Bi-directional effects of vitamin B 12 and methotrexate on Daphnia magna fitness and genomic methylation

Here we interrogated, using three separate but complementary experimental approaches, the impact of vitamin B12 availability and methotrexate exposure on Daphnia magna, which we hypothesised should have an opposite efect on One carbon metabolism (OCM). OCM is a vital biological process supporting a variety of physiological processes, including DNA methylation. Contrary to mammalian models, this process remains largely unexplored in invertebrates. The purpose of this study was to elucidate the impact of OCM short-term alteration on the ftness and epigenome of the keystone species, Daphnia. We used maternal age at reproduction, brood size and survival rates in combination with DNA methylation sensitive comet assay to determine the efects of vitamin B12 or MTX on ftness and the epigenome. Vitamin B12 had a positive infuence on Daphnia ftness and we provide evidence demonstrating that this may be associated with an increased level of genome-wide DNA methylation. Conversely, exposing D. magna to MTX negatively infuenced the ftness of the animals and was associated with loss of global DNA methylation, translating in decreased ftness. These results highlight the potential importance of OCM in invertebrates, providing novel evidence supporting a potential role for epigenetic modifcations to the genome in D. magna environmental adaptability.European Commission - Seventh Framework Programme (FP7)Norwegian Research Counci

Male infertility and DNA germ cell breaks affected by the epigenetic factor PRDM9

DNA-binding histone-3-lysine-4,36-trimethyltransferase PRDM9 specifies meiotic recombination hotspots in mice, rats and humans. Interallelic variation at the Prdm9 locus plays a role in hybrid male sterility. Sterile mouse F1 hybrid male offspring from the PWD × C57BL/6J (B6) cross exhibit meiosis breakdown reminiscent of that observed in Prdm9-deficient B6 sterile mice. However, reciprocal (B6 × PWD)F1 hybrids and some rodent models lacking PRDM9, i.e., PWD and SHR rat males execute meiotic recombination, produce sperm, raising the possibility that PRDM9's role may extend beyond meiosis. Here I demonstrate that PRDM9 is important for post- meiotic male gamete development and release. Unlike their parents, (B6 × PWD)F1 generated spermatozoa of lower quantity and motility but higher percentage of deformations, thus resembling oligoasthenoteratozoospermic (OAT) (semi)sterile men. Histopathological and (ultra)structural analysis revealed compromised spermiogenesis characterized by acrosome detachment and aberrant nucleus elongation in (B6 × PWD)F1 hybrids. Consequently, F1 spermatozoa had malformed acrosomes and nuclear DNA breaks with elevated base oxidation. While deletion of one Prdm9 copy improved sperm phenotypes in (B6 × PWD)F1, copy number gains of the surrounding genes had the opposite effect...

Samčí neplodnost a zlomy DNA v pohlavních buňkách ovlivněných epigenetickým faktorem PRDM9

(Czech) Histon-3-lysin-4,36-trimethyltransferasa PRDM9 s DNA-vazebnými doménami určuje místa meiotické rekombinace u myši, potkana a člověka. Interalelická variace genu Prdm9 hraje roli v hybridní sterilitě. Sterilní myší F1 samci z křížení PWD × C57BL/6J (B6) vykazují kompletní zástavu meiózy připomínající zástavu v Prdm9-deficientních B6 samcích. Reciprocí F1 C57BL/6J × PWD a Prdm9-deficientní samci kmene myši PWD a potkana SHR ale dokončují meiotickou rekombinaci a produkují spermie. To naznačuje, že by PRDM9 mohl ovlivňovat i vývoj spermií po meiotickém dělení. V této práci prokazuji, že je PRDM9 důležitý pro postmeiotický vývoj samčích pohlavních buněk a jejich uvolnění z varlat. Na rozdíl od rodičů nesou BPF1 méně spermií s vyššími procenty nepohyblivých a deformovaných. Fenotyp (B6 × PWD)F1 se tak podobá syndromu oligoasthenoteratozoospermia (OAT) semisterilních mužů. Histopathologické a (ultra)strukturální analýzy odhalily v (B6 × PWD)F1 abnormální spermiogenezi charakterizovanou odchlípením akrosomu a aberantní nukleární elongací. Následkem toho měly F1 spermatozoa malformované akrosomy a DNA zlomy s oxidovanými bázemi. Zatímco delece jedné kopie Prdm9 vylepšilo fenotypy (B6 × PWD)F1 spermií, zvýšení počtu kopií sousedních genů mělo opačný efekt. Hybridní sterilita je proto citlivá na...DNA-binding histone-3-lysine-4,36-trimethyltransferase PRDM9 specifies meiotic recombination hotspots in mice, rats and humans. Interallelic variation at the Prdm9 locus plays a role in hybrid male sterility. Sterile mouse F1 hybrid male offspring from the PWD × C57BL/6J (B6) cross exhibit meiosis breakdown reminiscent of that observed in Prdm9-deficient B6 sterile mice. However, reciprocal (B6 × PWD)F1 hybrids and some rodent models lacking PRDM9, i.e., PWD and SHR rat males execute meiotic recombination, produce sperm, raising the possibility that PRDM9's role may extend beyond meiosis. Here I demonstrate that PRDM9 is important for post- meiotic male gamete development and release. Unlike their parents, (B6 × PWD)F1 generated spermatozoa of lower quantity and motility but higher percentage of deformations, thus resembling oligoasthenoteratozoospermic (OAT) (semi)sterile men. Histopathological and (ultra)structural analysis revealed compromised spermiogenesis characterized by acrosome detachment and aberrant nucleus elongation in (B6 × PWD)F1 hybrids. Consequently, F1 spermatozoa had malformed acrosomes and nuclear DNA breaks with elevated base oxidation. While deletion of one Prdm9 copy improved sperm phenotypes in (B6 × PWD)F1, copy number gains of the surrounding genes had the opposite effect....Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Bi-directional efects of vitamin B12 and methotrexate on Daphnia magna ftness and genomic methylation

Here we interrogated, using three separate but complementary experimental approaches, the impact of vitamin B12 availability and methotrexate exposure on Daphnia magna, which we hypothesised should have an opposite efect on One carbon metabolism (OCM). OCM is a vital biological process supporting a variety of physiological processes, including DNA methylation. Contrary to mammalian models, this process remains largely unexplored in invertebrates. The purpose of this study was to elucidate the impact of OCM short-term alteration on the ftness and epigenome of the keystone species, Daphnia. We used maternal age at reproduction, brood size and survival rates in combination with DNA methylation sensitive comet assay to determine the efects of vitamin B12 or MTX on ftness and the epigenome. Vitamin B12 had a positive infuence on Daphnia ftness and we provide evidence demonstrating that this may be associated with an increased level of genome-wide DNA methylation. Conversely, exposing D. magna to MTX negatively infuenced the ftness of the animals and was associated with loss of global DNA methylation, translating in decreased ftness. These results highlight the potential importance of OCM in invertebrates, providing novel evidence supporting a potential role for epigenetic modifcations to the genome in D. magna environmental adaptability.European Commission - Seventh Framework Programme (FP7)Norwegian Research Counci

Rat PRDM9 shapes recombination landscapes, duration of meiosis, gametogenesis, and age of fertility

International audienceBackground: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear.Results: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants.Conclusions: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility