Frequency, morbidity and equity - the case for increased research on male fertility.

Currently, most men with infertility cannot be given an aetiology, which reflects a lack of knowledge around gamete production and how it is affected by genetics and the environment. A failure to recognize the burden of male infertility and its potential as a biomarker for systemic illness exists. The absence of such knowledge results in patients generally being treated as a uniform group, for whom the strategy is to bypass the causality using medically assisted reproduction (MAR) techniques. In doing so, opportunities to prevent co-morbidity are missed and the burden of MAR is shifted to the woman. To advance understanding of men's reproductive health, longitudinal and multi-national centres for data and sample collection are essential. Such programmes must enable an integrated view of the consequences of genetics, epigenetics and environmental factors on fertility and offspring health. Definition and possible amelioration of the consequences of MAR for conceived children are needed. Inherent in this statement is the necessity to promote fertility restoration and/or use the least invasive MAR strategy available. To achieve this aim, protocols must be rigorously tested and the move towards personalized medicine encouraged. Equally, education of the public, governments and clinicians on the frequency and consequences of infertility is needed. Health options, including male contraceptives, must be expanded, and the opportunities encompassed in such investment understood. The pressing questions related to male reproductive health, spanning the spectrum of andrology are identified in the Expert Recommendation.

DNA methylation dynamic in male rat germ cells during gametogenesis.

In mammals, a near complete resetting of DNA methylation (DNAme) is observed during germline establishment. This wave of epigenetic reprogramming is sensitive to the environment, which could impair the establishment of an optimal state of the gamete epigenome, hence proper embryo development. Yet, we lack a comprehensive understanding of DNAme dynamics during spermatogenesis, especially in rats, the model of choice for toxicological studies. Using a combination of cell sorting and DNA methyl-seq capture, we generated a stage-specific mapping of DNAme in nine populations of differentiating germ cells from perinatal life to spermiogenesis. DNAme was found to reach its lowest level at gestational day 18, the last demethylated coding regions being associated with negative regulation of cell movement. The following de novo DNAme displayed three different kinetics with common and distinct genomic enrichments, suggesting a non-random process. DNAme variations were also detected at key steps of chromatin remodeling during spermiogenesis, revealing potential sensitivity. These methylome datasets for coding sequences during normal spermatogenesis in rat provide an essential reference for studying epigenetic-related effects of disease or environmental factors on the male germline.

Epitranscriptome marks detection and localization of RNA modifying proteins in mammalian ovarian follicles.

BACKGROUND: Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3'UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization. RESULTS: The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that m6A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases ("writers"), binding proteins ("readers") and demethylases ("erasers") catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m5C-associated proteins were generally less abundant. In contrast, m6A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m5C mark in mice but both m5C and m6A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues. CONCLUSIONS: So far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period.

Reproductive toxicity of emerging plasticizers, flame retardants, and bisphenols, using culture of the rat fetal testis†.

The use of bis (2-ethylhexyl) phthalate (DEHP), 2,2'4,4'-tetrabromodiphenyl ether (BDE47), and bisphenol A (BPA), as plasticizers, flame retardants, and epoxy resins, respectively, has been regulated due to their endocrine disrupting activities. Replacements for these chemicals are found in human matrices, yet the endocrine disrupting potential of these emerging contaminants is poorly characterized. We compared the effects of legacy chemicals with those of their replacements using fetal rat testis organ culture. Fetal testes sampled at gestation day 15 were grown ex vivo, and the impact was evaluated after a 3-day exposure to 10 µM of each legacy chemical; two BPA analogs (bisphenol M and bisphenol TMC); three replacements for DEHP/MEHP (2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisononyl-phthalate, and diisodecyl adipate); or two replacements for BDE47 (tributoxyethyl phosphate and isopropylated triphenyl phosphate). We showed that only BPA and MEHP significantly decrease testosterone secretions after 24 h, while BPM and BPTMC have the opposite effect. Luteinizing hormone-stimulated testosterone was reduced by BPA and MEHP but was increased by BPTMC. After exposure, testes were used for immunofluorescent staining of germ cells, Sertoli cells, and Leydig cells. Interestingly, exposures to BPM or BPTMC induced a significant increase in the Leydig cell density and surface area. A decrease in germ cell density was observed only after treatment with MEHP or BDE47. MEHP also significantly decreased Sertoli cell proliferation. These studies show that some replacement chemicals can affect testicular function, while others appear to show little toxicity in this model. These findings provide essential information regarding the need for their regulation.

