Spontaneous Acrosome Reaction Research Articles

Essential role of germ cell glycerol-3-phosphate phosphatase for sperm health, oxidative stress control and male fertility in mice

ObjectivesObesity, diabetes and high-calorie diets are associated with defective sperm function and lowered male fertility. Mature spermatozoa primarily use fructose and glucose, and glucose and glycerol metabolism are important for sperm function. We recently discovered a novel mammalian enzyme, glycerol-3-phosphate (Gro3P) phosphatase (G3PP), and showed that it operates the glycerol shunt by hydrolyzing Gro3P to glycerol, and regulates glucose, lipid and energy metabolism in pancreatic β-cells and liver. We now observed that G3PP expression is the highest in the testis and spermatozoa, and investigated its role in male fertility. MethodsWe examined G3PP expression during spermatogenesis in mouse and assessed male fertility and spermatozoon function in conditional germ cell specific G3PP-KO (cG3PP-KO) mice and tamoxifen-inducible conditional germ cell G3PP-KO (icG3PP-KO) mice. We also determined the structural and metabolic parameters and oxidative stress in the spermatozoa from icG3PP-KO and control mice. ResultsG3PP expression in mouse spermatocytes and spermatids markedly increases during spermatogenesis. Male cG3PP-KO mice, in which germ cell G3PP is deleted from embryonic stage, are infertile due to dysfunctional sperm with reduced motility and capacitation, and elevated spontaneous acrosomal reaction and oxidative stress. However, icG3PP-KO male mice do not have altered fertility, due to the presence of ∼10% normal spermatozoa. icG3PP-KO spermatozoa display significantly reduced functionality and morphological and ultrastructural alterations. The icG3PP-KO spermatozoa show reduced glycerol production, elevated levels of Gro3P and reactive oxygen species (ROS), and oxidative stress that is associated with increased mitochondrial membrane potential. ConclusionsGerm cell G3PP deletion leads to the generation of spermatozoa that are functionally and structurally abnormal, likely due to the build-up of Gro3P that increases mitochondrial membrane potential, ROS, and oxidative stress and alters spermatozoa function. Overall, the results indicate that G3PP and the glycerol shunt are essential for normal spermatozoa function and male fertility.

Guinea pig spermatozoa adhesion to an immobilized fibronectin matrix alters their physiology and increases their survival.

Isthmus is the region of the oviduct considered a reservoir for spermatozoa, where they are retained and then released synchronously with ovulation. Integrins mediate this interaction, and it is suggested that they regulate the viability and capacitation of spermatozoa. Spermatozoa retained in the oviductal epithelial cells show specific characteristics: normal morphology, intact acrosome and plasma membrane, no DNA fragmentation, and low levels of intracellular Ca2+ and protein phosphorylation at Tyr. This work aimed to define spermatozoa's ability to adhere to an immobilized fibronectin matrix and its effects on their viability and capacitation. We found that guinea pig spermatozoa showed a high affinity for adhering to an immobilized fibronectin matrix but not to those made up of type 1 collagen or laminin. This interaction was mediated by integrins that recognize the RGD domain. Spermatozoa adhered to an immobilized fibronectin matrix were maintained in a state of low capacitation: low levels of intracellular concentration of Ca2+, protein phosphorylation in Tyr and F-actin. Also, spermatozoa kept their plasma membrane and acrosome intact, flagellum beating, and showed low activation of caspases 3/7. The spermatozoa adhered to the immobilized fibronectin matrix, gradually detached, forming rosettes and did not undergo a spontaneous acrosomal reaction but were capable of experiencing a progesterone-induced acrosomal reaction. In conclusion, the adhesion of spermatozoa to an immobilized fibronectin matrix alters the physiology of the spermatozoa, keeping them in a steady state of capacitation, increasing their viability in a similar way to what was reported for spermatozoa adhered to oviductal epithelial cells.

Role of genetic variations and protein expression of β-Microsemino protein in intrauterine insemination outcome of unexplained infertile men: A case-control study.

Intrauterine insemination (IUI) is often the first-line treatment for unexplained infertility. -Microsemino protein (MSMB) is an abundant protein in seminal plasma that has an inhibitory effect on spontaneous acrosome reaction. The present study aimed to evaluate MSMB gene variations and protein expression on IUI success rate in unexplained infertile men. A case-control study was performed on 100 unexplained infertile Iranian men referred to the Royan Institute, Tehran, Iran for IUI (50 men with IUI positive result [IUI+], and 50 men with IUI negative result [IUI-]). Couples with female infertility factors (such as hormonal disorders, infrequent menstrual period, abnormality in uterus, fallopian tubes, or ovaries) and men with infections of the male accessory glands, hypogonadotropic hypogonadism, clinical varicocele, retractile testis, genital trauma, drug use, or concurrent hormonal treatment Y chromosome microdeletions, and abnormal karyotype were excluded from the study. The polymerase chain reaction sequencing was performed for the promoter and the coding regions of MSMB functional domains. To study the protein expression, the total protein of sperm was extracted, and sandwich enzyme-linked immunosorbent assay was performed. 4 variations were detected (rs12770171, rs10993994, rs2075894, and rs4517463). None of them showed significant differences between the IUI+ and IUI- groups. The mean value of protein expression did not show any differences between the groups. In conclusion, there is no association between genetic variations of promoter and coding regions of MSMB functional domains as well as its expression with IUI success in unexplained infertile men.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Proteomic analysis of fipronil-induced molecular defects in spermatozoa

The phenylpyrazole insecticide fipronil has wide-ranging applications from agriculture to public health to control undesirable organisms. However, several studies have reported the residual environmental hazards of fipronil and demonstrated its harmful effects even in mammalian reproduction. Therefore, this study was conducted to demonstrate the mode of action of fipronil on mouse spermatozoa. We treated fipronil to spermatozoa and performed comprehensive function evaluations. Moreover, proteomic analyses were conducted to identify the alteration of protein expression levels in spermatozoa. Most of sperm motility and kinematic parameters and intracellular ATP levels were diminished, and the spontaneous acrosome reaction was promoted after treatment with fipronil. Proteomic analyses revealed altered expression levels of 14 proteins after treatment. These proteins have been reported to be associated with sperm-specific pathways, prominently the cytoskeleton of the sperm, “9 + 2” axoneme composition, metabolism, and fertility. Collectively, our results showed that fipronil alters sperm functional-related proteins and therefore influences male fertility. This study elucidates the possible reproductive toxic hazards associated with male infertility through aberrant suppression of sperm proteins.

Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function

To explore the mechanism of sperm dysfunction caused by dibutyl phthalate (DBP), the effects of DBP on intracellular [Ca2+] and [pH], reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, phosphorylation of protein kinase A (PKA) substrate proteins and phosphotyrosine (p-Tyr) proteins, sperm motility, spontaneous acrosome reaction, and tail bending were examined in mouse spermatozoa. At 100 µg/mL, DBP significantly increased tail bending and [Ca2+]i. Interestingly, DBP showed biphasic effects on [pH]i. DBP at 10–100 µg/mL significantly decreased sperm motility. Similarly, Ca2+ ionophore A23187 decreased [pH]i sperm motility, suggesting that DBP-induced excessive [Ca2+]i decreased sperm motility. DBP significantly increased ROS and LPO. DBP at 100 µg/mL significantly decreased mPTP closing, MMP, and ATP levels in spermatozoa, as did H2O2, indicative of ROS-mediated mitochondrial dysfunction caused by DBP. DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins. DBP at 1–10 µg/mL significantly increased the spontaneous acrosome reaction, suggesting that DBP can activate sperm capacitation. Altogether, DBP showed a biphasic effect on intracellular signaling in spermatozoa. At concentrations relevant to seminal ortho-phthalate levels, DBP activates [pH]i, protein tyrosine kinases and PKA via physiological levels of ROS generation, potentiating sperm capacitation. DBP at high doses excessively raises [Ca2+]i and ROS and disrupts [pH]i, impairing the mitochondrial function, tail structural integrity, and sperm motility.

