Fetal Growth Retardation and Lack of Hypotaurine in Ezrin Knockout Mice

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez^−/−) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis–time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez^−/− mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.     

BRCA1与DSB修复路径取向

乳腺癌易感基因1(BRCA1)是一个肿瘤抑制基因.BRCA1参与DNA末端切除、细胞周期调控以及染色体修饰等来维护基因组的稳定性.有研究表明,它能够促进正确的DNA双链断裂(DSBs)修复,如同源重组修复(HDR)和经典的非同源末端连接(C-NHEJ);而抑制错误性的DSB修复,如单链退火修复(SSA)和非经典的末端连接(A-EJ);其机制是通过与某些DNA修复相关蛋白质的相互作用来引导DSB修复.目前,BRCA1在DSB修复通路中的作用机制尚未完全明确,仍有待进一步的研究.本文主要阐述BRCA1在DSB各修复通路中是如何发挥其引导作用的.     

Increased Adiposity in Annexin A1-Deficient Mice

Production of Annexin A1 (ANXA1), a protein that mediates the anti-inflammatory action of glucocorticoids, is altered in obesity, but its role in modulation of adiposity has not yet been investigated. The objective of this study was to investigate modulation of ANXA1 in adipose tissue in murine models of obesity and to study the involvement of ANXA1 in diet-induced obesity in mice. Significant induction of ANXA1 mRNA was observed in adipose tissue of both C57BL6 and Balb/c mice with high fat diet (HFD)-induced obesity versus mice on chow diet. Upregulation of ANXA1 mRNA was independent of leptin or IL-6, as demonstrated by use of leptin-deficient ob/ob mice and IL-6 KO mice. Compared to WT mice, female Balb/c ANXA1 KO mice on HFD had increased adiposity, as indicated by significantly elevated body weight, fat mass, leptin levels, and adipocyte size. Whereas Balb/c WT mice upregulated expression of enzymes involved in the lipolytic pathway in response to HFD, this response was absent in ANXA1 KO mice. A significant increase in fasting glucose and insulin levels as well as development of insulin resistance was observed in ANXA1 KO mice on HFD compared to WT mice. Elevated plasma corticosterone levels and blunted downregulation of 11-beta hydroxysteroid dehydrogenase type 1 in adipose tissue was observed in ANXA1 KO mice compared to diet-matched WT mice. However, no differences between WT and KO mice on either chow or HFD were observed in expression of markers of adipose tissue inflammation.These data indicate that ANXA1 is an important modulator of adiposity in mice, with female ANXA1 KO mice on Balb/c background being more susceptible to weight gain and diet-induced insulin resistance compared to WT mice, without significant changes in inflammation.     

Circadian Influence on Ethanol-Related Intrauterine Growth Retardation in Mice

Circadian influences on growth and development in response to ethanol were studied in mice. On gestational day 10, pregnant animals received a single intraperitoneal injection of ethanol with the following dose levels: 1.0, 2.5 or 4.0 g/kg at one of four circadian phases (0700, 1300, 1900 or 0100 hr). 48 hrs after injection the embryonic weight and length, protein and DNA content and placental weight and protein were determined. Ethanol-related intrauterine growth retardation were shown to be dose- and circadian phase-dependent, the greatest susceptibility being seen during the dark phase. The variations observed are discussed with regard to changes in drug metabolism and tissue sensitivity.     

Circadian Influence on Ethanol-Related Intrauterine Growth Retardation in Mice

Circadian influences on growth and development in response to ethanol were studied in mice. On gestational day 10, pregnant animals received a single intraperitoneal injection of ethanol with the following dose levels: 1.0, 2.5 or 4.0 g/kg at one of four circadian phases (0700, 1300, 1900 or 0100 hr). 48 hrs after injection the embryonic weight and length, protein and DNA content and placental weight and protein were determined. Ethanol-related intrauterine growth retardation were shown to be dose- and circadian phase-dependent, the greatest susceptibility being seen during the dark phase. The variations observed are discussed with regard to changes in drug metabolism and tissue sensitivity.     