Impact of in Utero Rat Exposure to 17Alpha-Ethinylestradiol or Genistein on Testicular Development and Germ Cell Gene Expression.

Although the decline in male fertility is believed to partially result from environmental exposures to xenoestrogens during critical developmental windows, the underlying mechanisms are still poorly understood. Experimental in utero exposures in rodents have demonstrated the negative impact of xenoestrogens on reproductive development, long-term adult reproductive function and offspring health. In addition, transcriptomic studies have demonstrated immediate effects on gene expression in fetal reproductive tissues, However, the immediate molecular effects on the developing germ cells have been poorly investigated. Here, we took advantage of a transgenic rat expressing the green fluorescent protein specifically in germ cells allowing purification of perinatal GFP-positive germ cells. Timed-pregnant rats were exposed to ethinylestradiol (EE2, 2 µg/kg/d), genistein (GE, 10 mg/kg/d) or vehicle by gavage, from gestational days (GD) 13-19; testes were sampled at GD20 or post-natal (PND) 5 for histological analysis and sorting of GFP-positive cells. While EE2-exposed females gained less weight during treatment compared to controls, neither treatment affected the number of pups per litter, sex ratio, anogenital distance, or body and gonadal weights of the offspring. Although GE significantly decreased circulating testosterone at GD20, no change was observed in either testicular histology or germ cell and sertoli cell densities. Gene expression was assessed in GFP-positive cells using Affymetrix Rat Gene 2.0 ST microarrays. Analysis of differentially expressed genes (DEGs) (p < 0.05; fold change 1.5) identified expression changes of 149 and 128 transcripts by EE2 and GE respectively at GD20, and 287 and 207 transcripts at PND5, revealing an increased effect after the end of treatment. Only about 1% of DEGs were common to both stages for each treatment. Functional analysis of coding DEG revealed an overrepresentation of olfactory transduction in all groups. In parallel, many non-coding RNAs were affected by both treatments, the most represented being small nucleolar and small nuclear RNAs. Our data suggest that despite no immediate toxic effects, fetal exposure to xenoestrogens can induce subtle immediate changes in germ cell gene expression. Moreover, the increased number of DEGs between GD20 and PND5 suggests an effect of early exposures with latent impact on later germ cell differentiation.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Dynamics in the expression of epigenetic modifiers and histone modifications in perinatal rat germ cells during de novo DNA methylation†.

Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

Sperm DNA integrity in adult survivors of paediatric leukemia and lymphoma: A pilot study on the impact of age and type of treatment.

Childhood cancer survivors (CCS) are more likely than siblings to report low sperm count and to use assisted reproductive technologies. Yet, it is still unclear if the sperm produced many years after remission of cancer display DNA and chromatin damage linked to male infertility and poor embryo development. As well, the importance of the age at diagnosis in relation to puberty is poorly understood. In this pilot study, we compared reproductive parameters and sperm damage from adult survivors of childhood leukemia and lymphoma, sub-divided into those diagnosed before or after puberty, to men with no history of cancer. Our data indicate that CCS, independently of the age of diagnosis, have a high risk of low sperm count and when sperm are present, chances of DNA and chromatin abnormalities appear similar to those seen in the general population. Exposure to alkylating agents is correlated with low sperm count whereas exposure to anthracyclines, and doxorubicin in particular, could have long-term consequences on sperm integrity. This study highlights the need for further research on fertility among male CCS and the importance of informing families about the potential long-term impact of chemotherapy on male fertility regardless of age at diagnosis.

Sperm DNA Damage in Cancer Patients.

Fertility is a growing healthcare issue for a rising number of cancer survivors. In men, cancer itself and its treatment can negatively affect spermatogenesis by targeting the dividing spermatogonia and their cellular environment, ultimately leading to a reduction of testicular germ cells and sperm count. Experimental data and prospective longitudinal studies have shown that sperm production can recover after cancer treatment. But despite this, yet unpredictable, recovery in sperm production, cancer survivors are more at risk to produce sperm with aneuploidy, DNA damage, abnormal chromatin structure, and epigenetic defects even 2 years post-treatment. Sperm DNA alteration is of clinical concern, as these patients may father children or seek assisted reproduction technologies (ART) using gametes with damaged genome that could result in adverse progeny outcomes. Interestingly, large cohort studies revealed lower birth rate but no significant impact on the health of the children born from male cancer survivors (naturally or using ART). Nevertheless, a better understanding of how cocktail of chemotherapy and new anticancer agents affect spermatogenesis and sperm quality is needed to reduce side effects. Moreover, developing new fertility preservation strategies is essential as sperm cryopreservation before treatment is currently the only option but does not apply for prepubertal/young postpubertal patients.

In vitro study of doxorubicin-induced oxidative stress in spermatogonia and immature Sertoli cells.

Pediatric chemotherapy treatments can impair long-term male fertility. Unfortunately, no fertility preservation solution is available for pre-pubertal boys. Studies suggest that doxorubicin, used against pediatric cancers, induces oxidative stress in the testis. However, the targeted testicular cell types remain unknown. The goal of this study was to determine whether doxorubicin can induce oxidative stress in rat spermatogonia (GC-6Spg) and immature Sertoli (Ser-W3) cell lines, and to assess their protection by antioxidants. Using the MTT assay, we have shown that doxorubicin induces a time- and dose-dependent cytotoxicity in these two cell lines, Ser-W3 being more sensitive than GC-6Spg. After 3 h of treatment, reactive oxygen species and nuclear 8-oxo-deoxyguanosine increase in Ser-W3, but not in GC-6Spg. Moreover, after 6 h of treatment, intracellular reduced glutathione levels decrease significantly in Ser-W3 cells. These results show that doxorubicin induces oxidative stress in the Ser-W3 cell line. However, a depletion in glutathione does not affect their survival, and supplementation only offers a weak protection after exposure to doxorubicin, suggesting that the glutathione system is not essential for Ser-W3 cell line's defense against doxorubicin. On the other hand, among four antioxidants selected from the literature, none reduces the cytotoxicity of doxorubicin in Ser-W3 cells. Together, our data suggest that oxidative stress may not be a major pathway for doxorubicin's cytotoxicity in GC-6Spg and Ser-W3 lines. This study provides new insights in the mechanisms by which chemotherapies affect the pre-pubertal testis, with the long-term goal to help improve the quality of life of pediatric cancer survivors.

Fetal testis organ culture reproduces the dynamics of epigenetic reprogramming in rat gonocytes.

BACKGROUND: Epigenetic reprogramming is a critical step in male germ cell development that occurs during perinatal life. It is characterized by the remodeling of different epigenetic marks such as DNA methylation (5mC) and methylation of histone H3. It has been suggested that endocrine disruptors can affect the male germline epigenome by altering epigenetic reprogramming, but the mechanisms involved are still unknown. We have previously used an organ culture system that maintains the development of the different fetal testis cell types, to evaluate the effects of various endocrine disruptors on gametogenesis and steroidogenesis in the rat. We hypothesize that this culture model can reproduce the epigenetic reprogramming in gonocytes. Our aim was to establish the kinetics of three epigenetic marks throughout perinatal development in rats in vivo and compare them after different culture times. RESULTS: Using immunofluorescence, we showed that H3K4me2 transiently increased in gonocytes at 18.5 days post-coitum (dpc), while H3K4me3 displayed a stable increase in gonocytes from 18.5 dpc until after birth. 5mC progressively increased from 20.5 dpc until after birth. Using GFP-positive gonocytes purified from GCS-EGFP rats, we established the chronology of re-methylation of H19 and Snrpn in rat gonocytes. Most importantly, using testis explanted at 16.5 or 18.5 dpc and cultured for 2-4 days, we demonstrated that the kinetics of changes in H3K4me2, H3K4me3, global DNA methylation and on parental imprints can generally be reproduced ex vivo with the model of organ culture without the addition of serum. CONCLUSIONS: This study reveals the chronology of three epigenetic marks (H3K4me2, H3K4me3 and 5mC) and the patterns of methylation of H19 and Snrpn differentially methylated regions in rat gonocytes during perinatal development. Most importantly, our results suggest that the organ culture can reproduce the process of epigenetic reprogramming and can be used to study the impact of environmental chemicals on the establishment of the male germ cell epigenome.

Doxorubicin and vincristine affect undifferentiated rat spermatogonia.

