Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa.

STUDY QUESTION: Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? SUMMARY ANSWER: Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. WHAT IS KNOWN ALREADY: Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. STUDY DESIGN, SIZE, DURATION: Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5 mm, 1 h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 9000 genomic regions, 150-1000 bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. LIMITATIONS, REASONS FOR CAUTION: The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. WIDER IMPLICATIONS OF THE FINDINGS: The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. STUDY FUNDING/COMPETING INTEREST(S): Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.

Dyslipidemia alters sperm maturation and capacitation in LXR-null mice.

Lipid metabolism disorders (dyslipidemia) are causes of male infertility, but little is known about their impact on male gametes when considering post-testicular maturation events, given that studies concentrate most often on endocrine dysfunctions and testicular consequences. In this study, three-month-old wild-type (wt) and Liver-X-Receptors knock out (Lxrα;ß-/- ) males were fed four weeks with a control or a lipid-enriched diet containing 1.25% cholesterol (high cholesterol diet (HCD)). The HCD triggered a dyslipidemia leading to sperm post-testicular alterations and infertility. Sperm lipids were analyzed by LC-MS and those from Lxrα;ß-/- males fed the HCD showed higher chol/PL and PC/PE ratios compared to wt-HCD (P < 0.05) and lower oxysterol contents compared to wt (P < 0.05) or Lxrα;ß-/- (P < 0.05). These modifications impaired membrane-associated events triggering the tyrosine phosphorylation normally occurring during the capacitation process, as shown by phosphotyrosine Western blots. Using flow cytometry, we showed that a smaller subpopulation of spermatozoa from Lxrα;ß-/- -HCD males could raise their membrane fluidity during capacitation (P < 0.05 vs wt or wt-HCD) as well as their intracellular calcium concentration (P < 0.05 vs Lxrα;ß-/- and P < 0.001 vs wt). The accumulation of the major sperm calcium efflux pump (PMCA4) was decreased in Lxrα;ß-/- males fed the HCD (P < 0.05 vs Lxrα;ß-/- and P < 0.001 vs wt). This study is the first showing an impact of dyslipidemia on post-testicular sperm maturation with consequences on the capacitation signaling cascade. It may lead to the identification of fertility prognostic markers in this pathophysiological situation, which could help clinicians to better understand male infertilities which are thus far classified as idiopathic.

A novel antioxidant formulation designed to treat male infertility associated with oxidative stress: promising preclinical evidence from animal models.

STUDY QUESTION: Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? SUMMARY ANSWER: Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. WHAT IS KNOWN ALREADY: Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. STUDY DESIGN, SIZE, DURATION: The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. MAIN RESULTS AND ROLE OF CHANCE: Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). LIMITATIONS, REASONS FOR CAUTION: It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. WIDER IMPLICATIONS OF THE FINDINGS: Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

Deferasirox, an Iron-Chelating Agent, Improves Testicular Morphometric and Sperm Functional Parameters in a Rat Model of Varicocele.

Varicocele is characterized by testicular dysfunction that originates from hyperthermia and hypoxia, leading to defects in testicular tissue and altered spermatozoa structure and function. The varicocele testis is characterized by the presence of intracellular iron deposits that contribute to the associated oxidative stress. Therefore, we tested the hypothesis that administration of an iron-chelating agent, such as deferasirox (DFX), could potentially mitigate the consequences of varicocele on testicular tissue and spermatozoa. Using a well-established rat model of varicocele (VCL), we show that treatment with DFX partially improved the structure and function of the testis and spermatozoa. In particular, sperm motility was markedly restored whereas abnormal sperm morphology was only partially improved. No significant improvement in sperm count was observed that could be associated with the proapoptotic response observed following iron chelation treatment. No reduction in oxidative damage to spermatozoa was observed since lipid peroxidation and DNA integrity were not modified. This was suggested to be a result of increased oxidative stress. Finally, we also saw no indication of attenuation of the endoplasmic reticulum/unfolded protein (ER/UPR) stress response that we recently found associated with the VCL testis in rats.

Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity?

