Ayhan Kocer

Glutathione Peroxidases at Work on Epididymal Spermatozoa: An Example of the Dual Effect of Reactive Oxygen Species on Mammalian Male Fertilizing Ability

The mammalian glutathione peroxidase (GPx) gene family encodes bifunctional enzymes that can work either as classical reactive oxygen species (ROS) scavengers or as thiol peroxidases, thereby introducing disulfide bridges in thiol-containing proteins. These dual effects are nowhere better demonstrated than in epididymal maturing spermatozoa, where the concomitant actions of several GPx ensure the achievement of the structural maturation of sperm cells as well as their protection against ROS-induced damage. We review here the roles played by the sperm-associated forms of GPx4 (mitochondrial GPx4 and nuclear GPx4), the secreted GPx5 protein, and the epithelial proteins GPx1, GPx3, and cellular GPx4, all functioning in the mammalian epididymis at different stages of the sperms epididymal journey, and in different epididymis compartments.

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.

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

Dietary Cholesterol-Induced Post-Testicular Infertility

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr−/− animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce.

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.

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.

SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.

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.RésuméLes 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.

Recent knowledge concerning mammalian sperm chromatin organization and its potential weaknesses when facing oxidative challenge

Spermatozoa are the smallest and most cyto-differentiated mammalian cells. From a somatic cell-like appearance at the beginning of spermatogenesis, the male germ cell goes through a highly sophisticated process to reach its final organization entirely devoted to its mission which is to deliver the paternal genome to the oocyte. In order to fit the paternal DNA into the tiny spermatozoa head, complete chromatin remodeling is necessary. This review essentially focuses on present knowledge of this mammalian sperm nucleus compaction program. Particular attention is given to most recent advances that concern the specific organization of mammalian sperm chromatin and its potential weaknesses. Emphasis is placed on sperm DNA oxidative damage that may have dramatic consequences including infertility, abnormal embryonic development and the risk of transmission to descendants of an altered paternal genome.AbstractRésuméLe spermatozoïde est la cellule la plus petite et la plus cytologiquement différenciée chez les mammifères. D’une apparence proche de celle d’une cellule somatique au début de la spermatogenèse, la cellule germinale mâle va, au travers d’un processus hautement sophistiqué, atteindre une organisation finale entièrement dédiée à sa mission qui est de conduire le lot chromosomique paternel au sein de l’ovule. Afin de pouvoir accommoder l’ADN paternel dans la minuscule tête du spermatozoïde, un remodelage complet de la chromatine est nécessaire. Cette revue est essentiellement concentrée sur les aspects connus à ce jour de ce programme de condensation nucléaire spermatique. Une attention particulière est donnée aux avancées les plus récentes concernant l’organisation très spécifique du noyau spermatique et sur ses points de fragilité, en particulier face aux dommages radicalaires. Ces derniers peuvent avoir des conséquences dramatiques qui se posent en termes d’infertilité, de développements embryonnaires anormaux et de risque de transmission à la descendance d’un patrimoine génétique paternel altéré.