Anticancer drugs, such as alkylating agents, can affect male fertility by targeting the DNA of proliferative spermatogonial stem cells (SSC). Therefore, to reduce such side effects, other chemotherapeutics are used. However, less is known about their potential genotoxicity on SSC. Moreover, DNA repair mechanisms in SSC are poorly understood. To model treatments deprived of alkylating agents that are commonly used in cancer treatment, we tested the impact of exposure to doxorubicin and vincristine, alone or in combination (MIX), on a rat spermatogonial cell line with SSC characteristics (GC-6spg). Vincristine alone induced a cell cycle arrest and cell death without genotoxic impact. On the other hand, doxorubicin and the MIX induced a dose-dependent cell death. More importantly, doxorubicin and the MIX induced DNA breaks, measured by the COMET assay, at a non-cytotoxic dose. To elucidate which DNA repair pathway is activated in spermatogonia after exposure to doxorubicin, we screened the expression of 75 genes implicated in DNA repair. Interestingly, all were expressed constitutively in GC-6spg, suggesting great potential to respond to genotoxic stress. Doxorubicin treatments affected the expression of 16 genes (>1.5 fold change; P < 0.05) involved in cell cycle, base/nucleotide excision repair, homologous recombination and non-homologous end joining (NHEJ). The significant increase in CDKN1A and XRCC1 suggest a cell cycle arrest and implies an alternative NHEJ pathway in response to doxorubicin-induced DNA breaks. Together, our results support the idea that undifferentiated spermatogonia have the ability to respond to DNA injury from chemotherapeutic compounds and escape DNA break accumulation.

Protective role of zinc against the toxicity induced by exposure to cadmium during gestation and lactation on testis development.

To assess the effects of exposure to Cd and Zn on rat testicular development, offspring, from mothers receiving either tap water, Cd, Zn or Cd+Zn during gestation and lactation periods, were observed on gestational day (GD) 20 and on postnatal days (PND) 12, 21 and 35. During gestation, Cd induced maternal hypozincemia and less transfer of Zn to the fetus. During lactation, progressive Cd accumulation and Zn depletion in testis at PND12 and PND21 were noted. An increase of abnormal seminiferous tubules and a decrease in testis weight and plasmatic testosterone concentration were also observed at PND21 and PND35 respectively. Interestingly, Zn supply induced a significant protection against Cd toxicity. These results suggest that the toxic effects of Cd observed during development are mediated by the disruption of Zn metabolism, which is established in mothers during pregnancy causing Zn deficiency in fetuses and continues to become more pronounced during lactation.

Ozone treatment prevents the toxicity of an environmental mixture of estrogens on rat fetal testicular development.

Effluents from wastewater treatment plants contain a mixture of estrogens (MIX: 17ß-estradiol: E2, estrone: E1, estriol: E3 and 17α-ethinylestradiol EE2). High doses of estrogens have been shown to negatively impact fetal testicular development, but the impact of low doses of estrogens in mixture have yet to be elucidated. Using an organ culture system in which embryonic 15.5 day-old rat testes were grown ex vivo, we showed that exposure to the MIX at environmentally relevant concentrations reduces testis growth. No effect was observed on testosterone secretion, but we quantified a significant decrease in the number of Sertoli cells and gonocytes because of higher rates of apoptosis. As ozone (O3) can be used as a disinfectant during wastewater treatment, we confirmed by HPLC-MS analysis that it removes the four parent compounds. Interestingly, the negative effects of the MIX were not observed when testes were exposed to the MIX treated with O3.

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.

Case report: the use of annexin V coupled with magnetic activated cell sorting in cryopreserved spermatozoa from a male cancer survivor: healthy twin newborns after two previous ICSI failures.

PURPOSE: The aim of the study is to report successful outcome (live births) after sperm sorting with annexin V-MACS on cryopreserved spermatozoa with high level of sperm DNA fragmentation from a cancer patient survivor. METHODS: Cryopreserved spermatozoa were sorted with annexin V-MACS prior to ICSI. Sperm DNA fragmentation was evaluated by SCSA(®) and TUNEL. RESULTS: The couple had two previous IVF/ICSI cycles failures using sperm cryopreserved before cancer treatment. On third ICSI cycle attempt results were as follow: pre-annexin V-MACS sperm quality: 10 × 10(6)/ml, 3.3 % progressive motility, 1 % normal forms, TUNEL: 72.5 % positive cells, SCSA(®): 76.6 % DFI. Post-annexin V-MACS sperm quality: 2.8 × 10(6)/ml, 10 % progressive motility, TUNEL: 58.8 % positive cells. Eight metaphase II oocytes were collected, 4 fertilized, 2 embryos were transferred on day 3 and healthy twins were born (1 boy, 1 girl). CONCLUSIONS: Annexin V-MACS technique could be a potential tool to improve sperm quality on cryopreserved spermatozoa of cancer patient and improve ICSI outcome.

