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:While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by FASP digestion were analysed by LC-MS/MS with SWATH acquisition. A total of 4,304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus and cauda spermatozoa had strong enrichment for tubulins and included 4 histone proteins. The most significant proteomic remodelling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1,131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias towards protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.
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:This study includes raw data of sperm smallRNA profiles from treated (nABX & LPHS, with nABX: non-absorbable antibiotics ; LPHS: low-protein, high sugar diet) and control (CON) males across FVB and C57BL/6J genetic backgrounds and mature (76-78 weeks (>1yr)) and young (10-11 weeks) ages. Sequencing was conducted at 100 bp, single-end on a Nextseq2000, resulting in approximately 1-5 million reads for most samples.
Project description:This study includes raw data of blastocyst single-embryo SMART-seq profiles generated by IVF using sperm derived from mature (50-57 weeks) and young (9-11 weeks) males of C57BL/6N background. After SMART-seq library preparation of single embryos, indexed samples were pooled and sequenced on the NextSeq 2000 using a P3 flow cell in 50bp paired-end mode, targeting an average of 5 million reads per sample.