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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.

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.

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.

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.

Regucalcin Is Under―Expressed in Human Breast and Prostate Cancers: Effect of Sex Steroid Hormones

Regucalcin plays an important role in maintenance of intracellular Ca2+ homeostasis, suppresses cell proliferation, inhibits expression of oncogenes, and increases the expression of tumour suppressor genes. This suggests that regucalcin functions may be altered in cancer tissues. In this study the regucalcin expression in breast and prostate cancer cases was analysed by RT‐PCR and immunohistochemistry showing that the mRNA and/or protein are under‐expressed in these tumors. The effect of sex steroid hormones on regucalcin expression in breast and prostate cancer cells was determined by real‐time PCR. MCF‐7 and LNCaP cells were stimulated with 0, 1, and 10 nM of 17β‐estradiol (E2) or 5α‐dihydrotestosterone (DHT), respectively, for 0, 6, 12, 24, and 48 h. MCF‐7 cells were also stimulated with E2 conjugated to BSA (E2‐BSA). To explore the mechanisms underlying the sex steroid regulation of regucalcin expression, control treatments with ICI 182,780, flutamide and cyclohexamide were carried out. E2 effects regulating regucalcin expression were not abrogated in the presence of ICI 182,780, and were similar to those observed with E2‐BSA, which suggests the involvement of a membrane‐bound estrogen receptor. In LNCaP cells, DHT down‐regulated regucalcin expression, an effect inhibited by the presence of both flutamide and cyclohexamide, suggesting the involvement of androgen receptor and de novo protein synthesis. The loss of regucalcin expression in breast and prostate cancer cases and the regulation of its expression by sex steroid hormones suggest that it may be associated with development and progression of these human tumors. J. Cell. Biochem. 107: 667–676, 2009.

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.