The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment

Abstract

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 μM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.

Keywords: CellROX probes; lipid peroxidation; ovine; oxidative stress; reactive oxygen species; sperm.

Figure 1

Multiparametric flow cytometry analyses and motility and kinetic parameters of ram sperm from the four experimental groups [fresh in breeding season (BS), fresh in nonbreeding season (NBS), positive control treated with hydrogen peroxide (300 μM) (Control +) and cryopreserved (Cryo)]. (A) Viability (%), (B) apoptosis (%), (C) total motility (TM, %), (D) progressive motility (PM, %), (E) curvilinear velocity (VCL, μm/s), and (F) amplitude of the lateral displacement of the sperm head (ALH, μm). Significant differences (P < 0.05) among experimental groups are noted with different lowercase letters (a, b). The same seven males were analyzed in each experimental group.

Figure 2

Redox status analyses in ram sperm from the four experimental groups using different assays [fresh in breeding season (BS), fresh in nonbreeding season (NBS), positive control treated with hydrogen peroxide (300 μM) (Control +) and frozen-thawed (Cryo)]. (A) CellROX™ Deep Red-positive cells (%), (B) CellROX™ Green-positive cells (%), (C) MDA generation (nmol/mL), and (D) high DNA fragmentation index (hDFI, %). Significant differences (P < 0.05) among experimental groups are noted with different lowercase letters (a, b). The same seven males were analyzed in each experimental group.

Figure 3

Correlation matrix of all sperm quality markers evaluated highlighting the correlations between the CellROX™ Deep Red and CellROX™ Green fluorescent probes. The four experimental groups [fresh samples from breeding and nonbreeding seasons, frozen-thawed and positive control treated with hydrogen peroxide (300 μM)] were included to construct the correlation matrix. Viability (Zombie-negative cells), apoptosis (caspase-3/7-positive cells), TM (total motility), PM (progressive motility), VCL (curvilinear velocity), ALH (amplitude of the lateral displacement of the sperm head), CR Deep Red (CellROX™ Deep Red-positive cells), CR Green (CellROX™ Green-positive cells), MDA (malondialdehyde generation), and hDFI (high DNA fragmentation index). The R squared value between two parameters is noted in each cell. Blue indicates positive correlations, and red indicates negative correlations. The color intensity represents the strength of the correlation between two sperm quality parameters. A total of seven males were analyzed (the same number of samples is required for this analysis). Asterisks show significant correlations among sperm quality parameters. The number of asterisks (*) indicates the level of significance: (*) indicates P < 0.05, two asterisks (**) indicate P < 0.01, and three asterisks (***) indicate P < 0.001.

Figure 4

Intracellular reactive oxygen species localization by confocal microscopy in ram sperm samples. (A, B) Light field image of ram sperm samples. (C) Middle piece of the sperm stained red with the CellROX™ Deep Red fluorescent probe. (D) Sperm nucleus stained green with the CellROX™ Green fluorescent probe.

Figure 5

Flow cytometry analysis from viable sperm (CellROX™ Deep Red and Green), and intracellular reactive oxygen species localization by confocal microscopy in fresh (breeding season) and positive control [treated with hydrogen peroxide (300 μM)] ram sperm samples. (A) CellROX™ Deep Red positive viable sperm. (B) CellROX™ Green positive viable sperm. (C) Light field image of the BS sample. (D) Light field image of the positive control sample. (E) CellROX™ Deep Red fluorescent probe in the BS sample (middle piece stained red). (F) CellROX™ Deep Red fluorescent probe in the positive control sample (middle piece not stained). (G) CellROX™ Green fluorescent probe in the BS sample (nucleus not stained). (H) CellROX™ Green fluorescent probe in the positive control sample (nucleus stained green). The number of asterisks (*) indicates the level of significance: one asterisk (*) indicates P < 0.05, and three asterisks (***) indicate P < 0.001.