In utero exposure to arsenite contributes to metabolic and reproductive dysfunction in male offspring of CD-1 mice.

In utero exposure to arsenite (iAs) is known to increase disease risks later in life. We investigated the effect of in utero exposure to iAs in the drinking water on metabolic and reproductive parameters in male mouse offspring at postnatal and adult stages. Pregnant CD-1 mice were exposed to iAs (as sodium arsenite) in the drinking water at 0 (control), 10 ppb (EPA standard for drinking water), and 42.5 ppm (tumor-inducing dose in mice) from embryonic day (E) 10-18. At birth, pups were fostered to unexposed females. Male offspring exposed to 10 ppb in utero exhibited increase in body weight at birth when compared to controls. Male offspring exposed to 42.5 ppm in utero showed a tendency for increased body weight and a smaller anogenital distance. The body weight in iAs-exposed pups continued to increase significantly compared to control at 3 weeks and 11 weeks of age. At 5 months of age, iAs-exposed males exhibited greater body fat content and glucose intolerance. Male offspring exposed to 10 ppb in utero had higher circulating levels of leptin compared to control. In addition, males exposed to 42.5 ppm in utero exhibited decreased total number of pups born compared to controls and lower average number of litters sired over a six-month period. These results indicate that in utero exposure to iAs at either human relevant concentration or tumor-inducing concentration is a potential cause of developmental origin of metabolic and reproductive dysfunction in adult male mice.

Loss of Glis3 causes dysregulation of retrotransposon silencing and germ cell demise in fetal mouse testis.

Fetal germ cell development is regulated by an elaborate combination of cell-extrinsic and cell-intrinsic signals. Here we identify a novel role for the Krüppel-like transcription factor Gli-Similar 3 (Glis3) in male germ cell development in the mouse embryos. Glis3 is expressed in male germ cells during the brief window of time prior to initiation of piRNA-dependent retrotransposon surveillance. Disruption of Glis3 function led to a widespread reduction in retrotransposon silencing factors, aberrant retrotransposon expression and pronounced germ cell loss. Experimental induction of precocious Glis3 expression in vivo before its normal expression resulted in premature expression of several piRNA pathway members, suggesting that GLIS3 is necessary for the activation of the retrotransposon silencing programs. Our findings reveal an unexpected role for GLIS3 in the development of male germ cells and point to a central role for GLIS3 in the control of retrotransposon silencing in the fetal germline.

A new mouse line for cell ablation by diphtheria toxin subunit A controlled by a Cre-dependent FLEx switch.

Recombinase responsive mouse lines expressing diphtheria toxin subunit A (DTA) are well established tools for targeted ablation of genetically defined cell populations. Here we describe a new knock-in allele at the Gt(Rosa)26Sor locus that retains the best features of previously described DTA alleles-including a CAG promoter, attenuated mutant DTA cDNA, and ubiquitous EGFP labeling-with the addition of a Cre-dependent FLEx switch for tight control of expression. The FLEx switch consists of two pairs of antiparallel lox sites requiring Cre-mediated recombination for inversion of the DTA to the proper orientation for transcription. We demonstrate its utility by Cre-dependent ablation of both a broad domain in the embryonic nervous system and a discrete population of cells in the fetal gonads. We conclude that this new DTA line is useful for targeted ablation of genetically-defined cell populations.

Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice.

BACKGROUND: Mice exposed to high levels of arsenic in utero have increased susceptibility to tumors such as hepatic and pulmonary carcinomas when they reach adulthood. However, the effects of in utero arsenic exposure on general physiological functions such as reproduction and metabolism remain unclear. OBJECTIVES: We evaluated the effects of in utero exposure to inorganic arsenic at the U.S. Environmental Protection Agency (EPA) drinking water standard (10 ppb) and at tumor-inducing levels (42.5 ppm) on reproductive end points and metabolic parameters when the exposed females reached adulthood. METHODS: Pregnant CD-1 mice were exposed to sodium arsenite [none (control), 10 ppb, or 42.5 ppm] in drinking water from gestational day 10 to birth, the window of organ formation. At birth, exposed offspring were fostered to unexposed dams. We examined reproductive end points (age at vaginal opening, reproductive hormone levels, estrous cyclicity, and fertility) and metabolic parameters (body weight changes, hormone levels, body fat content, and glucose tolerance) in the exposed females when they reached adulthood. RESULTS: Arsenic-exposed females (10 ppb and 42.5 ppm) exhibited early onset of vaginal opening. Fertility was not affected when females were exposed to the 10-ppb dose. However, the number of litters per female was decreased in females exposed to 42.5 ppm of arsenic in utero. In both 10-ppb and 42.5-ppm groups, arsenic-exposed females had significantly greater body weight gain, body fat content, and glucose intolerance. CONCLUSION: Our findings revealed unexpected effects of in utero exposure to arsenic: exposure to both a human-relevant low dose and a tumor-inducing level led to early onset of vaginal opening and to obesity in female CD-1 mice.

Multifunctional regulators of cell growth are differentially expressed in anergic murine B cells.

Defective anergy is a major cause of failed tolerance and is amenable to therapeutic manipulation. To better define the molecular basis of anergy in B cells tolerized by matrix self-antigen, we used complementary approaches of representational difference analysis (RDA) and microarray to identify genes differentially transcribed in anergic as compared to non-tolerant B cells isolated from a well-characterized murine autoantibody transgenic model. Forty RDA clones representing 16 genes were isolated from receptor-stimulated B cells and independently confirmed as differentially expressed in tolerant cells using custom microarray, dot blotting and/or quantitative PCR. Differential expression was conserved in tolerant cells from two different transgenic founder lineages and from two genetically disparate backgrounds. Prominent among recovered gene fragments were genes encoding multifunctional proteins not previously implicated in B cell biology, but with roles in biologic processes fundamental to the tolerance phenotype, including cell growth, proliferation and differentiation. RDA also identified a novel transcript not previously reported in nucleic acid databases. To further explore dependence on receptor stimulation and to identify additional genes, commercial oligonucleotide arrays were probed with labeled B cell transcripts and analyzed for genes differentially expressed in resting as well as stimulated cells and in both B6 and MRL mouse strains. Arrays identified differential expression of a subset of RDA genes as well as 46 additional genes, including subsets engaged in signal transduction, transcriptional regulation, cell growth and apoptosis. Immunoblotting confirmed differential protein expression for galectin-3 and galectin-1, two interactive members of the galectin family, suggesting a novel role for galectins as regulators of immune tolerance.