49 research outputs found
KLC3 is involved in sperm tail midpiece formation and sperm function
AbstractKinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear.Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ÎHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa
Ovarian Aging-Like Phenotype in the Hyperandrogenism-Induced Murine Model of Polycystic Ovary
There are prominently similar symptoms, effectors, and commonalities in the majority of characteristics between ovarian aging and polycystic ovarian syndrome (PCOS). Despite the approved role of oxidative stress in the pathogenesis of PCOS and aging, to our knowledge, the link between the PCO(S) and aging has not been investigated yet. In this study we investigated the possible exhibition of ovarian aging phenotype in murine model of PCO induced by daily oral administration of letrozole (1âmg/kg body weight) for 21 consecutive days in the female Wistar rats. Hyperandrogenization showed irregular cycles and histopathological characteristics of PCO which was associated with a significant increase in lipid peroxidation (LPO) and reactive oxygen species (ROS) and decrease in total antioxidant capacity (TAC) in serum and ovary. Moreover, serum testosterone, insulin and tumor necrosis factor-alpha (TNF-α) levels, and ovarian matrix metalloproteinase-2 (MMP-2) were increased in PCO rats compared with healthy controls, while estradiol and progesterone diminished. Almost all of these findings are interestingly found to be common with the characteristics identified with (ovarian) aging showing that hyperandrogenism-induced PCO in rat is associated with ovarian aging-like phenotypes. To our knowledge, this is the first report that provides evidence regarding the phenomenon of aging in PCO
In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa
peer-reviewedThis study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: HolsteinâFriesian ejaculates (nâ=â10 bulls) were split across four treatments and processed: (1) NS fresh at 3âĂâ106 spermatozoa, (2) X-SS frozen at 2âĂâ106 spermatozoa, (3) X-SS fresh at 2âĂâ106 spermatozoa and (4) X-SS fresh at 1âĂâ106 spermatozoa. NS frozen controls of 20âĂâ106 spermatozoa per straw were sourced from previously frozen ejaculates (nâ=â3 bulls). Experiment 2: Aberdeen Angus ejaculates (nâ=â4 bulls) were split across four treatments and processed as: (1) NS fresh 3âĂâ106 spermatozoa, (2) Y-SS fresh at 1âĂâ106 spermatozoa, (3) Y-SS fresh at 2âĂâ106 spermatozoa and (4) X-SS fresh at 2âĂâ106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0â=âday of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (Pâ<â0.001), NS frozen treatments had the greatest PLM (Pâ<â0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (Pâ<â0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (Pâ<â0.01).ACCEPTEDpeer-reviewe
A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific
peer-reviewedBackground: Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique
nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA
methylation patterns during male germ cell differentiation have been associated with infertility in several species.Background: Spermatozoa have a remarkable epigenResults: The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA)
highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men.
Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation
of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias
in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To
map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and
monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA
followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome
coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells
were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated
sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that
were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program
(piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites
and rDNA repeats.
Conclusions: These results highlight the undermethylation of bull spermatozoa when compared with both bovine
somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in
bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the
cattle genome may deserve further attention.
