Unraveling the Molecular Impact of Sperm DNA Damage on Human Reproduction.

Semen analysis is the cornerstone in the investigation of fertility status of male partner. However, more advanced tests have emerged including the analysis of sperm chromatin integrity and DNA damage as markers of semen quality. This is of particular interest, as preserving the genetic information is essential to achieve a successful reproductive event. Moreover, the presence of unrepaired DNA lesions can affect cellular functions, resulting in the onset of pathological conditions associated with male infertility, and the transmission of diseases to the offspring. Hence, in this chapter, we aim to review the main factors leading to sperm DNA damage, along with the different types of damage which can occur. Furthermore, molecular mechanisms involved in DNA repair during spermatogenesis or after fertilization of the oocyte are described, and the laboratory techniques currently used in diagnostics and research, for the analysis of sperm DNA damage are also presented. Finally, the impact of sperm DNA damage on reproductive outcomes such as fertilization and pregnancy rates will be discussed with a focus on animal and human studies, along with the identification of new markers of sperm chromatin integrity.

Obesity-related genes are expressed in human Sertoli cells and modulated by energy homeostasis regulating hormones.

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.

Insights into leptin signaling and male reproductive health: the missing link between overweight and subfertility?

Obesity stands as one of the greatest healthcare challenges of the 21st century. Obesity in reproductive-age men is ever more frequent and is reaching upsetting levels. At the same time, fertility has taken an inverse direction and is decreasing, leading to an increased demand for fertility treatments. In half of infertile couples, there is a male factor alone or combined with a female factor. Furthermore, male fertility parameters such as sperm count and concentration went on a downward spiral during the last few decades and are now approaching the minimum levels established to achieve successful fertilization. Hence, the hypothesis that obesity and deleterious effects in male reproductive health, as reflected in deterioration of sperm parameters, are somehow related is tempting. Most often, overweight and obese individuals present leptin levels directly proportional to the increased fat mass. Leptin, besides the well-described central hypothalamic effects, also acts in several peripheral organs, including the testes, thus highlighting a possible regulatory role in male reproductive function. In the last years, research focusing on leptin effects in male reproductive function has unveiled additional roles and molecular mechanisms of action for this hormone at the testicular level. Herein, we summarize the novel molecular signals linking metabolism and male reproductive function with a focus on leptin signaling, mitochondria and relevant pathways for the nutritional support of spermatogenesis.

Molecular Mechanisms Controlled by mTOR in Male Reproductive System.

In recent years, the mammalian target of rapamycin (mTOR) has emerged as a master integrator of upstream inputs, such as amino acids, growth factors and insulin availability, energy status and many others. The integration of these signals promotes a response through several downstream effectors that regulate protein synthesis, glucose metabolism and cytoskeleton organization, among others. All these biological processes are essential for male fertility, thus it is not surprising that novel molecular mechanisms controlled by mTOR in the male reproductive tract have been described. Indeed, since the first clinical evidence showed that men taking rapamycin were infertile, several studies have evidenced distinct roles for mTOR in spermatogenesis. However, there is a lack of consensus whether mTOR inhibition, which remains the experimental approach that originates the majority of available data, has a negative or positive impact on male reproductive health. Herein we discuss the latest findings concerning mTOR activity in testes, particularly its role on spermatogonial stem cell (SSC) maintenance and differentiation, as well as in the physiology of Sertoli cells (SCs), responsible for blood-testis barrier maintenance/restructuring and the nutritional support of spermatogenesis. Taken together, these recent advances highlight a crucial role for mTOR in determining the male reproductive potential.

Is Technical-Grade Chlordane an Obesogen?

The prevalence of obesity has tripled in recent decades and is now considered an alarming public health problem. In recent years, a group of endocrine disruptors, known as obesogens, have been directly linked to the obesity epidemic. Its etiology is generally associated with a sedentary lifestyle, a high-fat diet and genetic predisposition, but environmental factors, such as obesogens, have also been reported as contributors for this pathology. In brief, obesogens are exogenous chemical compounds that alter metabolic processes and/or energy balance and appetite, thus predisposing to weight gain. Although this theory is still recent, the number of compounds with suspected obesogenic activity has steadily increased over the years, though many of them remain a matter of debate. Technical-grade chlordane is an organochlorine pesticide widely present in the environment, albeit at low concentrations. Highly lipophilic compounds can be metabolized by humans and animals into more toxic and stable compounds that are stored in fat tissue and consequently pose a danger to the human body, including the physiology of adipose tissue, which plays an important role in weight regulation. In addition, technical-grade chlordane is classified as a persistent organic pollutant, a group of chemicals whose epidemiological studies are associated with metabolic disorders, including obesity. Herein, we discuss the emerging roles of obesogens as threats to public health. We particularly discuss the relevance of chlordane persistence in the environment and how its effects on human and animal health provide evidence for its role as an endocrine disruptor with possible obesogenic activity.

Effect of Leptin in Human Sertoli Cells Mitochondrial Physiology.

Leptin is an adipose tissue hormone that acts as energy sensor and reproductive function regulator. Recent reports suggest that leptin is involved in mitochondrial biogenesis in different tissue cells. Herein, we hypothesized that leptin could also affect Sertoli cells mitochondrial dynamics and biogenesis. Human Sertoli cells (hSCs) were cultured in the presence of different leptin concentrations (5, 25 and 50 ng/mL) or vehicle for 24 h. The three different leptin concentrations were selected to mimic the circulating levels found either in normal weight, obese, and morbidly obese individuals, respectively. Leptin receptor (LEPR) expression was evaluated as well as mitochondrial membrane potential, complexes levels, complex II activity and basal respiration. Moreover, mitochondrial DNA copy number and expression of mitochondrial biogenesis markers were assessed. In hSCs, leptin concentrations similar to those found both in lean men decreased mitochondrial complex II protein levels, but no changes in its activity were observed. This is in agreement with basal respiration and mitochondrial membrane potential assessments, which indicate no alterations in mitochondrial fitness. Furthermore, no changes in mitochondrial biogenesis markers were observed upon leptin exposure, although SIRT1/3 levels were increased after exposure to the highest leptin concentration. Overall, the increase in SIRT1/3 levels suggests a role for leptin in glycolysis, which given the relevance of SCs glycolytic flux for germ cells nutritional support further reinforces that this mechanism can be linked to obesity-related subfertility/infertility.

Technical-grade chlordane compromises rat Sertoli cells proliferation, viability and metabolic activity.

Environmental contaminants are a daily presence in human routine. Multiple studies highlight the obesogenic activity of some chemicals. Moreover, these compounds have been suggested as a cause of male subfertility and/or infertility. Technical-grade chlordane (TGC) is classified as an endocrine-disruptor chemical, while its classification as obesogen is controversial. Herein, we studied the influence of TGC on Sertoli cells (SCs) metabolism. Rat Sertoli cells (rSCs) were cultured without and in the presence of increasing concentrations (1, 10 and 1000 nM) of TGC. The viability, proliferation, metabolic activity and the metabolic profile of rSCs was assessed. Expression of key glycolysis-related enzymes, transporters and biomarkers of oxidative damage were also evaluated. Our results show that exposure to higher concentrations of TGC decreases SCs proliferation and viability, which was accompanied by increased glucose consumption associated with an upregulation of Glut3 levels. As a result, pyruvate/lactate production were enhanced thus increasing the glycolytic flux in cells exposed to 1000 nM TGC, although lactate dehydrogenase expression and activity did not increase. Notably, biomarkers associated with oxidative damage remained unchanged after exposure to TGC. This is the first report showing that TGC alters glucose rSCs metabolism and the nutritional support of spermatogenesis with consequences for male fertility.

Carbonic anhydrases are involved in mitochondrial biogenesis and control the production of lactate by human Sertoli cells.

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 CO2 to HCO3- 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.