Dataset Information

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

ABSTRACT: Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 10⁶/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm-OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

SUBMITTER: Gimeno BF

PROVIDER: S-EPMC7823615 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Gimeno Brenda Florencia BF Bariani María Victoria MV Laiz-Quiroga Lucía L Martínez-León Eduardo E Von-Meyeren Micaela M Rey Osvaldo O Mutto Adrián Ángel AÁ Osycka-Salut Claudia Elena CE

Animals : an open access journal from MDPI 20210103 1

Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm ...[more]

PMID: 33401609

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Project description:Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Project description:We used a comprehensive quantitative proteomic profiling based on LC-MS/MS combined with TMT labeling strategy to analyze frozen-thawed Ashidan yak spermatozoa proteomic dynamic changes under three sequential conditions: density gradient centrifugation-based purification（FTH sperm）, incubation in capacitation medium(CAP sperm), and treatment with the calcium ionophore A23187 to induce the acrosome reaction process(AR sperm). FTH sperm Briefly, sperm-containing straws were thawed for 30 s at 37ºC prior to addition to a prepared 45-90% Percoll solution (Percoll P1644; 3.1mM KCl, 2.9mM NaH2PO4,80mM NaCl, 10mM HEPES, 2mM CaCl2·H2O, 0.4mM MgCl2·6H2O, 26mM Sodium DL-Lactate Solution, 25mM NaHCO3; pH 7.3-7.4) and diluted using modified Tyrode's Hepes buffered medium (Sp-TALPH, 114mM NaCl, 3.2mM KCl, 2mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 10mM HEPES, 3mg/ml BSA, 0.2mM Na pyruvate, 7.5x10-3mg/ml Gentamicin; pH 7.3-7.4) followed by centrifugation for 10 min at 700 xg to enable the isolation of highly motile sperm. To ensure that the gradient material was completely eliminated from these samples, the sperm cells collected from the bottom layer were washed two times using supplemented Sp-TALPH medium and centrifuged for 5 min at 300 xg. Sperm were then isolated and adjusted to a concentration of 100x106/mL using modified Tyrode's bicarbonate buffered medium (Sp-TALP; 114mM NaCl, 3.2mM KCl, 25mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 6mg/ml BSA, 0.2mM Na pyruvate, 5x10-3mg/ml Gentamicin; pH 7.3-7.4). A CASA approach was then used to assess sperm motility, with only those samples exhibiting >70% total motility being retained for further analyses, as per previously published protocols. An aliquot containing 100 million FTH sperm was taken from each sample for subsequent protein isolation, with the remaining sperm being incubated in capacitation medium. CAP sperm Capacitation medium was composed of Sp-TALP containing heparin (50 ug/ml) and caffeine (5 mM), and sperm were incubated in this medium for 30 min at 38.5ºC in a 5%CO2 incubator, as these conditions have previously been shown to be optimal for the induction of capacitation in frozen-thawed yak sperm. An aliquot containing 100 million CAP sperm was taken from each sample for subsequent protein isolation. AR sperm AR induction was achieved by treating 990 uL capacitated sperm samples with 10 µL of A23187 (1.0mM in DMSO, CSNpharm, IL, USA) to a final concentration of 10 µM, followed by a 15 min incubation at 38.5ºC, For individual replicates, aliquots of 100 million AR sperm were collected for subsequent protein analyses.

Dataset Information

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Publications

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Similar Datasets

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