Understanding the age-related alterations in the testis-specific proteome.

INTRODUCTION: Fertility rates in developing countries have declined over the past decades, and the trend of delayed fatherhood is rising as societies develop. The reasons behind the decline in male fertility with advancing age remain mysterious, making it a compelling and crucial area for further research. However, the limited number of studies dedicated to unraveling this enigma poses a challenge. Thus, our objective is to illuminate some of the upregulated and downregulated mechanisms in the male testis during the aging process. AREAS COVERED: Herein, we present a critical overview of the studies addressing the alterations of testicular proteome through the aging process, starting from sexually matured young males to end-of-life-expectancy aged males. The comparative studies of the proteomic testicular profile of men with and without spermatogenic impairment are also discussed and key proteins and pathways involved are highlighted. EXPERT OPINION: The difficulty of making age-comparative studies, especially of advanced-age study subjects, makes this topic of study quite challenging. Another topic worth mentioning is the heterogeneous nature and vast cellular composition of testicular tissue, which makes proteome data interpretation tricky. The cell type sorting and comorbidities testing in the testicular tissue of the studied subjects would help mitigate these problems.

L-Carnitine and Male Fertility: Is Supplementation Beneficial?

L-Carnitine, a natural antioxidant found in mammals, plays a crucial role in the transport of long-chain fatty acids across the inner mitochondrial membrane. It is used as a nutritional supplement by professional athletes, improving performance and post-exercise recovery. Additionally, its therapeutic applications, including those in male infertility, have been investigated, as it may act as a defense mechanism against the excessive production of reactive oxygen species (ROS) in the testis, a process that can lead to sperm damage. This effect is achieved by enhancing the expression and activity of enzymes with antioxidant properties. Nevertheless, the mechanisms underlying the benefits of L-Carnitine remain unknown. This review aims to consolidate the current knowledge about the potential benefits of L-Carnitine and its role in male (in)fertility. Considering in vitro studies with Sertoli cells, pre-clinical studies, and investigations involving infertile men, a comprehensive understanding of the effects of L-Carnitine has been established. In vitro studies suggest that L-Carnitine has a direct influence on somatic Sertoli cells, improving the development of germ cells. Overall, evidence supports that L-Carnitine can positively impact male fertility, even at a relatively low dose of 2 g/day. This supplementation enhances sperm parameters, regulates hormone levels, reduces ROS levels, and subsequently improves fertility rates. However, further research is needed to elucidate the underlying mechanisms and establish optimal doses. In conclusion, the role of L-Carnitine in the field of male reproductive health is highlighted, with the potential to improve sperm quality and fertility.

A Comprehensive Review of the Impact of Chromium Picolinate on Testicular Steroidogenesis and Antioxidant Balance.

Low testosterone (T) levels are a major cause of male infertility, as this hormone is crucial for several processes throughout the entire male reproductive tract. Leydig cells (LC) produce T through testicular steroidogenesis. Disrupted LC function can hinder steroid production and fertility. Among the factors that affect steroidogenesis, endocrine-disrupting chemicals (EDCs) raise concerns, as they disturb hormonal signaling. Chromium is classified as an EDC, and its main forms are hexavalent (Cr(VI)) and trivalent chromium (Cr(III)). While Cr(III) is controversially regarded as an essential metal, its compound Cr(III) picolinate (CrPic3) is used as a nutritional supplement due to its antidiabetic and antioxidant properties. This review aims to identify the possible effects of CrPic3 on testicular steroidogenesis and thus, on male fertility. The detriments caused by CrPic3 in LC include the inhibition of enzymes involved in steroidogenesis, and, as in other cells, the induction of mutagenesis and apoptosis. Remarkably, CrPic3 impacts male fertility through the alteration of reactive oxygen species (ROS), T levels, and sperm parameters (sperm motility and abnormal sperm count). However, gaps and inconsistencies exist in the literature concerning its effects on male fertility. Thus, further research is imperative to comprehend the underlying mechanisms of CrPic3 in the physiological processes relevant to male fertility, ensuring the supplement's safety for use by men.

Impact of Chromium Picolinate on Leydig Cell Steroidogenesis and Antioxidant Balance Using an In Vitro Insulin Resistance Model.