Selective induction of glutathione S-transferases in round spermatids from the Brown-Norway rat by the chemotherapeutic regimen for testicular cancer.

Chemotherapeutic drugs can affect DNA in male germ cells, thereby impacting on the integrity of the genome transmitted to offspring. Drug metabolizing enzymes can protect cells from xenobiotic insult. We analyzed the expression pattern of such enzymes in isolated round spermatids from rats exposed to drugs used to treat testicular cancer: bleomycin, etoposide, and cisplatin (BEP). The number of isozymes expressed and the overall relative expression values were highest for the glutathione S-transferases (GSTs). Moreover, BEP treatment significantly increased the expression of 8 GSTs and 3 aldehyde dehydrogenases. Increased expression of GST isozymes was confirmed by qRT-PCR and Western blot analysis. Although Gst genes can be targets for epigenetic modifications, promoter DNA methylation was not affected by BEP treatment. As GSTs are involved in drug resistance mechanisms, we hypothesize that BEP induction of GST expression may lead to the survival of damaged germ cells and the production of abnormal sperm.

Degradation of 17α-ethinylestradiol by ozonation--identification of the by-products and assessment of their estrogenicity and toxicity.

The presence of the synthetic estrogen 17α-ethinylestradiol (EE2) in waters at low levels is a concern due to its ability to act as an endocrine disruptor. Ozone (O(3)) is commonly used in water treatment and reacts with EE2 to form by-products having characteristics that are mostly unknown. The aim of this study was to identify the by-products of E2 and EE2 ozonation and determine their estrogenicity and toxicity relative to the parent compound. Ozonation by-products were identified via LC-MS analysis. The estrogenicity was measured using the YES assay, and toxicity was determined by monitoring effects on histology of fetal rat testes and testosterone secretion by these tissues. Two EE2 by-products were identified with open phenolic ring structures (masses 302 and 344 u). The Yeast Estrogen Screening (YES) assay showed a decreased but incomplete removal of estrogenicity after ozonation of EE2. Histological analysis of fetal testes revealed that neither E2 nor EE2, with or without ozonation, had any effect on seminiferous cord formation; however, a remarkable negative effect on testosterone secretion was observed, with EE2 by-products after ozonation showing the most rapid and extensive inhibition. These results show that the removal of EE2 via reaction with O(3) resulted in the formation of by-products that are less estrogenic (as demonstrated by the YES assay), but have a greater negative impact on testosterone secretion. Thus, the disappearance of the parent compound is not a sufficient endpoint, as the by-products created may be more toxic. Care should be taken when implementing oxidation applications such as ozone during waste water treatment.

Epigenetic alterations in sperm DNA associated with testicular cancer treatment.

DNA methylation, a key component of the epigenome involved in regulating gene expression, is initially acquired in the germ line at millions of sites across the genome. Altered sperm methylation patterns are associated with infertility and transgenerational effects in humans and rodents. Testicular cancer is the most common form of cancer among men of reproductive age and has a high cure rate associated with chemotherapy treatment with bleomycin, etoposide, and cis-platinum (BEP). Although these drugs result in improved survival, they also affect the number and quality of germ cells. Our goal was to assess germ cell methylation patterns in a rodent model emulating the BEP treatment regimens used in human testicular cancer treatment. Animals were treated with control, or 0.3× (low) or 0.6× (high) dose of BEP, where a 1× dose is equivalent to human treatment regimens. Both dose-dependent and germ cell-dependent DNA methylation alterations were found at numerous loci throughout the genome. Of about 3000 loci tested, 42 loci were affected by BEP at the round spermatid stage of germ cell development, whereas 101 loci were affected in spermatozoa; 15 loci were consistently altered in spermatozoa of all high dose-treated rats. Both hyper- and hypomethylation were detected, suggesting either an interference with normal methylation patterning or abnormal repair of damaged patterns during spermatogenesis. The results indicate that a combination chemotherapy regimen used for testicular cancer treatment can result in altered DNA methylation patterns in spermatozoa and that some loci are more susceptible to damage than others.

PABP interacting protein 2A (PAIP2A) regulates specific key proteins during spermiogenesis in the mouse.

During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2b double-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2a have established roles in reproduction, the roles of many others remain to be determined.