Protective Role of Peroxiredoxins against Reactive Oxygen Species in Neonatal Rat Testicular Gonocytes

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6−/− mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.


Introduction
Peroxiredoxins are found in all living organisms, from bacteria, plants, yeasts to animals, where they act as scavengers of hydrogen peroxide (H 2 O 2 ), 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 H 2 O 2 acting as a second messenger in the vicinity of the enzymes, which is achieved by the transient and localized inhibition of PRDXs [2,3].

Animals
PND2 newborn male Sprague Dawley rats were purchased from Charles Rivers Laboratories (Saint-Constant, Quebec, Canada). The pups were handled and euthanized according to the protocols approved by the McGill University Health Centre Animal Care Committee and the Canadian Council on Animal Care. USC Institutional Animal Care and Use Committee; Martine Culty protocol #20792-AM001 (Physiology and toxicology of male reproductive system).

Gonocyte Isolation and in Vitro Culture and Treatments
Neonatal gonocytes were isolated by performing sequential enzymatic tissue dissociation together with mechanical dissociation of the pooled testes from 40 PND3 pups per experiment. This was followed by a step of differential overnight adhesion at 37 • C in medium containing 5% fetal bovine serum (FBS), and cell separation of the non-adherent cells on a 2-4% bovine serum albumin (BSA) gradient in serum-free medium on the next morning [22,23]. Enriched gonocyte preparations at 70-80% purity were obtained by pooling fractions containing high proportions of gonocytes, according to size and appearance, while a gonocyte purity above 95% was used for gene array analysis [18]. Freshly isolated gonocytes were cultured at 20 to 30,000 cells per well in 500 µL of RPMI 1640 containing 2.5% FBS, antibiotics, alone or with the PRDX inhibitors conoidin A, MJ33 and ezatiostat, and/or H 2 O 2 , at different concentrations, for 2 to 18 h, in 3.5% CO 2 , at 37 • C. Cell differentiation was examined by treating the gonocytes with 10 −6 M retinoic acid (RA), in the absence or presence of the PRDX inhibitors.

Cell Viability
Cell viability was assessed using a Trypan blue exclusion assay, by counting live and trypan blue-positive gonocytes as previously described [19], and viability was expressed as the mean ± SEM of the percentage of live cells against the total number of gonocytes in 3 independent experiments, each performed with triplicates.

RNA Extraction and Real-Time Quantitative PCR (Q-PCR) Analysis
Total RNA was extracted from cell pellets using the PicoPure RNA isolaton kit (Arcturus, Mountain View, CA, USA) and digested with DNase I (Qiagen, Santa Clarita, CA, USA), followed by cDNA synthesis with a single-strand cDNA transcriptor synthesis kit (Roche Diagnostics, Indianapolis, IN, USA), as previously described [18,19]. Quantitative real-time PCR (qPCR) was performed using SYBRgreen PCR Master Mix kit (Bio-Rad, Hercules, CA, USA) on a LightCycler 480 (LC480, Roche Diagnostics) [18]. The forward and reverse primers used are provided in Table 1. The comparative Ct method was used to calculate the relative expression of the differentiation marker Stra8, and PRDX 1 to 6, using 18S rRNA as housekeeping gene for data normalization. Changes in Stra8 gene expression are expressed as percent of the control values; and given as means ± SEM from 3 or 4 experiments, each using triplicates. PRDX data are expressed in relative gene expression and shown as the means ± SEM from 3 experiments.

Gene Array Analysis
Briefly, the RNA samples isolated from three independent PND3 gonocytes and PND8 spermatogonia cell preparations, each made from multiple rat testes (each sample corresponding to the RNA of 60-90 pups for gonocytes, and 10 pup rats for spermatogonia) were analyzed using Illumina RatRef-12 Expression BeadChips, as previously described [18]. The relative levels of antioxidant genes were expressed in arbitrary units and presented as the means ± SEM of all data.
As a comparison, the RNA from an enriched germ cell population prepared from the pooled testes of 4 PND60 adult rats was also analyzed in the gene arrays, as described before [24].

Lipid Peroxidation Measurement by Bodipy Labelling
Lipid peroxidation was measured using the fluorescent lipid peroxidation sensor BODIPY 581/591 C 11 , a reporter fatty acid labelled with bodipy (4,4-difluoro-3a,4adiaza-s-indacene) fluorophore, which can enter the cells and is used as a surrogate for endogenous lipids (Bodipy; Life Technologies (Burlington, Ontario, ON, Canada)). The peroxidation of the reporter fatty acid leads to a shift in the fluorescence of BODIPY from red to green in the cells. Thus, cells presenting green fluorescence corresponds to cells in which lipid peroxidation occurred, which can be quantified by assessing their proportion in each sample. Following 1.5 h of gonocyte treatments with either medium, PRDXs inhibitors and/or H 2 O 2 , the Bodipy reagent was added to the wells at 20 µM final concentration for an additional 30 min at 37 • C. The reactions were stopped by collecting and centrifuging the cells at 425× g for 10 min at 4 • C. The pellets were washed with PBS, the cells fixed with paraformaldehyde (3.5% final) for 7 min, washed and further collected by cytospin centrifugation on microscopic slides. For each slide, 5 pictures were taken using FITC (oxidized Bodipy reporter) and Texas Red (non-oxidized reporter) fluorescence, on a Leica fluorescent microscope. Lipid peroxidation was measured as the percent of Bodipy-positive green fluorescent cells over the total gonocyte numbers. Data are shown as the means ± SEM of samples from 3 different experiments.