BACKGROUND: The Sperm Chromatin Structure Assay (SCSA®), in addition to identifying the DNA Fragmentation Index (DFI) also identifies High DNA satiability (HDS), supposed to reflect the nuclear compaction of spermatozoa. However, data on what exactly this parameter reveals, its relevance and usefulness are contradictory. In order to shed light on this situation, spermatozoa of a cohort (N = 397) of infertile men were subjected to the SCSA®, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling) and CMA3 (Chromomycin A3) tests. In a smaller subcohort (N = 100), aniline blue (AB) and toluidine blue (TB) staining were performed in addition. The objective of this study was thus to answer the question of whether HDS is a relevant and reliable parameter to be taken into account? RESULTS: HDS does not appear to be a reliable indicator of nuclear immaturity because it shows a weak correlation with the CMA3, AB and TB stains. The low correlation of HDS with sperm DNA fragmentation (TUNEL and SCSA®) and DNA condensation (CMA3, AB and TB) tests suggests that these two parameters could be decoupled. Unlike DFI and TUNEL, HDS has not been shown to correlate with classic clinical situations of male infertility (asthenozoospermia, teratozoospermia or astheno-teratozoospermia). CONCLUSION: HDS correlates poorly with most tests that focus specifically on the level of maturity of the sperm nucleus. To our knowledge, this study is the first to compare SCSA®, TUNEL, AB, TB and CMA3 assays on identical samples. It shows the potency, consistency and limitations of each test and the care that must be taken in their interpretation.

CONTEXTE: Le test SCSA® (Sperm Chromatin Structure Assay), en plus d'identifier l'indice de fragmentation de l'ADN (DFI = DNA fragmentation Index), identifie également la susceptibilté à la coloration à l'acridine orange de l'ADN (HDS: High DNA Stainability), censée refléter la compaction nucléaire des spermatozoïdes. Cependant, les données sur ce que révèle exactement ce paramètre, sa pertinence et son utilité sont contradictoires. Afin de faire la lumière sur cette situation, les spermatozoïdes d'une cohorte (N = 397) d'hommes stériles ont été soumis aux tests SCSA®, TUNEL et CMA3. Dans une sous-cohorte plus petite (N = 100), la coloration au bleu d'aniline (AB) et au bleu de toluidine (TB) a été effectuée en plus. L'objectif de cette étude était donc de répondre à la question de savoir si le HDS est. un paramètre pertinent et fiable à prendre en compte? RÉSULTATS: Le HDS ne semble pas être un indicateur fiable de l'intégrité nucléaire car il montre une faible corrélation avec les tests CMA3, AB et TB. La faible corrélation du HDS avec les tests de fragmentation de l'ADN du sperme (TUNEL et SCSA®) et de condensation de l'ADN (CMA3, AB et TB) suggère que ces deux paramètres pourraient être découplés. Contrairement au DFI et au TUNEL, il n'a pas été démontré que le HDS est. corrélé avec les situations cliniques classiques de l'infertilité masculine (asthénozoospermie, tératozoospermie ou asthéno-tératozoospermie). CONCLUSION: Le HDS présente une faible corrélation avec la plupart des tests qui se concentrent spécifiquement sur le niveau de maturité du noyau du sperme. À notre connaissance, cette étude est. la première à comparer les tests SCSA®, TUNEL, AB, TB et CMA3 sur des échantillons identiques. Elle montre la puissance, la cohérence et les limites de chaque test et le soin qui doit être apporté à leur interprétation.

Impairment of sperm maturation and capacitation due to diet-dependent cholesterol overload.

BACKGROUND: Lipid metabolic disorders (dyslipidemia) are constantly increasing in occidental societies and lead to the development of pathologies such as obesity, diabetes, and metabolic syndrome. It has been demonstrated that dyslipidemia can alter the reproductive function. Animal models have recently been used to show that the offspring of dyslipidemic males could also develop such pathologies and that the transgenerational transmission involved post-testicular sperm maturation. These data targeted the essential role of male gamete epididymal maturation and its importance for the health of the offspring. OBJECTIVES: This publication summarizes in the first place experimental data obtained using a mouse model of dyslipidemia-induced post-testicular infertility, knockout mice for the two isoforms of the 'Liver X Receptors' (Lxrα;ß-/- ), the major regulators of cholesterol homeostasis. The impact of a high cholesterol diet (HCD) on the protein YWHAZ (14-3-3 ζ or tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Zeta) was also investigated in our model. MATERIALS AND METHODS: In our mouse model, when young fertile Lxrα;ß-/- males aged three months were fed four weeks with a HCD, they developed an epididymal phenotype leading to infertility. The level of sperm YWHAZ was evaluated by Western blot and its tyrosine phosphorylation state by immunoprecipitation followed by Western blot. RESULTS: Our data revealed that sperm lipid composition and structure were altered, leading to defects of the capacitation-associated signaling pathway. They also showed that both the level and the tyrosine phosphorylation state of YWHAZ were affected by the HCD in sperm cells from Lxrα;ß-/- males. DISCUSSION AND CONCLUSION: YWHAZ could be a new important regulator of capacitation-associated tyrosine phosphorylation and a marker of dyslipidemia-induced infertility.

