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A Human Sperm-Zona Pellucida Binding Test Using Oocytes That Failed to Fertilize In Vitro
1266
MALE INFERTILITY
Temporal Changes in Motility Parameters Related to Acrosomal Status: Identification and Characterization of Populations of Hyperactivated Human Sperm L. ROBERTSON, D. P. WOLF AND J. s. TASH, Department of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas, and Oregon Regional Primate Research Center, Beaverton and Oregon Health Sciences University, Portland, Oregon Biol. Reprod., 39: 797-805, 1988 The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. In this study, video microscopy and digital image analysis were used to measure curvilinear (VCL) and straight line (VSL) velocity, average linearity of progression (LIN [100 x VSL/VCLJ), maximum and mean amplitude of lateral head displacement (ALH), beatcross-frequency (BCF), DANCE (VCL x meanALH) andDANCEMEAN (meanALH/(LIN/100)). These parameters were measured for sperm in semen and in the swim-up fraction of washed cells during incubation for up to 24 h under in vitro fertilization (IVF) conditions. Acrosomal loss was monitored in the same population of washed cells by an immunofluorescence end-point assay. The greatest increase in mean values of motility parameters was observed when seminal sperm were washed free of seminal plasma. Increases continued for up to 6 h of incubation. Two subpopulations of hyperactivated sperm were identified; one type, not found in semen, showed star-spin trajectories, and constituted 3.0, 3.8, 4.5, and 4.1 % of the swim-up population after 0, 3, 6 and 24 h of incubation. The second type, termed transitional showed a more progressive trajectory and constituted less than 1 % in semen. In total, hyperactivated cells constituted 0.8% of cells in semen, 14.5% of the swim-up population with no incubation, and 23.1, 22.7, and 19.4% after 3, 6, and 24 h of incubation, respectively. Acrosomal loss in the swim-up population was delayed during the first 3 h of incubation, then increased from near 5% at 3 h to 7 and 12% at 6 and 24 h, respectively. The kinetics of change in the extent of hyperactivation and in acrosomal loss, although measured in different cell populations, are consistent with an association between these two events.
Editorial Comment: Sperm require a period of maturation that normally occurs in the female reproductive tract, called capacitation, before they attain the ability to undergo an acrosome reaction and fertilize the egg. Capacitated sperm are not morphologically different from noncapacitated sperm. However, capacitated sperm exhibit a vigorous, less linear, form of movement termed hyperactivated motility. Hyperactivated motility is considered an integral part of the capacitation process and a prerequisite for fertilization in many mammalian species. The authors demonstrate that hyperactivation of motility occurs before the acrosome reaction, another key prefertilization event. Hyperactivation may prove to be a useful marker for the progression of capacitation before acrosomal loss. Although computer-assisted analysis of sperm motility seldom is helpful in the management of the subfertile man these systems currently provide the ability to study the motion of individual sperm populations in great detail. The quantification of hyperactivation in male factor infertility patients should provide additional insight into
functional defects present in men with decreased sperm production. John D. McConnell, M.D. A Human Sperm-Zona Pellucida Binding Test Using Oocytes That Failed to Fertilize In Vitro D. Y. LIU, A. LOPATA, W. I. H. JOHNSTONE AND H. W. G. BAKER, University of Melbourne, Royal Women's Hospital, Melbourne, Victoria, Australia Fertil. Steril., 50: 782-788, 1988 A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetramethylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.
Editorial Comment: Sperm binding to the zona pellucida is a critical step in human fertilization. Since the zona pellucida is the species specific barrier to sperm binding, it is digested enzymatically in assays that use the eggs of other species (for example hamster) to determine human sperm-egg interaction. Thus, the hamster oocyte penetration assay does not test the ability of a given infertile man's sperm to bind to the zona pellucida. Experiments in other mammalian species have demonstrated that inhibition of this binding step (for example by antibodies) can prevent fertilization. In theory, specific defects in sperm-zona pellucida binding could be present. The zona pellucida from oocytes that failed to fertilize during in vitro fertilization were used in an assay system to measure sperm-zona pellucida binding. Preliminary data in this study suggest that there is a subset of men who have decreased fertilization capacity because of abnormalities related to sperm-zona pellucida binding. Further study will be required to determine the predictive value of this test and, more importantly, to determine the exact biochemical or genetic defects present in the sperm head of men who have impaired zona pellucida binding. John D. McConnell, M.D. Restricted Lesions After Testicular Biopsies in Young and Adult Rats I. SJOGREN AND L. PLOEN, Department of Anatomy and His-
tology, Swedish University of Agricultural Sciences, Uppsala and Department of Toxicology, Kabi Vitrum AB, Stockholm, Sweden Int. J. Androl., 11: 525-531, 1988 In order to evaluate the possible harmful effects of surgical removal of a testicular biopsy, adult and immature rats were subjected to unilateral testicular biopsy and were studied 2-4
MALE INFERTILITY
Temporal Changes in Motility Parameters Related to Acrosomal Status: Identification and Characterization of Populations of Hyperactivated Human Sperm L. ROBERTSON, D. P. WOLF AND J. s. TASH, Department of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas, and Oregon Regional Primate Research Center, Beaverton and Oregon Health Sciences University, Portland, Oregon Biol. Reprod., 39: 797-805, 1988 The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. In this study, video microscopy and digital image analysis were used to measure curvilinear (VCL) and straight line (VSL) velocity, average linearity of progression (LIN [100 x VSL/VCLJ), maximum and mean amplitude of lateral head displacement (ALH), beatcross-frequency (BCF), DANCE (VCL x meanALH) andDANCEMEAN (meanALH/(LIN/100)). These parameters were measured for sperm in semen and in the swim-up fraction of washed cells during incubation for up to 24 h under in vitro fertilization (IVF) conditions. Acrosomal loss was monitored in the same population of washed cells by an immunofluorescence end-point assay. The greatest increase in mean values of motility parameters was observed when seminal sperm were washed free of seminal plasma. Increases continued for up to 6 h of incubation. Two subpopulations of hyperactivated sperm were identified; one type, not found in semen, showed star-spin trajectories, and constituted 3.0, 3.8, 4.5, and 4.1 % of the swim-up population after 0, 3, 6 and 24 h of incubation. The second type, termed transitional showed a more progressive trajectory and constituted less than 1 % in semen. In total, hyperactivated cells constituted 0.8% of cells in semen, 14.5% of the swim-up population with no incubation, and 23.1, 22.7, and 19.4% after 3, 6, and 24 h of incubation, respectively. Acrosomal loss in the swim-up population was delayed during the first 3 h of incubation, then increased from near 5% at 3 h to 7 and 12% at 6 and 24 h, respectively. The kinetics of change in the extent of hyperactivation and in acrosomal loss, although measured in different cell populations, are consistent with an association between these two events.
Editorial Comment: Sperm require a period of maturation that normally occurs in the female reproductive tract, called capacitation, before they attain the ability to undergo an acrosome reaction and fertilize the egg. Capacitated sperm are not morphologically different from noncapacitated sperm. However, capacitated sperm exhibit a vigorous, less linear, form of movement termed hyperactivated motility. Hyperactivated motility is considered an integral part of the capacitation process and a prerequisite for fertilization in many mammalian species. The authors demonstrate that hyperactivation of motility occurs before the acrosome reaction, another key prefertilization event. Hyperactivation may prove to be a useful marker for the progression of capacitation before acrosomal loss. Although computer-assisted analysis of sperm motility seldom is helpful in the management of the subfertile man these systems currently provide the ability to study the motion of individual sperm populations in great detail. The quantification of hyperactivation in male factor infertility patients should provide additional insight into
functional defects present in men with decreased sperm production. John D. McConnell, M.D. A Human Sperm-Zona Pellucida Binding Test Using Oocytes That Failed to Fertilize In Vitro D. Y. LIU, A. LOPATA, W. I. H. JOHNSTONE AND H. W. G. BAKER, University of Melbourne, Royal Women's Hospital, Melbourne, Victoria, Australia Fertil. Steril., 50: 782-788, 1988 A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetramethylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.
Editorial Comment: Sperm binding to the zona pellucida is a critical step in human fertilization. Since the zona pellucida is the species specific barrier to sperm binding, it is digested enzymatically in assays that use the eggs of other species (for example hamster) to determine human sperm-egg interaction. Thus, the hamster oocyte penetration assay does not test the ability of a given infertile man's sperm to bind to the zona pellucida. Experiments in other mammalian species have demonstrated that inhibition of this binding step (for example by antibodies) can prevent fertilization. In theory, specific defects in sperm-zona pellucida binding could be present. The zona pellucida from oocytes that failed to fertilize during in vitro fertilization were used in an assay system to measure sperm-zona pellucida binding. Preliminary data in this study suggest that there is a subset of men who have decreased fertilization capacity because of abnormalities related to sperm-zona pellucida binding. Further study will be required to determine the predictive value of this test and, more importantly, to determine the exact biochemical or genetic defects present in the sperm head of men who have impaired zona pellucida binding. John D. McConnell, M.D. Restricted Lesions After Testicular Biopsies in Young and Adult Rats I. SJOGREN AND L. PLOEN, Department of Anatomy and His-
tology, Swedish University of Agricultural Sciences, Uppsala and Department of Toxicology, Kabi Vitrum AB, Stockholm, Sweden Int. J. Androl., 11: 525-531, 1988 In order to evaluate the possible harmful effects of surgical removal of a testicular biopsy, adult and immature rats were subjected to unilateral testicular biopsy and were studied 2-4