochondrial potential, and mitochondrial complex expressions and activities), and the expression of androgen and corticosteroid receptors, as well as interleukine-6 (IL-6) and glutathione content. Corticosterone presented a biphasic impact on the extracellular fluxes of metabolites. Low sub-physiological corticosterone stimulated the glycolytic activity of SCs. Still, no alterations were perceived for lactate and alanine production. However, the lactate/alanine ratio was decreased in a dose-dependent mode, opposite to the mitochondrial complex II activity rise and concurrent with the decrease of IL-6 expression levels. Our results suggest that corticosterone finely tuned the energetic profile of mouse SCs, with sub-physiological concentrations promoting glycolytic expenditure, without translating into cell redox power and mitochondrial respiratory chain performance. Corticosterone deeply impacted the expression of the pro-inflammatory IL-6, which may alter cell-to-cell communication in the testis, in the last instance and impact of the spermatogenic performance., Competing Interests: The authors declare that they have no conflict of interest.

Cryopreservation is globally used as a method for long-term preservation, although freeze-thawing procedures may strongly impair the gamete function. The correct cryopreservation procedure is characterized by the balance between freezing rate and cryoprotective agents (CPAs), which minimizes cellular dehydration and intracellular ice formation. For this purpose, osmoregulation is a central process in cryopreservation. During cryopreservation, water and small solutes, including penetrating cryoprotective agents, cross the plasma membrane. Aquaporins (AQPs) constitute a family of channel proteins responsible for the transport of water, small solutes, and certain gases across biological membranes. Thirteen homologs of AQPs (AQP0-12) have been described. AQPs are widely distributed throughout the male and female reproductive systems, including the sperm and oocyte membrane. The composition of the male and female gamete membrane is of special interest for assisted reproductive techniques (ART), including cryopreservation. In this review, we detail the mechanisms involved in gamete cryopreservation, including the most used techniques and CPAs. In addition, the expression and function of AQPs in the male and female gametes are explored, highlighting the potential protective role of AQPs against damage induced during cryopreservation.

The strong hormonal dysregulation associated with obesity is responsible for the disruption of several reproductive events. Sertoli cells (SCs) function is dependent on energetic homeostasis and thus, directly associated with energy homeostasis regulating hormones. To further understand the influence of those hormones with SCs function and obesity, we hypothesize that human SCs express obesity-related genes (ORG; MC4R, GNPDA2, TMEM18, and FTO) and that they respond to energy homeostasis regulating hormones (leptin, ghrelin, and glucagon-like protein 1 [GLP-1]) stimuli. To test our hypothesis, SCs were cultured with increasing doses of leptin (0, 5, 25, or 50 ng/ml, for 24 h), ghrelin (0, 20, 100, and 500 pM, for 24 h), and GLP-1 (10, 1000, or 1 × 105 pM, for 6 h). The presence and abundance of ORG transcripts and proteins in SCs were accessed by polymerase chain reaction techniques, Western blot analysis, and immunofluorescence staining. Our results show that human SCs express MC4R, GNPDA2, TMEM18, and FTO in specific cellular locations. MC4R and FTO expression in human SCs was not responsive to the treatments. However, GNPDA2 and TMEM18 expression increased after exposure to the highest concentration of leptin and ghrelin, respectively. We highlight for the first time that human SCs express ORG and that these are responsive to energy homeostasis hormonal stimuli. GNPDA2 and TMEM18 expression respond in opposite directions according to overall energy status, mediated by energy homeostasis regulating hormones. Leptin and ghrelin control of ORG expression by human SCs can be associated with overweight-related infertility and subfertility in males., (© 2020 Wiley Periodicals LLC.)

