Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Summary

This protocol describes a detailed methodology for detecting H₂O₂ localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Abstract

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H₂O₂ and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H₂O₂ with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

Introduction

The reactive oxygen species production has been linked with sperm quality recently¹. Although ROS production in mitochondria can be considered a normal physiological process, oxidative stress by an excess of ROS production is a clear cause of damage in spermatozoa at different levels. In humans, oxidative stress is associated with male infertility, altering motility and the ability to undergo capacitation²; in mammals, change of DNA integrity in frozen sperm samples has been also related to synthesis of H₂O₂³.

Cryopreservation is a common technique....

Protocol

NOTE: Fluorochrome incubation and confocal analysis will take at least 2-3 h for a control and a treated sample. Data processing is not included in this time calculation. Required materials can be found in the Table of Materials. This protocol can be applied to fresh or cryopreserved spermatozoa. Solea senegalensis is a fish species that spawns in cold water, work always under cold conditions (4-7 °C). See Figure 1 for a general view of the protocol.

1. Preparatory Work Before the Experiment

1. Prepare a 1 mM mitochondria stain stock solution in analytical grade DMSO.

Results

Confocal microscopy is an ideal method for intracellular ROS evaluation in teleost sperm. The combination of the three fluorochromes (DAPI, a mitochondria stain and DCFH-DA) presented in this study (Figure 1) provides many useful information that can be applied in basic research and can have applications in improving procedures used in industrial aquaculture plants, such as cryopreservation protocols. Different types of analysis may be carried out in order to.......

Discussion

It is well known that mitochondria are key organelles for sperm motility and function. These organelles are concurrently directly involved in ROS production. Interestingly, controlled levels of ROS are needed for proper sperm function¹. Positive relationships between fertility and oxidative stress have been shown in mammals¹¹ but excessive levels affect sperm quality¹². One crucial factor that could be decisive towards a positive or negative effect i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA)	Sigma-Aldrich	D6883
4′,6-diamidino-2-phenylindole (DAPI)	Sigma-Aldrich	D9542
CaCl2	Sigma-Aldrich	C1016
Confocal Microscopy	Zeiss	LSM800
Cover slips	Thermo Fisher Scientific	12-541B
DMSO, Analytical Grade	Sigma-Aldrich	W387520
HEPES	Sigma-Aldrich	H3375
KCl	Sigma-Aldrich	P9541
Methanol, Analytical Grade	Sigma-Aldrich	34860
MitoTrackerDeep Red	Thermo Fisher Scientific	M22426
Microcentrifuge (refrigerated)	Thermo Fisher Scientific	75002441
NaCl	Sigma-Aldrich	S7653
Neubauerchamber	Sigma-Aldrich	BR717810
Slides	Thermo Fisher Scientific	10143562BEF