Leydig cells (LCs) play a pivotal role in male fertility, producing testosterone. Chromium (III) picolinate (CrPic3), a contentious supplement with antidiabetic and antioxidant properties, raises concerns regarding male fertility. Using a rodent LC line, we investigated the cytotoxicity of increasing CrPic3 doses. An insulin resistance (IR) model was established using palmitate (PA), and LCs were further exposed to CrPic3 to assess its antioxidant/antidiabetic activities. An exometabolome analysis was performed using 1H-NMR. Mitochondrial function and oxidative stress were evaluated via immunoblot. Steroidogenesis was assessed by quantifying androstenedione through ELISA. Our results uncover the toxic effects of CrPic3 on LCs even at low doses under IR conditions. Furthermore, even under these IR conditions, CrPic3 fails to enhance glucose consumption but restores the expression of mitochondrial complexes CII and CIII, alleviating oxidative stress in LCs. While baseline androgen production remained unaffected, CrPic3 promoted androstenedione production in LCs in the presence of PA, suggesting that it promotes cholesterol conversion into androgenic intermediates in this context. This study highlights the need for caution with CrPic3 even at lower doses. It provides valuable insights into the intricate factors influencing LCs metabolism and antioxidant defenses, shedding light on potential benefits and risks of CrPic3, particularly in IR conditions.

Effects of Age and Lifelong Moderate-Intensity Exercise Training on Rats' Testicular Function.

Aging is associated with testicular morphological and functional alterations, but the underlying molecular mechanisms and the impact of physical exercise are poorly understood. In this study, we examined the effects of age and lifelong moderate-intensity exercise on rat testis. Mature adults (35 weeks) and middle-aged (61 weeks) Wistar Unilever male rats were maintained as sedentary or subjected to a lifelong moderate-intensity treadmill training protocol. Testis weight and histology, mitochondrial biogenesis and function, and proteins involved in protein synthesis and stress response were evaluated. Our results illustrate an age-induced testicular atrophy that was associated with alterations in stress response, and mitochondrial biogenesis and function. Aging was associated with increased testicular levels of heat shock protein beta-1 (HSP27) and antioxidant enzymes. Aging was also associated with decreased mRNA abundance of the nuclear respiratory factor 1 (Nrf1), a key transcription factor for mitochondrial biogenesis, which was accompanied by decreased protein levels of the oxidative phosphorylation system (OXPHOS) complexes subunits in the testes of older animals. On the other hand, exercise did not protect against age-induced testicular atrophy and led to deleterious effects on sperm morphology. Exercise led to an even more pronounced decrease in the Nrf1 mRNA levels in testes of both age groups and was associated with decreased mRNA abundance of other mitochondrial biogenesis markers and decreased protein levels of OXPHOS complexes subunits. Lifelong moderate-intensity exercise training was also associated with an increase in testicular oxidative stress markers and possibly with reduced translation. Together, our results indicate that exercise did not protect against age-induced testicular atrophy and was not associated with beneficial changes in mitochondria and stress response, further activating mechanisms of protein synthesis inhibition.

Obesity-Related Genes Expression in Testes and Sperm Parameters Respond to GLP-1 and Caloric Restriction.

Aim: Calorie restriction (CR) diets and glucagon-Like Peptide-1 (GLP-1) analogs are known to alter energy homeostasis with the potential to affect the expression of obesity-related genes (ORGs). We hypothesized that CR and GLP-1 administration can alter ORGs expression in spermatozoa and testes, as well as the sperm parameters implicated in male fertility. Materials and Methods: Six-week-old adult male Wistar rats (n = 16) were divided into three groups, submitted either to CR (n = 6, fed with 30% less chow diet than the control rats), GLP-1 administration (n = 5, 3.5 pmol/min/kg intraperitoneal) for 28 days, or used as controls (n = 5, fed ad libitum). Selected ORGs expression, namely the fat mass and obesity-associated (FTO), melanocortin-4 receptor (MC4R), glucosamine-6-phosphate deaminase 2 (GNPDA2), and transmembrane protein 18 (TMEM18) were evaluated in testes and spermatozoa by a quantitative polymerase chain reaction (qPCR). Results: CR resulted in lower body weight gain and insulin resistance, but a higher percentage of sperm head defects. GLP-1 administration, despite showing no influence on body weight or glucose homeostasis, resulted in a lower percentage of sperm head defects. CR and GLP-1 administration were associated with a higher expression of all ORGs in the testes. Under CR conditions, the genes FTO and TMEM18 expression in the testes and the MC4R and TMEM18 transcripts abundance in sperm were positively correlated with the spermatozoa oxidative status. The abundance of FTO and TMEM18 in the spermatozoa of rats under CR were positively correlated with sperm concentration, while the testes' TMEM18 expression was also positively correlated with sperm vitality and negatively correlated with insulin resistance. Testes GNPDA2 expression was negatively correlated with sperm head defects. Conclusions: CR and GLP-1 administration results in higher ORGs expression in testes, and these were correlated with several alterations in sperm fertility parameters.