Aquaporins (AQPs) are a family of transmembrane channel proteins responsible for the transport of water and small uncharged molecules. Thirteen distinct isoforms of AQPs have been identified in mammals (AQP0-12). Throughout the male reproductive tract, AQPs greatly enhance water transport across all biological barriers, providing a constant and expeditious movement of water and playing an active role in the regulation of water and ion homeostasis. This regulation of fluids is particularly important in the male reproductive tract, where proper fluid composition is directly linked with a healthy and competent spermatozoa production. For instance, in the testis, fluid regulation is essential for spermatogenesis and posterior spermatozoa transport into the epididymal ducts, while maintaining proper ionic conditions for their maturation and storage. Alterations in the expression pattern of AQPs or their dysfunction is linked with male subfertility/infertility. Thus, AQPs are important for male reproductive health. In this review, we will discuss the most recent data on the expression and function of the AQPs isoforms in the human, mouse and rat male reproductive tract. In addition, the regulation of AQPs expression and dysfunction linked with male infertility will be discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel responsible for the direct transport of bicarbonate and chloride. CFTR-dependent ionic transport is crucial for pH regulation and fluid homeodynamics among epithelial surfaces. Particularly, CFTR performs an essential role in the male reproductive tract, which requires a tight regulation of water and electrolytes in order to produce healthy spermatozoa. The absence or malfunction of CFTR results in cystic fibrosis, the most common lethal disease among Caucasians, that is characterized by an impaired fluid and ionic homeostasis in the whole organism. Due to the wide expression and importance of CFTR, the male reproductive tract is highly affected by cystic fibrosis, resulting in male infertility. Although CFTR is not permeable to water, this protein acts as a regulator of other protein channels, such as aquaporins. In fact, CFTR acts as a molecular partner of aquaporins in epithelial cells, regulating fluid homeodynamics. Herein, up-to-date data concerning the regulation of aquaporin-mediated water transport by CFTR will be discussed, highlighting the role of both channels in the male reproductive tract., Competing Interests: Conflict of interest None to declare., (© 2020 Elsevier Inc. All rights reserved.)

Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17β-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.

In the male reproductive tract, ionic equilibrium is essential to maintain normal spermatozoa production and, hence, the reproductive potential. Among the several ions, HCO 3 - and H + have a central role, mainly due to their role on pH homeostasis. In the male reproductive tract, the major players in pH regulation and homeodynamics are carbonic anhydrases (CAs), HCO 3 - membrane transporters (solute carrier 4-SLC4 and solute carrier 26-SLC26 family transporters), Na + -H + exchangers (NHEs), monocarboxylate transporters (MCTs) and voltage-gated proton channels (Hv1). CAs and these membrane transporters are widely distributed throughout the male reproductive tract, where they play essential roles in the ionic balance of tubular fluids. CAs are the enzymes responsible for the production of HCO 3 - which is then transported by membrane transporters to ensure the maturation, storage, and capacitation of the spermatozoa. The transport of H + is carried out by NHEs, Hv1, and MCTs and is essential for the electrochemical balance and for the maintenance of the pH within the physiological limits along the male reproductive tract. Alterations in HCO 3 - production and transport of ions have been associated with some male reproductive dysfunctions. Herein, we present an up-to-date review on the distribution and role of the main intervenient on pH homeodynamics in the fluids throughout the male reproductive tract. In addition, we discuss their relevance for the establishment of the male reproductive potential.

Purpose: L-Theanine is the major free amino acid present in tea (Camellia sinensis L.). The effects of several tea constituents on male reproduction have been investigated, but L-theanine has been overlooked. Sertoli cells (SCs) are essential for the physical and nutritional support of germ cells. In this study, we aimed to investigate the ability of L-theanine to modulate important mechanisms of human SCs (hSCs) metabolism, mitochondrial function and oxidative profile, which are essential to prevent or counteract spermatogenesis disruption in several health conditions., Methods: We evaluated the effect of a dose of L-theanine attained by tea intake (5 μM) or a pharmacological dose (50 μM) on the metabolism (proton nuclear magnetic resonance and Western blot), mitochondrial functionality (protein expression of mitochondrial complexes and JC1 ratio) and oxidative profile (carbonyl levels, nitration and lipid peroxidation) of cultured hSCs., Results: Exposure of hSCs to 50 µM of L-theanine increased cell proliferation and glucose consumption. In response to this metabolic adaptation, there was an increase in mitochondrial membrane potential, which may compromise the prooxidant-antioxidant balance. Still, no alterations were observed regarding the oxidative damages., Conclusions: A pharmacological dose of L-theanine (50 µM) prompts an increase in hSCs proliferation and a higher glucose metabolization to sustain the pool of Krebs cycle intermediates, which are crucial for cellular bioenergetics and biosynthesis. This study suggests an interplay between glycolysis and glutaminolysis in the regulation of hSCs metabolism.

