Fluorimetric Techniques for the Assessment of Sperm Membranes

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 protocol^1,3.

1. Obtain approximately 1–6 mL of bull semen in a 15 mL tube at room temperature.
2. To each 1 mL of semen, add 6 mL of prewarmed (at 37 °C) NKM buffer (110 mM Na....

Results

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.

Discussion

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 others^17<.......

Materials

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