RESUMO
Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.
Assuntos
Mitocôndrias , Sêmen , Humanos , Masculino , Feminino , Animais , Camundongos , Espécies Reativas de Oxigênio , Espermatozoides , SuperóxidosRESUMO
Sperm capacitation is broadly defined as a suite of biochemical and biophysical changes resulting from the acquisition of fertilization ability. To gain insights into the regulation mechanism of crustacean sperm capacitation, 4D label-free quantitative proteomics was first applied to analyze the changes of sperm in Eriocheir sinensis under three sequential physiological conditions: seminal vesicles (X2), hatched with the seminal receptacle content (X3), and incubated with egg water (X5). In total, 1536 proteins were identified, among which 880 proteins were quantified, with 82 and 224 proteins significantly altered after incubation with the seminal receptacle contents and egg water. Most differentially expressed proteins were attributed to biological processes by Gene Ontology annotation analysis. As the fundamental bioenergetic metabolism of sperm, the oxidative phosphorylation, glycolysis, and the pentose phosphate pathway presented significant changes under the treatment of seminal receptacle contents, indicating intensive regulation for sperm in the seminal receptacle. Additionally, the seminal receptacle contents also significantly increased the oxidation level of sperm, whereas the enhancement of abundance in superoxide dismutase, peroxiredoxin 1, and glutathione S-transferase after incubation with egg water significantly improved the resistance against oxidation. These results provided a new perspective for reproduction studies in crustaceans.
Assuntos
Braquiúros , Proteômica , Capacitação Espermática , Espermatozoides , Animais , Masculino , Braquiúros/metabolismo , Braquiúros/fisiologia , Proteômica/métodos , Capacitação Espermática/fisiologia , Espermatozoides/metabolismoRESUMO
The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.
Assuntos
Transportador 1 de Glucose-Sódio , Motilidade dos Espermatozoides , Animais , Masculino , Camundongos , Glucose/metabolismo , Camundongos Knockout , Sêmen/metabolismo , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Pesticides serve as essential tools in agriculture and public health, aiding in pest control and disease management. However, their widespread use has prompted concerns regarding their adverse effects on humans and animals. This review offers a comprehensive examination of the toxicity profile of pesticides, focusing on their detrimental impacts on the nervous, hepatic, cardiac, and pulmonary systems, and their impact on reproductive functions. Additionally, it discusses how pesticides mimic hormones, thereby inducing dysfunction in the endocrine system. Pesticides disrupt the endocrine system, leading to neurological impairments, hepatocellular abnormalities, cardiac dysfunction, and respiratory issues. Furthermore, they also exert adverse effects on reproductive organs, disrupting hormone levels and causing reproductive dysfunction. Mechanistically, pesticides interfere with neurotransmitter function, enzyme activity, and hormone regulation. This review highlights the effects of pesticides on male reproduction, particularly sperm capacitation, the process wherein ejaculated sperm undergo physiological changes within the female reproductive tract, acquiring the ability to fertilize an oocyte. Pesticides have been reported to inhibit the morphological changes crucial for sperm capacitation, resulting in poor sperm capacitation and eventual male infertility. Understanding the toxic effects of pesticides is crucial for mitigating their impact on human and animal health, and in guiding future research endeavors.
Assuntos
Disruptores Endócrinos , Fertilidade , Praguicidas , Humanos , Praguicidas/toxicidade , Praguicidas/efeitos adversos , Masculino , Disruptores Endócrinos/toxicidade , Disruptores Endócrinos/efeitos adversos , Animais , Fertilidade/efeitos dos fármacos , Infertilidade Masculina/induzido quimicamente , Exposição Ambiental/efeitos adversos , Reprodução/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacosRESUMO
Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation.
Assuntos
Adenilil Ciclases , Septinas , Animais , Masculino , Camundongos , Adenilil Ciclases/metabolismo , Camundongos Knockout , Fosforilação , Septinas/química , Septinas/deficiência , Septinas/genética , Septinas/metabolismo , Capacitação Espermática , Espermatozoides/metabolismo , Técnicas de Introdução de GenesRESUMO
Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45 h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.
