Project description:Maternal caloric restriction during the last week of gestation resulted in low birth weight (LBW) and increased risk of LBW-associated metabolic diseases in adult life. The metabolic phenotypes transmitted to F2 generation by paternal manner without additional altered nutrition. To investigate the mechanism of this intergenerational inheritance, two Cohorts were exposed to different magnitudes of undernutrition both in utero during the last week of gestation and/or postnatal until weaning. We performed MeDIP-seq on the genomic DNA from sperm collected from these mice.
Project description:Maternal caloric restriction during the last week of gestation resulted in a low birth weight (LBW) and increased risk of LBW-associated metabolic diseases in adult life. The metabolic phenotypes transmitted to F2 generation by paternal manner without additional altered nutrition. To investigate the mechanism of this intergenerational inheritance, two Cohorts were exposed to different magnitudes of unternourition both in utero during the last week of gestation and/or post nataly until weaning. We performed small RNA-seq on non-coding RNA from sperm collected from these mice. Small RNA libraries were size-selected to be between 132 to 200 bp (including adapter) after library amplification.
Project description:Proteomic investigations of spermatozoa provide practical tools for distinguishing normal, functional spermatozoa from abnormal spermatozoa. Indeed, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) applies high-throughput industrial applications to identify sperm-specific proteins indicative of chemical exposure. As such, a direct comparison of protein expression profiles between control and exposed cells returned a set of protein markers. Because mature mammalian spermatozoa are virtually incapable of protein synthesis, the predicted protein biomarkers in spermatozoa offer considerable stability for use in clinical application. In the current study, we applied 2-DE coupled with ESI-MS/MS to investigate the modified protein profile in F1 capacitated spermatozoa due to gestational bisphenol-A (BPA) exposure to ascertain whether these proteomic modifications could explain the observed functional alterations in spermatozoa.
Project description:Prototypical micro RNAs (miRNAs) are 21~25-base-pair RNAs that regulate differentiation, carcinogenesis and pluripotency by eliminating mRNAs or blocking their translation, processes collectively termed RNA interference (RNAi). RNAi mediated by miRNAs regulates early development in zebrafish, and mouse embryos lacking the miRNA precursor processor, Dicer, are inviable. However, the role of miRNAs during mammalian fertilization is unknown. We here show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. Sperm contained a broad profile of miRNAs and a subset of potential mRNA targets were expressed in fertilizable, metaphase II (mII) oocytes. Oocytes contained transcripts for the RNAinduced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, levels of sperm-borne miRNA (measured by quantitative PCR) were apparently low relative to those of unfertilized, mII oocytes, and fertilization did not alter the part of the mII oocyte miRNA landscape that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs - to suppress miRNA function - did not perturb pronuclear activation or preimplantation development. Contrastingly, we provide evidence that nuclear transfer by microinjection alters the miRNA profile of enucleated oocytes. These data argue that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development. Keywords: miRNA profiling Seven samples were analyzed for the study.
Project description:Understanding molecular mechanisms of cellular pathways regularly requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography analyses of mouse and human sperm revealed key microtubule structures at 6.0 Å resolution via subtomogram averaging. The well-resolved secondary and tertiary structures allowed us to unbiasedly match novel densities discovered in our 3D reconstruction maps with 21615 protein models from the library of the mouse proteome generated by AlphaFold2. Additional biochemical and mass spectrometry analyses helped validate potential candidates. Not only was it possible to de novo identify novel mammal-specific sperm proteins using this label-free structural approach, but their cellular localizations and molecular interactions could be determined in situ. Specifically, the novel sperm proteins form an extensive interaction network crosslinking the lumen of microtubules, suggesting they could modulate the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella. This project includes global protein abundance data from five biochemical fractionations of mouse sperm cells.
Project description:We reported RNA profiles of mice spermatozoa, a total of 35,288,825 reads matching 33,039 transcripts, including 27,310 coding transcripts, were obtained. RNA profiles of the spermatozoa of 9-10 weeks adult mice were sequenced by RNA-seq,using Illumina GAIIx.