While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull
spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome
relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis
A comparison of low - versus high - fertility Holstein bulls for identification of fertility markers
Bibliography: p. 87-9
Development and application of sensitive genome-wide platforms to study the genetic and epigenetic (DNA methylation) makeup of gametes and early bovine embryos
Pour ce projet, nous avons dĂ©veloppĂ© une plateforme pour lâanalyse pangĂ©nomique de la mĂ©thylation de lâADN chez le bovin qui est compatible avec des Ă©chantillons de petites tailles. Cet outil est utilisĂ© pour Ă©tudier les caractĂ©ristiques gĂ©nĂ©tiques et Ă©pigĂ©nĂ©tiques (mĂ©thylation de l'ADN) des gamĂštes soumis aux procĂ©dures de procrĂ©ation mĂ©dicalement assisitĂ©e et des embryons prĂ©coces. Dans un premier temps, une plateforme dâanalyse de biopuces spĂ©cifiques pour lâĂ©tude de la mĂ©thylation de lâADN chez lâespĂšce bovine a Ă©tĂ© dĂ©veloppĂ©e. Cette plateforme a ensuite Ă©tĂ© optimisĂ©e pour produire des analyses pangĂ©nomiques de mĂ©thylation de l'ADN fiables et reproductibles Ă partir dâĂ©chantillons de trĂšs petites tailles telle que les embryons prĂ©coces (â„ 10 ng d'ADN a Ă©tĂ© utilisĂ©, ce qui correspond Ă 10 blastocystes en expansion). En outre, cet outil a permis dâĂ©valuer de façon simultanĂ©e la mĂ©thylation de lâADN et le transcriptome dans le mĂȘme Ă©chantillon, fournissant ainsi une image complĂšte des profils gĂ©nĂ©tiques et Ă©pigĂ©nĂ©tiques (mĂ©thylation de lâADN). Comme preuve de concept, les profils comparatifs de mĂ©thylation de l'ADN spermatique et de blastocystes bovins ont Ă©tĂ© analysĂ©s au niveau de l'ensemble du gĂ©nome. Dans un deuxiĂšme temps, grĂące Ă cette plateforme, les profils globaux de mĂ©thylation de l'ADN de taureaux jumeaux monozygotes (MZ) ont Ă©tĂ© analysĂ©s. MalgrĂ© quâils sont gĂ©nĂ©tiquement identiques, les taureaux jumeaux MZ ont des descendants avec des performances diffĂ©rentes. Par consĂ©quent, l'hypothĂšse que le profil de mĂ©thylation de l'ADN spermatique de taureaux jumeaux MZ est diffĂ©rent a Ă©tĂ© Ă©mise. Dans notre Ă©tude, des diffĂ©rences significatives entre les jumeaux MZ au niveau des caractĂ©ristiques de la semence ainsi que de la mĂ©thylation de lâADN ont Ă©tĂ© trouvĂ©es, chacune pouvant contribuer Ă lâobtention de performances divergentes incongrues des filles engendrĂ©es par ces jumeaux MZ. Dans la troisiĂšme partie de ce projet, la mĂȘme plateforme a Ă©tĂ© utilisĂ©e pour dĂ©couvrir les impacts dâune supplĂ©mentation Ă forte concentration en donneur de mĂ©thyle universel sur les embryons prĂ©coces bovins. La supplĂ©mentation avec de grandes quantitĂ©s d'acide folique (AF) a Ă©tĂ© largement utilisĂ©e et recommandĂ©e chez les femmes enceintes pour sa capacitĂ© bien Ă©tablie Ă prĂ©venir les malformations du tube neural chez les enfants. Cependant, plus rĂ©cemment, plusieurs Ă©tudes ont rapportĂ© des effets indĂ©sirables de lâAF utilisĂ© Ă des concentrations Ă©levĂ©es, non seulement sur le dĂ©veloppement de l'embryon, mais aussi chez les adultes. Au niveau cellulaire, lâAF entre dans le mĂ©tabolisme monocarbonĂ©, la seule voie de production de S-adĂ©nosyl mĂ©thionine (SAM), un donneur universel de groupements mĂ©thyles pour une grande variĂ©tĂ© de biomolĂ©cules, y compris lâADN. Par consĂ©quent, pour rĂ©soudre cette controverse, une forte dose de SAM a Ă©tĂ© utilisĂ©e pour traiter des embryons produits in vitro chez le bovin. Ceci a non seulement permis dâinfluencer le phĂ©notype des embryons prĂ©coces, mais aussi dâavoir un impact sur le transcriptome et le mĂ©thylome de lâADN. En somme, le projet en cours a permis le dĂ©veloppement d'une plateforme d'analyse de la mĂ©thylation de l'ADN Ă lâĂ©chelle du gĂ©nome entier chez le bovin Ă coĂ»t raisonnable et facile Ă utiliser qui est compatible avec les embryons prĂ©coces. De plus, puisque câest l'une des premiĂšres Ă©tudes de ce genre en biologie de la reproduction bovine, ce projet avait trois objectifs qui a donnĂ© plusieurs nouveaux rĂ©sultats, incluant les profils comparatifs de mĂ©thylation de l'ADN au niveau : i) blastocystes versus spermatozoĂŻdes ; ii) semence de taureaux jumeaux MZ et iii) embryons prĂ©coces traitĂ©s Ă de fortes doses de SAM versus des embryons prĂ©coces non traitĂ©s.In this project, we developed a bovine genome-wide DNA methylation platform compatible with small sample size to study genetic and epigenetic (DNA methylation) makeup of ART-treated bovine gametes and early embryos. Initially, a bovine-specific array-based DNA methylation analysis