Sperm Metabolism Research Articles

Metabolomics analysis of buck semen cryopreserved with trehalose.

Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study investigated the cryopreservation impact of trehalose on buck semen cryopreservation and finished metabolic profiling of freeze-thawed media by way of the GC–MS-based metabolomics for the first time. Metabolic pattern recognition and metabolite identification by means of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway topology analysis revealed the results of trehalose on buck sperm metabolism at some point of cryopreservation. The results confirmed that trehalose drastically progressed sperm motility parameters and structural integrity after thawing. PCA and PLS-DA analysis discovered that the metabolic patterns of the freezing-thawing media of buck semen cryopreserved with trehalose (T group) or without trehalose (G group, Control) were certainly separated. Using screening conditions of VIP >1.5 and p vaule <0.05, a total of 48 differential metabolites have been recognized, whithin l-isoleucine, L-leucine, L-threonine, and dihydroxyacetone were notably enriched in valine, leucine and isoleucine biosynthesis, glycerolipid metabolism, and aminoacyl-tRNA biosynthesis pathways. In brief, trehalose can efficiently improve membrane structural integrity and motion parameters in buck sperm after thawing, and it exerts a cryoprotective impact with the aid of changing sperm amino acid synthesis and the glycerol metabolism pathway.

Spermatozoan Metabolism as a Non-Traditional Model for the Study of Huntington's Disease.

Huntington’s Disease (HD) is a fatal autosomal dominant neurodegenerative disease manifested through motor dysfunction and cognitive deficits. Decreased fertility is also observed in HD animal models and HD male patients, due to altered spermatogenesis and sperm function, thus resulting in reduced fertilization potential. Although some pharmaceuticals are currently utilized to mitigate HD symptoms, an effective treatment that remedies the pathogenesis of the disease is yet to be approved by the FDA. Identification of genes and relevant diagnostic biomarkers and therapeutic target pathways including glycolysis and mitochondrial complex-I-dependent respiration may be advantageous for early diagnosis, management, and treatment of the disease. This review addresses the HD pathway in neuronal and sperm metabolism, including relevant gene and protein expression in both neurons and spermatozoa, indicated in the pathogenesis of HD. Furthermore, zinc-containing and zinc-interacting proteins regulate and/or are regulated by zinc ion homeostasis in both neurons and spermatozoa. Therefore, this review also aims to explore the comparative role of zinc in both neuronal and sperm function. Ongoing studies aim to characterize the products of genes implicated in HD pathogenesis that are expressed in both neurons and spermatozoa to facilitate studies of future treatment avenues in HD and HD-related male infertility. The emerging link between zinc homeostasis and the HD pathway could lead to new treatments and diagnostic methods linking genetic sperm defects with somatic comorbidities.

Integrated multi-omics analyses reveals molecules governing sperm metabolism potentially influence bull fertility

Bull fertility is of paramount importance in bovine industry because semen from a single bull is used to breed several thousands of cows; however, so far, no reliable test is available for bull fertility prediction. In the present study, spermatozoa from high- and low-fertility bulls were subjected to high-throughput transcriptomic, proteomic and metabolomic analysis. Using an integrated multi-omics approach the molecular differences between high- and low-fertility bulls were identified. We identified a total of 18,068 transcripts, 5041 proteins and 3704 metabolites in bull spermatozoa, of which the expression of 4766 transcripts, 785 proteins and 33 metabolites were dysregulated between high- and low-fertility bulls. At transcript level, several genes involved in oxidative phosphorylation pathway were found to be downregulated, while at protein level genes involved in metabolic pathways were significantly downregulated in low-fertility bulls. We found that metabolites involved in Taurine and hypotaurine metabolism were significantly downregulated in low-fertility bulls. Integrated multi-omics analysis revealed the interaction of dysregulated transcripts, proteins and metabolites in major metabolic pathways, including Butanoate metabolism, Glycolysis and gluconeogenesis, Methionine and cysteine metabolism, Phosphatidyl inositol phosphate, pyrimidine metabolism and saturated fatty acid beta oxidation. These findings collectively indicate that molecules governing sperm metabolism potentially influence bull fertility.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

Genetic Ablation of Na,K-ATPase α4 Results in Sperm Energetic Defects.