The process that allows cells to control their pH and bicarbonate levels is essential for ionic and metabolic equilibrium. Carbonic anhydrases (CAs) catalyse the conversion of CO 2 to HCO 3 - and H + and are thus essential for this process. Herein, we inhibited CAs with acetazolamide - ACT and SLC-0111 - to study their involvement in the metabolism, mitochondrial potential, mitochondrial biogenesis and lipid metabolism of human Sertoli cells (hSCs), obtained from biopsies from men with conserved spermatogenesis. We were able to identify three isoforms of CAs, one mitochondrial isoform (CA VB) and two cell membrane-bound isoforms (CA IX and CA XII) in hSCs. When assessing the expression of markers for mitochondrial biogenesis, we observed a decrease in HIF-1α, SIRT1, PGC1α and NRF-1 mRNAs after all CAs were inhibited, resulting in decreased mitochondrial DNA copy numbers. This was followed by an increased production of lactate and alanine in the same conditions. In addition, consumption of glucose was maintained after inhibition of all CAs in hSCs. These results indicate a reduced conversion of pyruvate to acetyl-coA, possibly due to decreased mitochondrial function, caused by CA inhibition in hSCs. Inhibition of CAs also caused alterations in lipid metabolism, since we detected an increased expression of hormone-sensitive lipase (HSL) in hSCs. Our results suggest that CAs are essential for mitochondrial biogenesis, glucose and lipid metabolism in hSCs. This is the first report showing that CAs play an essential role in hSC metabolic dynamics, being involved in mitochondrial biogenesis and controlling lactate production., (© 2019 Federation of European Biochemical Societies.)

High 17β-Estradiol (E2) levels are known to cause alterations of spermatogenesis and environments throughout the male reproductive tract. Sertoli cells (SCs) ensure an adequate environment inside the seminiferous tubule. Glycerol stands as essential for the maintenance of blood⁻testis barrier created by SCs, however, the role of E2 in this process is not known. Herein, we hypothesized that the effect of E2 on glycerol permeability in mouse SCs (mSCs) could be mediated by aquaglyceroporins. The expression of aquaglyceroporins was assessed by RT-PCR and qRT-PCR. Glycerol permeability was evaluated by stopped-flow light scattering. We were able to identify the expression of AQP3 and AQP9 in mSCs where AQP9 is more abundant than AQP3. Our results show that high E2 levels decrease AQP9 mRNA abundance with no influence on AQP3 in mSCs. Interestingly, high E2 levels decreased mSCs' permeability to glycerol, while downregulating AQP9 expression, thus suggesting a novel mechanism by which E2 modulates fluid secretion in the testis. In conclusion, E2 is an important regulator of mSCs physiology and secretion through changes in AQP9 expression and function. Thus, alterations in glycerol permeability induced by E2 may be the cause for male infertility in cases associated with the presence of high E2 levels.

The purity of the primary cultures of Sertoli cells is a factor for the validation of the studies using this methodological approach. There is a probability of contamination of these cultures with other testicular cellular types, such as peritubular cells and germ cells (that represent a large percentage of the volume of the seminiferous tubules).For the evaluation of the purity of cultures, the immunocytochemistry technique (immunoperoxidase or immunofluorescence) is frequently used to label a protein marker specific for the Sertoli cells within the testicular environment. The expression of several protein markers can be used, with different particularities, being that vimentin is often used as a marker for Sertoli cells. Vimentin is expressed independently of the differentiation state of the cells and is observed around the nuclei and in the cytoplasm of Sertoli cells.

The successful isolation and culture of Sertoli cells depend on a series of delicate processes of mechanical isolation and enzymatic digestion of the testicular tissue, taking advantage of an array of enzymes (such as DNAse, collagenase, and pancreatin) in order to digest the extracellular matrix components. The complexity of these processes may present some differences depending on the origin of the testicular sample (whole tissue or biopsy) and of the species in question. Rat and mouse Sertoli cells are obtained by a similar protocol, whereas bovine and human Sertoli cells require a more extensive mechanical and enzymatic processing.

Movement of water into and out of the cell is fundamental to life. In the male reproductive tract, at the level of the seminiferous epithelium, Sertoli cells are pivotal in mediating the movement of water into the luminal compartment, a process providing a means of transport of spermatozoa into the epididymal ducts. Here, we describe a stopped-flow light-scattering methodology to easily assess the Sertoli cell osmotic water permeability. The devised methodology is well suited for studying how the Sertoli cell's permeability changes following exogenous stimuli.

Pioglitazone is a synthetic agonist for the nuclear receptor peroxisome proliferator-activated receptor γ used to treat type 2 diabetes mellitus. Recently we reported that antidiabetic drugs regulate the nutritional support of spermatogenesis by Sertoli cells. Herein, we investigate the effects of pioglitazone on human Sertoli cells metabolism. Human Sertoli cells were cultured in the presence of pioglitazone (1, 10, 100μM). Protein levels of phosphofructokinase 1, lactate dehydrogenase, hexokinase, glucose transporters (GLUT1, GLUT2, GLUT3), monocarboxylate transporter 4 and oxidative phosphorylation complexes were determined by Western blot. Lactate dehydrogenase and alanine aminotransferase activity were assessed and metabolite production and consumption determined by proton nuclear magnetic resonance. Mitochondrial membrane potential was also determined. Glucose consumption more than doubled in human Sertoli cells stimulated with pioglitazone 100μM. Mitochondrial complex II protein levels increased 50% with exposure to pioglitazone (100μM) in human Sertoli cells, though mitochondrial membrane potential was decreased by 32%. The pharmacological concentration of pioglitazone (10μM) almost doubled lactate production and established crucial correlations among key intervenient of glycolysis. Moreover, in the same concentration, alanine aminotransferase decreased more than 80%. Our results suggest that pioglitazone (10μM) increases the efficiency of the glycolytic flux and lactate production by human Sertoli cells, which is essential to sustain and preserve the spermatogenic event. Thus, pioglitazone may improve male fertility and thus, be considered a suitable antidiabetic drug for men in reproductive age., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

