Treatment of cryopreserved bovine sperm with calcium ionophore A23187 increases in vitro embryo production.

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.

Effect of tyrode albumin lactate pyruvate and modified Krebs Ringers broth media on in vitro capacitation of HD-K75 boar spermatozoa at different period of incubation.

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.

The Role of Erythropoietin in Bovine Sperm Physiology.

Erythropoietin (EPO), a hormone secreted mainly by the kidney, exerts its biological function by binding to its cell-surface receptor (EpoR). The presence of EPO and EpoR in the male and female reproductive system has been verified. Therefore, some of the key properties of EPO, such as its antioxidant and antiapoptotic effects, could improve the fertilizing capacity of spermatozoa. In the present study, the effect of two different concentrations of EPO (10 mIU/µL and 100 mIU/µL) on bovine sperm-quality parameters was evaluated during a post-thawing 4-h incubation at 37 °C. EPO had a positive effect on sperm motility, viability, and total antioxidant capacity. Moreover, EPO inhibited apoptosis, as it reduced both BCL2-associated X apoptosis regulator (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and cleaved cysteine-aspartic proteases (caspases) substrate levels in a dose-dependent manner. In addition, EPO induced sperm capacitation and acrosome reaction in spermatozoa incubated in capacitation conditioned medeia. These results establish a foundation for the physiological role of EPO in reproductive processes and hopefully will provide an incentive for further research in order to fully decipher the role of EPO in sperm physiology and reproduction.

Inhibition of forward and reverse transport of Ca2+ via Na+/Ca2+ exchangers (NCX) prevents sperm capacitation.

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.

Reviewing mathematical models of sperm signaling networks.

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.

Ethylene oxide suppresses boar sperm function during capacitation.

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 µÐœ). 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.

Glucose prevents the acquisition of the capacitated state in pig spermatozoa.

BACKGROUND: Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species-specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction. OBJECTIVES: To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process. MATERIAL AND METHODS: The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content. RESULTS: The presence of glucose in the in vitro capacitating medium diminishes, in a concentration-dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non-metabolizable substrate (l-glucose) for 4 h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation-like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control. DISCUSSION AND CONCLUSIONS: Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization.

Influence of BMI, Cigarette Smoking and Cryopreservation on Tyrosine Phosphorylation during Sperm Capacitation.

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.

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

BACKGROUND: Exposure 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. RESULTS: Lead 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. CONCLUSIONS: Pb2+ 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.

Assessing the Risks of Pesticide Exposure: Implications for Endocrine Disruption and Male Fertility.

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.

Metabolic Shift in Porcine Spermatozoa during Sperm Capacitation-Induced Zinc Flux.

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.

Production of bovine embryos by piezo-ICSI using capacitated spermatozoa selected by fluorescence-activated cell sorting (FACS-piezo-ICSI).

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 2 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 (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). 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.

Characterization of expression patterns and dynamic relocation of Notch proteins during acrosome reaction of bull spermatozoa.

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Effect of exogenous sperm capacitation inducers on stallion sperm.

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.

A side-by-side comparison of different capacitation media in developing mouse sperm fertilizing ability.

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.

Effects of MnTBAP on Porcine Semen Cryopreservation and Capacitation.

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 µM of MnTBAP based on a Tris-egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 µM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 µM and 150 µM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 µM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 µM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 µM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze-thawing, improving the fertilization capacity, and leading to increased embryo development.

Sperm Incubation in Biggers-Whitten-Whittingham Medium Induces Capacitation-Related Changes in the Lizard Sceloporus torquatus.

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that ensure reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. We aimed to perform a sperm analysis to determine if capacitation-related changes were induced after incubation with capacitating media. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 µL using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 h. A fraction was retrieved hourly for ongoing sperm assessment. The sperm analysis included assessments of its motility, viability, the capacitation status using the chlortetracycline (CTC) assay, and the acrosome integrity with the lectin binding assay to detect changes during incubation. We found that total motility was maintained up to 2 h post incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The CTC assay indicated a reduction in the percentage of sperm showing the full (F) pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. Our observations support the notion that this process may also occur in saurian. While sperm analysis is a valuable method for assessing certain functional changes, additional approaches are required to validate this process.

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

BACKGROUND: 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. METHODS: 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. RESULTS: Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45 min 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 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (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 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). CONCLUSION: 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.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.