platform was developed. This platform was subsequently optimized to produce reliable and reproducible genome-wide DNA methylation analysis from very small sample sizes, e.g. bovine early embryos (â„ 10 ng gDNA input, corresponding to 10 expanded blastocysts). In addition, this platform permitted concurrent assessment of both DNA methylation and transcription in the same sample, thereby providing a very complete picture of genetic and epigenetic (DNA methylation) profiles. As proof of concept, for the first time, comparative DNA methylation profiles of bovine sperm and blastocysts were analysed at a genome-wide level. Using this platform, global DNA methylation profiles of monozygotic (MZ) twin bulls were analysed. Despite being geneticially identical, MZ twin bulls consistently have different progeny performance. Therefore, it was hypothesised that the DNA methylation profile of sperm from MZ twin bulls is different. In our study, there were significant differences between MZ twin for semen end points, as well as for the sperm epigenome (DNA methylation), all of which would be expected to contribute to incongruous divergent performances of daughters sired by MZ twins.In the next part of this project, using the developed platform, impacts of supplementation of a high-concentration global methyl donor on bovine early embryos was investigated. Supplementation with large amounts of folic acid (FA) has been extensively used and recommended in pregnant women for its well-established ability to prevent neural tube defects in children. However, more recently, several studies reported adverse effects of high FA concentrations, not only on embryo development, but also in adults. At the cellular level, FA enters one-carbon metabolism, the only pathway to produce S-adenosyl methionine (SAM) as the global methyl donor for a wide variety of biomolecules, including DNA. Therefore, to address this controversy, a high dose of SAM was used to treat in vitro -produced bovine embryos. This not only affected early embryo phenotypes, but also the transcritome and genome-wide DNA methylome. Overall, the current project resulted in development of a user-friendly and cost-effective bovine genome-wide DNA methylation analysis platform, which is compatible with small cell number such as early embryos. In addition, as one of the first studies of its kind in bovine reproductive biology, this project had three objectives which yielded several novel results, including comparative genome-wide DNA methylation profiles of: i) bovine blastocysts versus sperm; ii) sperm from monozygotic twin bulls and iii) high dose SAM-treated versus non-treated bovine early embryos
An integrated platform for bovine DNA methylome analysis suitable for small samples
Background: Oocytes and early embryos contain minute amounts of DNA, RNA and proteins, making the study of early mammalian development highly challenging. The study of the embryo epigenome, in particular the DNA methylome, has been made accessible thanks to the possibility of amplifying specific sequences according to their initial methylation status. This paper describes a novel platform dedicated to the genome-wide study of bovine DNA methylation, including a complete pipeline for data analysis and visualization. The platform allows processing and integrating of DNA methylome and transcriptome data from the same sample. Procedures were optimized for genome-wide analysis of 10 ng of DNA (10 bovine blastocysts). Bovine sperm and blastocysts were compared as a test of platform capability. Results: The hypermethylation of bovine sperm DNA compared to the embryo genome was confirmed. Differentially methylated regions were distributed across various classes of bovine sperm genomic feature including primarily promoter, intronic and exonic regions, non-CpG-island regions (shore, shelf and open-sea) and CpG islands with low-to-intermediate CpG density. The blastocyst genome bore more methylation marks than sperm DNA only in CpG islands with high CpG density. Long-terminal-repeat retrotransposons (LTR), LINE and SINE were more methylated in sperm DNA, as were low-complexity repetitive elements in blastocysts. Conclusions: This is the first early embryo compatible genome-wide epigenetics platform for bovine. Such platforms should improve the study of the potential epigenetic risks of assisted reproductive technologies (ART), the establishment sequence of embryonic cell lines and potential deviations in both gene expression and DNA methylation capable of having long-term impact.Natural Sciences and Engineering Research Council of Canad