Background Information: Infertile men often present deregulation of serum estrogen levels. Notably, high levels of estradiol (E2) are associated with low sperm production and quality. Sertoli cells (SCs) are responsible for spermatogenesis maintenance and are major targets for the hormonal signalling that regulates this complex process., Results: In this study, we used primary cultures of human SCs and studied the localisation, expression and functionality of the Na(+) -dependent HCO3 (-) transporters by confocal microscopy, immunoblot, epifluorescence and voltage clamp after 24 h of exposure to E2 (100 nM). All studied transporters were identified in human SCs. In E2-treated human SCs, there was an increase in NBCn1, NBCe1 and NDCBE protein levels, as well as an increase in intracellular pH and a decrease in transcellular transport., Conclusions: We report an association between increased levels of E2 and the expression/function of Na(+) -dependent HCO3 (-) transporters in human SCs. Our results provide new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis. These mechanisms may have an influence on male reproductive potential and help to explain male infertility conditions associated with estrogen deregulation., Significance: Exposure to E2 increased human SCs intracellular pH. E2 is a modulator of ionic transcellular transport in human SCs., (© 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Men with Klinefelter syndrome (KS) present severe hormonal dysregulation, particularly elevated serum estradiol concentration. Estrogens act through specific receptors and regulate testes development and spermatogenesis. Herein, we evaluated GPR30, ERα, and ERβ mRNA expression in testis of KS men and men with 46XY karyotype by reverse transcriptase and quantitative PCR. ERβ transcripts are the most abundant in testicular tissue of 46XY men. Notably, testicular GPR30 transcription in KS men was approximately 12 times higher. Since GPR30 is essential to mediate estrogen effects over steroidogenesis, our data illustrate that GPR30 may underpin the testicular alterations observed in KS men., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Human metabolism is an essential biological process that involves the consumption of different substrates to ensure the nutritional and energetic needs of cells. The disruption of this highly regulated system constitutes the onset of several disorders/dysfunctions such as diabetes mellitus, cardiovascular diseases and hypertension., Objective: In this review, we propose to discuss promising natural products that can act as modulators of cell metabolism and point towards possible targets to take into account in the development of new therapies against metabolic diseases., Methods: After having defined our main focus, we undertook an intensive search of bibliographic databases to select the peer-reviewed papers that fits within the review thematic. The information of the screened papers was described in an organized manner through the review and different types of studies were included., Results: Two hundred and seventy papers were included in the review, as well as one reliable website from the World Health Organization. Several articles described that pharmacological agents are commonly used to counteract metabolic disorders. However, in many cases these products are insufficient, represent high costs to health care systems and are associated with several undesirable effects, highlighting the need to search for new therapies. Notably, many papers reported the promising results of natural products in the treatment of several metabolic disorders, constituting a possible alternative or complementary strategy to pharmacological agents., Conclusion: The findings of this review confirm that the currently available treatments for metabolic disorders and its associated complications remain far below the expected results.

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 17β-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.