Of vessels and cells: the spatial organization of the epididymal immune system.

BACKGROUND: One third of infertility cases in couples worldwide has an exclusive male origin and immune disorders, essentially due to repetitive infections, are emerging an cause of male infertility. As the place of sperm maturation, epididymis must be preserved from excessive immune responses that may arise following infections of the male genital tract. At the same time, epididymis must set and maintain a tolerogenic environment in order not to destroy sperm cells that enter the tissue at puberty, long after the immune system has been taught to recognize self pathogens. The immune cells that populate the epididymis have raised growing interest over the last thirty years but they may be not sufficient to understand the immune balance existing in this organ, between immune response to pathogens and tolerance to spermatozoa. Indeed, immune cells are the most motile cells in the organism and need blood and lymphatic vessels to traffic between lymphoid organs and sites of infection to induce efficient responses. OBJECTIVES: To review the literature on the blood and lymphatic vessels, and on the immune cells present at steady state in the rodent epididymis (rat and mouse). MATERIALS AND METHODS: PubMed database was searched for studies reporting on the spatial organization of the rodent epididymal vasculature and immune cell types at steady state. This search was combined with recent findings from our team. RESULTS: At steady state, the rodent epididymis presents with dense blood and lymphatic networks, and a large panel of immune cells distributed across the interstitum and epithelium along the organ. CONCLUSIONS: The immune system of the rodent epididymis is highly organized. Exploring its functions, especially in an infectious context, is the essential coming step before any transposition to human.

Epididymal small non-coding RNA studies: progress over the past decade.

BACKGROUND: Small non-coding RNAs (sncRNAs) accomplish a huge variety of biological functions. Over the past decade, we have witnessed the substantial progress in the epididymal sncRNA studies. In the Epididymis 7, we had the true privilege of having a whole session to share our findings and exchange ideas on the epididymal sncRNA studies. OBJECTIVES: This mini-review attempts to provide an overview of what is known about the sncRNAs in the mammalian epididymis and discuss the future directions in this field. METHODS: We surveyed literature regarding the sncRNA studies in the mammalian epididymis, and integrated some of our unpublished findings as well. We focus on the progress in methodology and the advances in our understanding of the expression and functions of epididymal sncRNAs. RESULTS AND DISCUSSION: The applications of high-throughput approaches have made great contributions in the discovery of new sncRNA species and profiling their dynamics in the epithelial cells, the passing spermatozoa, and the luminal environment. The diverse classes of epididymal sncRNAs exert important biological functions from the in situ regulation of epididymal gene expression to the epigenetic inheritance in the offspring. CONCLUSION: Although still in its infancy, we believe that the research on epididymal sncRNAs will not only lead to a better understanding of their physiological and pathological functions, but also contribute to the whole landscape of the RNA field.

Identification of sperm antigens as a first step towards the generation of a contraceptive vaccine to decrease fossorial water vole Arvicola terrestris Scherman proliferations.

Immunocontraceptive strategies have proved to be efficient in controlling fertility of various mammalian species. In the present study we have made the first steps towards the identification of Arvicola terrestris sperm antigens that could be used as targets in the development of a contraceptive vaccine to limit the proliferations of this pest rodent. Rabbit-raised polyclonal antisera directed against complete A. terrestris spermatozoa were used to identify and characterize on 2D-gels coupled with a MALDI-TOF mass spectrometry analysis A. terrestris sperm proteins. Amongst the proteins pinpointed by this approach some were further investigated based on their tissue- and/or sperm-specific expression, and their relevance to fertility or sperm/egg interaction. In parallel, three proteins that have been already reported in the literature to be appropriate targets for the development of contraceptive vaccines in other mammalian species have also been looked for in A. terrestris. With the selected protein targets, a reverse-PCR approach using degenerate primers was employed to amplify corresponding A. terrestris cDNAs. After conceptual translation and sequence alignment, different proteins were studied to determine zones with sufficient sequence divergence and of antigenic/immunogenic nature that could be used in future assays to immunize animals.

Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?

