Progeny Of Mice Research Articles

Modulatory effects of feeding pregnant and lactating mice Rhodiola kirilowii extracts on the immune system of offspring.

Plants of Rhodiola genus are medicinal herbs that have a number of therapeutic properties, including anti-inflammatory and immunomodulatory activity. The present study aimed to determine whether the use Rhodiola kirilowii as an immunostimulant during pregnancy has an adverse effect on the development of the offspring immune system. Following mating, pregnant mice were placed in three groups that were fed during pregnancy and lactation with R. kirilowii aqueous extract (RKW; 20 mg/kg), R. kirilowii 50% hydro-alcoholic extract (RKW-A; 20 mg/kg) or water (control group), receiving water. Following birth, offspring were given six weeks to develop prior to evaluation of their immune system. Morphometric and morphological examination of the spleen did not reveal any abnormalities or differences between the experimental and control groups. However, both RKW and RKW-A splenic lymphocytes presented a diminished proliferative response to concanavalin A. RKW spleen lymphocytes demonstrated increased metabolic activity following phytohaemagglutinin (PHA) stimulation, which was associated with a higher percentage of cluster of differentiation 4 positive spleen cells and lower interleukin-17a (IL-17a) serum concentration. The RKW-A group exhibited a diminished proliferative response of spleen lymphocytes to PHA and lipopolysaccharide (LPS), and increased serum concentrations of IL-10 and tumor necrosis factor-α (TNF-α). The progeny of mice fed with RKW-A extract demonstrated a significantly lower level of anti-SRBC antibody following immunization compared with progeny of the control (P=0.0305) and RKW (P=0.0331) groups. In conclusion, caution is recommended in the use of RKW and RKW-A extracts as immunostimulants in pregnancy.

Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice

Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30mg/kg) were added, and dose-response of inherited germline mutations was analyzed. Male gpt delta transgenic mice treated with ENU in three dose groups were mated with untreated females 10 weeks after the last treatment, and offspring were obtained. The ENU-treated male mice showed dose-dependent increases in gpt mutant frequencies in their sperm, testis, and liver. gDNA of one family (parents and four offspring) from each dose group was used for whole-exome sequencing, and unique de novo mutations in the offspring were detected. Frequencies of inherited mutations increased with dosage more than 25-fold in the highest dose group. The mutation spectrum of the inherited mutations showed characteristics of ENU-induced mutations, such as A:T base substitutions. No confirmed mutations were observed in the control group. Filtering using the alternate reads ratio resulted in the mutation frequencies and spectra similar to those obtained by the Sanger sequencing confirmation. These results suggest that direct sequencing analysis may be a useful tool to investigate inherited germline mutations induced by environmental mutagens.

Feeding Pregnant and Lactating Mice Rhodiola kirilowii Extracts helps to Preserve Thymus Function of their Adult Progeny.

Plants belonging to Rhodiola kirilowii species, members of Rhodiola genus and Crassulaceae family, grow wildly in Tibet, Mongolia and China mountains and are traditionally used as adaptogens, antidepressants and anti-inflammatory remedies. Nowadays, R. kirilowii is cultivated in some countries, also in Poland. In our previous papers we reported immuno- and angio-modulatory effects of aqueous and hydro-alcoholic extracts of radix and rhizome of this plant in non-pregnant and pregnant mice. The aim of the present study was to evaluate the effect of feeding pregnant and further lactating mice these extracts on selected thymus function parameters in adult progeny. The counts of M-30+ apoptotic cells, in the thymuses obtained from progeny of mice fed during pregnancy and lactation water or 50% water-alcoholic extract of Rhodiola kirilowii, were significantly lower (p<0.05) than apoptotic cells counts observed in the control mice. No significant differences in the counts of IL-7-positive cells in the thymuses obtained from progeny of the control mice and mothers treated with water or hydro-alcohol extracts of Rhodiola kirilowii were observed.

Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine.