The Na,K-ATPase alpha 4 isoform (NKAα4) is expressed specifically in the male germ cells of the testes and is particularly abundant in mature spermatozoa. Genetic deletion of NKAα4 in mice (NKAα4 KO mice) results in complete infertility of male, but not female mice. The reduced fecundity of NKAα4 KO male mice is due to a series of defects, including a severe impairment in total and hyperactive sperm motility. In this work, we show that deletion of NKAα4 also leads to major defects in sperm metabolism and energetics. Thus, compared to wild-type sperm, sperm from NKAα4 KO mice display a significant reduction in the extracellular acidification rate (ECAR), indicative of impaired glycolytic flux. In addition, mitochondrial function is disrupted in sperm lacking NKAα4, as indicated by a reduction in the mitochondrial membrane potential and lower oxygen consumption rate (OCR). Moreover, the ratio between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD/NADH) is increased in NKAα4 KO sperm, indicating a shift in the cellular redox state. These metabolic changes are associated with augmented reactive oxygen species (ROS) production and increased lipid peroxidation in NKAα4 KO sperm. Altogether, these findings reveal a novel link between NKAα4 activity and sperm energetics, highlighting the essential role of this ion transporter in sperm physiology.

Melatonin mediates via melatonin receptor 1 in a temperature-dependent manner regulating ATP metabolism and antioxidative enzyme activity of boar spermatozoa in vitro

Researches indicated that melatonin (MLT) could ameliorate oxidative damage and increase sperm motility for conservation sperms, whereas the underlying mechanism remains unknown. And elucidating this, present study was designed to investigate the energy metabolism of sperm throughout preservation at 17 °C and activating with 37 °C. Herein, semen of ten Landrace boars (16∼18 months of age) were diluted and treated with gradient concentrations of MLT (0 nM, 1 nM, 10 nM, 1 μM, 1 mM and 5 mM), and kept away from light in the incubator of 17 °C for 5 days (preservation), and the sperm motility was evaluated after 37 °C incubation for 10 min (activating). The results showed that 1.0 μM MLT treated-sperms had higher motility than other treatments from the 2nd onward. On the 3rd day 17 °C storage, 1.0 μM MLT significantly decreased the ATP content and the mRNA abundance of mitochondrial DNA encoding genes, including NADH dehydrogenase subunits (NDs) 1–4 and 4L, cytochrome b (CYTB), cytochrome c oxidases (COXs) 1 and 3, ATP synthase 6, but increased the hexokinase activity (HK) and the malondialdehyde (MDA) content compared to that of the control. Whereas after 10 min 37 °C incubation, 1.0 μM MLT increased the ATP content, and the mRNA expressions of ND2 and 4L, CYTB, COX1, ATP6, and the activities of mitochondrial respiratory chain complex I and superoxide dismutase (SOD), but depressed the pyruvate kinase (PK) activity in contrast to that at 17 °C. Furthermore, 1.0 μM MLT treated sperms had a higher MLT receptor 1 (MT1) protein level at 17 °C storage than that after 10 min 37 °C incubation, no changes of the MT2 expression were observed at different temperature. This study suggests that MLT might mediate via MT1 in a temperature-dependent manner regulating sperm ATP generation and antioxidative enzyme activity in vitro.

