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Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.
Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.
Integrity and functionality of sperm membranes are a few of the factors indicating sperm viability and fertilization potential. The plasma membrane acts as a barrier between intracellular and extracellular compartments, thereby maintaining the cellular osmotic equilibrium1. Any stress that induces damage to the plasma membrane integrity might impair homeostasis, reduce viability and fertilization capacity, and increase cell death. For instance, cryopreservation reduces sperm viability due to damage to its plasma membrane, as a result of temperature changes and osmotic stress2. We previously reported that exposing bull sp....
All of the experiments were performed in accordance with the 1994 Israeli guidelines for animal welfare. Bovine sperm was supplied by commercial Israeli company for artificial insemination and breeding. Ejaculates of 11 bulls were evaluated in this study.
1. Sperm Sample Preparation
NOTE: The procedure is based on the Roth laboratory's protocol1,3.
Figure 1 shows simultaneous fluorimetric assessment of sperm membranes (plasma, acrosomal and mitochondrial) using PI, DAPI, FITC-PSA and JC-1. Assessment of sperm membranes using simultaneous staining with four fluorescent probes allows, for example, evaluating the proportion of sperm in each category—live vs. dead; high vs. low ΔΨm; intact vs. damaged acrosome—simultaneously for each spermatozoon.
Sperm fertilization potential depends on multiple factors reflecting its quality. A high concentration of spermatozoa and a high proportion of highly progressively motile spermatozoa might be considered high-quality semen. Nevertheless, such an evaluation does not take into account other cellular and functional parameters. The use of 'bench-top' microcapillary flow cytometer can be easily adapted to evaluation of various sperm structures using fluorescent probes, as previously shown by others17<.......
The authors would like to thank "SION" Israeli company for artificial insemination and breeding (Hafetz-Haim, Israel) for their help and cooperation, and Ms. Li Na (IMV Technologies, L'Aigle, France) for assistance with the instrument setup and training.
....Name | Company | Catalog Number | Comments |
NaCl | Sigma | S5886 | |
KCl | Sigma | P5405 | |
MOPS [3-N-morphilino propanesulfonic acid] | Sigma | M1254 | |
PBS | Sigma | P5493 | |
DMSO | Sigma | D2438 | |
Ethanol absolute | Sigma | 64-17-5 | |
Hemacytometer | Neubauer Germany | hemocytometer | |
DAPI (4',6-diamidino-2-phenylindole) | Sigma | D9542 | fluorescent probe |
PI (propidium iodide ) | Sigma | P4170 | fluorescent probe |
FITC-PSA (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin ) | Sigma | L0770 | fluorescent probe |
JC-1 (5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide) | ENZOBiochem, New York, NY, USA | ENZ52304 | fluorescent probe |
Annexin V conjugated to FITC | MACS, Miltenyi Biotec | 130-093-060 | fluorescent probe |
Annexin V binding buffer 20X stock solution | MACS, Miltenyi Biotec | 130-092-820 | buffer |
Nikon Eclipse, TE-2000-u | Nikon, Tokyo, Japan | inverted fluorescence microscope | |
Nis Elements | Nikon, Tokyo, Japan | software | |
Nikon DXM1200F | Nikon, Tokyo, Japan | digital camera | |
Guava EasyCyte Plus | IMV Technologies, L'Aigle, France | microcapillary sperm flow cytometer | |
CytoSoft | Guava Technologies Inc., Hayward, CA, USA; distributed by IMV Technologies | software | |
Buffered solution for cytometry | IMV Technologies, L'Aigle, France | 023862 | buffer |
Viability and concentration kit | IMV Technologies, L'Aigle, France | 024708 | kit for viability assessment |
Mitochondrial activity kit | IMV Technologies, L'Aigle, France | 024864 | kit for mitochondrial activity assessment |
Viability & acrosome integrity kit | IMV Technologies, L'Aigle, France | 025293 | kit for acrosome integrity assessment |
JMP-13 | SAS Institute Inc., 2004, ary, NC, USA | software | |
Bovine sperm | "SION", Israeli company for artificial insemination and dreeding, Hafetz-Haim, Israel |
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