Assuntos
Sêmen , Espermatozoides , Coelhos , Masculino , Animais , Bovinos , Espermatozoides/metabolismo , Fertilização in vitro , Acrossomo , Capacitação Espermática , Imunoglobulina G , FertilizaçãoRESUMO
Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K+ currents in human sperm (IKSper ) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca2+ , K+ , Cl- , and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K+ ]i , [Cl- ]i , and pHi , but a decrease in [Ca2+ ]i . Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K+ ]i , [Cl- ]i , and pHi , and the decrease in [Ca2+ ]i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.
Assuntos
Reação Acrossômica , Capacitação Espermática , Humanos , Masculino , Capacitação Espermática/fisiologia , Reação Acrossômica/fisiologia , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Canais de Potássio/metabolismo , HomeostaseRESUMO
Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.
Assuntos
Catequina , Masculino , Suínos , Animais , Catequina/farmacologia , Molibdênio/metabolismo , Sêmen , Fertilização , Espermatozoides/metabolismo , Fertilização in vitroRESUMO
Previous studies demonstrated that hamster sperm hyperactivation is suppressed by extracellular Na+ by lowering intracellular Ca2+ levels, and Na+/Ca2+-exchanger (NCX) specific inhibitors canceled the suppressive effects of extracellular Na+. These results suggest the involvement of NCX in the regulation of hyperactivation. However, direct evidence of the presence and functionality of NCX in hamster spermatozoa is still lacking. This study aimed to reveal that NCX is present and is functional in hamster spermatozoa. First, NCX1 and NCX2 transcripts were detected via RNA-seq analyses of hamster testis mRNAs, but only the NCX1 protein was detected. Next, NCX activity was determined by measuring the Na+-dependent Ca2+ influx using the Ca2+ indicator Fura-2. The Na+-dependent Ca2+ influx was detected in hamster spermatozoa, notably in the tail region. The Na+-dependent Ca2+ influx was inhibited by the NCX inhibitor SEA0400 at NCX1-specific concentrations. NCX1 activity was reduced after 3 h of incubation in capacitating conditions. These results, together with authors' previous study, showed that hamster spermatozoa possesses functional NCX1 and that its activity was downregulated upon capacitation to trigger hyperactivation. This is the first study to successfully reveal the presence of NCX1 and its physiological function as a hyperactivation brake.
Assuntos
Sêmen , Espermatozoides , Animais , Cricetinae , Masculino , Sêmen/metabolismo , RNA Mensageiro , Espermatozoides/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Cálcio/metabolismoRESUMO
The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H2O2), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H2O2 and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation.
Assuntos
Antioxidantes , Peróxido de Hidrogênio , Humanos , Masculino , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Diamida/metabolismo , Motilidade dos Espermatozoides , Sêmen/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo , Peroxirredoxinas/metabolismoRESUMO
Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pHi), but the mechanisms controlling this process are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in pHi modulation. Here, we employed an image-based flow cytometry technique combined with pharmacological approaches to study pHi dynamics at the subcellular level during capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pHi increases require the initial uptake of HCO3-, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel (also known as HVCN1) and cAMP-activated protein kinase (protein kinase A, PKA) antagonists impair alkalization mainly in the principal piece. Na+/HCO3- cotransporter (NBC) and cystic fibrosis transmembrane regulator (CFTR) antagonists impair alkalization only mildly, predominantly in the head. Motility measurements indicate that inhibition of alkalization in the principal piece prevents the development of hyperactivated motility. Altogether, our findings shed light on the complex control mechanisms of pHi and underscore their importance during human sperm capacitation.This article has an associated First Person interview with the first author of the paper.
Assuntos
Capacitação Espermática/genética , Espermatozoides/metabolismo , Humanos , MasculinoRESUMO
Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.