Reserve‐adaptive potential of sperm plasma in stallions of different ages

During the aerobic metabolism of sperm, various reactive oxygen species are produced. Sperm cells are extremely vulnerable to oxidative stress (OS), which causes loss of membrane integrity, resulting in increased cell permeability, enzyme inactivation, structural DNA damage and cellular apoptosis. The aim of this study was to assess the reserve‐adaptive potential (RAP) of sperm plasma in stallions of various ages. RAP is the difference between the level of spontaneous and induced oxidative protein modification (OMP), reflecting the degree of OS. Spontaneous OMP reflects the accumulation of OS products in vivo. To determine the level of spontaneous OMP, a sperctrophotometric analysis of 2,4‐dinitrophenylhydrazones (DNPH) formed during the interaction of carbonyl derivatives of sperm plasma proteins with 2,4‐dinitrophenylhydrazine was carried out (spectrophotometer “SF‐2000”, Russia). R.L. Levine, modified by E.E. Dubinina. Measurements of DNPH derivatives were carried out at 14 wavelengths: in the range 260‐280nm for the registration of aldehyde dinitrophenylhydrazones of a neutral nature (ADNPHn), of a basic nature (ADNPHb) – 258‐264 and 428‐520nm, for the determination of ketone‐dinitrophenylhydrazones of a neutral nature (CDNPHn) 363‐370nm, basic character (CDNFGb) – 430‐434 and 524‐535nm. After that, the total amount of carbonyl derivatives was found. Induced OMP was determined in a similar way, but after adding iron (II) sulfate and hydrogen peroxide solutions to the reaction mixture. This provoked the formation of new carbonyl groups. The more RAP, the higher the ability of the biological material to resist the OS.Two groups of stallions were formed: the first group consisted of 15 stallions aged 13 to 20 years (average age 15.2±1.8 years), the second group consisted of 15 stallions aged 3 to 6 years (average age 4.0±0.5 years). The spontaneous OMP of sperm plasma in aged stallions was 495.6 Eod/ g protein, the induced one – 573.4 Eod / g protein. In the group of young stallions, spontaneous OMP – 347.2 Eod / g protein, induced – 485.3 Eod / g protein. The OMP level of sperm plasma in older stallions is initially higher than in young stallions (p = 0.04), which indicates the accumulation of carbonyl derivatives in vivo as a result of the prevalence of OS over the antioxidant capabilities of the organism. But when additional oxidizing components are added to the reaction mixture, the OMP of aged stallions does not increase as significantly as in young ones. RAP of sperm plasma of old stallions is 13.56%, of young stallions – 28.75%. The ability to withstand OS, the reserve of antioxidant capabilities of sperm plasma in young stallions is 2.1 times higher than in older stallions.

Effects of Mineral Salts on Chilled Canine Sperm Quality

Mineral ions have essential roles for maintaining osmotic balance, forming parts of primary enzymes relating to the sperm metabolism and function. This study was conducted to evaluate the effects of mineral salts as supplement to semen extender on chilled canine sperm quality during 10 days of storage. The sperm motility was performed by computer assisted sperm analysis (CASA). The plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential parameters were determined using a fluorescent staining combination. The results showed that although the percentage of sperm motility and sperm plasma membrane integrity in the mineral salts and the control extenders were not markedly different during the first 6 days of storage (P&gt;0.05), the acrosome membrane integrity, mitochondrial membrane potential and the healthy sperm parameters in the mineral salts extender was substantially higher than that in the control extender during this period (P&lt;0.05). Notably, the sperm quality of sperm in the mineral salts extender decreased rapidly after the first 6 days and evidently lower than that in the rest extender (P&lt;0.05). In conclusion, the mineral salts extender can improve sperm quality in chilled canine sperm during 6 days of storage.

The Stallion Spermatozoa: A Valuable Model to Help Understand the Interplay Between Metabolism and Redox (De)regulation in Sperm Cells.

Significance: Proper functionality of the spermatozoa depends on the tight regulation of their redox status; at the same time these cells are highly energy demanding and in the energetic metabolism, principally in the electron transport chain in the mitochondria, reactive oxygen species are continuously produced, in addition to that observed in the Krebs cycle and during the β-oxidation of fatty acids. Recent Advances: In addition, in glycolysis, elimination of phosphate groups from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate results in the byproducts glyoxal (G) and methylglyoxal (MG); these products are 2-oxoaldehydes. The presence of adjacent carbonyl groups makes them strong electrophiles that react with nucleophiles in proteins, lipids, and DNA, forming advanced glycation end products. Critical Issues: This mechanism is behind subfertility in diabetic patients; in the animal breeding industry, commercial extenders for stallion semen contain a supraphysiological concentration of glucose that promotes MG production, constituting a potential model of interest. Future Directions: Increasing our knowledge of sperm metabolism and its interactions with redox regulation may improve current sperm technologies in use, and shall provide new clues to understanding infertility in males. Moreover, stallion spermatozoa due to its accessibility, intense metabolism, and suitability for proteomics/metabolomic studies may constitute a suitable model for studying regulation of metabolism and interactions between metabolism and redox homeostasis. Antioxid. Redox Signal. 37, 521-537.

Sperm metabolic rate predicts female mating frequency across Drosophila species.