Immunohistochemistry (IHC)/Immunocytochemistry (ICC)
For IHC, PND3 testis samples were fixed with 4% paraformaldehyde solution (PFA), paraffin embedded, and 4 to 6 µm thick sections were made. IHC and ICC analyses were performed as previously described [18,19,23,25]. Briefly, the slides were dewaxed, rehydrated, treated for antigen retrieval, and then with a blocking reagent in PBS. The slides were then incubated overnight at 4 • C in Anti PRDX6 primary antibody diluted (1:100) in PBS containing 0.1% Triton X-100 and serum. This was followed by washes and 1-h incubation with a biotinylated secondary antibody (dilution 1:100) in 0.1% Triton X-100 and 10% BSA, at room temperature, then 15 min of treatment with Streptavidin-horse radish peroxidase and 15 min with an AEC Chromogen solution. Mayer's hematoxylin was used for counterstaining, and Clear-Mount for coating. The slides were examined using bright-field microscopy. As negative controls, some slides were treated with non-specific immunoglobulin G instead of specific antibody.
For ICC, the protein expression of PRDX1, 2, and 6 were examined in low purity gonocyte fractions pooled, washed with PBS, and fixed with 3.5% paraformaldehyde right after the BSA gradient. The fixed cells were collected by cytospin centrifugation, the slides dried and treated with acetone:methanol (60:40), followed by the antigen retrieval solution. The ICC reactions were similar to those described above for IHC, except for the use of fluorescent secondary antibodies. DAPI was used as a nuclear signal. Rabbit and mouse IgG were used as negative controls and gave no fluorescent signal (data not shown). Pictures of fluorescent signals and bright field were taken, using the same time of exposure for the specific antibodies and non-specific IgGs. Representative samples are shown.

Statistical Analysis
Statistical analysis was performed using the unpaired Student's t test or one-way ANOVA with post-hoc Tukey multiple comparison analysis, using Prism version 5.04 (GraphPad Software, San Diego, CA, USA). Changes with p values ≤ 0.05 were considered significant.

PRDXs Are among the Highest Expressed Antioxidant Genes in Neonatal Gonocytes and in Spermatogonia
Gene array analysis of antioxidant genes in highly purified rat PND3 gonocytes and PND8 spermatogonia showed that, at both ages, Prdxs were among the most abundant antioxidant genes ( Figure 1A, Table S1). Comparing the signal intensities of all genes in the arrays showed that thioredoxin 1 (Txn1), Prdx1 and superoxide dismutase (Sod) 1 were among the 1% most abundant genes in these cells, comprising relative signal intensities around and above 2000. Next, Prdx2, Prdx5, Prdx6, Sod2, Gstp1, and Gpx4 were among the most abundant genes, with relative signal intensities from 600 to 1200 (Table S1). Other highly expressed genes (signal intensities between 300 and 600) included Gsto1, Prdx3, Txnl1, Mgst1, Txnrd1, Gstp2, Gpx1, and Nrf2. As a comparison, these genes had much higher signal intensities than 65 percent of the genes in the cells, which presented intensities below or around 20, levels found for Sod3 and Catalase. The ranking of expression for PRDXs were Prdx1 > Prdx2 > Prdx5 > Prdx6 > Prdx3, in both germ cell types. Measurement of the relative expression of the PRDXs by qPCR in PND3 gonocytes indicated a similar ranking of expression levels, where Prdx1 and Prdx2 were the most abundant, followed by Prdx6, Prdx5 and Prdx3, and finally Prdx4 present at very low levels ( Figure 1B). The similarities in expression profiles of antioxidant genes between gonocytes and spermatogonia suggested a conserved antioxidant machinery between the two phases of germ cell development. For ICC, the protein expression of PRDX1, 2, and 6 were examined in low purity gonocyte fractions pooled, washed with PBS, and fixed with 3.5% paraformaldehyde right after the BSA gradient. The fixed cells were collected by cytospin centrifugation, the slides dried and treated with acetone:methanol (60:40), followed by the antigen retrieval solution. The ICC reactions were similar to those described above for IHC, except for the use of fluorescent secondary antibodies. DAPI was used as a nuclear signal. Rabbit and mouse IgG were used as negative controls and gave no fluorescent signal (data not shown). Pictures of fluorescent signals and bright field were taken, using the same time of exposure for the specific antibodies and non-specific IgGs. Representative samples are shown.

Statistical Analysis
Statistical analysis was performed using the unpaired Student's t test or one-way ANOVA with post-hoc Tukey multiple comparison analysis, using Prism version 5.04 (GraphPad Software, San Diego, CA, USA). Changes with p values 0.05 were considered significant.

PRDXs Are among the Highest Expressed Antioxidant Genes in Neonatal Gonocytes and in Spermatogonia
Gene array analysis of antioxidant genes in highly purified rat PND3 gonocytes and PND8 spermatogonia showed that, at both ages, Prdxs were among the most abundant antioxidant genes ( Figure 1A, Table S1). Comparing the signal intensities of all genes in the arrays showed that thioredoxin 1 (Txn1), Prdx1 and superoxide dismutase (Sod) 1 were among the 1% most abundant genes in these cells, comprising relative signal intensities around and above 2000. Next, Prdx2, Prdx5, Prdx6, Sod2, Gstp1, and Gpx4 were among the most abundant genes, with relative signal intensities from 600 to 1200 (Table S1). Other highly expressed genes (signal intensities between 300 and 600) included Gsto1, Prdx3, Txnl1, Mgst1, Txnrd1, Gstp2, Gpx1, and Nrf2. As a comparison, these genes had much higher signal intensities than 65 percent of the genes in the cells, which presented intensities below or around 20, levels found for Sod3 and Catalase. The ranking of expression for PRDXs were Prdx1 > Prdx2 > Prdx5 > Prdx6 > Prdx3, in both germ cell types. Measurement of the relative expression of the PRDXs by qPCR in PND3 gonocytes indicated a similar ranking of expression levels, where Prdx1 and Prdx2 were the most abundant, followed by Prdx6, Prdx5 and Prdx3, and finally Prdx4 present at very low levels ( Figure 1B). The similarities in expression profiles of antioxidant genes between gonocytes and spermatogonia suggested a conserved antioxidant machinery between the two phases of germ cell development. This was different from the levels found in enriched PND60 adult rat germ cells, in which the relative gene expression of Gstm5 (6235) was the highest, followed by Gpx4 (5507). The immunological analysis of the protein expression of PRDX1, 2, and 6 in mixed suspensions of PND3 gonocytes, Sertoli and myoid cells confirmed that the three PRDX proteins were expressed in gonocytes, Sertoli, and myoid cells, but at lower levels in gonocytes than in most of the somatic cells ( Figure 2A). PRDX1 showed a slightly stronger signal than PRDX2, both being higher than PRDX6 in gonocytes, in agreement with the transcript levels. PRDX6 protein was also visible by IHC analysis in the cytoplasm of gonocytes in PND3 testis sections, but it was lower than the levels observed in Sertoli cells and some interstitial cells ( Figure 2B). The immunological analysis of the protein expression of PRDX1, 2, and 6 in mixed suspensions of PND3 gonocytes, Sertoli and myoid cells confirmed that the three PRDX proteins were expressed in gonocytes, Sertoli, and myoid cells, but at lower levels in gonocytes than in most of the somatic cells ( Figure 2A). PRDX1 showed a slightly stronger signal than PRDX2, both being higher than PRDX6 in gonocytes, in agreement with the transcript levels. PRDX6 protein was also visible by IHC analysis in the cytoplasm of gonocytes in PND3 testis sections, but it was lower than the levels observed in Sertoli cells and some interstitial cells ( Figure 2B).

PRDXs Are Required for PND3 Gonocyte Survival in Basal Conditions
In view of the abundance of PRDXs in gonocytes, and knowing the importance of these peroxidases for spermatozoa viability and function, we examined whether inhibiting PRDXs would have an impact on the survival of PND3 gonocytes. For that, we performed dose-response and timecourse studies in basal conditions where only endogenous ROS may be formed, using either the