Exenatide and Dapagliflozin Combination Enhances Sertoli Cell Secretion of Key Metabolites for Spermatogenesis.

The incidence of metabolic diseases such as type 2 diabetes mellitus (DM) and obesity has been increasing dramatically. Both diseases are closely linked and new approaches for type 2 DM treatment aim to enable weight loss. A combined therapy of dapagliflozin and exenatide has been used against type 2 DM, influencing allbody glucose dynamics. Spermatogenesis is highly dependent on the metabolic cooperation established between Sertoli cells (SCs) and developing germ cells. To study the effects of dapagliflozin and exenatide on SC metabolism, mouse SCs were treated in the presence of sub-pharmacologic, pharmacologic, and supra-pharmacologic concentrations of dapagliflozin (50, 500, 5000 nM, respectively) and/or exenatide (2.5, 25, 250 pM, respectively). Cytotoxicity of these compounds was evaluated and the glycolytic profile, glycogen content assay, and lipid accumulation of SCs were determined. Dapagliflozin treatment decreased fat cellular deposits, demonstrating its anti-obesity properties at the cellular level. Polytherapy of exenatide plus dapagliflozin increased lactate production by SCs, which has been reported to improve sperm production and quality. Thus, the results herein suggest that the use of these two pharmacological agents can protect male fertility, while improving their glucose homeostasis and inducing weight loss.

Antioxidants Present in Reproductive Tract Fluids and Their Relevance for Fertility.

Nowadays, infertility is classified as a disease of the reproductive system. Although it does not compromise the life of the individual, it can have detrimental effects on the physiological and psychological health of the couple. Male fertility evaluation is mainly focused on the analysis of sperm parameters. However, the ejaculated fluid is also composed of seminal plasma, and the study of this fluid can provide crucial information to help in the assessment of male fertility status. Total antioxidant capacity of the seminal plasma has been positively correlated with the fertility of men. Moreover, evidence highlights to a similar importance as that of female reproductive tract fluid antioxidant capabilities and female fertility. Herein, we describe the functions of seminal plasma and female reproductive tract fluids, as well as their main antioxidant components and their relationships with fertility outcomes. Additionally, this review contains the most up to date information regarding the mechanisms of the interaction between the male and the female reproductive fluids and the importance of proper antioxidant capacity for fertilization.

Expression of obesity-related genes in human spermatozoa affects the outcomes of reproductive treatments.

OBJECTIVE: To study the abundance of obesity-related gene (ORG) mRNA in human spermatozoa and its association with sperm quality parameters, embryonic development, and pregnancy rates after assisted reproduction treatment (ART). DESIGN: Cross-sectional study of spermatozoa ORG mRNA expression, and sperm and embryonic development parameters of infertile couples attending a single ART center. SETTING: University, in collaboration with a medically assisted reproduction center. PATIENT(S): One hundred six couples seeking fertility treatment and receiving ART. INTERVENTION(S): Expression of spermatozoa ORG mRNA was assessed by quantitative reverse transcription-polymerase chain reaction. Sperm and embryonic development parameters were measured by board-certified embryologists. Serum ß-human chorionic gonadotropin levels and fetal heartbeat detection on ultrasound were used to document biochemical and clinical pregnancy, respectively. MAIN OUTCOME MEASURE(S): Correlations between the abundance of ORG transcripts in spermatozoa and sperm quality, embryonic development, and achievement of pregnancy. RESULTS: The abundance of spermatozoa FTO mRNA was positively correlated with total sperm count (r = 0.5030), fertilization rate (r = 0.4854), embryo cleavage rate (r = 0.5705), and high-quality embryo rate (r = 0.6982). The abundance of spermatozoa MC4R transcript was negatively correlated with sperm viability (r = -0.3111) and positively correlated with biochemical pregnancy (r = 0.4420). The abundance of MC4R and GNPDA2 transcripts was higher in spermatozoa of men with asthenozoospermia and teratozoospermia than in those with normozoospermia. CONCLUSION: To our knowledge, this is the first report showing that the abundance of MC4R and FTO transcripts in spermatozoa is associated with sperm and embryo quality parameters, as well as pregnancy rates. Overall, these results further support the view that male factors beyond classic sperm quality parameters, namely the abundance of ORG transcripts, also affect the outcome of ART.

