Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

Abstract

The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5-/- epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5-/- male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability.

Figure 1. Generation of Gpx5-deficient mice.

(A) Schematic representation of mouse Gpx5 gene organization. Top: Gray boxes (E1 to E5) indicate the 5 GPX5 coding exons. Black arrows point to in-frame ATG translational start sites. Arrowheads indicate the relative positions of the various primers used in PCR amplification. Numbers (in bp) at the boundaries of each exon correspond to the GenBank Gpx5 sequence (accession number NM_010343), with “1” indicating the cap site. Bottom: A scheme of the targeting vector and the resulting allele is shown. In vivo Cre-mediated excision of the Gpx5 exon 2 led to a premature translation frameshift. (B) PCR experiments carried out on genomic DNA extracted from animal fingers. In the left panel, lanes were run on the same gel but were noncontiguous. (C) RT-PCR assays carried out with mRNA extracted from caput epididymal tissues. In B and C, the bp numbers indicate the size of the amplified products, and the primer pairs used are indicated at the bottom of each panel (see A). (D) Western blot assays carried out with caput epididymidis protein extracts and a polyclonal anti-GPX5 antibody directed against a C-ter synthetic peptide (31). The kDa number in the left margin indicates the size of the GPX5 full-length protein. +/+, +/–, and –/– indicate animals that were WT, KO heterozygous, or homozygous for GPX5. The H₂O lanes correspond to a control amplification carried out on water.

Figure 2. Impact of GPX5 deletion on mouse fertility.

(A) Litter sizes (each gray or white square corresponds to an independent litter for Gpx5 KO or WT males), with respect to the age of the males. Female mice had a WT genetic background. (B) Mortality of pups from matings between WT female mice and Gpx5^–/– male mice aged 13 months and 16 months. Data are mean ± SEM. (C) Examples of aberrant development in 2 distinct cases of pregnancies reported in B (from matings with Gpx5^–/– males aged 13 months).

Figure 3. Evaluation of spermatozoa integrity by cytometry.

(A) Evaluation of spermatozoa viability. Histograms show the incorporation of propidium iodide (PI) in spermatozoa from caput or cauda epididymides. (B) Disulfide bonds/free thiol quantification by mBrB staining. Histograms show the incorporation of mBrB. (C) Protamine association with sperm chromatin, as determined by chromomycin A3 (CMA3) staining. **P < 0.05; ***P < 0.01. (D) Evaluation of sperm DNA fragmentation using the Halomax detection assay, based on the sperm chromatin dispersion test (55). Data are mean ± SEM. n = 4.

Figure 4. Cauda-retrieved spermatozoa of Gpx5^–/– animals suffer oxidative damages.

(A) Immunodetection of the nuclear adduct 8-oxodG in cauda epididymidis-retrieved spermatozoa preparations from 6-month-old WT, KO, or hydrogen peroxide–treated WT male mice. Images are representative of several distinct detections. (B) Histograms showing the percentage of 8-oxodG–positive spermatozoa in cauda epididymidis-retrieved spermatozoa preparations, from 6-month-old WT, KO, and positive control (WT spermatozoa treated with hydrogen peroxide) male mice. Data are mean ± SEM. n = 3. **P < 0.05.

Figure 5. GPX5 deficiency leads to cauda epididymidis oxidative response.

(A and B) Quantitative RT-PCR estimations of Gpx1, Gpx3, and Gpx4 mRNA accumulations in caput (A) and cauda (B) epididymidis samples from WT and KO male mice. Data are mean ± SEM. n = 2. (C) Analysis of global GPX activity using either hydrogen peroxide or tert-butyl hydroperoxide (t-BOOH) as a substrate. Data are mean ± SEM. **P < 0.05; ***P < 0.01.

Figure 6. Catalase, but not eSOD3, participates in the antioxidant response of the Gpx5^–/– cauda epdidymidis.

(A) Schematic representation of the ROS recycling pathway using the catalytic triad of scavengers. (B) Quantitative RT-PCR estimations of eSOD3 and catalase mRNA accumulations in caput and cauda epididymis samples from WT and KO male mice, using the cyclophilin transcript as a reference. Data are mean ± SEM. n = 2. **P < 0.05.

Figure 7. The cauda epididymidis epithelium of Gpx5^–/– animals suffers from oxidative damage.

(A) Detection of the nuclear 8-oxodG modification in cauda epididymidis epithelium sections of 14-month-old WT and Gpx5^–/– male mice. Note the strong and uniform nuclear labeling in the Gpx5^–/– section. L, lumen; E, epithelium; ITS, intertubular space. (B) MDA measurements on cauda-collected spermatozoa samples from 6-month-old WT and Gpx5^–/– animals. Data are mean ± SEM.