OPJ  Vol.3 No.1 , March 2013
Fluorometric Viability Assessment of Capacitated and Acrosome-Reacted Boar Spermatozoa by Flow Cytometry
Abstract: Sperm capacitation involves functional changes, such as the removal or appearance of specific molecules and changes in the plasma membrane; the acrosome reaction (AR) is an exocytotic event induced by calcium influx, enabling the spermatozoa to penetrate the zona pellucida. These processes can be achieved only if the spermatozoa have good viability; indeed, determination of sperm viability is used for the assessment of semen quality. Membrane integrity and mitochondrial activity are important viability parameters of spermatozoa and fluorescent techniques based on membrane permeability to dyes have been developed to determine these parameters. The aim of this work was to determine the viability of boar sperm (fresh, one hour of capacitation induction and 20 min of AR induction) by flow cytometry using propidium iodide (PI) (1.25 μg/mL) and rhodamine 123 (R123) (0.20 μg/mL). Aliquots of 5 × 105 sperm were incubated with each fluorochrome separately and simultaneously for 10 or 20 min, respectively, at 38℃. The proportion of labeled spermatozoa and their fluorescence intensities were measured using a flow cytometer. The fluorescence index (FI) with PI gradually increased during the incubation and we found significant differences between all the groups. With R123, the FI increased in the capacitated sperm but decreased in the acrosome-reacted sperm, with significant differences between the fresh and capacitated spermatozoa. Our results suggest that the increase in the R123 fluorescence intensity in capacitated spermatozoa is due to changes in the mitochondrial membrane activity because the spermatozoa experienced changes in membrane fluidity and flagellar activation during capacitation. The use of fluorochromes and flow cytometry is a good tool for monitoring many markers of sperm function. Although capacitation and AR processes have been well studied, there is still much information to be elucidated with regard to these complex processes.
Cite this paper: R. Fierro, H. González-Márquez, R. Ortiz, J. Chevrier and B. Foliguet, "Fluorometric Viability Assessment of Capacitated and Acrosome-Reacted Boar Spermatozoa by Flow Cytometry," Optics and Photonics Journal, Vol. 3 No. 1, 2013, pp. 40-44. doi: 10.4236/opj.2013.31007.

[1]   R. Yanagimachi, “Mechanisms of Fertilization in Mammals,” In: L. Mastroianni and J. D. Biggers, Eds., Fertilization and Embryonic Development in Vitro, Plenum Press, New York, 1981, pp. 81-182.

[2]   R. Yanagimachi, “Mammalian Fertilization,” In: E. Knobil and J. D. Neill, Eds., The Physiology of Reproduction, Vol. 1, Raven Press, New York, 1994, pp. 189-317.

[3]   P. Talbot and R. Chacon, “A Triple Stain Technique for Evaluating Normal Acrosome Reactions of Human Sperm,” Journal of Experimental Zoology, Vol. 215, No. 2, 1981, pp. 201-211. doi:10.1002/jez.1402150210

[4]   R. Fierro, M. C. Bene, B. Foliguet, G. C. Faure and G. Grignon, “Evaluation of Human Sperm Acrosome Reaction and Viability by Flow Cytometry,” Italian Journal of Anatomy and Embryology, Vol. 103, No. 41998, pp. 75-84.

[5]   D. L. Garner, D. Pinkel, L. A. Johnson and M. M. Pace, “Assessment of Spermatozoal Function Using Dual Fluorescent Staining and Flow Cytometric Analyses,” Biology of Reproduction, Vol. 34, No. 1, 1986, pp. 127-138. doi:10.1095/biolreprod34.1.127

[6]   L. V. Johnson, M. L. Walsh and L. B. Chen, “Localization of Mitochondria in Living Cells with Rhodamine 123,” Proceedings of the National Academy of Sciences USA, Vol. 77, No. 2, 1980, pp. 990-994. doi:10.1073/pnas.77.2.990

[7]   D. P. Evenson, Z. Darzynkiewicz and M. R. Melamed, “Simultaneous Measurement by Flow Cytometry of Sperm Cell Viability and Mitochondrial Membrane Potential Related to Cell Motility,” The Journal of Histochemistry & Cytochemistry, Vol. 30, No. 3, 1982, pp. 279-280. doi:10.1177/30.3.6174566

[8]   M. A. Van Dilla, B. L. Gledhill, S. Lake, P. N. Dean, J. W. Gray, V. Kachel, et al., “Measurement of Mammalian Sperm Deoxyribonucleic Acid by Flow Cytometry. Problems and Approaches,” The Journal of Histochemistry & Cytochemistry, Vol. 25, No. 7, 1977, pp. 763-773. doi:10.1177/25.7.70455

[9]   M. Kerker, D. S. Wang and H. W. Chew, “An Optical Model for Fluorescence of Mammalian Sperm in Flow Cytometry,” Cytometry, Vol. 1, No. 2, 1980, pp. 161-167. doi:10.1002/cyto.990010212

[10]   R. Fierro, B. Foliguet, G. Grignon, M. Daniel, M. C. Bene, G. C. Faure, et al., “Lectin-Binding Sites on Human Sperm During Acrosome Reaction: Modifications Judged by Electron Microscopy/Flow Cytometry,” Archives of Andrology, Vol. 136, No. 3, 1996, pp. 187-196. doi:10.3109/01485019608987095

[11]   D. L. Garner and E. S. E. Hafez, “Spermatozoa and Seminal Plasma,” In: E. S. E. Hafez, Ed., Reproduction in Farm Animals, Lea & Febiger, Philadelphia, 1993, pp. 165-187.

