**1. Introduction**

Peroxiredoxins are found in all living organisms, from bacteria, plants, yeasts to animals, where they act as scavengers of hydrogen peroxide (H2O2), lipid peroxides and peroxynitrite. Mammalian Peroxiredoxins (PRDXs) are important, not only as antioxidant enzymes preventing reactive oxygen species (ROS)-induced cell damage, but also as physiological regulators and sensors in a variety of cell and tissue types [1]. Indeed, the activation of several phosphatases, kinases, and tumor suppressor proteins have been shown to require a certain level of H2O2 acting as a second messenger in the vicinity of the enzymes, which is achieved by the transient and localized inhibition of PRDXs [2,3]. PRDXs are classified depending on the cysteine residues (Cys) in their active site, that will react with peroxides. They comprise the 2-Cys PRDX1 to 4, the atypical 2-Cys PRDX5, and the 1-Cys PRDX6, which has the particularity of being bifunctional, with both peroxidase and calcium-independent phospholipase A2 (iPLA2) activities [4]. The 2-Cys PRDX1 to 4 are homodimers in which the thiol of a cysteine residue of one PRDX subunit gets oxidized, then further reacts with the thiol group of the catalytic cysteine of the other subunit, forming a disulfide bond between the two subunits. By contrast, in the atypical PRDX5, 2 Cys of the same chain react upon oxidation to form an intrasubunit disulfide bond. Inactive PRDXs are then reactivated by a reduction of the disulfide bonds by thioredoxin (TRX), itself further reactivated by TRX reductase (TRD), using NADPH as a reducing equivalent. In the case of PRDX6, since the enzyme has only one catalytic Cys, the oxidized thiol will be reduced by the glutathione-GSH-transferase P1 (GSTP1) system [4–6].

Studies in PRDXs knockout mice have support the understanding of the diverse roles of these enzymes by highlighting the di fferent defects in mice deficient for a specific PRDX [7]. In particular, spermatozoa have been shown to express the six PRDX isoforms [4], which act as ROS scavengers and are required to maintain viability as well as fertilizing competence [3,6,8–10]. While ROS are needed for sperm capacitation, due to their regulatory role in the phosphorylation of key proteins, their levels must be tightly controlled to prevent damaging oxidative stress, mainly by PRDX1 and 6 in rat [8–10]. In mice, PRDX6 deficiency or inhibition of its PLA2 activity were found to impair in vitro sperm fertilizing competence [11]. Low levels of PRDX6 were observed in infertile men, positioning PRDX6 as the first line of defense against oxidative stress in human spermatozoa [12]. Although spermatogenesis occurs in the testes of PRDX6 KO mice, these animals are subfertile, with defective and underperforming spermatozoa, suggesting potential alterations of some of the processes leading to sperm formation. While the importance of PRDXs on sperm integrity and function is clear, little is known on the role of PRDXs in germ cells from primordial germ cells to spermatids.

The goal of this study was to examine the expression and role of PRDXs in neonatal gonocytes (also called pre-/pro-spermatogonia), the direct precursors of spermatogonial stem cells and first wave spermatogonia [13,14]. Gonocytes di fferentiate from primordial germ cells in the fetal gonad primordium, and undergo distinct phases of development, including successive phases of proliferation and quiescence in the fetus, resuming mitosis at postnatal day (PND) 3 in the rat, and simultaneously migrating toward the basement membrane of the seminiferous tubules where they di fferentiate to spermatogonia around PND6 [15]. We have previously shown that rat neonatal gonocyte di fferentiation is regulated by all trans-retinoic acid (RA) [16,17]. Extensive cell remodeling takes place during the proliferation, relocation and di fferentiation of neonatal gonocytes, in part regulated by the ubiquitin proteasome system [18]. We recently reported that neonatal gonocytes express high levels of cyclooxygenase 2 (COX2) and produce prostaglandins [19]. While COX2 and prostaglandins were reported to regulate ROS production in Sertoli cells [20], in other cell types such as the kidney mesanglial cell, ROS were shown to regulate COX2 expression and prostaglandin synthesis [21]. However, nothing is known on ROS formation and the antioxidant machinery in neonatal gonocytes. The present study demonstrates that PRDXs are essential for maintaining ROS homeostasis and cell viability in neonatal gonocytes, and that the iPLA2 activity of PRDX6 in these cells is not as critical as it is in spermatozoa, suggesting di fferential role for these antioxidant enzymes at di fferent phases of germ cell development.