BACKGROUND: In humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs. METHODS: Forty Nellore bulls were divided into three age groups: 1.8 to 2 years - young bulls; 3.5 to 7 years - adult bulls; and 8 to 14.3 years - aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA). RESULTS: We report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05). CONCLUSION: These results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

CONTEXTE: Chez l'homme, de nombreuses données indiquent maintenant que l'avancée de l'âge du père est associée à une réduction de l'intégrité de l'ADN des spermatozoïdes et aux échecs de développement embryonnaire. D'un autre côté, il est aussi rapporté que les jeunes pères, tels les adolescents, sont porteurs d'un risque accru d'issue défavorable de la grossesse. Ces observations suggèrent que, au moins chez l'humain, il existe une tranche d'âge dans laquelle l'intégrité de l'ADN des spermatozoïdes est optimale. Chez les bovins, on dispose de peu de connaissances sur la qualité de l'ADN des spermatozoïdes des jeunes taureaux et sur son mode d'évolution avec l'âge. La présente étude a pour but de combler ce manque car il peut être important, pour l'industrie bovine, de savoir à quelle période précise un taureau est un reproducteur optimal pour les programmes de reproduction. MATÉRIEL ET MÉTHODES: Quarante taureaux Nellore ont été répartis en trois groupes d'âge: 1, 8 à 2 ans ­ jeunes taureaux; 3,5 à 7 ans ­ taureaux adultes; et 8 à 14,3 ans ­ taureaux âgés. Trois éjaculats ont été collectés par taureau, cryopréservés et évalués pour différents paramètres incluant l'analyse assistée du sperme par ordinateur (CASA), l'intégrité des membranes plasmique et acrosomique, le potentiel mitochondrial, la protamination du noyau, l'altération oxydative de l'ADN et l'évaluation de la structure de la chromatine du noyau du spermatozoïde (SCSA). RÉSULTATS: Nous rapportons ici que les jeunes taureaux présentent des valeurs supérieures de la mobilité des spermatozoïdes et de l'intégrité des membranes plasmique et acrosomique, ainsi qu'un potentiel mitochondrial élevé. Cependant, les jeunes taureaux présentent aussi des valeurs plus élevées d'anomalies morphologiques des spermatozoïdes que celles des groupes adulte et âgé (p < 0.05). De plus, les jeunes taureaux ont une protamination plus défectueuse que celle des taureaux plus âgés. Les taureaux les plus âgés présentent plus d'altérations oxydatives du noyau que les jeunes taureaux alors que les deux groupes -jeunes et âgés- sont plus susceptibles d'avoir une dénaturation de l'ADN nucléaire comme indiqué par le SCSA (p < 0.05). CONCLUSIONS: Ces résultats indiquent que les spermatozoïdes des jeunes taureaux survivent le mieux au processus de congélation, suivis par les adultes puis les âgés. Toutefois, les jeunes taureaux et les âgés sont plus susceptibles d'avoir une altération de l'ADN, causée respectivement par une protamination déficiente et une oxydation. Par conséquent, bien que les jeunes taureaux aient des paramètres spermatiques corrects à l'évaluation classique, nos résultats indiquent que leurs spermatozoïdes peuvent présenter un certain degré d'immaturité structurale nucléaire.

Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

Les spermatozoïdes sont des cellules particulièrement complexes et très spécialisées comparées aux cellules somatiques. Leur rôle est de délivrer dans l'ovule le patrimoine génétique paternel ainsi qu'un lot de protéines et d'ARNs de façon à initier un nouvel individu. Le succès reproductif qui recouvre les aspects de développement embryonnaire harmonieux et de santé de la descendance repose en partie sur l'intégrité de la chromatine spermatique. Les dommages à l'ADN spermatique sont clairement associés aux échecs reproductifs que ce soit en reproduction naturelle et en procréation médicalement assistée (PMA). Cette revue présente les derniers développements concernant l'organisation très particulière de la chromatine spermatique et ses impacts sur le succès reproductif quand cette organisation est perturbée, en particulier sur le développement embryonnaire et les risques trangénérationnels.

Developmental regulation of a silkworm gene encoding multiple GATA-type transcription factors by alternative splicing.

Gene BmGATA beta of the silkworm Bombyx mori was previously shown to encode factor BCFI, which regulates the expression of a class of chorion genes expressed during the late stages of choriogenesis. We now show that the expression of the BmGATA beta gene is spatially and temporally regulated by alternative splicing that generates two major (BmGATA beta 1 and BmGATA beta 2) and one minor (BmGATA beta 3) mRNA isoforms of non-identical tissue distribution. The three isoforms differ in the organization of the DNA-binding domains of the corresponding polypeptides. While all three isoforms are expressed in ovarian follicular cells and in testes, only one of them, BmGATA beta 1, is gonad-specific. BmGATA beta 2 is expressed in a variety of other larval and pupal tissues, while BmGATA beta 3 is detected in some pupal but none of the larval tissues. Analysis of RNA isolated from follicular cells of developing ovarian follicles has shown that the onset of ovarian transcription for all three mRNA isoforms occurs during late vitellogenesis, and that the level of accumulated mRNA declines significantly at the onset of choriogenesis. Coincident with the onset of late chorion gene expression, we have observed a significant change in the preference of splice site selection in favour of the one that results in the generation of BmGATA beta 1 mRNA. The transcriptional activation of the BmGATA beta gene in follicular cells during late vitellogenesis correlates with the previously demonstrated initial accumulation of factor BCFI in the cytoplasm of follicular cells and its appearance in follicular cell nuclei only during the late stages of choriogenesis. The relationship between factor BCFI and the different polypeptides encoded by the three BmGATA beta mRNA isoforms is discussed.

Cloning of the mouse gene encoding plasma glutathione peroxidase: organization, sequence and chromosomal localization.

Using a reverse transcription coupled to PCR amplification strategy, with degenerated primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, we have generated, from mouse epididymal RNA, a cDNA fragment which was subsequently used to isolate a genomic clone encoding mouse plasma GPX (GPX3). GPX3 is a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma. We confirm here that the mouse epididymis is a new site of expression of GPX3 and report, together with the sequence, the structural analysis and the chromosomal localization of the mouse GPX3 single-copy gene to chromosome 11.

Nuclear oxysterol receptors, LXRs, are involved in the maintenance of mouse caput epididymidis structure and functions.

In this study we looked at the epididymides and spermatozoa of mice knocked-out for nuclear oxysterol receptors (LXR). We have shown that LXR-deficient mice exhibited upon ageing a severe disruption of their caput epididymides associated with abnormal accumulation of neutral lipids. The epididymis defaults were correlated with sperm head fragility and infertility. In agreement with the observed caput defect in transgenic animals in which both LXRalpha and LXRbeta isoforms were disrupted, we have shown here that both receptors are expressed in caput and cauda epididymides regions. LXRbeta was predominantly expressed throughout the mouse epididymis while the expression of LXRalpha was weaker. In addition, the expression of selected genes that can be considered as markers of adult epididymis function was monitored via Northern blots in the different single and double LXR-deficient backgrounds. Altogether, the data presented here suggest that LXR receptors are important actors in epididymis function.

Antioxidant strategies in the epididymis.

Spermatozoa are very specialized cells, dedicated to fertilization of the oocyte. The attainment of this biological role is partly due to the fusogenic properties of the sperm plasma membrane, which is particularly rich in polyunsaturated fatty acids (PUFA). This predominance of PUFA renders spermatozoa highly susceptible to lipid peroxidation due to attacks from reactive oxygen species (ROS). These attacks ultimately lead to the impairment of sperm function through oxidative stress. Despite such disruptive effects, it should be also emphasized that these molecules also play an important positive, physiological role in the regulation of sperm physiology through their participation in apoptosis and the signal transduction cascades that control sperm maturation and capacitation. In this article, the different sources of ROS are examined and then the antioxidant strategies that protect these cells during epididymal transit are reviewed. While the major focus is on the involvement of glutathione peroxidase in this process, consideration will also be given to a range of additional antioxidant enzymes (catalase, indolamine dioxygenase and superoxide dismutase) that have evolved to protect spermatozoa during this extremely vulnerable phase in their life history. Besides the classical enzymatic roles of these enzymes in recycling ROS, additional features are discussed in the light of contraceptive development.

Characterization of an androgen response element within the promoter of the epididymis-specific murine glutathione peroxidase 5 gene.

We have shown in earlier studies, using a mouse model, that the expression of the glutathione peroxidase 5 protein (GPX5) is restricted to the epididymis and that the accumulation of its corresponding mRNA is hormonally, spatially and temporally regulated throughout postnatal development. We report here, using run-on assays, transient expression experiments as well as gel-shift and footprinting analyses on the findings that at least part of the androgenic control of the GPX5 expression is exerted at the transcriptional level via an androgen response element localized in the distal promoter region of the GPX5 gene. The gpx5 androgen response element (ARE) is found to be consistent with the consensus palindromic steroid-receptor target sequence 5'-AGWACWnnnTGTYCT-3' but exhibits a quite weak conservation in the left half site. The data presented here further expand the diversity of sequence able to confer androgen responsiveness.