PRDXs Are Required for PND3 Gonocyte Survival in Basal Conditions
In view of the abundance of PRDXs in gonocytes, and knowing the importance of these peroxidases for spermatozoa viability and function, we examined whether inhibiting PRDXs would have an impact on the survival of PND3 gonocytes. For that, we performed dose-response and time-course studies in basal conditions where only endogenous ROS may be formed, using either the inhibitor of 2-Cys PRDXs, Conoidin A; the inhibitor of GSTP1 (that promotes PRDX6 glutathione-dependent re-activation), Ezatiostat; or the inhibitor of the phospholipase A 2 activity of PRDX6, MJ33. Conoidin A induced a rapid dose-dependent decrease in gonocyte viability with concentrations of 5-10 µM, inducing 50% cell death after one hour of treatment ( Figure 3A). There were no surviving cells with conoidin A concentrations of 1-10 µM after 18 h of treatments. By contrast, after 18 h of treatment with MJ33, there was no or minimally detrimental effects on cell viability with concentration from 1 to 20 µM ( Figure 3B). However, at 50 µM, MJ33 did induce cell death after 18 h. Eztiostat dose responses were performed for two hours of treatment, because preliminary studies showed that longer time-periods induced the loss of a large amount of cells. Eztiostat inhibited cell survival in a dose-dependent manner ( Figure 3C), with a concentration of 50 µM inducing nearly 50% of cell death. These data suggest that the peroxidase activity of 2-cys PRDXs and PRDX6 play a critical role in gonocyte protection against excessive endogenous ROS, and that inhibiting these enzymes even for a short time is sufficient to induce significant gonocyte cell death. Thus, PRDXs are required to maintain ROS homeostasis in neonatal gonocytes. By contrast, inhibiting the PLA2 activity of PRDX6 with MJ33 did not exert rapid deleterious effects on cell viability.
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 13 inhibitor of 2-Cys PRDXs, Conoidin A; the inhibitor of GSTP1 (that promotes PRDX6 glutathionedependent re-activation), Ezatiostat; or the inhibitor of the phospholipase A2 activity of PRDX6, MJ33. Conoidin A induced a rapid dose-dependent decrease in gonocyte viability with concentrations of 5-10 μM, inducing 50% cell death after one hour of treatment ( Figure 3A). There were no surviving cells with conoidin A concentrations of 1-10 μM after 18 h of treatments. By contrast, after 18 h of treatment with MJ33, there was no or minimally detrimental effects on cell viability with concentration from 1 to 20 μM ( Figure 3B). However, at 50 μM, MJ33 did induce cell death after 18 h. Eztiostat dose responses were performed for two hours of treatment, because preliminary studies showed that longer time-periods induced the loss of a large amount of cells. Eztiostat inhibited cell survival in a dose-dependent manner ( Figure 3C), with a concentration of 50 μM inducing nearly 50% of cell death. These data suggest that the peroxidase activity of 2-cys PRDXs and PRDX6 play a critical role in gonocyte protection against excessive endogenous ROS, and that inhibiting these enzymes even for a short time is sufficient to induce significant gonocyte cell death. Thus, PRDXs are required to maintain ROS homeostasis in neonatal gonocytes. By contrast, inhibiting the PLA2 activity of PRDX6 with MJ33 did not exert rapid deleterious effects on cell viability. PRDXs are required to prevent oxidative stress-induced cell death in PND3 gonocytes. Oxidative stress can be generated by external factors, as a result of exposure of the animals and their reproductive system to oxidative agents, or through the production of ROS by other testicular cell types in the vicinity of the gonocytes. Thus, we tested the ability of PRDXs to protect PND3 gonocytes from exogenous ROS, using H2O2 as an oxidative agent. The treatment of gonocytes with H2O2 at 100 and 200 μM for two hours induced a dose-dependent decrease in cell viability, with a 40% and 50% reduction in viability, respectively (Figure 4).
These damaging effects were significantly worsened by treating the cells with PRDXs inhibitors, with MJ33 having the mildest and ezatiostat the worst effects ( Figure 4). These results indicate that the oxidative stress generated by H2O2 exerts an adverse effect on gonocyte viability, and that 2 Cys PRDX and PRDX6 peroxidase activities are required to protect gonocytes from oxidative stress. This further implies that other antioxidant proteins expressed in gonocytes, even those present at high levels such as Txn1, Sod1, and Sod2, are not able to rescue gonocytes from exposure to exogenous oxidants. Moreover, the fact that ezatiostat and MJ33 exacerbated the adverse effect of H2O2 on gonocyte viability suggests that both the peroxidase and phospholipase A2 activity of PRDX6 are important for gonocyte survival. PRDXs are required to prevent oxidative stress-induced cell death in PND3 gonocytes. Oxidative stress can be generated by external factors, as a result of exposure of the animals and their reproductive system to oxidative agents, or through the production of ROS by other testicular cell types in the vicinity of the gonocytes. Thus, we tested the ability of PRDXs to protect PND3 gonocytes from exogenous ROS, using H 2 O 2 as an oxidative agent. The treatment of gonocytes with H 2 O 2 at 100 and 200 µM for two hours induced a dose-dependent decrease in cell viability, with a 40% and 50% reduction in viability, respectively (Figure 4).
These damaging effects were significantly worsened by treating the cells with PRDXs inhibitors, with MJ33 having the mildest and ezatiostat the worst effects ( Figure 4). These results indicate that the oxidative stress generated by H 2 O 2 exerts an adverse effect on gonocyte viability, and that 2 Cys PRDX and PRDX6 peroxidase activities are required to protect gonocytes from oxidative stress. This further implies that other antioxidant proteins expressed in gonocytes, even those present at high levels such as Txn1, Sod1, and Sod2, are not able to rescue gonocytes from exposure to exogenous oxidants. Moreover, the fact that ezatiostat and MJ33 exacerbated the adverse effect of H 2 O 2 on gonocyte viability suggests that both the peroxidase and phospholipase A 2 activity of PRDX6 are important for gonocyte survival. Antioxidants 2020, 9,

H2O2 Induces Lipid Peroxidation, and PRDXs Prevent Endogenous ROS-Induced Lipid Peroxidation in PND3 Gonocytes
Lipid peroxidation has been known for decades to be one of the sources of spermatozoa damage in infertile men [26]. Thus, we examined whether lipid peroxidation could also play a role in the deleterious effects of H2O2 and PRDX inhibitors on gonocyte viability, using a fluorescent fatty acid reporter system that allowed quantifying the proportion of cells where lipid peroxidation had occurred. While lipid peroxidation was detectable in 40% of the control gonocytes, a condition corresponding to 80% cell viability, treatment with H2O2 (a condition decreasing cell viability by 50%) doubled the numbers of gonocytes presenting lipid peroxidation to nearly 80% of positive cells in gonocytes ( Figure 5). To examine the effects of PRDX inhibitors, we used concentrations of conoidin A and ezatiostat decreasing viability by ~50% (10 and 50 μM respectively), and a concentration of MJ33 that did not significantly alter cell viability compared to controls (20 μM). Treatments with conoidin A alone had the same effect as H2O2 on lipid peroxidation, doubling the percent of gonocytes presenting lipid peroxidation, while MJ33 and ezatiostat induced significant but lower increases in lipid peroxidation, reaching 70% of positive cells, a 1.55 increase over the levels in control cells ( Figure 5).

H 2 O 2 Induces Lipid Peroxidation, and PRDXs Prevent Endogenous ROS-Induced Lipid Peroxidation in PND3 Gonocytes
Lipid peroxidation has been known for decades to be one of the sources of spermatozoa damage in infertile men [26]. Thus, we examined whether lipid peroxidation could also play a role in the deleterious effects of H 2 O 2 and PRDX inhibitors on gonocyte viability, using a fluorescent fatty acid reporter system that allowed quantifying the proportion of cells where lipid peroxidation had occurred. While lipid peroxidation was detectable in 40% of the control gonocytes, a condition corresponding to 80% cell viability, treatment with H 2 O 2 (a condition decreasing cell viability by 50%) doubled the numbers of gonocytes presenting lipid peroxidation to nearly 80% of positive cells in gonocytes ( Figure 5). To examine the effects of PRDX inhibitors, we used concentrations of conoidin A and ezatiostat decreasing viability by~50% (10 and 50 µM respectively), and a concentration of MJ33 that did not significantly alter cell viability compared to controls (20 µM). Treatments with conoidin A alone had the same effect as H 2 O 2 on lipid peroxidation, doubling the percent of gonocytes presenting lipid peroxidation, while MJ33 and ezatiostat induced significant but lower increases in lipid peroxidation, reaching 70% of positive cells, a 1.55 increase over the levels in control cells ( Figure 5).

H2O2 Induces Lipid Peroxidation, and PRDXs Prevent Endogenous ROS-Induced Lipid Peroxidation in PND3 Gonocytes
Lipid peroxidation has been known for decades to be one of the sources of spermatozoa damage in infertile men [26]. Thus, we examined whether lipid peroxidation could also play a role in the deleterious effects of H2O2 and PRDX inhibitors on gonocyte viability, using a fluorescent fatty acid reporter system that allowed quantifying the proportion of cells where lipid peroxidation had occurred. While lipid peroxidation was detectable in 40% of the control gonocytes, a condition corresponding to 80% cell viability, treatment with H2O2 (a condition decreasing cell viability by 50%) doubled the numbers of gonocytes presenting lipid peroxidation to nearly 80% of positive cells in gonocytes ( Figure 5). To examine the effects of PRDX inhibitors, we used concentrations of conoidin A and ezatiostat decreasing viability by ~50% (10 and 50 μM respectively), and a concentration of MJ33 that did not significantly alter cell viability compared to controls (20 μM). Treatments with conoidin A alone had the same effect as H2O2 on lipid peroxidation, doubling the percent of gonocytes presenting lipid peroxidation, while MJ33 and ezatiostat induced significant but lower increases in lipid peroxidation, reaching 70% of positive cells, a 1.55 increase over the levels in control cells ( Figure 5).  Combining PRDX inhibitors with H 2 O 2 did not further increase lipid peroxidation, which already affected most of the cells with individual treatments (data not shown). These results show that an Antioxidants 2020, 9, 32 9 of 14 oxidative stressor such as H 2 O 2 induces similar levels of lipid peroxidation as endogenous ROS accumulating in the absence of PRDX activities in PND3 gonocytes. This suggests that lipid peroxides are produced physiologically in gonocytes, and that PRDXs are critical for preventing excessive levels that could affect gonocyte survival. The data also imply that PRDXs protect gonocytes from excessive endogenous ROS-induced lipid peroxidation. Moreover, the fact that 40% of control gonocytes showed lipid peroxidation without affecting their viability, and that lipid peroxidation was not proportional to viability in cells treated with PRDX inhibitors, suggest that gonocytes can tolerate a certain level of lipid peroxidation. The finding that MJ33 treatment increased lipid peroxidation suggests that the PRDX6 iPLA 2 activity also plays a role in repairing oxidized membranes [27].

PRDX Inhibition Blocks RA-Induced Differentiation in PND3 Gonocytes
Considering the physiological roles played by ROS in many tissues and cell types, including in spermatozoa, and our finding that inhibiting PRDXs increases endogenous ROS formation in PND3 gonocytes, we examined whether blocking PRDXs could affect RA-induced gonocytes differentiation. First, we determined cell viability in cells treated for two hours with RA alone or together with two different concentrations of PRDX inhibitors, in order to select concentrations of inhibitors that would not overly decrease cell survival. RA at 1 µM did not have an effect on cell survival. Similarly, concentrations of 20 µM MJ33 or 10 µM ezatiostat did not affect cell viability ( Figure 6). While 0.5 µM conoidin A decreased cell viability by 9% when used alone, and 17% when added with RA in comparison to control and RA treatments, these was still relatively minor effects.
Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 13 Combining PRDX inhibitors with H2O2 did not further increase lipid peroxidation, which already affected most of the cells with individual treatments (data not shown). These results show that an oxidative stressor such as H2O2 induces similar levels of lipid peroxidation as endogenous ROS accumulating in the absence of PRDX activities in PND3 gonocytes. This suggests that lipid peroxides are produced physiologically in gonocytes, and that PRDXs are critical for preventing excessive levels that could affect gonocyte survival. The data also imply that PRDXs protect gonocytes from excessive endogenous ROS-induced lipid peroxidation. Moreover, the fact that 40% of control gonocytes showed lipid peroxidation without affecting their viability, and that lipid peroxidation was not proportional to viability in cells treated with PRDX inhibitors, suggest that gonocytes can tolerate a certain level of lipid peroxidation. The finding that MJ33 treatment increased lipid peroxidation suggests that the PRDX6 iPLA2 activity also plays a role in repairing oxidized membranes [27].

PRDX Inhibition Blocks RA-Induced Differentiation in PND3 Gonocytes
Considering the physiological roles played by ROS in many tissues and cell types, including in spermatozoa, and our finding that inhibiting PRDXs increases endogenous ROS formation in PND3 gonocytes, we examined whether blocking PRDXs could affect RA-induced gonocytes differentiation. First, we determined cell viability in cells treated for two hours with RA alone or together with two different concentrations of PRDX inhibitors, in order to select concentrations of inhibitors that would not overly decrease cell survival. RA at 1 μM did not have an effect on cell survival. Similarly, concentrations of 20 μM MJ33 or 10 μM ezatiostat did not affect cell viability ( Figure 6). While 0.5 μM conoidin A decreased cell viability by 9% when used alone, and 17% when added with RA in comparison to control and RA treatments, these was still relatively minor effects. Figure 6. Effects of trans-retinoic acid (RA) and PRDX inhibitors on PND3 gonocyte viability. RA (1 μM) and PRDX inhibitors at the indicated concentrations were added alone or together for two hours on gonocytes. Cell viability was determined by the trypan blue exclusion method. Results represent the means ± SEM from at least 3 experiments. Statistical significance: * p ≤ 0.05, *** p ≤ 0.001, treatment against control; &&& p ≤ 0.001, treatment against RA alone.
Next, we measured the mRNA expression of Stra8, previously found to be a good marker for assessing neonatal gonocyte differentiation [15][16][17]. While two hours of treatment with RA significantly increased Stra8 expression by over three-fold, there was no effect of conoidin A, MJ33 or ezatiostat added alone to the cells (Figure 7). However, both conoidin A and ezatiostat significantly repressed RA-induced Stra8 increases, indicating inhibitory effects of PRDX inhibitors on gonocytes differentiation. However, MJ33 did not alter RA effect on Stra8 levels. These data suggest that the oxidative stress resulting from PRDXs inhibition is detrimental to gonocytes differentiation, whereas PRDX6 iPLA activity does not appear to be required for gonocyte differentiation. and PRDX inhibitors at the indicated concentrations were added alone or together for two hours on gonocytes. Cell viability was determined by the trypan blue exclusion method. Results represent the means ± SEM from at least 3 experiments. Statistical significance: * p ≤ 0.05, *** p ≤ 0.001, treatment against control; &&& p ≤ 0.001, treatment against RA alone.
Next, we measured the mRNA expression of Stra8, previously found to be a good marker for assessing neonatal gonocyte differentiation [15][16][17]. While two hours of treatment with RA significantly increased Stra8 expression by over three-fold, there was no effect of conoidin A, MJ33 or ezatiostat added alone to the cells (Figure 7). However, both conoidin A and ezatiostat significantly repressed RA-induced Stra8 increases, indicating inhibitory effects of PRDX inhibitors on gonocytes differentiation. However, MJ33 did not alter RA effect on Stra8 levels. These data suggest that the oxidative stress resulting from PRDXs inhibition is detrimental to gonocytes differentiation, whereas PRDX6 iPLA activity does not appear to be required for gonocyte differentiation. Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 13

Discussion
The goal of the present study was to determine whether peroxiredoxins play a role in the survival of neonatal male germ cells, as it is the case in spermatozoa. We hypothesized that neonatal germ cells and spermatozoa would likely require different antioxidant machineries, in view of their considerable molecular, morphological and functional differences, as well as their distinct cellular environments. Indeed, the most abundant antioxidants genes in PND3 gonocytes were Txn1, Prdx1, Sod1, Prdx2, Prdx6, and Prdx5, whereas a mixed population of adult germ cells expressed Sod1 at a similar level as these genes, followed by mGst1, Prdx1, Prdx6, Txn1, Prdx2 and Prdx5 at lower levels. These data indicate that neonatal gonocytes express very high levels of antioxidant genes relative to adult germ cells, although one cannot exclude that some of these genes might be highly expressed in discreet subsets of adult spermatogonia, spermatocytes, or spermatids. The requirement for high levels of antioxidant genes in neonatal gonocytes might reflect the production of ROS during their multiple functions, including DNA methylation, cell proliferation, migration, and differentiation, all processes requiring energy and the generation of ROS [13,14].
Indeed, our finding that the direct inhibition of 2-Cys PRDX with conoidin A and the specific inhibition of GSTP1, enzyme necessary for the re-activation of PRDX6 peroxidase activity, with ezatiostat [28][29][30][31] induce rapid and extensive cell death in PND3 gonocytes implies that high levels of ROS are formed in these cells in physiological conditions. This further suggests that PRDXs play an essential role in maintaining ROS at levels required for physiological functions, but not high enough to induce cell damage. Our results showing that lipid peroxidation is greatly increased by these inhibitors and is associated with increased cell death suggest that lipid peroxides are in part responsible for the adverse effects on gonocytes. In this context, high levels of antioxidant genes, in particular PRDXs, protect neonatal gonocytes from damaging levels of ROS. Moreover, the data clearly show that other antioxidant genes are not capable of rescuing gonocytes from the deleterious effects of ROS accumulation in the absence of PRDXs. In this regard, PRDXs are clearly essential to the maintenance on ROS homeostasis and gonocytes survival, as they are in spermatozoa [4,6].
Recently, it was reported that as human PRXD6, targeted to the yeast mitochondrial matrix, elicited glutathione disulfide (GSSG) formation upon treatment of cells with H2O2 [32]. Because yeast lack GSTP1, it was suggested that other enzymes may re-activate the peroxidase activity of PRDX6. Although we observed that glutaredoxin 1 (GRX1) and other GSTs were present in gonocytes, the

