Mouse Germ Cells Research Articles

GPR62 constitutively activates cAMP signaling but is dispensable for male fertility in mice.

G-protein-coupled receptors (GPCRs) participate in diverse physiological functions and are promising targets for drug discovery. However, there are still over 140 orphan GPCRs whose functions remain to be elucidated. Gpr62 is one of the orphan GPCRs that is expressed in the rat and human brain. In this study, we found that Gpr62 is also expressed in male germ cells in mice, and its expression increases along with sperm differentiation. GPR62 lacks the BBXXB and DRY motifs, which are conserved across many GPCRs, and it was able to induce cAMP signaling in the absence of a ligand. These structural and functional features are conserved among mammals, and the mutant analysis of GPR62 has revealed that lacking of these motifs is involved in the constitutive activity. We also found that GPR62 can homooligomerize, but it is not sufficient for its constitutive activity. We further investigated its physiological function by using Gpr62 knockout (Gpr62-/-) mice. Gpr62-/- mice were born normally and did not show any abnormality in growth and behavior. In addition, both male and female Gp62-/- mice were fertile, and the differentiation and motility of spermatozoa were normal. We also found that Gpr61, the gene most similar to Gpr62 in the GPCR family shows a constitutive activity and an expression pattern similar to those of Gpr62 Our results suggest that GPR62 constitutively activates the cAMP pathway in male germ cells but is dispensable for male fertility, which is probably due to its functional redundancy with GPR61.

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc.

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis.

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during spermatogenesis.

Ssm1b expression and function in germ cells of adult mice and in early embryos.

Ssm1b (Strain-specific modifier of DNA methylation 1b) is a Krüppel-associated box (KRAB) zinc finger gene that promotes CpG methylation in the mouse transgene HRD (Heavy chain enhancer, rearrangement by deletion). We report here that Ssm1b expression and concomitant HRD methylation are also present in the male and female germ cells of adult mice. Ssm1b is expressed in both diploid (2N) and haploid (1N) oocytes, as well as in 1N spermatids and spermatozoa, but not in 2N spermatogonia. Interestingly, Ssm1b mRNA is not detected in any other adult mouse organ examined, although Ssm1-family mRNAs are highly expressed in the heart. Reflecting strain specificity, Ssm1b expression and HRD methylation are not observed in early-stage C3H/HeJ mouse embryos; however, an Ssm1b-like gene that closely resembles an Ssm1b-like gene previously found in wild-derived mice is expressed in cultured embryonic stem cells derived from C3H/HeJ embryos, suggesting that culture conditions affect its expression. Collectively, this work demonstrates that HRD methylation by Ssm1b is more temporally restricted during spermatogenesis compared to oogenesis, and is altered when embryonic stem cells are cultured from C3H/HeJ inner cell mass cells.

Combination of 2-methoxyestradiol (2ME) and cyclophosphamide (CP) inhibits tumour progression in S-180 mouse tumour model system

2-Methoxyestradiol (2ME), an anti-neoplastic and anti-angiogenic agent, has been used in preclinical research for cancer treatment. Cyclophosphamide (CP), the anti-neoplastic and alkaloid drug, has long been used as a traditional chemotherapeutic drug against different human neoplastic diseases. Our earlier studies showed that 2ME inhibit angiogenesis and cell proliferation rate differently in vivo and in vitro tumour models. But little work has been done on the combination effect of 2ME and CP on tumour model system. Therefore, the present study has been oriented to evaluate the efficacy and safety of combination effect of 2ME and CP on Sarcoma-180 tumour model system. In the present paper, an attempt has also been taken to find out whether the combination effect of 2ME and CP is less toxic than the traditional chemotherapeutic drug-CP, considering S-180 tumour bearing mouse bone marrow and germ cell toxicity as parameters and chromosomal aberrations, metaphase index and sperm head abnormality as biological endpoints. The result of these studies showed significant regression in tumour growth with inhibition of viable cancer cell population and less toxicity in the somatic and germinal cell of tumour bearing mouse.

Gold nanoparticles: Mutagen, antimutagen, or comutagen?

The effect of ultrasmall gold nanoparticles (GNPs), both direct and in combination with the alkylating mutagen dipin, on the genetic structures of male germ cells in mice were studied using the meiotic micronucleus assay. It was shown for the first time that GNPs can exhibit mutagenic, antimutagenic, and comutagenic activity depending on the experimental conditions.

Induction of experimental autoimmune orchitis by immunization with xenogenic testicular germ cells in mice

We previously showed that immunization of mice with syngeneic or allogeneic testicular germ cells (TGC) alone induces autoimmune inflammation in the testes without using any adjuvant. In the present study, we examined testicular autoimmune response against xenogenic TGC antigens in mice. The mice were immunized with murine, rat or guinea pig TGC and then the histopathology, delayed type hypersensitivity (DTH) response and humoral autoimmunity were investigated. The results showed that immunization with not only murine but also rat TGC caused experimental autoimmune orchitis (EAO) with hypospermatogenesis in mice, while that with guinea pig TGC could not. The DTH response to murine TGC was significantly elevated in mice that had been immunized with murine or rat but not guinea pig TGC. Serum autoantibody to murine TGC was immunohistochemically detected in the mice immunized with either murine, rat or guinea pig TGC, however, the level of autoantibody detected by ELISA revealed significant elevation when mice were immunized with murine and rat TGC. With the immunoblotting after electrophoresis, the murine TGC proteins at molecular masses around 55kDa and 70kDa can be detected when incubated with sera from m-TGC and r-TGC groups. These results represent the cross-reactivity among TGC of the mouse, the rat and the guinea pig at the levels of humoral immunity and also demonstrate that the rat TGC could elicit significant DTH response to murine TGC with the resultant EAO. This is the first to succeed in EAO induction by the use of xenogenic TGC.

