An integrated overview on the regulation of sperm metabolism (glycolysis-Krebs cycle-oxidative phosphorylation)

An overview of the sperm metabolism is presented; using the stallion as a model we review glycolysis, Krebs Cycle and oxidative phosphorylation, paying special attention to the interactions among them. In addition, metabolism implies a series of coordinated oxidation-reduction reactions and in the course of these reactions reactive oxygen species (ROS) and reactive oxoaldehydes are produced ; the electron transport chain (ETC) in the mitochondria is the main source of the anion superoxide and hydrogen peroxide, while glycolysis produces 2-oxoaldehydes such as methylglyoxal as byproducts; due to the adjacent carbonyl groups are strong electrophiles (steal electrons oxidizing other compounds). Sophisticated mechanisms exist to maintain redox homeostasis, because ROS under controlled production also have important regulatory functions in the spermatozoa. The interactions between metabolism and production of reactive oxygen species are essential for proper sperm function, and deregulation of these processes rapidly leads to sperm malfunction and finally death. Lastly, we briefly describe two techniques that will expand our knowledge on sperm metabolism in the coming decades, metabolic flow cytometry and the use of the “omics” technologies, proteomics and metabolomics, specifically the micro and nano proteomics/metabolomics. A better understanding of the metabolism of the spermatozoa will lead to big improvements in sperm technologies and the diagnosis and treatment of male factor infertility

Differences in the proteome of stallion spermatozoa explain stallion-to-stallion variability in sperm quality post-thaw†

The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those with a fold change > 3, a P = 8.2e-04, and a q = 0.074 (equivalent to False discovery rate (FDR)) were selected, and the following proteins were identified in fresh samples as discriminant variables of good motility post-thaw: F6YTG8, K9K273, A0A3Q2I7V9, F7CE45, F6YU15, and F6SKR3. Other discriminant variables were also identified as predictors of good mitochondrial membrane potential and viability post-thaw. We concluded that proteomic approaches are a powerful tool to improve current sperm biotechnologies

Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation, and stallion sperm function

Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C; P < 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C; P < 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient tricarboxylic acid cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids, and DNA, causing sperm damage

Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI

The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions

Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our laboratory. Aliquots of the ejaculate were separated and stored at −80 °C until further proteomic analysis. Semen analysis was performed using computer assisted sperm analysis and flow cytometry. Significant differences in proteome composition of seminal plasma were observed in the group of stallions showing better motility post thaw. 3116 proteins were identified, and of these, 34 were differentially expressed in stallions with better motility post thaw, 4 of them were also differentially expressed in stallions with different percentages of linearly motile sperm post thaw and 1 protein, Midasin, was expressed in stallions showing high circular velocity post thaw. Seminal plasma proteins may play a major role in sperm functionality; being vehiculated through extracellular vesicles and participating in sperm physiology. Bioinformatic analysis identifies discriminant proteins able to predict the outcome of cryopreservation, identifying potential new biomarkers to assess ejaculate quality

ANGPTL-4 is associated with obesity and lipid profile in children and adolescents

Angiopoietin-like protein 4 (ANGPTL-4) regulates lipidic metabolism and a_ects energy homeostasis. However, its function in children with obesity remains unknown. We investigated plasma ANGPTL-4 levels in children and its relationship with body mass index (BMI) and di_erent lipidic parameters such as free fatty acids (FFA). Plasma ANGPTL-4 levels were analyzed in two di_erent cohorts. In the first cohort (n = 150, age 3-17 years), which included children with normal weight or obesity, we performed a cross-sectional study. In the second cohort, which included only children with obesity (n = 20, age 5-18 years) followed up for two years after an intervention for weight loss, in which we performed a longitudinal study measuring ANGPTL-4 before and after BMI-loss. In the cross-sectional study, circulating ANGPTL-4 levels were lower in children with obesity than in those with normal weight. Moreover, ANGPTL-4 presented a negative correlation with BMI, waist circumference, weight, insulin, homeostasis model assessment of insulin resistance index (HOMA index), triglycerides, and leptin, and a positive correlation with FFA and vitamin-D. In the longitudinal study, the percent change in plasma ANGPTL-4 was correlated with the percent change in FFA, total-cholesterol and high-density lipoprotein cholesterol. This study reveals a significant association of ANGPTL-4 with pediatric obesity and plasma lipid profile

Seminal plasma proteins as potential biomarkers for sperm motility and velocities

Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm analysis (CASA), while protein composition of the seminal plasma was studied using UHPLC-MS/MS. Specific proteins were more abundant in samples with poorer percentages of total motility, average path velocity and circular velocity, and were: Secreted phosphoprotein 1, Fructose-bisphosphate aldolase (p = 1,95E-09; q = 0.0005) and Malate dehydrogenase 1 (p = 1,41E-11; q = 0.002), to the contrary samples with better straight-line velocity values were enriched in Glutathione peroxidase (p=0.00013; q=0.04) and Triosephosphate isomerase (p=0.00015; q=0.04)

The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility

Endometrial area of the blood flow as a marker of endometritis in equine

In this study, uterine blood flow area (BFA) has been evaluated for the first time using power Doppler ultrasound (PD) as a marker of endometritis in mares and jennies. The uterine BFA in healthy mares was greater in oestrus than in diestrus (p <.001). However, differences in endometrial blood flow between oestrus and diestrus were not observed in mares with endometritis. The uterine blood flow in healthy jennies is not affected by the oestrus cycle. Both species showed an increase in endometrial BFA in pathological uterine conditions compared to controls. BFA was a good marker of endometritis with an area under curve (AUC) (estrus:0.94 (p <.001) diestrus:0.98 (p <.001) in mares and AUC (0.91 (p <.0001) in jennies. The results of this preliminary study suggest that PD ultrasound in combination with computerized image analysis has the potential to be a very useful tool in the diagnosis of endometritis

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. Significance: Proteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions