RESUMEN
After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.
Asunto(s)
Calcimicina , Ionóforos de Calcio , Criopreservación , Fertilización In Vitro , Preservación de Semen , Capacitación Espermática , Espermatozoides , Animales , Bovinos , Masculino , Ionóforos de Calcio/farmacología , Criopreservación/veterinaria , Criopreservación/métodos , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , Calcimicina/farmacología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Capacitación Espermática/efectos de los fármacos , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Femenino , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/efectos de los fármacosRESUMEN
Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteómica , Capacitación Espermática , Espermatozoides , Ubiquitina , Capacitación Espermática/fisiología , Animales , Masculino , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Porcinos , Espermatozoides/metabolismo , Espermatozoides/fisiología , Proteómica/métodos , Proteoma/metabolismoRESUMEN
Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately -30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa.
Asunto(s)
Fosfatidilinositoles , Monoéster Fosfórico Hidrolasas , Motilidad Espermática , Espermatozoides , Animales , Masculino , Espermatozoides/metabolismo , Espermatozoides/fisiología , Fosfatidilinositoles/metabolismo , Ratones , Motilidad Espermática/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Capacitación Espermática/fisiología , Técnicas de Sustitución del Gen , Humanos , MutaciónRESUMEN
BACKGROUND: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated. RESULTS: Partial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation. CONCLUSIONS: Inhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.
Asunto(s)
Calcio , Intercambiador de Sodio-Calcio , Capacitación Espermática , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/efectos de los fármacos , Calcio/metabolismo , Porcinos , Espermatozoides/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Motilidad Espermática/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
Dave Garbers' work significantly contributed to our understanding of sperm's regulated motility, capacitation, and the acrosome reaction. These key sperm functions involve complex multistep signaling pathways engaging numerous finely orchestrated elements. Despite significant progress, many parameters and interactions among these elements remain elusive. Mathematical modeling emerges as a potent tool to study sperm physiology, providing a framework to integrate experimental results and capture functional dynamics considering biochemical, biophysical, and cellular elements. Depending on research objectives, different modeling strategies, broadly categorized into continuous and discrete approaches, reveal valuable insights into cell function. These models allow the exploration of hypotheses regarding molecules, conditions, and pathways, whenever they become challenging to evaluate experimentally. This review presents an overview of current theoretical and experimental efforts to understand sperm motility regulation, capacitation, and the acrosome reaction. We discuss the strengths and weaknesses of different modeling strategies and highlight key findings and unresolved questions. Notable discoveries include the importance of specific ion channels, the role of intracellular molecular heterogeneity in capacitation and the acrosome reaction, and the impact of pH changes on acrosomal exocytosis. Ultimately, this review underscores the crucial importance of mathematical frameworks in advancing our understanding of sperm physiology and guiding future experimental investigations.
Asunto(s)
Reacción Acrosómica , Transducción de Señal , Capacitación Espermática , Motilidad Espermática , Espermatozoides , Masculino , Espermatozoides/metabolismo , Espermatozoides/fisiología , Humanos , Reacción Acrosómica/fisiología , Capacitación Espermática/fisiología , Transducción de Señal/fisiología , Animales , Motilidad Espermática/fisiología , Modelos Biológicos , Modelos TeóricosRESUMEN
In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán et al., Theriogenology, 198, 2023 and 231; Oberlender et al., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo et al., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on in vitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.
Asunto(s)
Capacitación Espermática , Espermatozoides , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Porcinos , Medios de Cultivo/farmacología , Motilidad Espermática/efectos de los fármacos , Análisis de Semen/veterinariaRESUMEN
Oridonin, a natural terpenoid isolated from the leaves of Isodon rubescens (Hemsley) H.Hara, is widely used in oriental medicine for its anticancer properties across various cancer types. Despite its prevalent use, the toxic effects of oridonin on male reproduction, particularly its impact on sperm functions and the mechanisms involved, are not well understood. This study aimed to explore the effects and underlying mechanisms of oridonin on sperm functions. We initially treated Duroc boar spermatozoa with varying concentrations of oridonin (0, 5, 50, 75, 100, and 150⯵M) and incubated them to induce capacitation. We then assessed cell viability and several sperm functions, including sperm motility and motion kinematics, capacitation status, and ATP levels. We also analyzed the expression levels of proteins associated with the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathway and phosphotyrosine proteins. Our results indicate that oridonin adversely affects most sperm functions in a dose-dependent manner. We observed significant decreases in AKT, p-AKT (Thr308), phosphatase and tensin homolog (PTEN), p-PDK1, and p-PI3K levels following oridonin treatment, alongside an abnormal increase in phosphotyrosine proteins. These findings suggest that oridonin may disrupt normal levels of tyrosine-phosphorylated proteins by inhibiting the PI3K/PDK1/AKT signaling pathway, which is crucial for cell proliferation, metabolism, and apoptosis, thus potentially harming sperm functions. Consequently, we recommend considering the reproductive toxicity of oridonin when using it as a therapeutic agent.
Asunto(s)
Diterpenos de Tipo Kaurano , Transducción de Señal , Motilidad Espermática , Espermatozoides , Animales , Masculino , Adenosina Trifosfato/metabolismo , Supervivencia Celular/efectos de los fármacos , Diterpenos de Tipo Kaurano/efectos adversos , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/efectos de los fármacos , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , PorcinosRESUMEN
Ethylene oxide (E.O) is an epoxide compound, and it has been utilized as a sterilizer or production of ether compounds in several industries. Although the toxic effects of E.O on bacteria and mammals have been reported, its effects on male reproductive toxicity during sperm capacitation are not fully understood. Therefore, this study was designed to evaluate the effects of E.O exposure during sperm capacitation. Boar spermatozoa were treated with various E.O concentrations (0, 0.1, 1, 10, and 100⯵Ð). After exposure, sperm motility, motion kinematics, capacitation status, intracellular ATP levels, cell viability, expression levels of protein kinase A (PKA) activation, and tyrosine phosphorylation were evaluated. Results revealed that E.O exposure significantly decreased sperm motility, motion kinematics, and intracellular ATP levels but significantly increased the capacitated spermatozoa. In addition, the PKA activation and tyrosine phosphorylation were abnormally changed. According to our results, E.O may cause toxic effects on sperm function during capacitation, which induces male reproductive toxicity. Consequently, we suggest that male reproductive toxicity should be considered when using E.O.
Asunto(s)
Adenosina Trifosfato , Proteínas Quinasas Dependientes de AMP Cíclico , Capacitación Espermática , Motilidad Espermática , Espermatozoides , Masculino , Animales , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Porcinos , Fosforilación , Adenosina Trifosfato/metabolismo , Supervivencia Celular/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2â¯hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MßCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca2+) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50â¯%, 10â¯%, and 20â¯%, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5â¯%) than Heparin-LCL (10â¯%) and the control group (15.2â¯%). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species.
Asunto(s)
Citometría de Flujo , Capacitación Espermática , Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides , Animales , Masculino , Bovinos/embriología , Capacitación Espermática/efectos de los fármacos , Citometría de Flujo/veterinaria , Espermatozoides/fisiología , Espermatozoides/efectos de los fármacos , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/métodos , Femenino , Heparina/farmacologíaRESUMEN
Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.
Asunto(s)
Infertilidad Masculina , Ratones Noqueados , Motilidad Espermática , Animales , Humanos , Masculino , Ratones , Astenozoospermia/genética , Astenozoospermia/metabolismo , Astenozoospermia/patología , Sistemas CRISPR-Cas , Dineínas/metabolismo , Dineínas/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Ratones Endogámicos C57BL , Capacitación Espermática/genética , Motilidad Espermática/genética , Espermatozoides/metabolismo , Contactina 2/genética , Contactina 2/metabolismoRESUMEN
Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn2+) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn2+ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans.
Asunto(s)
Capacitación Espermática , Espermatozoides , Zinc , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Zinc/metabolismo , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Porcinos , Metaboloma , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de MasasRESUMEN
Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.
Asunto(s)
Fertilidad , Hexoquinasa , Infertilidad Masculina , Ratones Noqueados , Capacitación Espermática , Espermatozoides , Tirosina , Animales , Hexoquinasa/genética , Hexoquinasa/metabolismo , Masculino , Ratones , Fosforilación , Espermatozoides/metabolismo , Capacitación Espermática/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Fertilidad/genética , Tirosina/metabolismo , Femenino , Testículo/metabolismo , Motilidad Espermática/genética , Glucólisis , Espermatogénesis/genéticaRESUMEN
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.
Asunto(s)
Disruptores Endocrinos , Fertilidad , Plaguicidas , Humanos , Plaguicidas/toxicidad , Plaguicidas/efectos adversos , Masculino , Disruptores Endocrinos/toxicidad , Disruptores Endocrinos/efectos adversos , Animales , Fertilidad/efectos de los fármacos , Infertilidad Masculina/inducido químicamente , Exposición a Riesgos Ambientales/efectos adversos , Reproducción/efectos de los fármacos , Capacitación Espermática/efectos de los fármacosRESUMEN
Capacitation involves tyrosine phosphorylation (TP) as a key marker. Lifestyle-related factors, such as obesity and smoking, are recognized for their adverse effects on semen quality and male fertility, yet the underlying mechanisms, including their potential impact on TP, remain unclear. Moreover, the effect of sperm cryopreservation on TP at the human sperm population level is unexplored. Flow cytometry analysis of global TP was performed on pre-capacitated, post-capacitated and 1- and 3-hours' incubated fresh and frozen-thawed samples from sperm donors (n = 40). Neither being overweight nor smoking (or both) significantly affected the percentage of sperm showing TP. However, elevated BMI and smoking intensity correlated with heightened basal TP levels (r = 0.226, p = 0.003) and heightened increase in TP after 3 h of incubation (r = 0.185, p = 0.017), respectively. Cryopreservation resulted in increased global TP levels after capacitation but not immediately after thawing. Nonetheless, most donors' thawed samples showed increased TP levels before and after capacitation as well as after incubation. Additionally, phosphorylation patterns in fresh and frozen-thawed samples were similar, indicating consistent sample response to capacitation stimuli despite differences in TP levels. Overall, this study sheds light on the potential impacts of lifestyle factors and cryopreservation on the dynamics of global TP levels during capacitation.
Asunto(s)
Índice de Masa Corporal , Criopreservación , Capacitación Espermática , Espermatozoides , Tirosina , Humanos , Criopreservación/métodos , Masculino , Fosforilación , Tirosina/metabolismo , Espermatozoides/metabolismo , Adulto , Fumar Cigarrillos/efectos adversos , Preservación de Semen/métodos , Análisis de SemenRESUMEN
To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.
Asunto(s)
Reacción Acrosómica , Medios de Cultivo , Fertilización In Vitro , Capacitación Espermática , Espermatozoides , Animales , Capacitación Espermática/efectos de los fármacos , Masculino , Ratones , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Espermatozoides/metabolismo , Fertilización In Vitro/métodos , Femenino , Reacción Acrosómica/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Fosforilación , Fertilización , Desarrollo Embrionario/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismoRESUMEN
Context The Rsa I polymorphism of the melatonin receptor MTNR1A gene affects seasonal reproduction in sheep, but its effect on ram spermatozoa and their response to melatonin is unknown. Aims This study aims to evaluate whether Rsa I polymorphism of the MTNR1A gene influences the response of ram spermatozoa to in vitro added melatonin. Methods Spermatozoa from rams carrying different Rsa I allelic variants were incubated with melatonin in a TALP medium or a capacitation-triggering medium during the reproductive and non-reproductive seasons. After incubation, sperm motility, membrane integrity, mitochondria activity, oxidative damage, apoptotic markers and capacitation status were assessed. Key results In the reproductive season, the T/T genotype was related to some adverse effects of melatonin when spermatozoa were incubated in TALP medium, whereas the C/C genotype was linked with adverse effects when the hormone was added in a capacitation-triggering medium. The decapacitating effect of melatonin on spermatozoa was also different depending on genotype. Conclusions The melatonin effect on spermatozoa from rams carrying different Rsa I genotypes differed depending on the season and the medium. Implications The knowledge of the Rsa I allelic variant of the MTNR1A gene of rams could be helpful when carrying out in vitro reproductive techniques in the ovine species.
Asunto(s)
Melatonina , Estaciones del Año , Motilidad Espermática , Espermatozoides , Melatonina/farmacología , Animales , Masculino , Espermatozoides/efectos de los fármacos , Ovinos , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/genética , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismo , Polimorfismo Genético , Alelos , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/genética , GenotipoRESUMEN
In brief: Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. Abstract: To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. ß-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.
Asunto(s)
Capacitación Espermática , Motilidad Espermática , Espermatozoides , Taurina , Animales , Cricetinae , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Mesocricetus , Especies Reactivas de Oxígeno/metabolismo , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.
Asunto(s)
AMP Cíclico , Capacitación Espermática , Espermatozoides , Proteína que Contiene Valosina , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Proteína que Contiene Valosina/metabolismo , Proteína que Contiene Valosina/genética , Espermatozoides/metabolismo , Ratones , AMP Cíclico/metabolismo , Fosforilación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.
Asunto(s)
Capacitación Espermática , Espermatozoides , Animales , Capacitación Espermática/efectos de los fármacos , Masculino , Caballos/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Calcio/metabolismo , Zona Pelúcida/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , FosforilaciónRESUMEN
Nirmatrelvir (NMV) is a recently developed selective inhibitor of the main protease of Sars-Cov-2 that reduces the severity of infection. Despite its widespread use and various side effects, NMV's effect on male fertility is still unclear. This study was thus established to investigate how NMV affects male fertility. For experiments, Duroc spermatozoa were incubated with various concentrations of NMV (0, 0.1, 1, 10, 50, and 100 µM). Then, sperm motility, motion kinematics, capacitation status, intracellular ATP level, and cell viability were evaluated. In addition, the expression levels of phospho-PKA substrates, tyrosine-phosphorylated proteins, and PI3K/PDK1/AKT signaling pathway-related proteins were measured by western blotting. Our results showed that sperm motility, motion kinematics, proportion of capacitated spermatozoa, and intracellular ATP level were significantly decreased by NMV in a dose-dependent manner. Moreover, PKA activation was significantly suppressed by NMV, and expression levels of PI3K, phospho-PDK1, AKT, and phospho-AKT (Thr308 and Ser473) were significantly increased in a dose-dependent manner. Combining these findings, it is suggested that NMV has detrimental effects on sperm function by inducing abnormal changes in the PI3K/PDK1/AKT signaling pathway, resulting in PKA deactivation. Therefore, there is a need to pay particular attention to its male reproductive toxicity when NMV is administered.