Intake of saffron reduces γ-radiation-induced genotoxicity and oxidative stress in mice

Saffron (SAF), the dried stigmas of Crocus sativus, is commonly used for flavoring and coloring food. Studies on bioactivity of SAF have demonstrated its in vivo antioxidant activity. The aim of our study was to assess the impact of SAF intake on γ-radiation (RAD) induced (a) chromosomal damage, (b) oxidative stress in liver and brain, and (c) histopathological effects in the intestinal cells and male germ cells in mice. Freeze-dried aqueous extract of SAF was used for the experiments. Our preliminary cell-free DNA nicking assay using pBR322 DNA revealed protective effects of freeze-dried SAF extract against hydroxyl radical induced DNA damage. For the in vivo investigations, freeze-dried SAF extract in distilled water was administered by gavage (40 mg/kg b.w.) to male Swiss albino mice for six consecutive days. On the sixth day, the animals were exposed to RAD (1 or 2 Gy) and sacrificed 24 h later to collect bone marrow cells for assessing chromosomal damage by measuring micronucleated polychromatic erythrocytes (MnPCEs). Liver and brain samples from animals exposed to 2 Gy RAD were used for evaluating lipid peroxidation and activity of antioxidant enzymes. The testis and intestine were used for histopathological analysis. Our results demonstrated significant protective effects of SAF against RAD-induced genotoxic damage. SAF pretreatment reduced the level of lipid peroxidation with concomitant increase in glutathione content and activity of glutathione S-transferase, glutathione peroxidase, and catalase. The histopathological analysis showed minimal impact of SAF on RAD-induced damage in the intestinal cells and male germ cells.

Meioc maintains an extended meiotic prophase I in mice.

The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase—as early as the preleptotene stage—proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts.

Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

Spermatogenesis associated 22 is required for DNA repair and synapsis of homologous chromosomes in mouse germ cells.

Analysis of the N-ethyl-N-nitrosourea (ENU)-induced repro42 mutation previously identified spermatogenesis associated 22 (Spata22) as a gene required for meiotic progression and fertility in both male and female mice, but its specific contribution to the process was unclear. Here, we report on a novel, null allele of Spata22 (Spata22Gt ) and confirm its requirement for germ cell development. Similar to repro42 mutant mice, histological and mating analyses indicate that gametogenesis is profoundly affected in Spata22Gt/Gt males and females, resulting in infertility. Cytological examination confirms that germ cells do not progress beyond zygonema and meiotic arrest is linked to impairment of both synapsis and DNA repair. Analysis of SPATA22 distribution reveals that it localizes to foci associated with meiotic chromosomes during prophase I and that the number of foci peaks at zygonema; there are also more SPATA22 foci in oocytes than in spermatocytes. Furthermore, SPATA22 co-localizes with a number of proteins involved in meiotic recombination, including RAD51, DMC1, and MLH1, and is present until mid-pachynema, suggesting a role in resolution of recombination intermediates. In fact, SPATA22 co-localizes with MLH1 in more than 20% of foci at pachynema. Analysis of Spata22Gt/Gt meiocytes confirms that SPATA22 is required for localization of MEIOB but not RPA (two proteins known to interact with SPATA22), and immunoblotting corroborates that production of MEIOB is indeed decreased in the absence of SPATA22. Together, these data suggest that SPATA22 is required for both meiotic recombination and synapsis during meiosis in mice.

In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n-ethyl-n-nitrosourea (ENU), 6-mercaptopurine (6-MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration-dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration-related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6-MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real-time PCR results showed that the mRNA level expression of p53 increased and bcl-2 decreased significantly with the increasing ENU, 6-MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6-MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6-MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99-107, 2017. © 2017 Wiley Periodicals, Inc.

MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice

In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75–8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (α subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis.

Non-Canonical and Sexually Dimorphic X Dosage Compensation States in the Mouse and Human Germline

SummarySomatic X dosage compensation requires two mechanisms: X inactivation balances X gene output between males (XY) and females (XX), while X upregulation, hypothesized by Ohno and documented in vivo, balances X gene with autosomal gene output. Whether X dosage compensation occurs in germ cells is unclear. We show that mouse and human germ cells exhibit non-canonical X dosage states that differ from the soma and between the sexes. Prior to genome-wide reprogramming, X upregulation is present, consistent with Ohno's hypothesis. Subsequently, however, it is erased. In females, erasure follows loss of X inactivation, causing X dosage excess. Conversely, in males, erasure leads to permanent X dosage decompensation. Sex chromosomally abnormal models exhibit a “sex-reversed” X dosage state: XX males, like XX females, develop X dosage excess, while XO females, like XY males, develop X dosage decompensation. Thus, germline X dosage compensation states are determined by X chromosome number, not phenotypic sex. These unexpected differences in X dosage compensation states between germline and soma offer unique perspectives on sex chromosome infertility.

Atp7a and Atp7b regulate copper homeostasis in developing male germ cells in mice.

The maintenance of copper homeostasis is critical for all cells. As learned from mice with disturbed copper metabolism, this trace element is also important for spermatogenesis. The experiments conducted in yeasts have demonstrated that appropriate copper level must be preserved to enable meiosis progression; however, increased copper level is toxic for cells. This study aims to analyze the expression profile of Atp7a and Atp7b and other genes encoding copper-related proteins during spermatogenesis in mice. Using the transcripts and protein detection techniques, we demonstrate that within seminiferous tubuli, ATP7A is mainly present in early meiotic germ cells (leptotene to pachytene spermatocytes) and in Sertoli cells (SCs). During spermatogenesis, the progression Atp7a expression profile corresponds to Slc31a1 (encoding copper importer CTR1) and Atox1 (encoding chaperon protein, which delivers copper from CTR1 to ATP7A and ATP7B) expression, suggesting that male germ cells retrieve copper and ATP7A protects them from copper overdose. In contrast, ATP7B protein is observed in SCs and near elongated spermatids; thus, its function seems to be related to copper extraction during spermiogenesis. This is the first study to give a comprehensive view on the activity of copper-related genes during spermatogenesis in mice.

Prenatal and early postnatal NOAEL-dose clothianidin exposure leads to a reduction of germ cells in juvenile male mice.

Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; no-observed-adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life.

Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells.

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that β-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of β-TrCP2 in male germ cells of β-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The β-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in β-TrCP-deficient testes. DMRT1 contains a consensus β-TrCP degron sequence that was found to bind β-TrCP. Overexpression of β-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in β-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that β-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation.

Speedy A–Cdk2 binding mediates initial telomere–nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation

Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere-NE attachment. Deletion of Spdya in mice disrupts telomere-NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2's localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2-mediated telomere-NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere-NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.

Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse.

Does oxidative stress compromise the protein expression of heat shock protein A2 (HSPA2) in the developing germ cells of the mouse testis? Oxidative stress leads to the modification of HSPA2 by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system. Previous work has revealed a deficiency in HSPA2 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in HSPA2 remains to be established, we have recently shown that the HSPA2 expressed in the spermatozoa of normozoospermic individuals is highly susceptible to adduction, a form of post-translational modification, by the lipid aldehyde 4HNE that has been causally linked to the degradation of its substrates. This modification of HSPA2 by 4HNE adduction dramatically reduced human sperm-egg interaction in vitro. Moreover, studies in a mouse model offer compelling evidence that the co-chaperone BCL2-associated athanogene 6 (BAG6) plays a key role in regulating the stability of HSPA2 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation. Dose-dependent studies were used to establish a 4HNE-treatment regime for primary culture(s) of male mouse germ cells. The influence of 4HNE on HSPA2 protein stability was subsequently assessed in treated germ cells. Additionally, sperm lysates from infertile patients with established zona pellucida recognition defects were examined for the presence of 4HNE and ubiquitin adducts. A minimum of three biological replicates were performed to test statistical significance. Oxidative stress was induced in pachytene spermatocytes and round spermatids isolated from the mouse testis, as well as a GC-2 cell line, using 50-200 µM 4HNE or hydrogen peroxide (H2O2), and the expression of HSPA2 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of HSPA2 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and HSPA2 was examined in response to 4HNE exposure via proximity ligation assays. HSPA2 protein levels were significantly reduced compared with controls after 4HNE treatment of round spermatids (P < 0.01) and GC-2 cells (P < 0.001) but not pachytene spermatocytes. Using GC-2 cells as a model, HSPA2 was shown to be both adducted by 4HNE and targeted for ubiquitination in response to cellular oxidative stress. Inhibition of the proteasome with MG132 prevented HSPA2 degradation after 4HNE treatment indicating that the degradation of HSPA2 is likely to occur via a proteasomal pathway. Moreover, our assessment of proteasome activity provided evidence that 4HNE treatment can significantly increase the proteasome activity of GC-2 cells (P < 0.05 versus control). Finally, 4HNE exposure to GC-2 cells resulted in the dissociation of HSPA2 from its regulatory co-chaperone BAG6, a key mediator of HSPA2 stability in male germ cells. While these experiments were performed using a mouse germ cell-model system, our analyses of patient sperm lysate imply that these mechanisms are conserved between mouse and human germ cells. This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of HSPA2 in the spermatozoa of a specific sub-class of infertile males. In doing so, this work enhances our understanding of failed sperm-egg recognition and may assist in the development of targeted antioxidant-based approaches for ameliorating the production of cytotoxic lipid aldehydes in the testis in an attempt to prevent this form of infertility. Not applicable. This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare.