Joelle Henry-Berger

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.

DNA oxidative damage in mammalian spermatozoa: where and why is the male nucleus affected?

Gamete DNA integrity is one key parameter conditioning reproductive success as well as the quality of life for the offspring. In particular, damage to the male nucleus can have profound negative effects on the outcome of fertilization. Because of the absence of repair activity of the quiescent mature spermatozoa it is easily subjected to nuclear damage, of which oxidative damage is by far the most prominent. In relation to the organization of the mammalian sperm nucleus we show here that one can correlate the nuclear regions of lower compaction with areas preferentially showing oxidative damage. More precisely, we show that oxidative DNA damage targets primarily histone-rich and nuclear matrix-attached domains located in the peripheral and basal regions of the mouse sperm nucleus. These particular sperm DNA domains were recently shown to be enriched in genes of paramount importance in postfertilization DNA replication events and in the onset of the embryonic developmental program. We propose that monitoring of sperm DNA oxidation using the type of assay presented here should be considered in clinical practice when one wants to estimate the integrity of the paternal nucleus along with more classical assays that essentially analyze DNA fragmentation and nucleus compaction.

Deficient Tryptophan Catabolism along the Kynurenine Pathway Reveals That the Epididymis Is in a Unique Tolerogenic State

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1−/−-deficient mice. In the caput epididymis of Ido1−/− animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.

Indoleamine 2,3-dioxygenase 1 (ido1) is involved in the control of mouse caput epididymis immune environment.

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed.

A NETWORK-BASED APPROACH FOR PREDICTING HSP27 KNOCK-OUT TARGETS IN MOUSE SKELETAL MUSCLES

Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Here, we have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions, e.g. members of Hsp families (Hsp20, Cryab, Hsp70a1a, and Hsp90aa1), regulators of apoptosis (Fas, Chuk, and caspase-3), translation factors (Eif4E, and Eif4G1), cytoskeletal proteins (Desmin) and antioxidants (Sod1). The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness.

Oxidative DNA damage in mouse sperm chromosomes: Size matters.

Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

Rôle des récepteurs nucléaires des oxystérols LXR dans la régulation de l’homéostasie du cholestérol au niveau de l’appareil reproducteur mâle

ResumeLes récepteurs nucléaires des oxystérols LXR (Liver X receptor) α et LXRß sont des facteurs de transcription appartenant à la superfamille des récepteurs nucléaires. Ils sont activés par une série particulière d’oxystérols. Des antagonistes naturels ont été également identifiés comme les acides gras poly-insaturés ou certains sulfates de cholestérol plasmatiques. L’étude des souris déficientes en LXRs a permis de les associer à la régulation de nombreux métabolismes (cholestérol, acides gras, glucose, stéroïdes).Les LXRs et leur partenaire RXR (récepteur de l’acide rétinoïque 9-cis) sont exprimés dans le tractus génital mâle et les testicules, et leurs ligands y sont à des concentrations physiologiquement actives. Dans ces organes, l’homéostasie du cholestérol doit être strictement régulée car 1) le cholestérol est un précurseur indispensable pour la synthèse des stéroïdes testiculaires; 2) pendant la maturation épididymaire, la membrane plasmique des spermatozoïdes subit des changements de composition notamment la diminution de cholestérol et de lécithines.L’analyse des souris déficientes en récepteurs LXR α et LXRß a mis en évidence une déstructuration de la couche épithéliale du segment 2 de la tête de l’épididyme, ainsi qu’une fragilité des spermatozoïdes recueillis. Au total, les analyses de physiologie intégrative et moléculaire mettent en évidence le rôle des récepteurs nucléaires LXRs dans la physiologie de la reproduction chez le mâle.AbstractNuclear oxysterol receptors, LXRα and LXRß, are transcription factors that belong to the nuclear receptor superfamily. They bind and are activated by a specific class of oxysterols. Natural antagonists have also been described, such as polyunsaturated fatty acids or plasma sulfated oxysterols.Phenotypic analysis of mice lacking LXRα and/or LXRß demonstrated their roles in various physiologic processes and metabolisms (lipid or glucose homeostasis). LXR, as well as their heterodimeric partner RXR, the nuclear receptor for 9-cis retinoic acid, were shown to be expressed in male genital tracts and testes, and their respective ligands were found at physiologically active concentrations. In these organs, cholesterol homeostasis must be strictly regulated, as: 1) cholesterol is involved in androgen synthesis, and 2) during epididymal maturation of spermatozoa, the plasma membrane undergoes various modifications, mainly exchanges between cholesterol and phospholipids.We recently described that knock-out mice for both LXR encoding genes presented structural abnormalities of the epithelium of the head of the epididymis. These mice also presented fragile spermatozoa. Integrative and molecular physiology studies demonstrate a new role of these nuclear receptors in male reproductive physiology.

Comprehensive overview of murine epididymal mononuclear phagocytes and lymphocytes: Unexpected populations arise

Despite increasing evidence that epididymal immune disorders can lead to infertility, the cells and mechanisms underlying epididymal immunity remain poorly understood. In this study, we propose a rather exhaustive overview of innate and adaptive immune cells present in the murine caput and cauda epididymis. Using flow cytometry and a wide set of markers, we screened the broadest panel of immune cells ever, in this organ. For the first time, we unequivocally quantified the innate populations of monocytes, macrophages, and dendritic cells subtypes. We also revealed the presence of B cells, gamma delta T cells, and double negative T cells in the murine epididymis. They were localized by immunofluorescence stainings, and appeared to be all present in the interstitium and epithelium along the organ, but with respective preferential regional distribution. Altogether, these findings provide new insights on the actors and potential mechanisms involved in the immune responses against genital tract ascending pathogens and in the setting and maintenance of tolerance toward the sperm cells.