Marco G. Alves

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.

Hormonal control of Sertoli cell metabolism regulates spermatogenesis

Hormonal regulation is essential to spermatogenesis. Sertoli cells (SCs) have functions that reach far beyond the physical support of germ cells, as they are responsible for creating the adequate ionic and metabolic environment for germ cell development. Thus, much attention has been given to the metabolic functioning of SCs. During spermatogenesis, germ cells are provided with suitable metabolic substrates, in a set of events mediated by SCs. Multiple signaling cascades regulate SC function and several of these signaling pathways are hormone-dependent and cell-specific. Within the seminiferous tubules, only SCs possess receptors for some hormones rendering them major targets for the hormonal signaling that regulates spermatogenesis. Although the mechanisms by which SCs fulfill their own and germ cells metabolic needs are mostly studied in vitro, SC metabolism is unquestionably a regulation point for germ cell development and the hormonal control of these processes is required for a normal spermatogenesis.

Molecular mechanisms beyond glucose transport in diabetes-related male infertility

Diabetes mellitus (DM) is one of the greatest public health threats in modern societies. Although during a few years it was suggested that DM had no significant effect in male reproductive function, this view has been challenged in recent years. The increasing incidence of DM worldwide will inevitably result in a higher prevalence of this pathology in men of reproductive age and subfertility or infertility associated with DM is expected to dramatically rise in upcoming years. From a clinical perspective, the evaluation of semen parameters, as well as spermatozoa deoxyribonucleic acid (DNA) integrity, are often studied due to their direct implications in natural and assisted conception. Nevertheless, recent studies based on the molecular mechanisms beyond glucose transport in testicular cells provide new insights in DM-induced alterations in male reproductive health. Testicular cells have their own glucose sensing machinery that react to hormonal fluctuations and have several mechanisms to counteract hyper- and hypoglycemic events. Moreover, the metabolic cooperation between testicular cells is crucial for normal spermatogenesis. Sertoli cells (SCs), which are the main components of blood-testis barrier, are not only responsible for the physical support of germ cells but also for lactate production that is then metabolized by the developing germ cells. Any alteration in this tied metabolic cooperation may have a dramatic consequence in male fertility potential. Therefore, we present an overview of the clinical significance of DM in the male reproductive health with emphasis on the molecular mechanisms beyond glucose fluctuation and transport in testicular cells.

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.

The Warburg Effect Revisited—Lesson from the Sertoli Cell

Otto Warburg observed that cancerous cells prefer fermentative instead of oxidative metabolism of glucose, although the former is in theory less efficient. Since Warburgs pioneering works, special attention has been given to this difference in cell metabolism. The Warburg effect has been implicated in cell transformation, immortalization, and proliferation during tumorigenesis. Cancer cells display enhanced glycolytic activity, which is correlated with high proliferation, and thus, glycolysis appears to be an excellent candidate to target cancer cells. Nevertheless, little attention has been given to noncancerous cells that exhibit a “Warburg‐like” metabolism with slight, but perhaps crucial, alterations that may provide new directions to develop new and effective anticancer therapies. Within the testis, the somatic Sertoli cell (SC) presents several common metabolic features analogous to cancer cells, and a clear “Warburg‐like” metabolism. Nevertheless, SCs actively proliferate only during a specific time period, ceasing to divide in most species after puberty, when they become terminally differentiated. The special metabolic features of SC, as well as progression from the immature but proliferative state, to the mature nonproliferative state, where a high glycolytic activity is maintained, make these cells unique and a good model to discuss new perspectives on the Warburg effect. Herein we provide new insight on how the somatic SC may be a source of new and exciting information concerning the Warburg effect and cell proliferation.

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.

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.

Metformin and male reproduction: effects on Sertoli cell metabolism.

Metformin is commonly used to treat type 2 diabetes (T2D). While new clinical applications have been ascribed to metformin, including treatment of anovulatory infertility, its effects on male reproduction have not been investigated. The Sertoli cell (SC) is crucial for germ cell development, exerting metabolic control of spermatogenesis, therefore, we investigated the effects of metformin on SC metabolism.