In utero exposure of mouse progeny to alcohol (ethanol, EtOH) and methamphetamine (METH) causes substantial postnatal neurodevelopmental deficits. One emerging pathogenic mechanism underlying these deficits involves fetal brain production of reactive oxygen species (ROS) that alter signal transduction, and/or oxidatively damage cellular macromolecules like lipids, proteins, and DNA, the latter leading to altered gene expression, likely via non-mutagenic mechanisms. Even physiological levels of fetal ROS production can be pathogenic in biochemically predisposed progeny, and ROS formation can be enhanced by drugs like EtOH and METH, via activation/induction of ROS-producing NADPH oxidases (NOX), drug bioactivation to free radical intermediates by prostaglandin H synthases (PHS), and other mechanisms. Antioxidative enzymes, like catalase in the fetal brain, while low, provide critical protection. Oxidatively damaged DNA is normally rapidly repaired, and fetal deficiencies in several DNA repair proteins, including oxoguanine glycosylase 1 (OGG1) and breast cancer protein 1 (BRCA1), enhance the risk of drug-initiated postnatal neurodevelopmental deficits, and in some cases deficits in untreated progeny, the latter of which may be relevant to conditions like autism spectrum disorders (ASD). Risk is further regulated by fetal nuclear factor erythroid 2-related factor 2 (Nrf2), a ROS-sensing protein that upregulates an array of proteins, including antioxidative enzymes and DNA repair proteins. Imbalances between conceptal pathways for ROS formation, versus those for ROS detoxification and DNA repair, are important determinants of risk. Birth Defects Research (Part C) 108:108-130, 2016. © 2016 Wiley Periodicals, Inc.

Changes in Differentiation of Thymic T Cells in the Progeny of Female Mice Subjected to Immune System Stimulation during Early Pregnancy.

Differentiation of T cells was studied in the progeny of female C57Bl/6 mice subjected to immunostimulation by administration of concanavalin A or adoptive transfer of concanavalin A-activated splenocytes. Acceleration of T cell maturation during the prepubertal period and decelerated differentiation during the pubertal and post-pubertal periods were revealed.

Efficient chimeric mouse production using a novel embryonic stem cell line

Embryonic stem cells are commonly used for generating transgenic mice. Embryonic stem cells could participate in the development of chimeric animals after being injected into a blastocyst. Injection of genetically modified embryonic stem cells could lead to the transmission of the germ lines of a transgene or genomic modification in chimeric mice. Such founders are used to produce transgenic lines of mice. There are several projects dedicated to producing knock-out mouse lines (KOMP Repository, EUCOMM, Lexicon Genetics). Nevertheless, there is a need for complex genome modifications, such as large deletions, reporter genes’ insertion into the 3' gene regulatory sequence, or site-specific modifications of the genome. To do this, researchers need an embryonic stem cell line that is able to participate in the formation of chimeric animals even after prolonged cultivation in vitro. Several lines of embryonic stem cells of mice were produced in the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics (ICG), Siberian Branch, Russian Academy of Sciences (SB RAS). We tested the DGES1 cell line (2n = 40, XY) (of the 129S2/SvPasCrl genetic background) for chimeric mice production at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS. Embryonic stem cells were injected into 136 blastocysts (of the B6D2F1 genetic background), which were transplanted into CD-1 mice. Among 66 progeny, 15 were chimeric, 4 of which were more than 80% chimeric judged by the coat color. All chimeras were males without developmental abnormalities. 10 of 15 males were fertile. Microsatellite analysis of the progeny of chimeric mice revealed the contribution of the embryonic stem cell line DGES1 to the gamete formation. Thus, a novel DGES1 embryonic stem cell line could be efficiently used for transgenic mouse production using the B6D2F1 blastocysts and CD-1 recipients.

Reproductive effects of the tumor necrosis factor (TNF) deficiency in mice

TNF is a multifunctional cytokine that, at physiological concentrations, maintains the balance between apoptosis and survival of male germ cells and, at higher concentrations, has adverse effects on various stages of the reproductive process. Although ant-cytokine therapies have been used in millions of patients, the consequences of cytokine deficiency for reproductive functions are poorly understood and need attention. In this work, we have studied behavioral interactions between males and females, spermatogenesis, male fertility, and embryonic developmental characteristics of the progeny in TNFα knockout mice (TNF-/-). We have demonstrated that TNF is involved in the regulation of sexual behavior, spermatogenesis, pre- and postimplantation development. Complete TNF deficiency led to decreased reproductive efficiency: a lower number of viable embryos were observed in TNF-/- mice than in wild-type mice. The decrease in fertility was caused by preimplantation embryo loss in TNF-/- mice. Preimplantation loss in females might be caused by asospermia in TNF-/- males. Additionally, the sensitivity of reproductive functions to female stimuli was different between TNF-/- mice and wild-type mice, while interactions with females increased the concentrations of sper­matozoids in both TNF-/- and wild-type mice. Still higher levels were observed in knockout animals, which led to increase in the number of immature spermatozoids in epididymides.