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.

Author Correction: Alterations in seminal plasma proteomic profile in men with primary and secondary infertility.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alterations in seminal plasma proteomic profile in men with primary and secondary infertility.

Proteome of seminal plasma provides profound information related to the male reproductive health. This pilot study was conducted to characterize proteomic profile of seminal plasma from men with primary, or secondary infertility and compare it with proven fertile men. Study participants (n = 59) were recruited at the Cleveland Clinic and divided according to their fertility status: proven fertile (n = 39); primary infertility (n = 11) and secondary infertility (n = 9). Proteomic shotgun analysis revealed a total of 515 peptides common to primary infertility and control group; whereas 523 peptides were common to secondary infertility and control group. Bioinformatic analysis revealed dysregulation of biological processes such as cell secretion and vesicle mediated transport in primary infertility, whereas immune system response, regulation of proteolysis and iron homeostasis were dysregulated in secondary infertility. Western blot validation showed overexpression of ANXA2 and CDC42, and underexpression of SEMG2 proteins in primary infertility; and overexpression of ANXA2 and APP proteins in secondary infertility. This study elucidates the potential role of differentially expressed proteins in the seminal plasma as diagnostic biomarker for primary and secondary infertility. Furthermore, our results suggest maturation failure and immune reaction response as the main cause of infertility in men with primary and secondary infertility, respectively. Additional validation of the proteins involved in the above pathways is warranted.

Alterations of Spermatozoa Proteomic Profile in Men with Hodgkin's Disease Prior to Cancer Therapy.

PURPOSE: Hodgkin's disease (HD) is a type of cancer affecting men in the reproductive age with potential consequences on their fertility status. This study aims to analyze sperm parameters, alterations in proteomic profiles and validate selected protein biomarkers of spermatozoa in men with HD undergoing sperm banking before cancer therapy. MATERIALS AND METHODS: Semen analysis was carried out in healthy fertile donors (control, n=42), and patients diagnosed with HD (patients, n=38) before cancer therapy. We compared proteomic profiles of spermatozoa from donors (n=3) and patients (n=3) using LTQ-Orbitrap Elite hybrid MS system. RESULTS: A total of 1,169 proteins were identified by global proteomic in both groups. The ingenuity pathway analysis revealed that differentially expressed proteins involved in capacitation, acrosome reaction, binding of sperm to the zona pellucida, sperm motility, regulation of sperm DNA damage, and apoptosis were significantly downregulated in HD patients. Validation of proteins implicated in sperm fertility potential by Western Blot demonstrated that peroxiredoxin 2 (PRDX 2) was underexpressed (p=0.015), and transferrin (p=0.045) and SERPIN A5 (p=0.010) protein levels were overexpressed in spermatozoa of men with HD. CONCLUSIONS: Findings of this study indicates that the key proteins involved in sperm fertility potential are significantly altered in men with HD, which provides substantial explanation for the observed low sperm quality in HD subjects prior to cancer therapy. Furthermore, our results suggest PRDX 2, transferrin and SERPIN A5 as possible candidate proteins for assessing sperm quality in HD patients prior to cancer therapy.

Caloric restriction alters the hormonal profile and testicular metabolome, resulting in alterations of sperm head morphology.