[12]   L. A. Johnson, J. G. Aalbers, C. M. T. Willems, J. H. M. Rademaker and C. E. Rexroad Jr., “Use of Boar Spermatozoa for Artificial Insemination. III. Fecundity of Boar Spermatozoa Stored in Beltsville Liquid and Kiev Extenders for Three Days at 18?C,” Journal of Animal Science, Vol. 54, No. 1, 1982, pp. 132-136.

[13]   J. Conejo-Nava, R. Fierro, C. G. Gutierrez and M. Betancourt, “Membrane Status and in Vitro Capacitation of Porcine Sperm Preserved in Long-Term Extender at 16?C,” Archives of Andrology, Vol. 49, No. 4, 2003, pp. 287-295.

[14]   E. Bonilla, A. Amador and M. Betancourt, “In Vitro Capacitation of Boar Sperm in a Protein-Free Medium Supplemented with Histidine and Cysteine,” Medical Science Research, Vol. 22, 1994, pp. 725-726.

[15]   M. Betancourt, R. Fierro and D. Ambriz, “In Vitro Fertilization of Pig Oocytes Matured in Vitro,” Theriogenology, Vol. 40, 1993, pp. 1155-1160. doi:10.1016/0093-691X(93)90286-E

[16]   I. Jiménez, H. Gonzalez-Marquez, R. Ortiz, M. Betancourt, J. Herrera and R. Fierro, “Expression of Lectin Receptors on the Membrane Surface of Sperm of Fertile and Subfertile Boars by Flow Cytometry,” Archives of Andrology, Vol. 48, No. 2, 2002, pp. 159-166. doi:10.1080/014850102317267481

[17]   T. Berger, “Pisum Sativun Agglutinen Used as an Acrosomal Stain of Parene and Craprine Sperm,” Theriogenology, Vol. 33, 1990, pp. 689-695. doi:10.1016/0093-691X(90)90546-6

[18]   J. A. Herrera, R. Fierro, H. Zayas, A. García, J. Conejo, I. Jiménez, et al., “Acrosome Reaction in Fertile and Subfertile Boar Sperm,” Archives of Andrology, Vol. 48, No. 2, 2002, pp. 161-167. doi:10.1080/014850102317267445

[19]   L. B. Emilson, K. A. Dougherty, A. T. Cockett and R. L. Urry, “Simultaneous Determination of Human Sperm Morphology and Viability: Simple Office Technique,” Urology, Vol. 11, No. 5, 1978, pp. 488-491. doi:10.1016/0090-4295(78)90165-6

[20]   M. R. Bakst, “Fertilizing Capacity and Morphology of Fowl and Turkey Spermatozoa in Hypotonic Extender,” Journal of Reproduction and Fertility, Vol. 60, No. 1, 1980, pp. 121-127. doi:10.1530/jrf.0.0600121

[21]   A. Medrano and W. V. Holt, “Protective Effects of Glycerol during Cold Shock in Boar Spermatozoa. A Cryomicroscope Study Using Propidium Iodide and SYBR-14,” Reproduction in Domestic Animals, Vol. 31, No. 1, 1995, pp. 281-282. doi:10.1111/j.1439-0531.1995.tb00052.x

[22]   J. K. Graham, E. Kunze and R H. Hammerstedt, “Analysis of Sperm Cell Viability, Acrosomal Integrity, and Mitochondrial Function Using Flow Cytometry,” Biology of Reproduction, Vol. 43, No. 1, 1990, pp. 55-64. doi:10.1095/biolreprod43.1.55

[23]   D. J. McLean, N. Korn, B. S. Perez and R. J. Thurston, “Isolation and Characterization of Mitochondria from Turkey Spermatozoa,” Journal of Andrology, Vol. 14, No. 6, 1993, pp. 433-438.

[24]   B. Marquez and S. S. Suarez, “Different Signaling Pathways in Bovine Sperm Regulate Capacitation and Hyperactivation,” Biology of Reproduction, Vol. 70, No. 6, 2004, pp. 1626-1633. doi:10.1095/biolreprod.103.026476

[25]   L. Gillan, G. Evans and W. M. C. Maxwell, “Flow Cytometric Evaluation of Sperm Parameters in Relation to Fertility Potential,” Theriogenology, Vol. 63, No. 2, 2005, pp. 445-457. doi:10.1016/j.theriogenology.2004.09.024.