Spontaneously immortalized epithelial cells from mouse caput epididymidis.

We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

The silkmoth homolog of the Drosophila ecdysone receptor (B1 isoform): cloning and analysis of expression during follicular cell differentiation.

To understand the role that 20-hydroxy-ecdysone (20E) plays during ovarian development in Bombyx mori, we have undertaken the cloning of the silkworm ecdysone receptor (EcR) and a study of its expression during follicular cell differentiation. We have cloned a cDNA that contains a complete open reading frame for a 68.1 kDa polypeptide that shares extensive similarities with the B1 isoform of the Drosophila EcR. The presumed silkmoth EcR (BmEcR) is encoded by a single copy gene whose length is in excess of 23 kb. A portion of this gene encompassing seven exons that constitute the cloned BmEcR cDNA was also characterized. Employment of monoclonal antibodies, directed against the DNA binding domain of the Drosophila EcR, in Western blot analyses revealed the presence of a major 70 kDa polypeptide in extracts of follicular cells and other silkmoth tissues. The mRNA and protein encoded by BmEcR are present in constant amounts in follicular cells throughout vitellogenesis but disappear transiently at the onset of choriogenesis and reappear during the later stages of choriogenesis. The down-regulation of BmEcR in follicular cells during oogenesis suggest a complex relationship between 20E, the induction of the program of chorion gene expression in follicular cells during mid-vitellogenesis and the execution of this program at the end of vitellogenesis.

Mammalian glutathione peroxidases control acquisition and maintenance of spermatozoa integrity.

In mammals, posttesticular epididymal sperm maturation is considered an essential step in the transformation of immature testicular gametes to mature spermatozoa capable of fertilization. Reactive oxygen species (ROS) have been shown to be key actors in this maturation process, and it is now clear that ROS are central for sperm physiology in processes such as sperm maturation and capacitation. However, during epididymal maturation and storage and until the onset of fertilization, oxidative damage is a threat spermatozoa must face more than any other cells. Spermatozoa were found to be extremely sensitive to oxidative attacks correlated with lipid peroxidation, DNA damage, and impaired sperm motility, all affecting fertilization. To control the quantity of H(2)O(2) in the vicinity of male gametes, mammalian epididymis uses a panel of nonenzymatic and enzymatic scavengers, among which the glutathione peroxidase (GPx) family is largely represented. Among the various GPx proteins expressed in the mammalian epididymis, GPx4 and GPx5 occupy unique positions and functions that are reviewed in this paper. This paper underlines the importance of the GPx protein family in determining the fertilizing potential of mammalian spermatozoa. This is particularly relevant in the field of mammalian fertility and infertility as well as in the development of assisted medical procreation technologies and male gamete preservation techniques that are extensively used in human and animal reproduction programs.

GATA-type zinc finger motif-containing sequences and chorion gene transcription factors of the silkworm Bombyx mori.

To characterize a DNA-binding protein, BCFI, which regulates the expression of silkmoth chorion genes through binding to gene promoter elements identical to those recognized by the GATA family of transcription factors, we have carried out polymerase chain reaction amplifications of Bombyx mori genomic DNA using degenerate primers derived from the conserved DNA binding domain of mammalian GATA factors. Two single copy genes, BmGATA alpha and BmGATA beta, were identified, which encode sequences containing GATA-type zinc finger motifs. The BmGATA beta gene is expressed in follicular and Bm5 tissue culture cells, the two cell types that contain BCFI. No BmGATA alpha gene transcripts were detectable in the tissues that were tested. Upon overexpression in Escherichia coli, a peptide encompassing the BmGATA beta zinc finger motif was able to bind specifically to the BCFI recognition motif of the chorion gene promoters. A polyclonal antibody directed against the zinc finger domain of BmGATA beta was also used in gel retardation assays to confirm that factor BCFI is indeed encoded by the BmGATA beta gene. Conceptual translation of a complete cDNA clone encoding the BmGATA beta protein revealed that this protein has a size similar to that of an immunoreactive protein, presumably BCFI, which is present in follicular cell extracts.