Assuntos
Infertilidade Masculina , Desnutrição , Adulto , Animais , Feminino , Fertilidade , Humanos , Infertilidade Masculina/etiologia , Lactação , Masculino , Desnutrição/complicações , Camundongos , Gravidez , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/fisiologiaRESUMO
Glucose is a key substrate for supporting sperm energy production and function. Previous studies have demonstrated that sperm glucose uptake is facilitated by several isoforms of the glucose transporters (GLUT). Here, we report that sperm also expresses the Na+-dependent sodium glucose cotransporter (SGLT). This was first suggested by our observation that genetic deletion of the testis-specific Na,K-ATPase α4, which impairs the sperm plasma membrane Na+ gradient, reduces glucose uptake and ATP production. Immunoblot analysis revealed the presence of an SGLT in sperm, with specific expression of isoform 1 (SGLT-1), but not of isoform 2 (SGLT-2). Immunocytochemistry identified SGLT-1 in the mid- and principal piece of the sperm flagellum. Inhibition of SGLT-1 with the isotype-selective inhibitor phlorizin significantly reduced glucose uptake, glycolytic activity, and ATP production in noncapacitated and capacitated sperm from wild-type mice. Phlorizin also decreased total sperm motility, as well as other parameters of sperm movement. In contrast, inhibition of SGLT-1 had no significant effect on sperm hyperactivation, protein tyrosine phosphorylation, or acrosomal reaction. Importantly, phlorizin treatment impaired the fertilizing capacity of sperm. Altogether, these results demonstrate that mouse sperm express a functional SGLT transport system that is important for supporting sperm energy production, motility, and fertility.
Assuntos
ATPase Trocadora de Sódio-Potássio , Motilidade dos Espermatozoides , Trifosfato de Adenosina/metabolismo , Animais , Fertilidade , Glucose/metabolismo , Masculino , Camundongos , Florizina/metabolismo , Florizina/farmacologia , Isoformas de Proteínas/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Transportador 1 de Glucose-Sódio , ATPase Trocadora de Sódio-Potássio/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.
Assuntos
Fertilização in vitro , Sêmen , Cavalos , Animais , Feminino , Masculino , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Espermatozoides , Blastocisto , Capacitação Espermática , Oócitos , Penicilamina , EpinefrinaRESUMO
During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.
Assuntos
Oxirredutases , Capacitação Espermática , Espermatozoides , Reação Acrossômica/fisiologia , Animais , Cálcio/metabolismo , Feminino , Masculino , Camundongos , Oxirredutases/metabolismo , Sêmen/metabolismo , Glândulas Seminais/enzimologia , Espermatozoides/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-ß-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.
Assuntos
Sêmen , Injeções de Esperma Intracitoplásmicas , Gravidez , Feminino , Bovinos , Masculino , Animais , Injeções de Esperma Intracitoplásmicas/veterinária , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/fisiologia , Reação Acrossômica , Oócitos/fisiologia , Ditiotreitol/farmacologiaRESUMO
In newly improved MACS-Up method, magnetic field has been applied to separate non-apoptotic spermatozoa directly from the neat semen. The spermatozoa during passing through a viscous layer, located on the neat semen, contacted with progesterone and induced for the capacitation. Then, a clean population of non-apoptotic, and capacitated spermatozoa were selected in the pure culture media. Selected spermatozoa may be useful for use in ART. The 80 semen samples from normozoospermic individuals were divided separately into 4 attempts. Semen analysis, SCSA (sperm chromatin structure assay), FLICA (fluorescein-labelled inhibitors of caspase) methods, immunoassay of phosphorylation of tyrosine residues of sperm proteins, nuclear DNA integrity, caspase 3 activity and sperm capacitation rate were all performed for evaluation of sperm parameters respectively. To examine all aspects, the MACS-Up method compared with DGC (density gradient centrifuging) and MACS-DGC methods separately. This method can isolate non-apoptotic spermatozoa directly from the neat semen, which has similar performance compared to the MACS-DGC method. Movement and passing spermatozoa through the viscous layer, and contact with progesterone, significantly induced spermatozoa for capacitation compared with the control group. Also, the MACS-Up in comparison with routine DGC method could select spermatozoa with significantly higher total and progressive motility, DNA integrity, induced sperm population for capacitation and normal morphology. MACS-Up can be developed as an effective, short-time, and ease of performing method and used practically to select functional spermatozoa as novel sperm selection procedure. However, for clinical use of MACS-Up, all clinical aspects of this method should be considered and evaluated.
