José E. Cavaco

Metabolic regulation is important for spermatogenesis

Male factor infertility is increasing in developed countries, and several factors linked to lifestyle have been shown to negatively affect spermatogenesis. Sertoli cells are pivotal to spermatogenesis, providing nutritional support to germ cells throughout their development. Sertoli cells display atypical features in their cellular metabolism; they can metabolize various substrates, preferentially glucose, the majority of which is converted to lactate and not oxidized via the tricarboxylic acid cycle. Why Sertoli cells preferentially export lactate for germ cells is not entirely understood. However, lactate is utilized as the main energy substrate by developing germ cells and has an antiapoptotic effect on these cells. Several biochemical mechanisms contribute to the modulation of lactate secretion by Sertoli cells. These include the transport of glucose through the plasma membrane, mediated by glucose transporters; the interconversion of pyruvate to lactate by lactate dehydrogenase; and the release of lactate mediated by monocarboxylate transporters. Several factors that modulate Sertoli cell metabolism have been identified, including sex steroid hormones, which are crucial for maintenance of energy homeostasis, influencing the metabolic balance of the whole body. In fact, energy status is essential for normal reproductive function, since the reproductive axis has the capacity to respond to metabolic cues.

Tubular Fluid Secretion in the Seminiferous Epithelium: Ion Transporters and Aquaporins in Sertoli Cells

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K+ than the blood and express various membrane and water transporters (Na+/K+-ATPase; Ca2+-ATPase; V-type ATPase; Cl− channels; CFTR Cl− channels; K+ channels; L-, T- and N-type Ca2+ channels; Na+/H+ exchangers; Na+-driven HCO3−/Cl− exchangers (NDCBEs); Na+/HCO3− cotransporters (NBCes); Na+–K+–2Cl− cotransporter; Na+/Ca2+ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.

Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells.

BACKGROUND Sertoli cells metabolize glucose producing lactate for developing germ cells. As insulin regulates glucose uptake and its disturbance/insensitivity is associated with diabetes mellitus, we aimed to determine the effect of insulin deprivation in human Sertoli cell (hSC) metabolism and metabolism-associated gene expression. METHODS hSC-enriched primary cultures were maintained in the absence/presence of insulin and metabolite variations were determined by (1)H-NMR. mRNA expression levels of glucose transporters (GLUT1, GLUT3), lactate dehydrogenase (LDHA) and monocarboxylate transporter (MCT4) were determined by RT-PCR. RESULTS Insulin deprivation resulted in decreased lactate production and in decrease of glucose consumption that was completely reverted after 6h. Cells of both groups consumed similar amounts of glucose. In insulin-deprived cells, transcript levels of genes associated to lactate metabolism (LDHA and MCT4) were decreased. Transcript levels of genes involved in glucose uptake exhibited a divergent variation: GLUT3 levels were decreased while GLUT1 levels increased. Insulin-deprived hSCs presented: 1) altered glucose consumption and lactate secretion; 2) altered expression of metabolism-associated genes involved in lactate production and export; 3) an adaptation of glucose uptake by modulating the expression of GLUT1 and GLUT3. GENERAL SIGNIFICANCE This is the first report regarding the effect of insulin-deprivation on hSC metabolism.

High-energy diets may induce a pre-diabetic state altering testicular glycolytic metabolic profile and male reproductive parameters

Diabetes mellitus is a metabolic disorder that may arise from diet habits and is growing to epidemic proportions. Young male diabetic patients present high infertility/subfertility prevalence resulting from impaired reproductive function and poor semen quality. We aimed to evaluate the effects of a high‐energy diet (HED) on glucose tolerance/insulin levels and correlate the observed effects on male reproductive function with overall testicular metabolism. After 1 month, HED fed rats showed increased glycaemic levels, impaired glucose tolerance and hypoinsulinaemia. Moreover, an imbalance of intratesticular and serum testosterone levels was observed, whereas those of 17β‐estradiol were not altered. High‐energy diet also affected the reproductive parameters, with HED rats exhibiting a significant increase in abnormal sperm morphology. Glycolytic metabolism was favoured in testicles of HED rats with an increased expression of both glucose transporters 1 (GLUT1) and 3 (GLUT3) and the enzyme phosphofrutokinase 1. Moreover, lactate production and the expression of metabolism‐associated genes and proteins involved in lactate production and transport were also enhanced by HED. Alanine testicular content was decreased and thus intratesticular lactate/alanine ratio in HED rats was increased, suggesting increased oxidative stress. Other energetic substrates such as acetate and creatine were not altered in testis from HED rats, but intratesticular glycine content was increased in those animals. Taken together, these results suggest that HED induces a pre‐diabetic state that may impair reproductive function by modulating overall testicular metabolism. This is the first report on testicular metabolic features and mechanisms related with the onset of a pre‐diabetic state.