Oxidative Stress and Acrosomal Status of Human Spermatozoa Subjected to Hydrophobic Carbon Soot Treatments.

The fourth industrial revolution extensively reshapes the reality we are living in by blurring the boundaries of physical, digital and biological worlds. A good example is the previously unthinkable incursion of nanoscale waste materials, such as soot, into the technologies for assisted reproduction. Although the rapeseed oil soot may efficiently enhance the progressive motility of human spermatozoa, it is yet unknown whether this material induces undesirable oxidative stress and premature acrosome reaction, endangering the sperm-oocyte fusion and blastocyst formation. In an attempt to clarify this issue, we reveal that the three-hour incubation of human semen mixed with three main types of soot does not cause oxidative stress and spontaneous acrosome reaction of the sperm. These unique findings are attributed to synchronous elimination and stabilization of the oxidants via hydrogen bonding to the acidic groups of the soot (i.e., C=O and/or C-O-C) and electron donation by its basic chemical sites (i.e., C-OH and/or COOH). Moreover, the soot nanoparticles are electrostatically attracted by discrete positively charged areas on the sperm head, increasing its negative charge and in some cases interfering the acrosome reaction. Such novel mechanistic insights emphasize the credibility of rapeseed oil soot to confidently shift from the purely diagnostic and therapeutic phases in reproductive medicine to research dealing with the effect of carbon nanomaterials on the embryo development and implantation.

Mechanisms That Protect Mammalian Sperm from the Spontaneous Acrosome Reaction

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR.

Effects of Benzo[a]pyrene on Human Sperm Functions: An In Vitro Study.

Benzo(a)pyrene (BaP) is considered one of the most dangerous air pollutants for adverse health effects, including reproductive toxicity. It is found both in male and female reproductive fluids likely affecting spermatozoa after the selection process through cervical mucus, a process mimicked in vitro with the swim-up procedure. In vitro effects of BaP (1, 5, 10 µM) were evaluated both in unselected and swim-up selected spermatozoa after 3 and 24 h of incubation. BaP reduced total, progressive and hyperactivated motility and migration in a viscous medium both in swim-up selected and unselected spermatozoa. Viability was not significantly affected in swim-up selected but was reduced in unselected spermatozoa. In swim-up selected spermatozoa, increases in the percentage of spontaneous acrosome reaction and DNA fragmentation were observed after 24 h of incubation, whereas no differences between the control and BaP-treated samples were observed in caspase-3 and -7 activity, indicating no effects on apoptotic pathways. ROS species, evaluated by staining with CellROX® Orange and Dihydroethidium, did not differ in viable spermatozoa after BaP treatment. Conversely, the percentage of unviable ROS-positive spermatozoa increased. Our study suggests that BaP present in male and female genital fluids may heavily affect reproductive functions of human spermatozoa.

Effect of glucose and reactive oxygen species on boar sperm induced-acrosome exocytosis

Ejaculated boar spermatozoa can be liquid preserved for several days and be easily activated to produce physiological changes. One of the major changes is acrosome exocytosis that is physiologically related to capacitation. Glycolysis and reactive oxygen species (ROS) were studied regarding several boar sperm functions, but data available about their effect on boar sperm acrosome exocytosis are scarce. The objective of this work was to evaluate the effect of glucose and ROS on boar sperm acrosome exocytosis. We evaluated acrosome exocytosis by progesterone induction of capacitated sperm and assess viability, kinematics parameters, ROS levels, ATP content and Protein Kinase A activity in media with or without glucose and hydrogen peroxide or potassium chromate, as source of ROS. Our results show that glucose has no effect on acrosome exocytosis and also, it is not necessary for boar sperm capacitation, although it has a positive effect in the presence of ROS. On the other hand, ROS effects are related to spontaneous acrosome reaction. We conclude that glycolysis may function as a metabolic pathway that provides sustain but is not directly involved in boar sperm acrosome exocytosis and capacitation. Also, ROS do not promote capacitation in boar sperm, but affect spontaneous acrosome exocytosis.