Assuntos
Progesterona , Motilidade dos Espermatozoides , Humanos , Masculino , Análise do Sêmen , Capacitação Espermática , Espermatozoides/metabolismoRESUMO
Low motility is one of the causes of male infertility. In this study, the effects of progesterone solid lipid nanoparticles (SLNs) on sperm capacitation, acrosome reaction, oxidative stress and expression of SPACA1 and MAPK way genes were investigated. Progesterone SLNs were synthesized using the solvent emulsification evaporation method. Twenty asthenozoospermia samples were selected, and sperm and acrosome membrane integrity, acrosome reaction, sperm motility, viability, total antioxidant capacity (TAC), total oxidative status tests and PKA, PTK, P38MAPK and SPACA1 gene expressions were assessed. The synthesized nanoparticles were prepared with the size (187.6 nm), PDI (0.184), EE (85.82%), LP (3.43%) and ZP (-23.5mV). Progesterone SLNs increased sperm and acrosome membrane integrity and TAC (p < .05). Also, the expression of P38MAPK, PKA, PTK, and SPACA1 genes in this group showed a significant increase (p < .001). Progesterone SLNs increased acrosome reaction, sperm capacitation and TAC. Also, it increased the expression of PTK PKA, SPACA1 and P38MAPK genes.
Assuntos
Astenozoospermia , Nanopartículas , Acrossomo , Reação Acrossômica , Astenozoospermia/tratamento farmacológico , Astenozoospermia/genética , Humanos , Lipossomos , Masculino , Progesterona/farmacologia , Capacitação Espermática , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
PURPOSE: To investigate the effects of recombinant human oviduct-specific glycoprotein (rHuOVGP1) alone and in combination with progesterone (P4) on intracellular Ca2+ concentration [Ca2+]i and to investigate if rHuOVGP1 in combination with P4 can further enhance tyrosine phosphorylation (pY) of sperm proteins during human sperm capacitation. METHODS: Fluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca2+]i in human sperm during capacitation. Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca2+]i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca2+]i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein. RESULTS: rHuOVGP1 increases [Ca2+]i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1. CONCLUSION: The present study revealed that rHuOVGP1 may work with P4 to upregulate [Ca2+]i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.
Assuntos
Cálcio , Progesterona , Humanos , Masculino , Cálcio/metabolismo , Cálcio/farmacologia , Progesterona/farmacologia , Progesterona/metabolismo , Motilidade dos Espermatozoides , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio/farmacologia , Sêmen/metabolismo , Capacitação Espermática , Espermatozoides/metabolismo , Tirosina/metabolismo , Glicoproteínas/metabolismoRESUMO
Deficient sperm motility is a frequent cause of the age-related male sub-/infertility. Since the protein sirtuin 1 (SIRT1) develops anti-aging action and participates in sperm motility and ATP synthesis in mitochondria, we investigated its role in the acquisition of hyperactivated motility during capacitation. For this, the dynamics of sperm subpopulations were studied, using males of Sirt1+/- heterozygous mutant mice. After 2 hr of capacitation, we observed reduced percentage of hyperactivated spermatozoa in Sirt1+/- males. Interestingly, prior to capacitation, Sirt1+/- spermatozoa showed higher mitochondrial superoxide levels, which could render mitochondrial injury and thereby motility defects. Accordingly, the fertilization rate of Sirt1+/- males after mating was decreased. We elucidated that SIRT1 male insufficiency underlies posterior sperm defects to hyperactivate during capacitation and propose Sirt1+/- males as a model for the study of the age-related infertility.