Female mating rates vary widely, even among closely related species, but the reasons for this variation are not fully understood. Across Drosophila species, female mating frequencies are positively associated with sperm length. This association may be due in part to sperm limitation, with longer-spermed species transferring fewer sperm, or to cryptic female choice. However, a previously overlooked factor is sperm metabolic rate, which may correlate with sperm length. If faster-metabolizing sperm accumulate age-related cellular damage more quickly, then females should remate sooner to obtain fresh sperm. Alternatively, frequent female mating may select for increased sperm competitiveness via increased metabolism. Here, we measure sperm metabolism across 13 Drosophila species and compare these measures to published data on female mating rate and on sperm length. Using fluorescent lifetime imaging microscopy, we quantify NAD(P)H metabolism ex vivo, in intact organs. Phylogenetically controlled regression reveals that sperm metabolic rate is positively associated with sperm length and with female mating frequency. Path analysis shows sperm length driving sperm metabolism and sperm metabolism either driving or being driven by female mating rate. While the causal directionality of these relationships remains to be fully resolved, and the effect of sperm metabolism on sperm aging and/or sperm competitiveness remains to be established, our results demonstrate the importance of sperm metabolism in sexual selection.

L-carnitine added to post-thawed semen acts as an antioxidant and a stimulator of equine sperm metabolism.

The objective of this study was to enhance the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen by the addition of l-carnitine (LC) to post-thawed semen. Different concentrations of LC were added to thawed samples to obtain four treatments control and 0.5, 1 and 2mM LC. In the in vitro experiments, sperm motility and kinematics, membrane integrity and intracellular calcium ion concentration ([Ca2+ ]i ) were investigated, and the antioxidant bioactivity of LC was assessed by measuring hydrogen peroxide and nitrite concentrations (NO2 - ). The fertility rate was assessed via the artificial insemination of mares. The treatment with 1mM LC increased sperm [Ca2+ ]i (60.6±0.05 AU), reduced nitrite concentration (39.1±14.9µM/µg protein), increased the sperm straightness percentage (STR: 78.3±5.3%) and increased the pregnancy rate (75%) as compared to the control ([Ca2+ ]i 48.4±0.05 AU, NO2 - concentration 63.1±14.4µM/µg protein, STR 67.5±7.9%, 12.5% pregnancy rate, p<0.05). These results suggest that 1mM LC acts as an antioxidant and stimulator of sperm metabolism in post-thawed equine semen, increasing the fertility rate. Thus, addition of LC might be an alternative to improve the fertility of poor quality post-thawed equine semen.

A Comparative Analysis of the Altered Levels of Human Seminal Plasma Constituents as Contributing Factors in Different Types of Male Infertility.

(1) Background: The relationships between the biochemical and immunological components in seminal plasma and their physiological effects on male reproductive system have been underreported. In this study, we evaluated the potential of several seminal plasma biochemical and immunological markers in the pathophysiological developments of the infertile male patients. The study was designed to identify and assess different markers that may be associated with semen functions in different types of male infertility. (2) Methods: A total of 50 infertile male patients who underwent checkup for fertility assessment and 50 fertile controls were included in this study. The complete medical history of each recruited participant was reviewed. The infertile sub-groups (non-obstructive azoospermia (NOA), asthenozoospermia (AS), normozoospermic infertile (NI), and oligozoospermia (OZ)) were characterized based on sperm motility and concentration, while NI patients were included after a thorough check up of their female partners as well. We investigated each sample for 21 different analytes, enzymes, trace elements, and immunological markers to find crucial markers posing as contributing factors to a specific type of male infertility. (3) Results: The levels of 15 out of 21 markers, assayed from the seminal plasma of infertile males, were significantly altered in comparison to fertile controls (p < 0.05). For the first time, microprotein levels were also analyzed. The presence of monocytes, lymphocytes, and granulocytes was limited to semen from NOA patients, while a significant increase in the level of platelets was observed in AS. Hierarchical clustering and ROC-AUC analysis identified the three most significant markers (zinc, LDH, and TG) for the healthy control group and asthenozoospermic group (AUC, of 0.92 and 0.81, respectively). (4) Conclusions: The altered levels of biochemical and immunological markers in seminal plasma might be associated with the different male infertility profiles and could be required for the sperm metabolism and maintenance. However, a larger sample size and follow up analysis is required for establishing the hypothesized panel of markers as biomarkers at clinical stage.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis

Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis.

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.

Effect of glucose concentration and cryopreservation on mitochondrial functions of bull spermatozoa and relationship with sire conception rate

Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P < 0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to “non-productive” oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.

Is there a role for glucagon-like peptide-1 receptor agonists in the treatment of male infertility?