O-233 Follicular fluid extracellular vesicles as a potential tool for human spermatozoa selection in vitro

Abstract Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa.

Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5mm), pyruvate (10mm), and lactate (10mm) for 0.5 to 4h. Response to calcium ionophore A23187 (5μm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Incubation with lactate alone for 2h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.

Sperm interaction with bacteria induces the spontaneous acrosome reaction

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria.

Phosphodiesterase 10A inhibitor PF-2545920 as a prospective agent for the clinical promotion of sperm motility.

Phosphodiesterase (PDE) inhibitors can improve sperm motility in patients with asthenozoospermia. However, the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity. We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil. After seminal plasma was discarded, several semen samples were subjected to four treatments (control, PF-2545920, pentoxifylline, and sildenafil) to evaluate their ability to affect motility, viability, and spontaneous acrosome reactions. Intracellular calcium and adenosine triphosphate (ATP), mitochondrial membrane potential, and penetration through viscous medium were assessed by flow cytometry, luciferase, and hyaluronic acid after treatment with PF-2545920. Statistical analyses were performed using the analysis of variance statistical test. PF-2545920 elevated the percentage of motile spermatozoa compared to the control, pentoxifylline, and sildenafil groups at 10 µmol l -1 ( P < 0.01). It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions ( P < 0.05). PF-2545920 also increased mitochondrial membrane potential ( P < 0.001) and altered intracellular calcium ( P < 0.05) in a dose-dependent manner, including increasing sperm hyaluronic acid penetrating ability ( P < 0.05). Therefore, PF-2545920 might be an excellent choice for stimulating the sperm motility.

Search for genetic associations with semen morphology after cryopreservation in bulls.

Sperm morphology is an important indicator of sperm quality. We calculated the rank correlation coefficient between fertility and the number of cells with damaged acrosomes, which was r=-0.6, which is relatively high. The aim of the study was to find genetic associations with semen morphology after cryopreservation in bulls. The quality of spermatozoa was assessed by CASA and GWAS was used for genotype analysis. Calculations were carried out using PLINK and EMMAX programs and a Manhattan-type plot was obtained using the qqman package in R software. The localization of SNPs was determined and candidate genes were identified using the NCBI and Ensembl genomic browsers. The semen of 130 bulls was evaluated. Total sperm motility varied from 25 to 65% and progressive motility ranged from 15 to 60%. There were 13.6 to 39.2% of cells with damaged acrosomes and 1.2 to 14.1% of cells with damage in the tail and neck in frozen bovine semen. There were several significant SNPs associated with the number of missing acrosomes after cryopreservation. For example, a significant SNP on the fourth chromosome, BTB-00566744 (P-value=2.89E-15), is an intron variant of the POU6F2 gene, somatic mutations of which have been identified in human glaucoma and prolactinoma. Prolactin, in turn, significantly reduces the spontaneous acrosomal reaction. There were genomic associations with the percentage of swollen acrosomes after cryopreservation. Thus, the LPCAT4 gene is involved in the biosynthesis and metabolism of lipids and phospholipids. The identified genomic associations with disorders in the tail and neck region mostly repeated genes associated with disorders in the head region. These are the genes: FOXF1, ZBTB40, WNT4, FRMD4A, FAM107B, PTPRU, GLRA3, RABGAP1, EPC2, MBD5, TTK, BCKDHB, PPP3CA, MAP2K5, MAP2K6, CD59, DIP2B, AIG1, ADAT2, TTC29, POU4F2, ENDRA, GRIK1, NEDD4, C10H15orf61, WNT5A, RIMS1, RAD51B, KLHL1, PAPSS1, DKK2, RGS17, U6, ORC4. Thus, the RIM1α isoform can compensate for the lack RIM2α in calcium-induced acrosomal reaction. As a result, a large number of genome-wide associations that affect the preservation of spermatozoa after freezing-thawing have been identified. Authors acknowledge financial support from Russian Science Foundation, Grant No: 18-16-00071.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 μM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 μM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.