Energy homeostasis is crucial for all physiological processes. Thus, when there is low energy intake, negative health effects may arise, including in reproductive function. We propose to study whether caloric restriction (CR) changes testicular metabolic profile and ultimately sperm quality. Male Wistar rats (n = 12) were randomized into a CR group fed with 30% fewer calories than weight-matched, ad libitum-fed animals (control group). Circulating hormonal profile, testicular glucagon-like peptide-1 (GLP-1), ghrelin and leptin receptors expression, and sperm parameters were analyzed. Testicular metabolite abundance and glycolysis-related enzymes were studied by NMR and Western blot, respectively. Oxidative stress markers were analyzed in testicular tissue and spermatozoa. Expressions of mitochondrial complexes and mitochondrial biogenesis in testes were determined. CR induced changes in body weight along with altered GLP-1, ghrelin, and leptin circulating levels. In testes, CR led to changes in receptor expression that followed those of the hormone levels; modified testicular metabolome, particularly amino acid content; and decreased oxidative stress-induced damage in testis and spermatozoa, although sperm head defects increased. In sum, CR induced changes in body weight, altering circulating hormonal profile and testicular metabolome and increasing sperm head defects. Ultimately, our data highlight that conditions of CR may compromise male fertility.

Assessment of Sertoli Cell Proliferation by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide and Sulforhodamine B Assays.

The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short and simple protocol to isolate and culture these cells is provided. The other protocols discussed herein represent two different procedures, somewhat complementary, to assess SC proliferation. In brief, the sulforhodamine B assay allows the investigator to dye healthy fixed SCs maintained in culture. In the MTT assay, on the other hand, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by live SCs. These methods are mostly used to evaluate how SC proliferative activity responds to exposure to compounds such as toxicants or hormones. © 2019 by John Wiley & Sons, Inc.

Round cells do not contaminate or mask human sperm proteome in proteomic studies using cryopreserved samples.

Semen contains leucocytes and round cells, besides spermatozoa. The objective of this study was to identify whether the proteins from round cells and leucocytes affect the proteomic analysis of spermatozoa. Cryopreserved human sperm samples were divided into four groups: (1) samples with ≥1 × 106 /ml leucocytes unprocessed; (2) samples with ≥1 × 106 /ml leucocytes processed by 65% density centrifugation; (3) samples with round cells <1 × 106 /ml unprocessed; and (4) samples with round cells <1 × 106 /ml processed by 65% density centrifugation. Samples from each group (1, 2, 3 and 4) were pooled (n = 5) for quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparative analysis revealed nine differentially expressed proteins (DEPs) groups 1 and 2. Moreover, five DEPs were identified between groups 3 and 4. We observed that cylicin-1, Atlastin-1 and vesicle transport protein SFT2B are specific to spermatozoa, and none of them were associated with leucocytes. The number of DEPs in spermatozoa of processed and unprocessed cryopreserved semen samples was negligible. Our results indicate that the presence of round cells (<1 × 106 /ml) in the seminal ejaculation does not interfere in the accurate detection of spermatozoa proteome by LC-MS/MS.

Oxidation reduction potential: a new biomarker of male infertility.

Oxidative stress is considered a major etiology for male infertility, more specifically idiopathic infertility. The causes of seminal oxidative stress can be intrinsic, such as varicocele or due to the presence of active leukocytes and immature germ cells. Reported external causes are smoking, alcohol or exposure to environmental toxins. Traditional methods to determine the seminal oxidative stress do not evaluate this status directly, but rather measure its components or intermediate products indirectly, instead. The major disadvantages of the traditional methods are related with time and cost as these methods are extremely time consuming and require expensive equipment, consumables and highly skilled laboratory personnel. To overcome these drawbacks, the MiOXSYS® system, a method which directly measures the oxidation-reduction potential (ORP), was developed. The evaluation of the ORP using MiOXSYS® is cost-effective, easy and quick. However, this newly introduced method to evaluate the oxidative status of semen still requires validation in different andrology laboratory settings across the world.

Molecular Pathways Associated with Sperm Biofunction Are Not Affected by the Presence of Round Cell and Leukocyte Proteins in Human Sperm Proteome.