Glucagon‐like peptide‐1 receptor agonists (GLP1‐RAs) are novel anti‐hyperglycemic drugs efficacious on glucose control, weight loss, and cardiovascular prevention. These drugs may also be effective in modulating testicular function. In fact, they increase serum testosterone levels in diabetic and/or obese patients with functional hypogonadism on a dysmetabolic basis. Although part of this effect can be ascribed to weight loss, some evidence suggests that there is a direct effect at the testicular level. Indeed, human Leydig, Sertoli, and germ cells express GLP1 receptors. GLP1‐RAs improve sperm metabolism, motility, and insulin secretion in vitro. Likewise, GLP1‐RAs exert positive effects on the metabolism of human Sertoli cells in vitro. Finally, GLP1 is secreted by mouse Leydig cells and this suggests the presence of a paracrine mechanism by which these cells could support the metabolism of Sertoli cells. Therefore, the widespread use of GLP1‐RAs in clinical practice may reveal an important role in the management of male infertility in obese and/or diabetic patients given the negative impact of these diseases on testicular steroidogenesis and spermatogenesis. This should suggest the design of randomized controlled studies aimed at evaluating the effects of these drugs on testicular function.

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 48h at 18°C; a commercial extender containing 67mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1mM) and high pyruvate (10mM) (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 48h 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 48h 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.

PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice.

Orderly and stage‐specifically expressed proteins are essential for spermatogenesis, and proteases play a key role in protein activation and function. The present study aimed to investigate serine protease 55 (PRSS55), which was reported to play a role in sperm‐uterotubal junction (UTJ) migration and sperm‐zona pellucida (ZP) binding. We found that PRSS55 was specifically expressed in testicular spermatids and epididymal spermatozoa. By constructing knockout mice targeting all transcripts of Prss55, we demonstrated that deletion of Prss55 resulted in a serious decline of male fertility, with significantly increased sperm malformation and decreased sperm motility. In Prss55 −/− mice, increased structural abnormality, including deficient “9 + 2” microtubules, damaged peripheral dense fibre, and defective mitochondrial cristae, were found in sperm. In addition, sperm showed decreased expression of electron transfer chain molecules and lower ATP contents. These could be the potential causes of the astheno/teratozoospermia phenotype of the Prss55 −/− mice, and provided new evidence for the previously reported impaired sperm‐UTJ migration. Moreover, preliminary studies allowed us to speculate that PRSS55 might function by activating type II muscle myosin in the testis, which is involved in many processes requiring motivation and cytoskeleton translocation. Thus, PRSS55 is essential for the structural differentiation and energy metabolism of sperm, and might be a potential pathogenic factor in astheno/teratozoospermia. Our results provide an additional explanation for the male sterility of Prss55 −/− mice, and further reveal the role of PRSS55.

149 Effects of managing mature beef bulls on divergent planes of nutrition on motility and kinematic properties of fresh and frozen-thawed sperm

Abstract Fifteen mature beef bulls (BW = 800.4 ± 17.4 kg) were used in a 112-d experiment to evaluate effects of divergent planes of nutrition on motility and kinematic properties of fresh and frozen-thawed semen. Bulls were ranked by BW and randomly assigned to one of two treatments: 1) managed on a positive plane of nutrition (POS, n = 8), or 2) managed on a negative plane of nutrition (NEG, n = 7). Bulls were fed a common diet, adjusted biweekly to achieve targeted weight loss or gain of 12.5% of original BW. On d 112, electroejaculation was used to collect 2 ejaculates from each bull, which were combined. An aliquot of fresh semen was evaluated via computer-assisted semen analysis (CASA; IVOS II, Hamilton Thorne, Beverly, MA, USA) for motility and kinematic properties. Remaining semen was extended and frozen. Frozen semen was thawed for 40 s and held in a heating block at 37°C, then evaluated via CASA at 0 and 3 h post-thaw. Data were analyzed in the MIXED procedure of SAS, with data for post-thaw analysis evaluated as repeated measures in time. Treatment did not influence ejaculate volume or concentration (P ≤ 0.19). In fresh ejaculates no impacts (P ≤ 0.29) of treatment were observed for motility or kinematic properties. In frozen-thawed ejaculates, however, bulls in the NEG treatment had greater (P ≤ 0.02) proportions of motile and progressively motile sperm compared with POS. In sperm classified as motile or progressively motile, NEG had greater (P ≤ 0.002) average path and straight line velocities, and greater (P ≤ 0.05) amplitude of lateral head displacement than POS. Treatment impacts observed in frozen, but not fresh, indicate that sperm metabolism, mitochondrial function, antioxidant capacity, or other factors may be influenced by plane of nutrition resulting in altered motility and kinematic properties.