Developmental Defects and Male Sterility in Mice Lacking the Ubiquitin-Like DNA Repair Gene mHR23B

mHR23B encodes one of the two mammalian homologs of Saccharomyces cerevisiae RAD23, a ubiquitin-like fusion protein involved in nucleotide excision repair (NER). Part of mHR23B is complexed with the XPC protein, and this heterodimer functions as the main damage detector and initiator of global genome NER. While XPC defects exist in humans and mice, mutations for mHR23A and mHR23B are not known. Here, we present a mouse model for mHR23B. Unlike XPC-deficient cells, mHR23B(-/-) mouse embryonic fibroblasts are not UV sensitive and retain the repair characteristics of wild-type cells. In agreement with the results of in vitro repair studies, this indicates that mHR23A can functionally replace mHR23B in NER. Unexpectedly, mHR23B(-/-) mice show impaired embryonic development and a high rate (90%) of intrauterine or neonatal death. Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility. Such abnormalities are not observed in XPC and other NER-deficient mouse mutants and point to a separate function of mHR23B in development. This function may involve regulation of protein stability via the ubiquitin/proteasome pathway and is not or only in part compensated for by mHR23A.     

Reproductive Isolation in Hybrid Mice Due to Spermatogenesis Defects at Three Meiotic Stages