Involvement of metabolic pathway in the sperm spontaneous acrosome reaction

In order to fertilize the egg, spermatozoa must undergo a series of biochemical processes in the female reproductive tract collectively called capacitation. Only capacitated sperm can interact with the egg resulting in the acrosome reaction (AR), allowing egg penetration and fertilization. Sperm can undergo spontaneous AR (sAR) before reaching the egg, preventing successful fertilization. Here we investigated the metabolic pathways involved in sperm capacitation and sAR. Inhibition of glycolysis or oxidative phosphorylation did not affect capacitation or sAR levels; however, when both systems were inhibited, no capacitation occurred, and there was a significant increase in sAR. Under such ATP-starvation, the increase in sAR is triggered by Ca2+ influx into the sperm via the CatSper cation channel. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation; there was no change in its activity when a single metabolic system was inhibited, while complete inhibition of was observed when the two systems were inhibited. Protein tyrosine phosphorylation (PTP), also known to occur in sperm capacitation, was partially reduced by inhibition of one metabolic system, and completely blocked when the two metabolic systems were inhibited. We conclude that ATP, PKA and PTP are involved in the mechanisms protecting sperm from sAR.

Protein acetylation protects sperm from spontaneous acrosome reaction

In order to penetrate the egg, spermatozoa must undergo the acrosome reaction in close proximity to the egg. This process can take place only after a series of biochemical changes in the sperm, collectively termed capacitation, occur in the female reproductive tract. Sperm cells can undergo spontaneous-acrosome reaction(sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect sperm from undergoing sAR, and all of them are involved in proper capacitation. Here, we describe the involvement of protein acetylation in the mechanism that protects bovine spermatozoa from sAR. Incubation of bovine sperm under non-capacitation conditions revealed a strong increase in sAR that was significantly reduced in the presence of deacetylase inhibitors. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation, and its inhibition results in high sAR. The reduction in sAR by hyperacetylation was independent of PKA activity. We previously demonstrated that calmodulin-kinase II (CaMKII) activity protects sperm from sAR, and here we show that its activity is essential for reduction in sAR by hyperacetylation. We further show that the ‘exchange protein directly activated by Camp’ (EPAC) mediates both protein lysine acetylation and the reduced rate of sAR caused by hyperacetylation. In conclusion, these results suggest a PKA-independent and EPAC-CaMKII dependent hyperacetylation mechanism that protects sperm from sAR.

The role of bicarbonate in the modulation of capacitation, spontaneous acrosome reaction and motility of equine fresh and frozen spermatozoa

In this study, we defined the composition of the culture medium that yield a significant percentage of alive and functional equine spermatozoa during enough time before artificial insemination. The effects of sodium bicarbonate were analyzed in fresh and frozen semen in respect to sperm viability, capacitation, spontaneous acrosome reaction and several kinetic parameters such as total motility, progressive motility, VCL, VSL, ALH, BCF, LIN. Moreover, employing Bayk-6844 and Nifedipine, the involvement of L-type voltage-gated calcium channels in the modulation of intracellular calcium concentrations was investigated. Results evidenced an immediate effect of sodium bicarbonate in reducing fresh sperm viability and in increasing capacitation and spontaneous acrosome reaction of fresh and frozen spermatozoa. Furthermore, it affected total and progressive motility of fresh and frozen semen. Because of the sudden effects of the compound, we think that a buffer lacking sodium bicarbonate is more suitable to preserve the viability and the functional state of equine spermatozoa required to undergo at the right time those modifications necessary for fertilization. We also demonstrated that intracellular calcium modifications induced by Bayk-8644 and Nifedipine are not involved in signals related to vitality, capacitation, spontaneous acrosome reaction and motility.