Pre-diabetes alters testicular PGC1-α/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress

Pre-diabetes, a risk factor for type 2 diabetes development, leads to metabolic changes at testicular level. Peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) and Sirtuin 3 (Sirt3) are pivotal in mitochondrial function. We hypothesized that pre-diabetes disrupts testicular PGC-1α/Sirt3 axis, compromising testicular mitochondrial function. Using a high-energy-diet induced pre-diabetic rat model, we evaluated testicular levels of PGC-1α and its downstream targets, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), mitochondrial transcription factor A (TFAM) and Sirt3. We also assessed mitochondrial DNA (mtDNA) content, mitochondrial function, energy levels and oxidative stress parameters. Protein levels were quantified by Western Blot, mtDNA content was determined by qPCR. Mitochondrial complex activity and oxidative stress parameters were spectrophotometrically evaluated. Adenine nucleotide levels, adenosine and its metabolites (inosine and hypoxanthine) were determined by reverse-phase HPLC. Pre-diabetic rats showed increased blood glucose levels and impaired glucose tolerance. Both testicular PGC-1α and Sirt3 levels were decreased. NRF-1, NRF-2 and TFAM were not altered. Testicular mtDNA content was decreased. Mitochondrial complex I activity was increased, whereas mitochondrial complex III activity was decreased. Adenylate energy charge was decreased in pre-diabetic rats, as were ATP and ADP levels. Conversely, AMP levels were increased, evidencing a decreased ATP/AMP ratio. Concerning to oxidative stress pre-diabetes decreased testicular antioxidant capacity and increased lipid and protein oxidation. In sum, pre-diabetes compromises testicular mitochondrial function by repressing PGC-1α/Sirt3 axis and mtDNA copy number, declining respiratory capacity and increasing oxidative stress. This study gives new insights into overall testicular bioenergetics at this prodromal stage of disease.

Influence of 5α-dihydrotestosterone and 17β-estradiol on human Sertoli cells metabolism

Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17β-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17β-estradiol and suppressed by insulin deprivation

BACKGROUND Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. METHODS hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. RESULTS hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. GENERAL SIGNIFICANCE Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.

Estrogen Receptors α and β in Human Testis: Both Isoforms are Expressed

Currently, clinical and experimental evidence point to an essential role of estrogens and estrogen receptors in male fertility. The expression of estrogen receptor α (ERα) and β (ERβ) in human testis has been described. However, some studies were unable to detect ERα, while others report the expression of both isoforms, with ERβ presenting a wide distribution within somatic and germinal testicular cells. This has suggested that estrogens may exert their testicular effects exclusively through ERβ. The present work aims to study the expression of ERα and ERβ in testicular biopsies of men with conserved and disrupted spermatogenesis, in order to better clarify the positive cell populations. Human testicular tissue was obtained from 10 men undergoing testicular biopsy for infertility relief due to azoospermia: two patients had secondary obstructive azoospermia with conserved spermatogenesis, five had Sertoli cell-only syndrome, two had hypospermatogenesis and one had meiotic arrest. Reverse-transcription polymerase chain reaction (RT-PCR) allowed the detection of both ERα and ERβ mRNAs in all samples. Immunohistochemistry revealed that ERα was present in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, round spermatids and elongated spermatids/spermatozoa, while ERβ was present in the same cell types except spermatogonia and Sertoli cells. This study demonstrates ERα mRNA expression in human testis and describes its localization in somatic and germ cell subtypes. These findings suggest that both ER isoforms are involved in the control of testicular function.

High-energy diets: a threat for male fertility?

Male fertility is declining in developed countries, as well as in developing countries. External factors linked to lifestyle, such as eating disorders, negatively affect spermatogenesis, both at central and gonadal levels. The overconsumption of high‐energy diets (HED) alters the functioning of the male reproductive axis and consequently affects the testicular physiology, disrupting its metabolism and bioenergetic capacity. Testicular metabolism presents unique characteristics, partly because of its cellular heterogeneity and to the specific functions that each cell type plays within the testicular environment. Disruption of the tightly regulated metabolic pathways leads to adverse reproductive outcomes, such as inefficient energy supply to germ cells, sperm defects or spermatogenesis arrest. Testicular metabolic alterations induced by HED intake may also lead to mitochondrial dysfunction, which is closely associated to reactive oxygen species (ROS) overproduction and oxidative stress. ROS easily target spermatozoa DNA and lipids, contributing to decreased sperm quality. Thus, understanding the detrimental effects of HED overconsumption on the pathways underlying testicular metabolism and sperm production is imperative; otherwise, one may favour a transgenerational amplification of subfertility. Herein, we present an up‐to‐date overview of the effects of HED on testicular metabolism, sperm parameters and the subsequent consequences for male fertility.

Diabetes, insulin-mediated glucose metabolism and Sertoli/blood-testis barrier function

Blood testis barrier (BTB) is one of the tightest blood-barriers controlling the entry of substances into the intratubular fluid. Diabetes Mellitus (DM) is an epidemic metabolic disease concurrent with falling fertility rates, which provokes severe detrimental BTB alterations. It induces testicular alterations, disrupting the metabolic cooperation between the cellular constituents of BTB, with dramatic consequences on sperm quality and fertility. As Sertoli cells are involved in the regulation of spermatogenesis, providing nutritional support for germ cells, any metabolic alteration in these cells derived from DM may be responsible for spermatogenesis disruption, playing a crucial role in fertility/subfertility associated with this pathology. These cells have a glucose sensing machinery that reacts to hormonal fluctuations and several mechanisms to counteract hyper/hypoglycemic events. The role of DM on Sertoli/BTB glucose metabolism dynamics and the metabolic molecular mechanisms through which DM and insulin deregulation alter its functioning, affecting male reproductive potential will be discussed.