In human sperm proteomic experiments, leukocyte and round cell proteins may contaminate the sperm proteome and affect the bioinformatic results. The main objective of this study was to identify the possible interference of these proteins, especially from leukocytes, in identification of sperm functional pathways through proteomic and bioinformatic tools. We have evaluated the sperm proteome by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in four groups: (1) neat semen with round cells and leukocytes ≥1 × 106/mL; (2) samples with round cells and leukocytes ≥1 × 106/mL processed by 65% density gradient centrifugation; (3) neat semen with round cells <1 × 106/mL; and (4) samples with round cells <1 × 106/mL processed by 65% density gradient centrifugation. Pure leukocyte culture was used as a control group. The difference in the conserved DEPs (common to both sperm and leukocytes) between the sperm samples with leukocytes ≥1 × 106/mL and round cells <1 × 106/mL was negligible. Comparative analysis between groups 1, 2, 3, and 4 with the control group revealed that the presence of leukocyte proteins does not significantly alter the activation z-score of the identified canonical pathways or biological functions in sperm proteome. Our experimental results demonstrate that the presence of round cell and leukocyte proteins do not affect the identification of the molecular pathways associated with human spermatozoa protein function. Hence, the use of neat frozen semen samples for proteomic studies showed no significant impact on the downstream bioinformatic analysis.

Presence of Round Cells Proteins do not Interfere with Identification of Human Sperm Proteins from Frozen Semen Samples by LC-MS/MS.

In sperm proteomic experiments round cells and leukocyte proteins are profiled along with sperm proteome. The influence of round cell and leukocyte proteins on the sperm proteome has not been investigated. The objective of this study was to identify if the proteins from round cells, including leukocytes, interfere with the proteomic analysis of spermatozoa in frozen semen samples. Proteomic profiling of sperm was performed using liquid chromatography-tandem mass spectrometry in four groups: Group 1 contained neat semen with round cells and leukocytes ≥ 1 × 106/mL, group 2 contained neat semen with round cells ≥ 1 × 106/mL that was processed by 65% density gradient to remove the round cells and leukocytes, group 3 contained neat semen with round cells < 1 × 106/mL, and group 4 contained neat semen with round cells < 1 × 106/mL that was processed by 65% density gradient to remove the round cells. Pure leukocyte culture was used as control group. A total of 1638, 1393, 1755, and 1404 proteins were identified in groups 1, 2, 3, and 4, respectively. Comparative analysis of group 1 vs. 3 revealed 26 (1.18%) differentially expressed proteins (DEPs). On the other hand, only 6 (0.31%) DEPs were observed with group 2 vs. 4. Expression of these DEPs were either absent or very low in the control group. The results of our proteomics analysis failed to show any influence of non-spermatogenic round cell proteins on sperm proteome identification. These results validate the use of neat semen samples for sperm proteomic studies.

Metabolic dynamics of human Sertoli cells are differentially modulated by physiological and pharmacological concentrations of GLP-1.

Obesity incidence has pandemic proportions and is expected to increase even further. Glucagon-like peptide-1 (GLP-1) based therapies are well-established pharmacological resources for obesity treatment. GLP-1 regulates energy and glucose homeostasis, which are also crucial for spermatogenesis. Herein, we studied the GLP-1 effects in human Sertoli cells (hSCs) metabolism and mitochondrial function. hSCs were cultured in absence or exposed to increasing doses of GLP-1 mimicking physiological post-prandial (0.01 nM) levels or equivalent to pharmacological levels (1 and 100 nM) used for obesity treatment. We identified GLP-1 receptor in hSCs. Consumption/production of extracellular metabolites were assessed, as well as protein levels or activities of glycolysis-related enzymes and transporters. Mitochondrial membrane potential and oxidative damage were evaluated. Glucose consumption decreased, while lactate production increased in hSCs exposed to 0.01 and 1 nM GLP-1. Though lactate dehydrogenase (LDH) protein decreased after exposure to 100 nM GLP-1 its activity increased in hSCs exposed to the same concentration of GLP-1. Mitochondrial membrane potential decreased in hSCs exposed to 100 nM of GLP-1, while formation of carbonyl groups was decreased in those cells. Those effects were followed by an increase in p-mammalian target of rapamycin (mTOR) Ser(2448). Overall, the lowest concentrations of GLP-1 increased the efficiency of glucose conversion to lactate, while GLP-1 concentration of 100 nM induces mTOR phosphorylation, decreases mitochondrial membrane potential and oxidative damage. GLP-1 regulates testicular energy homeostasis and pharmacological use of GLP-1 analogues could be valuable to counteract the negative impact of obesity in male reproductive function.