Testis-specific peroxiredoxin 4 variant is not absolutely required for spermatogenesis and fertility in mice

Takujiro Homma,Naoki Ishii,Toshihiro Kurahashi,Nobuyuki Shirasawa,Junichi Fujii

doi:10.1038/s41598-020-74667-9

Takujiro Homma, Naoki Ishii + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-74667-9

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Abstract

PRDX4, a member of peroxiredoxin family, is largely concentrated in the endoplasmic reticulum (ER) and plays a pivotal role in the redox relay during oxidative protein folding as well as in peroxidase reactions. A testis-specific PRDX4 variant transcript (PRDX4t) lacks the conventional exon 1, which encodes the signal peptide that is required for entry into the ER lumen, but instead carries alternative exon 1, which is transcribed from the upstream promoter in a testis-specific manner and results in the PRDX4t protein being localized in the cytosol. However, the potential roles of PRDX4t in male genital action remain unknown. Using a CRISPR/Cas9 system, we first disrupted the testis-specific promoter/exon 1 and generated mice that were specifically deficient in PRDX4t. The resulting PRDX4t knockout (KO) mice underwent normal spermatogenesis and showed no overt abnormalities in the testis. Mating PRDX4t KO male mice with wild-type (WT) female mice produced normal numbers of offspring, indicating that a PRDX4t deficiency alone had no effect on fertility in the male mice. We then generated mice lacking both PRDX4 and PRDX4t by disrupting exon 2, which is communal to these variants. The resulting double knockout (DKO) mice were again fertile, and mature sperm isolated from the epididymis of DKO mice exhibited a normal fertilizing ability in vitro. In the meantime, the protein levels of glutathione peroxidase 4 (GPX4), which plays an essential role in the disulfide bond formation during spermatogenesis, were significantly increased in the testis and caput epididymis of the DKO mice compared with the WT mice. Based on these results, we conclude that the disruption of the function of PRDX4t in the spermatogenic process appears to be compensated by other factors including GPX4.

Highlights

Peroxiredoxin (PRDX) catalyzes the reductive removal of hydrogen peroxides using thioredoxin as an electron donor and plays multiple roles in redox reactions including intracellular signaling[1,2]
Western blot analyses indicated that the PRDX4t protein was produced in the testis at 28 days after birth (P28) and the levels were further increased in parallel with sexual maturation with an increase in sperm count whereas the conventional PRDX4 remained unchanged (Fig. 1A–C)
Consistent with previous r eports[12,13], we confirmed that PRDX4t was produced exclusively in round spermatids, elongating spermatids, and spermatozoa in WT mouse testes by immunofluorescence analyses (Fig. 1D and Supplementary Figure S2), suggesting that it has a potential role in mouse spermatogenesis

Summary

Introduction

Peroxiredoxin (PRDX) catalyzes the reductive removal of hydrogen peroxides using thioredoxin as an electron donor and plays multiple roles in redox reactions including intracellular signaling[1,2]. The genetic knockout (KO) of the systemic promoter/exon[1] of the Prdx[4] gene results in the complete inhibition of PRDX4 production in most tissues in mice[14], but the PRDX4t protein continues to be present in a certain stage of spermatogenic cells, to a slightly decreased e xtent[9,13] This appears to be associated with a delay in the sexual maturation, adult PRDX4 KO male mice are fertile and show nearly normal phenotypes in most t issues[14]. The protamines of primates and rodents contain multiple cysteine residues that are oxidized to form disulfide bridges This formation furnishes chromatin for resistance against oxidative stress and compacts the sperm nucleus, suggesting that enzymes responsible for the crosslinking could play a pivotal role in producing functional spermatozoa. Given the role of PRDX4 in oxidative protein folding in the ER, it is possible that PRDX4t is involved in the introduction of disulfide bridges in protamines during oxidative chromatin packaging in s perm[20]

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