Early in the process of speciation, reproductive failures occur in hybrid animals between genetically diverged populations. The sterile hybrid animals are often males in mammals and they exhibit spermatogenic disruptions, resulting in decreased number and/or malformation of mature sperms. Despite the generality of this phenomenon, comparative study of phenotypes in hybrid males from various crosses has not been done, and therefore the comprehensive genetic basis of the disruption is still elusive. In this study, we characterized the spermatogenic phenotype especially during meiosis in four different cases of reproductive isolation: B6-ChrX^MSM, PGN-ChrX^MSM, (B6 × Mus musculus musculus-NJL/Ms) F₁, and (B6 × Mus spretus) F₁. The first two are consomic strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrX^MSM, the genetic background is the laboratory strain C57BL/6J (predominantly M. m. domesticus), while in PGN-ChrX^MSM the background is the PGN2/Ms strain purely derived from wild M. m. domesticus. The last two cases are F₁ hybrids between mouse subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or apoptosis at either one or two of three distinct meiotic stages: premeiotic stage, zygotene-to-pachytene stage of prophase I, and metaphase I. This study shows that the sterility in hybrid males is caused by spermatogenic disruptions at multiple stages, suggesting that the responsible genes function in different cellular processes. Furthermore, the stages with disruptions are not correlated with the genetic distance between the respective parental strains.WHEN animals from genetically diverged populations hybridize, complete or partial sterility is often observed in the F₁ hybrids or in their descendants. This phenomenon belonging to postzygotic reproductive isolation accelerates irreversible genetic divergence by preventing free gene flow across the two diverging populations, and thereby plays a pivotal role in speciation. Sexual dimorphism is a general feature of reproductive isolation (Wu and Davis 1993; Laurie 1997; Orr 1997; Kulathinal and Singh 2008). In mammals, impairment is much more severe in males than in females, and in general the heterogametic sex is more sensitive to interspecific and intersubspecific genetic incompatibility. This phenomenon is well known as Haldane''s rule (Haldane 1922; Laurie 1997; Orr 1997).In many animals, the reproductive isolation is caused by spermatogenic disruptions characterized by reduced number of germ cells and small testis size. These animals include Drosophila (Joly et al. 1997), stickleback fish Pungitius (Takahashi et al. 2005), caviomorph rodent Thrichomys (Borodin et al. 2006), house musk shrew Suncus (Borodin et al. 1998), wallaby Petrogale (Close et al. 1996), and genus Mus (Forejt and Iványi 1974; Matsuda et al. 1992; Hale et al. 1993; Yoshiki et al. 1993; Kaku et al. 1995; Gregorová and Forejt 2000; Elliott et al. 2001, 2004; Good et al. 2008). Although reproductive isolation by spermatogenic impairment is a well-known phenomenon, its underlying genetic mechanism and molecular basis have remained elusive. The Dobzhansky–Muller model, which infers that hybrid sterility or inviability is caused by deleterious epistatic interactions between nuclear genes derived from their respective parent species or subspecies (Dobzhansky 1936; Muller 1942), is widely accepted in animals and plants and is also applicable to the sterility of hybrid animals in F₂ or backcross generations, so-called hybrid breakdown, in which the genes causing postzygotic reproductive isolation are partially recessive (Orr 2005).The genetic incompatibility between house mouse subspecies is an ideal animal model for studying the early stage of speciation. Two subspecies of mouse, Mus musculus domesticus and M. m. musculus, diverged from their common ancestor 0.3–1.0 MYA (Yonekawa et al. 1980; Moriwaki 1994; Bonhomme and Guénet 1996; Boursot et al. 1996; Din et al. 1996). M. m. domesticus ranges across western Europe and the Middle East, whereas M. m. musculus ranges throughout eastern Europe and northern Asia (Bonhomme and Guénet 1996). The two subspecies meet in a narrow hybrid zone, which is most likely maintained by a balance between dispersal and selection against hybrids (Hunt and Selander 1973; Bonhomme and Guénet 1996; Payseur et al. 2004). M. m. domesticus also displays reproductive isolation from the Japanese wild mouse, M. m. molossinus, which originated from hybridization of M. m. castaneus and M. m. musculus and its nuclear genome is predominantly derived from M. m. musculus (Yonekawa et al. 1980, 1988; Moriwaki 1994; Sakai et al. 2005). To investigate the reproductive isolation between M. m. domesticus and M. m. molossinus, we previously constructed a consomic strain B6-ChrX^MSM (Oka et al. 2004). This strain has the X chromosome from the MSM/Ms strain, which is derived from M. m. molossinus, in the genetic background of the laboratory strain C57BL/6J (B6), which is predominantly derived from M. m. domesticus (Moriwaki 1994). F₁ hybrid animals between B6 and MSM/Ms strains are fully fertile. On the contrary, B6-ChrX^MSM shows male-specific sterility characterized by a reduced sperm number and dysfunction of the sperm, including abnormal morphology and low motility, indicating that B6-ChrX^MSM is a model of hybrid breakdown in animals (Oka et al. 2004, 2007). Our previous study indicated that the abnormal morphology of the sperm head results from the genetic incompatibility between MSM/Ms-derived X-linked genes and B6 genes on autosomes including chromosomes 1 and 11 (Oka et al. 2007).In this study, to understand the genetic mechanism of reproductive isolation in mice, we first undertook in-depth characterization of phenotype for each B6-ChrX^MSM male especially during meiosis. Meiosis is a special cell division that produces four haploid cells after one round of chromosome replication and two rounds of chromosome segregation. During meiosis, homologous chromosomes pair, synapse, undergo crossing over, and achieve bipolar attachment to the spindle to segregate one set of chromosomes to each daughter cell. Homologous recombination is initiated during the leptotene stage of meiotic prophase I with the formation of DNA double-strand breaks (DSBs), which are repaired immediately during the zygotene stage or after crossing over of homologous chromosomes during the pachytene stage (Roeder 1997; Tarsounas and Moens 2001).During the first wave of spermatogenesis, most mitotic spermatogonia in the B6-ChrX^MSM testes fail to initiate meiotic DNA replication. Some proportion of those spermatogonia that enter into meiosis are again arrested and eliminated by apoptosis at the pachytene stage, resulting in the production of a small number of sperms. We extended the same analysis to three other cases of reproductive isolation. Another consomic strain PGN-ChrX^MSM has an MSM/Ms-derived X chromosome in the genetic background of the PGN2/Ms strain derived from wild mice (M. m. domesticus). PGN-ChrX^MSM males produce a small number of dysfunctional sperms as was the case with B6-ChrX^MSM males, but the former males show apoptosis mainly at metaphase of meiosis I. Furthermore, we examined F₁ hybrid males from intersubspecific cross of (B6 × M. m. musculus-NJL/Ms) and interspecific cross of (B6 × M. spretus). These F₁ hybrid males exhibited apoptosis at metaphase I and at the zygotene-to-pachytene stage of prophase I. As a whole, the postzygotic reproductive isolation in mice is caused by disruptions at a minimum of three different spermatogenic stages.     

A DNA-Damage Selective Role for BRCA1 E3 Ligase in Claspin Ubiquitylation,CHK1 Activation,and DNA Repair

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

Highlights? BRCA1 E3 ligase activity is essential for a subset of DNA damage responses ? BRCA1 V26A mutant cells are sensitive to topoisomerase inhibitors but not mitomycin C ? BRCA1 selectively ubiquitylates claspin after topoisomerase inhibition ? BRCA1 E3 ligase inactivation selectively impairs CHK1 activation     

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10⁻³) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10⁻³). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.     