Discussion
The goal of the present study was to determine whether peroxiredoxins play a role in the survival of neonatal male germ cells, as it is the case in spermatozoa. We hypothesized that neonatal germ cells and spermatozoa would likely require different antioxidant machineries, in view of their considerable molecular, morphological and functional differences, as well as their distinct cellular environments. Indeed, the most abundant antioxidants genes in PND3 gonocytes were Txn1, Prdx1, Sod1, Prdx2, Prdx6, and Prdx5, whereas a mixed population of adult germ cells expressed Sod1 at a similar level as these genes, followed by mGst1, Prdx1, Prdx6, Txn1, Prdx2 and Prdx5 at lower levels. These data indicate that neonatal gonocytes express very high levels of antioxidant genes relative to adult germ cells, although one cannot exclude that some of these genes might be highly expressed in discreet subsets of adult spermatogonia, spermatocytes, or spermatids. The requirement for high levels of antioxidant genes in neonatal gonocytes might reflect the production of ROS during their multiple functions, including DNA methylation, cell proliferation, migration, and differentiation, all processes requiring energy and the generation of ROS [13,14].
Indeed, our finding that the direct inhibition of 2-Cys PRDX with conoidin A and the specific inhibition of GSTP1, enzyme necessary for the re-activation of PRDX6 peroxidase activity, with ezatiostat [28][29][30][31] induce rapid and extensive cell death in PND3 gonocytes implies that high levels of ROS are formed in these cells in physiological conditions. This further suggests that PRDXs play an essential role in maintaining ROS at levels required for physiological functions, but not high enough to induce cell damage. Our results showing that lipid peroxidation is greatly increased by these inhibitors and is associated with increased cell death suggest that lipid peroxides are in part responsible for the adverse effects on gonocytes. In this context, high levels of antioxidant genes, in particular PRDXs, protect neonatal gonocytes from damaging levels of ROS. Moreover, the data clearly show that other antioxidant genes are not capable of rescuing gonocytes from the deleterious effects of ROS accumulation in the absence of PRDXs. In this regard, PRDXs are clearly essential to the maintenance on ROS homeostasis and gonocytes survival, as they are in spermatozoa [4,6].
Recently, it was reported that as human PRXD6, targeted to the yeast mitochondrial matrix, elicited glutathione disulfide (GSSG) formation upon treatment of cells with H 2 O 2 [32]. Because yeast lack GSTP1, it was suggested that other enzymes may re-activate the peroxidase activity of PRDX6. Although we observed that glutaredoxin 1 (GRX1) and other GSTs were present in gonocytes, the level of their expression was lower than that of GSTP1 ( Figure 1; Table S1). This finding and the decrease in gonocyte survival due to the inhibition of GSTP1 by ezatiostat, indicate that the re-activation of PRDX6 peroxidase activity is probably accomplished by GSTP1 and GSH in gonocytes, as described in other mammalian cells [33].
The finding, that conoidin A and ezatiostat both greatly aggravated H 2 O 2 adverse effects, suggests that 2-Cys PRDXs, likely PRDX1 and 2 which were the most abundant at the protein level, as well as PRDX6 are the peroxidases that actively remove ROS such as H 2 O 2 from neonatal gonocytes exposed to oxidative stress. Taken together with the fact that Sertoli cells produce H 2 O 2 [20], these data suggest that the exposure of neonatal gonocytes to ROS produced by Sertoli cells might be part of a physiological process, as it happens in various tissues and cell types [8,[34][35][36]. However, as shown by the effects of PRDX inhibitors, ROS levels need to be tightly regulated in order to maintain ROS at non-toxic levels in gonocytes.
The fact that the PRDX inhibitors conoidin A and ezatiostat both decrease RA-induced gonocyte differentiation suggests that PRDXs are essential to control ROS levels, preventing cell death and allowing some of the cells to undergo gonocyte differentiation. Moreover, the finding that PRDX inhibition promoted high levels of ROS, which prevented gonocyte differentiation without impairing viability, suggests that one way by which the Sertoli cells could control the timing of gonocyte differentiation would be by producing ROS at levels that would prevent or allow gonocyte differentiation. Further studies are necessary to confirm this possibility. Considering recent studies showing that gonocytes are heterogeneous, similarly to spermatogonia [14,37,38], it would be interesting to study whether neonatal testes contain sub-sets of gonocytes with different PRDX activities, corresponding to different functional states.
One of the main differences we found with our studies on PRDXs in spermatozoa is related to the role of the PRDX6 iPLA 2 activity. While this activity is critical for the survival and fertilizing ability of spermatozoa by repairing oxidized membranes [10][11][12], our results with MJ33 suggest that the PLA 2 activity of PRDX6 plays a role in gonocyte survival mainly in the presence of exogenous oxidative stress. Yet, the increased cell death observed after treating gonocytes for 18 h with 50 µM MJ33 suppressing PRDX6 iPLA 2 activity implies that impairing the repair of oxidized membranes in gonocytes can jeopardize PND3 gonocyte survival over time. We recently reported that exogenous addition of arachidonic acid or lysophosphatidic acid (LPA) prevented cell death in spermatozoa treated with MJ33 [39]. The addition of H 2 O 2 to cells where PRDX6 iPLA 2 is inactive and possibly arachidonic acid levels were depleted is sufficient to induce a large increase in cell death in only two hours. Thus, excessive levels of ROS combined with an increased lipid peroxidation and depletion of arachidonic acid and/or LPA appear to aggravate the fate of the cells.
Interestingly, we recently reported that neonatal gonocytes express COX2 and other enzymes of the prostaglandin pathway, and produce prostaglandins (PG) E2 and PGF2a [19]. Moreover, blocking PGE2 and PGF2a synthesis with ibuprofen for 24 h correlated with a partial decrease in RA-induced differentiation in PND3 gonocytes [19]. However, inhibiting the PRDX6 iPLA 2 activity for 2 h does not seem to affect gonocyte differentiation. Since arachidonic acid is the precursor of prostaglandins, the present data suggest that a short two-hour treatment with MJ33 may not deplete arachidonic acid sufficiently to compromise cell differentiation, or that this process involves other PLA 2 enzymes expressed in gonocytes [19].

Conclusions
In conclusion, the present study identified PRDX1, 2, 6, and 5 as major antioxidant genes and proteins in neonatal gonocytes, essential for the survival of the cells, especially under conditions of oxidative stress. Moreover, gonocytes were more sensitive to Conoidin A effects than spermatozoa, whereas they were less sensitive to the inhibition of the PLA 2 activity of PRDX6 than spermatozoa, suggesting a difference in the antioxidant machinery of gonocyte and spermatozoa. Further studies will be needed to examine the possibility that ROS, endogenous or produced by Sertoli cells, at non-toxic levels, might play a role in the regulation of neonatal gonocyte differentiation.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/1/32/s1, Table S1: Relative gene expression of antioxidant genes expressed in rat PND3 gonocytes and PND8 spermatogonia. The results represent the mean ± SEM of 3 independent RNA samples for each age, each made from cells isolated in multiple animals. There was no significant difference between the two types of germ cells for any of the genes. A few genes presenting a signal intensity below 20 were not included.
Author Contributions: A.B. performed the experiments presented in Figures 2-7