Conditional depletion of methyl-CpG-binding protein 2 in astrocytes depresses the hypercapnic ventilatory response in mice.

Mice that are deficient in the transcription factor methyl-CpG-binding protein 2 (MeCP2) have a depressed hypercapnic ventilatory response (HCVR). The expression of MeCP2 can be selectively removed from astrocytes or neurons, thus offering a tool to dissect the role of this transcription factor in astrocytes from that in neurons. Studies were carried out in the progeny of mice that were a cross between those harboring a tamoxifen (TAM)-inducible Cre recombinase transgene driven by the human astrocytic glial fibrillary acidic protein (hGFAP) promoter, or Cre recombinase under control of the synapsin promoter, with mice containing a Cre-excisable exon III in the Mecp2 gene. The TAM-conditional excision of the Mecp2 exon allowed the respiratory CO2 response to be studied in the same animals before and after selective depletion of MeCP2 in astrocytes. Immunohistochemistry showed that following TAM treatment only ∼20% of GFAP-labeled cells in the retrotrapazoid nucleus and in the raphé magnus were positive for MeCP2. The slope of the relative increase in minute ventilation as a function of 1, 3, and 5% inspired CO2 was depressed in mice with depleted astrocyte MeCP2 compared with wild-type littermates. In contrast, selective depletion of MeCP2 in neurons did not significantly affect slope. While neurons which constitute the respiratory network ultimately determine the ventilatory response to CO2, this study demonstrates that loss of MeCP2 in astrocytes alone is sufficient to result in a dramatic attenuation of the HCVR. We propose that the glial contribution to HCVR is under the control of the MeCP2 gene.

Inherited Alopecia Areata in Mice as a Result of Adoptive Transfer of In Vivo Mitogen-Activated Splenic Cells to Females During Early Period of Gestation.

Postnatal morphogenesis of the skin and its appendages were studied in the progeny of mice subjected during early gestation period to adoptive transfer of in vivo activated splenic cells. Reversible focal hair loss was observed on postnatal day 17 in the progeny. The number of hair follicles and sebaceous glands, the level of extracellular matrix, and the content of cells in the skin, including mast cells in the derma, decreased, while neutrophils prevailed. Alopecia areata was found in the subsequent generations after mating of male and female progeny. Hence, stimulation of the maternal immune system during early pregnancy led to disorders of the pre- and postnatal morphogenesis of the skin in the progeny and to development of alopecia areata that became an inherited sign.

Long-Term Changes in Proliferative Capacity of Thymocytes and Splenocytes in Mouse Progeny after Stimulation of Maternal Lymphocyte Proliferation at Early Terms of Pregnancy.

We studied the possibility of prenatal programming of proliferative potential of thymocytes and splenocytes in the offspring of C57Bl/6 mice via concanavalin A mitogen stimulation of lymphocyte proliferation in maternal organism at the early terms of pregnancy before the formation of immune system organs in the embryo. The offspring demonstrated elevated proliferative activity of thymocytes and splenocytes, enhanced lymphocyte proliferation, and increased production of IL-2 in in vitro response to concanavalin A during the prepubertal period and puberty. Mitogen stimulation of the maternal immune system in early pregnancy results in long-term enhancement of the proliferative potential of thymus and splenic cells in the offspring.

N-ethyl-N-Nitrosourea (ENU) induced mutations within the klotho gene lead to ectopic calcification and reduced lifespan in mouse models.

Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established.

Adaptive Modifications of Muscle Phenotype in High-Altitude Deer Mice Are Associated with Evolved Changes in Gene Regulation.

