The ribonuclease III endonuclease, Dicer1 (also known as Dicer), is essential for the synthesis of the 19-25 nucleotide noncoding RNAs known as micro-RNAs (miRNAs). These miRNAs associate with the RNA-induced silencing complex to regulate gene expression posttranscriptionally by base pairing with 3'untranslated regions of complementary mRNA targets. Although it is established that miRNAs are expressed in the reproductive tract, their functional role and effect on reproductive disease remain unknown. The studies herein establish for the first time the reproductive phenotype of mice with loxP insertions in the Dicer1 gene (Dicer1fl/fl) when crossed with mice expressing Cre-recombinase driven by the anti-müllerian hormone receptor 2 promoter (Amhr2Cre/+). Adult female Dicer1fl/fl;Amhr2Cre/+ mice displayed normal mating behavior but failed to produce offspring when exposed to fertile males during a 5-month breeding trial. Morphological and histological assessments of the reproductive tracts of immature and adult mice indicated that the uterus and oviduct were hypotrophic, and the oviduct was highly disorganized. Natural mating of Dicer1fl/fl;Amhr2Cre/+ females resulted in successful fertilization as evidenced by the recovery of fertilized oocytes on d 1 pregnancy, which developed normally to blastocysts in culture. Developmentally delayed embryos were collected from Dicer1fl/fl; Amhr2Cre/+ mice on d 3 pregnancy when compared with controls. Oviductal transport was disrupted in the Dicer1fl/fl;Amhr2Cre/+ mouse as evidenced by the failure of embryos to enter the uterus on d 4 pregnancy. These studies implicate Dicer1/miRNA mediated posttranscriptional gene regulation in reproductive somatic tissues as critical for the normal development and function of these tissues and for female fertility.
BackgroundDuring the process of spermatogenesis, male germ cells undergo dramatic chromatin reorganization, whereby most histones are replaced by protamines, as part of the pathway to compact the genome into the small nuclear volume of the sperm head. Remarkably, approximately 90 % (human) to 95 % (mouse) of histones are evicted during the process. An intriguing hypothesis is that post-translational modifications (PTMs) decorating histones play a critical role in epigenetic regulation of spermatogenesis and embryonic development following fertilization. Although a number of specific histone PTMs have been individually studied during spermatogenesis and in mature mouse and human sperm, to date, there is a paucity of comprehensive identification of histone PTMs and their dynamics during this process.ResultsHere we report systematic investigation of sperm histone PTMs and their dynamics during spermatogenesis. We utilized “bottom-up” nanoliquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) to identify histone PTMs and to determine their relative abundance in distinct stages of mouse spermatogenesis (meiotic, round spermatids, elongating/condensing spermatids, and mature sperm) and in human sperm. We detected peptides and histone PTMs from all four canonical histones (H2A, H2B, H3, and H4), the linker histone H1, and multiple histone isoforms of H1, H2A, H2B, and H3 in cells from all stages of mouse spermatogenesis and in mouse sperm. We found strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, little conservation was observed between H1, H2A, and H2B. Importantly, across eight individual normozoospermic human semen samples, little variation was observed in the relative abundance of nearly all histone PTMs.ConclusionIn summary, we report the first comprehensive and unbiased analysis of histone PTMs at multiple time points during mouse spermatogenesis and in mature mouse and human sperm. Furthermore, our results suggest a largely uniform histone PTM signature in sperm from individual humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0072-6) contains supplementary material, which is available to authorized users.
Highlights d High-throughput screening identifies key epigenetic proteins driving senescence d Depletion of the histone acetyltransferase p300 delays senescence d p300 induces the formation of de novo super enhancers in senescence d Depletion of p300 suppresses senescence-related gene expression
Bisphenol-A (BPA), a ubiquitous environmental endocrine disrupting chemical, is a component of polycarbonate plastic and epoxy resins. Because of its estrogenic properties, there is increasing concern relative to risks from exposures during critical periods of early organ differentiation. Prenatal BPA treatment in sheep results in low birth weight, hypergonadotropism, and ovarian cycle disruptions. This study tested the hypothesis that gestational exposure to bisphenol A, at an environmentally relevant dose, induces early perturbations in the ovarian transcriptome (mRNA and microRNA). Pregnant Suffolk ewes were treated with bisphenol A (0.5 mg/kg, sc, daily, produced ∼2.6 ng/mL of unconjugated BPA in umbilical arterial samples of BPA treated fetuses approaching median levels of BPA measured in maternal circulation) from days 30 to 90 of gestation. Expression of steroidogenic enzymes, steroid/gonadotropin receptors, key ovarian regulators, and microRNA biogenesis components were measured by RT-PCR using RNA derived from fetal ovaries collected on gestational days 65 and 90. An age-dependent effect was evident in most steroidogenic enzymes, steroid receptors, and key ovarian regulators. Prenatal BPA increased Cyp19 and 5α-reductase expression in day 65, but not day 90, ovaries. Fetal ovarian microRNA expression was altered by prenatal BPA with 45 down-regulated (>1.5-fold) at day 65 and 11 down-regulated at day 90 of gestation. These included microRNAs targeting Sry-related high-mobility-group box (SOX) family genes, kit ligand, and insulin-related genes. The results of this study demonstrate that exposure to BPA at an environmentally relevant dose alters fetal ovarian steroidogenic gene and microRNA expression of relevance to gonadal differentiation, folliculogenesis, and insulin homeostasis.
Genomic imprinting affects a subset of genes in mammals, such that they are expressed in a monoallelic, parent-of-origin-specific manner. These genes are regulated by imprinting control regions (ICRs), cis-regulatory elements that exhibit allele-specific differential DNA methylation. Although genomic imprinting is conserved in mammals, ICRs are genetically divergent across species. This raises the fundamental question of whether the ICR plays a species-specific role in regulating imprinting at a given locus. We addressed this question at the H19/insulin-like growth factor 2 (Igf2) imprinted locus, the misregulation of which is associated with the human imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). We generated a knock-in mouse in which the endogenous H19/Igf2 ICR (mIC1) is replaced by the orthologous human ICR (hIC1) sequence, designated H19 hIC1 . We show that hIC1 can functionally replace mIC1 on the maternal allele. In contrast, paternally transmitted hIC1 leads to growth restriction, abnormal hIC1 methylation, and loss of H19 and Igf2 imprinted expression. Imprint establishment at hIC1 is impaired in the male germ line, which is associated with an abnormal composition of histone posttranslational modifications compared with mIC1. Overall, this study reveals evolutionarily divergent paternal imprinting at IC1 between mice and humans. The conserved maternal imprinting mechanism and function at IC1 demonstrates the possibility of modeling maternal transmission of hIC1 mutations associated with BWS in mice. In addition, we propose that further analyses in the paternal knockin H19 +/hIC1 mice will elucidate the molecular mechanisms that may underlie SRS.G enomic imprinting is a conserved, epigenetic process in mammals that regulates the expression of a small number of genes in a monoallelic, parent-of-origin-specific manner. Typically clustered within domains, the parental-specific expression of imprinted genes is controlled by a cis-regulatory element, the imprinting control region (ICR). During gametogenesis, ICRs acquire differential DNA methylation patterns according to the sex of the germ cells. This DNA methylation is maintained in somatic cells after fertilization but is erased in primordial germ cells, allowing the establishment of sex-specific imprints in mature gametes. The proper establishment, maintenance, and erasure of imprints are crucial for the correct expression of imprinted genes. Misregulation of imprinted genes is associated with human imprinting disorders, including Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder, and Silver-Russell syndrome (SRS), an undergrowth disorder (1-3).Mouse models have been valuable to the study of imprinting at the H19/insulin-like growth factor 2 (Igf2) locus, serving as a proxy for the orthologous human locus. On distal mouse chromosome 7, reciprocal imprinting of the paternally expressed fetal growth factor gene, Igf2, and the maternally expressed noncoding RNA, H19, is regulated by the ICR located between H...
Highlights d ATAC-seq localizes retained nucleosomes to promoters and repetitive DNA in sperm d Gcn5-mediated histone acetylation is necessary for proper spermiogenesis d Gcn5 loss alters chromatin dynamics leading to increased histone retention in sperm d Gcn5 is necessary for normal sperm formation and male fertility in mice
Dicer is an RNAse III endonuclease that is essential for the biogenesis of microRNAs and small interfering RNAs. These small RNAs post-transcriptionally regulate mRNA gene expression through several mechanisms to affect key cellular events including proliferation, differentiation, and apoptosis. Recently, the role of Dicer function in female reproductive tissues has begun to be elucidated through the use of knock-out mouse models. Loss of Dicer within ovarian granulosa cells, luteal tissue, oocyte, oviduct, and potentially the uterus render females infertile. This review discusses these early studies and other data describing the current understanding of microRNAs and small interfering RNAs in female reproduction.
e During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.
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