- Email: [email protected]
Interaction of bovine sperm with cervical mucins
Abstracts / Animal Reproduction Science 169 (2016) 99–135
lular oxidative effect. The predictive models provided the effective concentration (EC50% ) of 122.7 M in an average time (T1/2 ) of 3.28 h. Interestingly, such EC50% and T1/2 have no effect on sperm motility. For the intracellular assays, both probes DHE and CellRoxGreen were able to detect variations on oxidative stress according to the used concentrations of peroxide. However, DHE showed low relative intensity when compared to that observed by CellRoxGreen. CellRoxGreen revealed two distinct populations, while the intensity of DHE increases the incubation time increases, indicating that the EC50% have a medium effect at 2.42 h. In conclusion, both probes are potential fluorescent assay systems for detecting oxidative status of boar sperm samples. Also, these probes consistently showed an adequate response according to the concentration of the oxidizing agent and the time of incubation.
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labelled sperm to assess sperm–mucin interactions. In addition, sperm–mucin interactions were assessed by Fluorescent Activated Cell Sorting (FACS). Results: Sperm associated strongly with the follicular and luteal mucin. In comparison, sperm had a much lower level of binding to oestrous mucin. Conclusion: Sperm interact with mucins and display differential binding with mucins from different stages of the oestrous cycle. This underlines a role for mucin–sperm interactions in cervical mucus function. As cervical mucus properties change during the cycle, this may modulate any sperm–mucin interaction. At oestrous, these would facilitate sperm mucus penetration while during other parts of the cycle, they may impede sperm. While electrostatic properties may play a role in this, it is also possible that sperm may have specific binding proteins that mediate interactions with mucins.
http://dx.doi.org/10.1016/j.anireprosci.2016.03.038 P20 Interaction of bovine sperm with cervical mucins Hazem Al Mhanna, Mary Gallagher, Stephen Carrington, Colm Reid ∗ School of Veterinary Medicine, College of Health & Agricultural Sciences, University College Dublin, Dublin, Ireland E-mail address: [email protected] (C. Reid).
http://dx.doi.org/10.1016/j.anireprosci.2016.03.039 P21 Characterization of the rheotaxis response of bull sperm using a microfluidic device L. Martinez-Fresneda 1,∗ , J. Costelloe 1,2 , A. O’Hara 1 , A. Lynch 1 , S. Monsonis-Centelles 1 , D. Newport 2 , S. Fair 1 1
Introduction: In cows, the cervical mucosa consists of longitudinal folds lined by a mucus-secreting epithelium. These folds, along with the secreted mucus, form a protective barrier between the external environment and the uterus. The cervix presents an open conformation at oestrus to allow sperm entry and a closed conformation outside of oestrus to protect the uterus from infection. Mucus properties are also modulated with thin and watery secretions during oestrus, but thick and viscoelastic secretions at other times. Mucus is comprised of water, salts and lipids as well as heavily glycosylated structural proteins called mucins. Mucins consist of a peptide backbone rich in serine and threonine, which undergo O-glycosylation. The resulting glycan chains are terminated in peripheral sugars, which may be neutral or charged. Typically however, the presence of sialic acid residues imparts an overall negative charge, which may influence trans-cervical transit. Objectives: We hypothesise that ‘sperm interact directly with cervical mucins’ and ‘sperm display differential binding to mucins from non-oestrous and oestrous mucus’. To test this, we determined whether sperm could bind to purified cervical mucin and compared sperm interactions between mucin from follicular, oestrous and luteal phases of the oestrous cycle. Methods: Mucus was collected from the bovine cervix at follicular (n = 10) oestrous (n = 3) and luteal (n = 10) stages of the oestrous cycle. Mucins were purified by density gradient centrifugation and gel filtration and their identity confirmed by lectin staining. Mucins were coated on microscope slides and probed with fluorescently
Department of Life Sciences, University of Limerick, Ireland 2 Department of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick, Ireland E-mail address: [email protected] (L. Martinez-Fresneda).
Sperm rheotaxis is characterized by sperm orientating and swimming against a flow and is thought to aid sperm navigation towards the site of fertilization in the oviducts. The aim of this study was to assess the swimming behaviour and rheotaxis response of bull sperm within a microfluidic device with varying flow velocities, different media viscosities and while undergoing hyperactivation. Sperm distribution across the channel was also assessed in order to quantify sperm wall tracking behaviour. A microfluidic device (300 m wide, 100 m deep and 30 mm in length) was manufactured using polymethyl methacrylate. Following priming of the device, a pool of frozen-thawed sperm from three Holstein Friesian bulls (50 L at a concentration of 20 × 106 sperm/mL), was exposed to a flow. Rheotaxis response was assessed as the distance the tenth sperm had travelled from the sample inlet at 2, 4, 6, 8, 10, 12 and 14 min using an inverted microscope. Rheotaxis was assessed in response to Experiment 1: A range of flow velocities (0, 10, 20, 30 50, 70 and 100 m/s). Experiment 2: Varying media viscosity (1, 5 and 10 mPa s). Experiment 3: Sperm hyperactivation (5 mM caffeine). Experiment 4: Sperm distribution across the channel as assessed by the percentage of sperm
lular oxidative effect. The predictive models provided the effective concentration (EC50% ) of 122.7 M in an average time (T1/2 ) of 3.28 h. Interestingly, such EC50% and T1/2 have no effect on sperm motility. For the intracellular assays, both probes DHE and CellRoxGreen were able to detect variations on oxidative stress according to the used concentrations of peroxide. However, DHE showed low relative intensity when compared to that observed by CellRoxGreen. CellRoxGreen revealed two distinct populations, while the intensity of DHE increases the incubation time increases, indicating that the EC50% have a medium effect at 2.42 h. In conclusion, both probes are potential fluorescent assay systems for detecting oxidative status of boar sperm samples. Also, these probes consistently showed an adequate response according to the concentration of the oxidizing agent and the time of incubation.
109
labelled sperm to assess sperm–mucin interactions. In addition, sperm–mucin interactions were assessed by Fluorescent Activated Cell Sorting (FACS). Results: Sperm associated strongly with the follicular and luteal mucin. In comparison, sperm had a much lower level of binding to oestrous mucin. Conclusion: Sperm interact with mucins and display differential binding with mucins from different stages of the oestrous cycle. This underlines a role for mucin–sperm interactions in cervical mucus function. As cervical mucus properties change during the cycle, this may modulate any sperm–mucin interaction. At oestrous, these would facilitate sperm mucus penetration while during other parts of the cycle, they may impede sperm. While electrostatic properties may play a role in this, it is also possible that sperm may have specific binding proteins that mediate interactions with mucins.
http://dx.doi.org/10.1016/j.anireprosci.2016.03.038 P20 Interaction of bovine sperm with cervical mucins Hazem Al Mhanna, Mary Gallagher, Stephen Carrington, Colm Reid ∗ School of Veterinary Medicine, College of Health & Agricultural Sciences, University College Dublin, Dublin, Ireland E-mail address: [email protected] (C. Reid).
http://dx.doi.org/10.1016/j.anireprosci.2016.03.039 P21 Characterization of the rheotaxis response of bull sperm using a microfluidic device L. Martinez-Fresneda 1,∗ , J. Costelloe 1,2 , A. O’Hara 1 , A. Lynch 1 , S. Monsonis-Centelles 1 , D. Newport 2 , S. Fair 1 1
Introduction: In cows, the cervical mucosa consists of longitudinal folds lined by a mucus-secreting epithelium. These folds, along with the secreted mucus, form a protective barrier between the external environment and the uterus. The cervix presents an open conformation at oestrus to allow sperm entry and a closed conformation outside of oestrus to protect the uterus from infection. Mucus properties are also modulated with thin and watery secretions during oestrus, but thick and viscoelastic secretions at other times. Mucus is comprised of water, salts and lipids as well as heavily glycosylated structural proteins called mucins. Mucins consist of a peptide backbone rich in serine and threonine, which undergo O-glycosylation. The resulting glycan chains are terminated in peripheral sugars, which may be neutral or charged. Typically however, the presence of sialic acid residues imparts an overall negative charge, which may influence trans-cervical transit. Objectives: We hypothesise that ‘sperm interact directly with cervical mucins’ and ‘sperm display differential binding to mucins from non-oestrous and oestrous mucus’. To test this, we determined whether sperm could bind to purified cervical mucin and compared sperm interactions between mucin from follicular, oestrous and luteal phases of the oestrous cycle. Methods: Mucus was collected from the bovine cervix at follicular (n = 10) oestrous (n = 3) and luteal (n = 10) stages of the oestrous cycle. Mucins were purified by density gradient centrifugation and gel filtration and their identity confirmed by lectin staining. Mucins were coated on microscope slides and probed with fluorescently
Department of Life Sciences, University of Limerick, Ireland 2 Department of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick, Ireland E-mail address: [email protected] (L. Martinez-Fresneda).
Sperm rheotaxis is characterized by sperm orientating and swimming against a flow and is thought to aid sperm navigation towards the site of fertilization in the oviducts. The aim of this study was to assess the swimming behaviour and rheotaxis response of bull sperm within a microfluidic device with varying flow velocities, different media viscosities and while undergoing hyperactivation. Sperm distribution across the channel was also assessed in order to quantify sperm wall tracking behaviour. A microfluidic device (300 m wide, 100 m deep and 30 mm in length) was manufactured using polymethyl methacrylate. Following priming of the device, a pool of frozen-thawed sperm from three Holstein Friesian bulls (50 L at a concentration of 20 × 106 sperm/mL), was exposed to a flow. Rheotaxis response was assessed as the distance the tenth sperm had travelled from the sample inlet at 2, 4, 6, 8, 10, 12 and 14 min using an inverted microscope. Rheotaxis was assessed in response to Experiment 1: A range of flow velocities (0, 10, 20, 30 50, 70 and 100 m/s). Experiment 2: Varying media viscosity (1, 5 and 10 mPa s). Experiment 3: Sperm hyperactivation (5 mM caffeine). Experiment 4: Sperm distribution across the channel as assessed by the percentage of sperm