Structurally Normal Corneas in Aldehyde Dehydrogenase 3a1-Deficient Mice

We have constructed an ALDH3a1 null mouse to investigate the role of this enzyme that comprises nearly one-half of the total water-soluble protein in the mouse corneal epithelium. ALDH3a1-deficient mice are viable and fertile, have a corneal epithelium with a water-soluble protein content approximately half that of wild-type mice, and contain no ALDH3a1 as determined by zymograms and immunoblots. Despite the loss of protein content and ALDH3a1 activity, the ALDH3a1(-/-) mouse corneas appear indistinguishable from wild-type corneas when examined by histological analysis and electron microscopy and are transparent as determined by light and slit lamp microscopy. There is no evidence for a compensating protein or enzyme. Even though the function of ALDH3a1 in the mouse cornea remains unknown, our data indicate that its enzymatic activity is unnecessary for corneal clarity and maintenance, at least under laboratory conditions.     

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3''-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.     

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation

1. Download high-res image (219KB)
2. Download full-size image

     

BRCA1 participates in DNA decatenation

The tumor suppressor BRCA1 has an important function in the maintenance of genomic stability. Increasing evidence suggests that BRCA1 regulates cell cycle checkpoints and DNA repair after DNA damage. However, little is known about its normal function in the absence of DNA damage. Here we show that BRCA1 interacts and colocalizes with topoisomerase IIalpha in S phase cells. Similar to cells treated with the topoisomerase IIalpha inhibitor ICRF-193, BRCA1-deficient cells show lagging chromosomes, indicating a defect in DNA decatenation and chromosome segregation. More directly, BRCA1 deficiency results in defective DNA decatenation in vitro. Finally, topoisomerase IIalpha is ubiquitinated in a BRCA1-dependent manner, and topoisomerase IIalpha ubiquitination correlates with higher DNA decatenation activity. Together these results suggest an important role of BRCA1 in DNA decatenation and reveal a previously unknown function of BRCA1 in the maintenance of genomic stability.     

Impairment of Visual Function and Retinal ER Stress Activation in Wfs1-Deficient Mice

Wolfram syndrome is an early onset genetic disease (1/180,000) featuring diabetes mellitus and optic neuropathy, associated to mutations in the WFS1 gene. Wfs1−/− mouse model shows pancreatic beta cell atrophy, but its visual performance has not been investigated, prompting us to study its visual function and histopathology of the retina and optic nerve. Electroretinogram and visual evoked potentials (VEPs) were performed in Wfs1^−/− and Wfs1^+/+ mice at 3, 6, 9 and 12 months of age. Fundi were pictured with Micron III apparatus. Retinal ganglion cell (RGC) abundance was determined from Brn3a immunolabeling of retinal sections. RGC axonal loss was quantified by electron microscopy in transversal optic nerve sections. Endoplasmic reticulum stress was assessed using immunoglobulin binding protein (BiP), protein disulfide isomerase (PDI) and inositol-requiring enzyme 1 alpha (Ire1α) markers. Electroretinograms amplitudes were slightly reduced and latencies increased with time in Wfs1^−/− mice. Similarly, VEPs showed decreased N+P amplitudes and increased N-wave latency. Analysis of unfolded protein response signaling revealed an activation of endoplasmic reticulum stress in Wfs1^−/− mutant mouse retinas. Altogether, progressive VEPs alterations with minimal neuronal cell loss suggest functional alteration of the action potential in the Wfs1^−/− optic pathways.     

Partial Absence of Pleuropericardial Membranes in Tbx18- and Wt1-Deficient Mice

The pleuropericardial membranes are fibro-serous walls that separate the pericardial and pleural cavities and anchor the heart inside the mediastinum. Partial or complete absence of pleuropericardial membranes is a rare human disease, the etiology of which is poorly understood. As an attempt to better understand these defects, we wished to analyze the cellular and molecular mechanisms directing the separation of pericardial and pleural cavities by pleuropericardial membranes in the mouse. We found by histological analyses that both in Tbx18- and Wt1-deficient mice the pleural and pericardial cavities communicate due to a partial absence of the pleuropericardial membranes in the hilus region. We trace these defects to a persisting embryonic connection between these cavities, the pericardioperitoneal canals. Furthermore, we identify mesenchymal ridges in the sinus venosus region that tether the growing pleuropericardial membranes to the hilus of the lung, and thus, close the pericardioperitoneal canals. In Tbx18-deficient embryos these mesenchymal ridges are not established, whereas in Wt1-deficient embryos the final fusion process between these tissues and the body wall does not occur. We suggest that this fusion is an active rather than a passive process, and discuss the interrelation between closure of the pericardioperitoneal canals, lateral release of the pleuropericardial membranes from the lateral body wall, and sinus horn development.