Caffeine is a widely consumed substance present in several beverages. There is an increasing consumption of energetic drinks, rich in caffeine, among young individuals in reproductive age. Caffeine has been described as a modulator of cellular metabolism. Hence, we hypothesized that it alters human Sertoli cells (hSCs) metabolism and oxidative profile, which are essential for spermatogenesis. For that purpose, hSCs were cultured with increasing doses of caffeine (5, 50, 500 μM). Caffeine at the lowest concentrations (5 and 50 μM) stimulated lactate production, but only hSCs exposed to 50 μM showed increased expression of glucose transporters (GLUTs). At the highest concentration (500 μM), caffeine stimulated LDH activity to sustain lactate production. Notably, the antioxidant capacity of hSCs decreased in a dose-dependent manner and SCs exposed to 500 μM caffeine presented a pro-oxidant potential, with a concurrent increase of protein oxidative damage. Hence, moderate consumption of caffeine appears to be safe to male reproductive health since it stimulates lactate production by SCs, which can promote germ cells survival. Nevertheless, caution should be taken by heavy consumers of energetic beverages and food supplemented with caffeine to avoid deleterious effects in hSCs functioning and thus, abnormal spermatogenesis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Many health benefits have been attributed to tea (Camellia sinensis (L.)), and tea infusions are used as dietary agent and included in food supplements. Herein, we report the effect of a white tea (WTEA) extract in Sertoli cell (SC) metabolism. The SC is responsible for the nutritional support of the developing germ cells., Methods: An aqueous WTEA extract was prepared and analyzed by (1)H-NMR. Rat SCs were cultured with or without the WTEA extract. mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by qPCR and western blot. LDH activity was assessed and metabolite production/consumption determined by (1)H-NMR., Results: WTEA-exposed SCs presented decreased protein and mRNA levels of GLUT1 and decreased glucose uptake. However, intracellular LDH activity was increased and SC lactate production was stimulated by the presence of the WTEA extract. Interestingly, alanine production was also found to be stimulated in WTEA extract-exposed SCs., Conclusion: WTEA extract altered the glycolytic profile of cultured SCs, stimulating lactate production. Since lactate is used as metabolic substrate and has an anti-apoptotic effect in the developing germ cells, the supplementation with WTEA extract may be advantageous to improve male reproductive health.

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., (© 2014 International Union of Biochemistry and Molecular Biology.)

The maintenance of pH homeostasis in the male reproductive tract is kept through the involvement of several mechanisms, among which is included the transmembranous movement of H(+) ions. Na(+)-H(+) exchangers (SLC9, solute carrier 9 family members) are among the membrane transporters known to participate in intracellular and extracellular pH regulation but also have important roles in salt and water absorption across epithelia and in the regulation of cell volume. The presence of several Na(+)-H(+) exchangers has been reported in the male reproductive tract. Their involvement in the processes that ensure the correct pursuance of the spermatogenetic event and spermatozoa maturation has been suggested. Indeed, the formation of mature spermatozoa is highly dependent on the maintenance of adequate ductal luminal milieu pH and ionic balance. Perturbations in these processes result in reduced male reproductive potential and consequently male subfertility and/or infertility. Thus, it is imperative to understand H(+) transport dynamics in order to identify and counteract possible alterations associated with reduced male fertility caused by pathological conditions. Herein, we will discuss the expression pattern and physiological roles of SLC9 family members in the cells of the male reproductive tract as well as the molecular basis of H(+) transport and its involvement in male reproductive potential., (© 2014 by the Society for the Study of Reproduction, Inc.)

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO₃⁻) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pH(i)) values. Diabetes mellitus alters pHi regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO₃⁻-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO₃⁻ transporters of the SLC4 family [anion exchanger 2 (AE2), Na⁺-driven Cl⁻/HCO₃⁻ exchanger (NDCBE), electrogenic Na⁺/HCO₃⁻ cotransporter 1 (NBCe1), electroneutral Na⁺/HCO₃⁻ cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO₃⁻ transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO₃⁻ transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats.

Bicarbonate (HCO₃⁻) membrane transport systems are crucial players in the physiology of several tissues. The molecular basis of HCO₃⁻ membrane transport is of major physiological relevance since this ion is involved in the establishment of intracellular and extracellular ionic composition, osmolariy and pH. The membrane HCO₃⁻ transporters are divided in two main families: solute carrier 4 (SLC4) and solute carrier 26 (SLC26), although HCO₃⁻ concentration can also be regulated by the cystic fibrosis transmembrane regulator (CFTR). In most tissues the SLC4 family represents the majority of HCO₃⁻ transporters members, which can be divided in two subgroups: the Na⁺-dependent and the Na⁺-independent transporters. The SLC26 family consists of ten members that can transport diverse ions besides HCO₃⁻. In the male reproductive tract, HCO₃⁻ transport occurs in several processes in order to assure a correct pursuance of the spermatogenetic event and spermatozoa capacitation, being also necessary for egg fertilization. Indeed, the formation of competent spermatozoa, the maintenance of an adequate ductal luminal milieu and spermatozoa capacitation are highly dependent of ionic balance and pH. Perturbations in these processes result in reduced male reproductive health and consequently male subfertility and/or infertility. Thus, it is imperative to understand HCO₃⁻ transport dynamics in order to identify and counteract possible alterations related with reduced male fertility caused by pathological conditions. Herein, we will review the major families and subfamilies of HCO₃⁻ membrane transport, discussing the molecular basis of HCO₃⁻ transport in the male reproductive tract and its role in male-associated subfertility and/or infertility.