At high-altitude, small mammals are faced with the energetic challenge of sustaining thermogenesis and aerobic exercise in spite of the reduced O2 availability. Under conditions of hypoxic cold stress, metabolic demands of shivering thermogenesis and locomotion may require enhancements in the oxidative capacity and O2 diffusion capacity of skeletal muscle to compensate for the diminished tissue O2 supply. We used common-garden experiments involving highland and lowland deer mice (Peromyscus maniculatus) to investigate the transcriptional underpinnings of genetically based population differences and plasticity in muscle phenotype. We tested highland and lowland mice that were sampled in their native environments as well as lab-raised F1 progeny of wild-caught mice. Experiments revealed that highland natives had consistently greater oxidative fiber density and capillarity in the gastrocnemius muscle. RNA sequencing analyses revealed population differences in transcript abundance for 68 genes that clustered into two discrete transcriptional modules, and a large suite of transcripts (589 genes) with plastic expression patterns that clustered into five modules. The expression of two transcriptional modules was correlated with the oxidative phenotype and capillarity of the muscle, and these phenotype-associated modules were enriched for genes involved in energy metabolism, muscle plasticity, vascular development, and cell stress response. Although most of the individual transcripts that were differentially expressed between populations were negatively correlated with muscle phenotype, several genes involved in energy metabolism (e.g., Ckmt1, Ehhadh, Acaa1a) and angiogenesis (Notch4) were more highly expressed in highlanders, and the regulators of mitochondrial biogenesis, PGC-1α (Ppargc1a) and mitochondrial transcription factor A (Tfam), were positively correlated with muscle oxidative phenotype. These results suggest that evolved population differences in the oxidative capacity and capillarity of skeletal muscle involved expression changes in a small suite of coregulated genes.

L-Carnitine reverses maternal cigarette smoke exposure-induced renal oxidative stress and mitochondrial dysfunction in mouse offspring.

Maternal smoking is associated with metabolic disorders, renal underdevelopment, and a predisposition to chronic kidney disease in offspring, yet the underlying mechanisms are unclear. By exposing female Balb/c mice to cigarette smoke for 6 wk premating and during gestation and lactation, we showed that maternal smoke exposure induced glucose intolerance, renal underdevelopment, inflammation, and albuminuria in male offspring. This was associated with increased renal oxidative stress and mitochondrial dysfunction at birth and in adulthood. Importantly, we demonstrated that dietary supplementation of l-carnitine, an amino acid shown to increase antioxidant defenses and mitochondrial function in numerous diseases, in smoke-exposed mothers during pregnancy and lactation significantly reversed the detrimental maternal impacts on kidney pathology in these male offspring. It increased SOD2 and glutathione peroxidase 1, reduced ROS accumulation, and normalized levels of mitochondrial preprotein translocases of the outer membrane, and oxidative phosphorylation complexes I-V in the kidneys of mouse progeny after intrauterine cigarette smoke exposure. These findings support the hypothesis that oxidative stress and mitochondrial dysfunction are closely linked to the adverse effects of maternal smoking on male offspring renal pathology. The results of our study suggest that l-carnitine administration in cigarette smoke-exposed mothers mitigates these deleterious renal consequences.

TGF-B3 Dependent Modification of Radiosensitivity in Reporter Cells Exposed to Serum From Whole-Body Low Dose-Rate Irradiated Mice.

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.

HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice.

Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR.

Conditional deletion of α-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning.

New granule cells are continuously integrated into hippocampal circuits throughout adulthood, and the fine-tuning of this process is likely important for efficient hippocampal function. During development, this integration process is critically regulated by the α-calcium/calmodulin-dependent protein kinase II (α-CaMKII), and here we ask whether this role is conserved in the adult brain. To do this, we developed a transgenic strategy to conditionally delete α-CaMKII from neural progenitor cells and their progeny in adult mice. First, we found that the selective deletion of α-CaMKII from newly generated dentate granule cells led to an increase in dendritic complexity. Second, α-CaMKII deletion led to a reduction in number of mature synapses and cell survival. Third, consistent with altered morphological and synaptic development, acquisition of one-trial contextual fear conditioning was impaired after deletion of α-CaMKII from newly generated dentate granule cells. Previous work in Xenopus identified α-CaMKII as playing a key role in the stabilization of dendritic and synaptic structure during development. The current study indicates that α-CaMKII plays a plays a similar, cell-autonomous role in the adult hippocampus and, in addition, reveals that the loss of α-CaMKII from adult-generated granule cells is associated with impaired hippocampus-dependent learning.

The nutritional sins of the mother...

In Utero Effects Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science , this issue p. [1255903][1] [1]: http://www.sciencemag.org/content/345/6198/1255903.full

In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism.

Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

AB0005 F1 Hybrid of NZW and Fcg Riib-Deficient B6 Mice Developed Phenotype Conversion from Rheumatoid Arthritis to Murine Lupus

BackgroundFcγRIIB negatively regulates BCR-mediated activation signals. We previously established an FcγRIIB-deficient B6-congenic mouse strain (KO1) by gene targeting in 129-derived embryonic stem cells followed by backcrossing to B6 mice. KO1...

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span.

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.