F1 Generation Mice Research Articles

Precise modelling of mitochondrial diseases using optimized mitoBEs.

The development of animal models is crucial for studying and treating mitochondrial diseases. Here we optimized adenine and cytosine deaminases to reduce off-target effects on the transcriptome and the mitochondrial genome, improving the accuracy and efficiency of our newly developed mitochondrial base editors (mitoBEs)1. Using these upgraded mitoBEs (version 2 (v2)), we targeted 70 mouse mitochondrial DNA mutations analogous to human pathogenic variants2, establishing a foundation for mitochondrial disease mouse models. Circular RNA-encoded mitoBEs v2 achieved up to 82% editing efficiency in mice without detectable off-target effects in the nuclear genome. The edited mitochondrial DNA persisted across various tissues and was maternally inherited, resulting in F1 generation mice with mutation loads as high as 100% and some mice exhibiting editing only at the target site. By optimizing the transcription activator-like effector (TALE)binding site, we developed a single-base-editing mouse model for the mt-Nd5 A12784G mutation. Phenotypic evaluations led to the creation of mouse models for the mt-Atp6 T8591C and mt-Nd5 A12784G mutations, exhibiting phenotypes corresponding to the reduced heart rate seen in Leigh syndrome and the vision loss characteristic of Leber's hereditary optic neuropathy, respectively. Moreover, the mt-Atp6 T8591C mutation proved to be more deleterious than mt-Nd5 A12784G, affecting embryonic development and rapidly diminishing through successive generations. These upgraded mitoBEs offer a highly efficient and precise strategy for constructing mitochondrial disease models, laying a foundation for further research in this field.

Effect of Bisphenol F on Reproductive Function in F1 Generation Male Mice and its Potential Mechanisms.

Bisphenol F (BPF) is an environmental endocrine disruptor capable of crossing the placental barrier and affecting the growth and development of offspring. Despite its potential impact, systematic research about effects of BPF on the reproductive function of male offspring remains limited. In this study, pregnant female mice were exposed to BPF at doses of 40, 400, and 4000 μg/kg during gestation and lactation, respectively, to evaluate its impact on testicular damage, testosterone levels, and spermatogenesis of male offspring (F1 generation), and further explore the mechanisms using transcriptomics. First, the study demonstrated that BPF induces testicular damage in F1 generation mice, leading to decreased testosterone levels and sperm quality. Second, transcriptomic analysis revealed that BPF affected spermatogenesis in F1 generation mice by disrupting retinol metabolism. Third, transcriptomic analysis revealed that BPF reduce the capacity for testosterone synthesis in F1 generation mice by diverting the testosterone precursor dehydroepiandrosterone (DHEA) towards the synthesis of 16α-hydroxydehydroepiandrosterone rather than testosterone. Finally, it was confirmed that BPF hinder cholesterol transport to mitochondria by inhibiting the cAMP signaling pathway, thereby impacting testosterone synthesis. In summary, the results of this study suggest that gestational exposure to BPF can lead to reproductive dysfunction in F1 generation male mice.

Co-exposure of microcystin and nitrite enhanced spermatogenic disorders: The role of mtROS-mediated pyroptosis and apoptosis

Microcystins (MCs) and nitrites are coexisted in the environment and have reproductive toxicity. The combined toxic effect and mechanism of MCs and nitrite on spermatogenesis remain largely unclear. In the present study, co-exposure to microcystin-leucine arginine (MC-LR) and sodium nitrite (NaNO2) aggravated testicular damage of Balb/c mice and mitochondrial impairment of spermatogonia, Sertoli cells, and sperm. Furthermore, MC-LR and NaNO2 reduced sperm density with a synergistic effect. In addition, MC-LR and NaNO2 synergistically induced oxidative stress in the reproductive system by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels and increasing levels of mitochondrial reactive oxygen species (mtROS) and reactive oxygen species (ROS). More importantly, mitoquidone mesylate (MitoQ), an inhibitor of mtROS, blocked MC-LR and NaNO2-induced spermatogonia and Sertoli cell apoptosis by inhibiting high expression of Bax, Fadd, Caspase-8, and cleaved-Caspase-3. On the other hand, MitoQ suppressed pyroptosis of Sertoli cells by inhibiting the expression of NLRP3, N-GSDMD, and cleaved-Caspase-1. Additionally, MitoQ alleviated co-exposure-induced sperm density reduction and organ index disorders in F1 generation mice. Together, co-exposure of MC-LR and NaNO2 can enhance spermatogenic disorders by mitochondrial oxidative impairment-mediated germ cell death. This study emphasizes the potential risks of MC-LR and NaNO2 on reproduction in realistic environments and highlights new insights into the cause and treatment of spermatogenic disorders.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Genetically engineered mouse model of HPV16 E6-E7 with vaginal-cervical intraepithelial neoplasia and decreased immunity

ObjectiveTo construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function. MethodsUsing CRISPR/Cas9, the expression vector HPV16-E6-E7-Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8+ T cell proportions in the spleen were evaluated. ResultsTwo F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous HPV16-E6-E7-engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8+ T cells decreased. ConclusionHPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.

IVF exposure induced intergenerational effects on metabolic phenotype in mice

Research questionWhat is the potential transmission of metabolic phenotype from IVF offspring to the subsequent generation? DesignAn IVF mouse model was established. The F1 generation mice were produced though IVF or natural mating and the F2 generation was obtained through the mating of F1 generation males with normal females. Their metabolic phenotype, including systemic and hepatic glucolipid metabolism, was examined. ResultsIt was found that IVF F1 males exhibited metabolic changes. Compared with the control group, the IVF F1 generation showed increased body weight, elevated fasting glucose and insulin, and increased serum triglyceride concentrations. IVF F1 mice also showed an increased expression of hepatic lipogenesis and autophagy genes. Moreover, IVF F1 males transmitted some metabolic changes to their own male progeny (IVF F2) in the absence of a dietary challenge. IVF F2 mice had increased peri-epididymal and subcutaneous fat and decreased insulin sensitivity. Under the ‘second hit’ of a high-fat diet, IVF F2 mice further showed increased hepatic lipid deposition with unaltered autophagy levels. ConclusionThis research demonstrates the impact of IVF on hepatic glucose–lipid metabolism in two successive generations of offspring, highlighting the need for additional investigation. Enhanced understanding of the mechanisms underlying the transmission of multigenerational effects induced by IVF could potentially lead to the advancement of therapeutic interventions for individuals experiencing infertility.

Environmentally relevant concentrations of benzophenones exposure disrupt intestinal homeostasis, impair the intestinal barrier, and induce inflammation in mice

The aim of this study is to investigate the adverse effects of benzophenones (BPs) on the intestinal tract of mice and the potential mechanism. F1-generation ICR mice were exposed to BPs (benzophenone-1, benzophenone-2, and benzophenone-3) by breastfeeding from birth until weaning, and by drinking water after weaning until maturity. The offspring mice were executed on postnatal day 56, then their distal colons were sampled. AB-PAS staining, HE staining, immunofluorescence, Transmission Electron Microscope, immunohistochemistry, Western Blot and RT-qPCR were used to study the effects of BPs exposure on the colonic tissues of offspring mice. The results showed that colonic microvilli appeared significantly deficient in the high-dose group, and the expression of tight junction markers Zo-1 and Occludin was significantly down-regulated and the number of goblet cells and secretions were reduced in all dose groups, and the expression of secretory cell markers MUC2 and KI67 were decreased, as well as the expression of intestinal stem cell markers Lgr5 and Bmi1, suggesting that BPs exposure caused disruption of intestinal barrier and imbalance in the composition of the intestinal stem cell pool. Besides, the expression of cellular inflammatory factors such as macrophage marker F4/80 and tumor necrosis factor TNF-α was elevated in the colonic tissues of all dose groups, and the inflammatory infiltration was observed, which means the exposure of BPs caused inflammatory effects in the intestinal tract of F1-generation mice. In addition, the contents of Notch/Wnt signaling pathway-related genes, such as Dll-4, Notch1, Hes1, Ctnnb1and Sfrp2 were significantly decreased in each high-dose group (P < 0.05), suggesting that BPs may inhibit the regulation of Notch/Wnt signaling pathway. In conclusion, exposure to BPs was able to imbalance colonic homeostasis, disrupt the intestinal barrier, and trigger inflammation in the offspring mice, which might be realized through interfering with the Notch/Wnt signaling pathway.

Evaluation of gadolinium-based contrast agents in pregnant CD-1 mice and subsequent in utero exposure of the developing offspring, including behavioral evaluations.

The offspring of CD-1 mice exposed during pregnancy to one of seven gadolinium-based contrast agents (GBCAs) were evaluated for potential effects on postnatal development and behavior. The GBCAs, comprising four linear (gadopentetate dimeglumine, gadodiamide, gadobenate dimeglumine, and gadoxetate disodium) and three macrocyclic (gadoterate meglumine, gadoteridol, and gadobutrol), were administered via intravenous injection once daily from Gestation Day 6 through 17 following confirmed mating (Day 0) at doses of at least twice the human equivalent recommended clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). All dams were allowed to deliver naturally. F0 generation females were monitored for maternal toxicity and gadolinium (Gd) levels in blood and brain. Offspring were evaluated for Gd levels in blood and brain at birth and on Day 70 postpartum. F1 generation mice were evaluated for survival and growth preweaning. Selected pups/litter were evaluated postweaning for sexual maturation, growth, and behavior. Gd was quantifiable in the brain of the F1 offspring on PND 1, with levels declining over time. There was no long-term effect of any GBCA on the growth and development of any offspring. There was no impact on neurodevelopment, as assessed by brain histology and validated neurobehavioral tests, including a battery of functional observational tests, motor activity, and learning and memory as evaluated in the Morris water maze. At the end of the postweaning period, the highest dose tested was considered the no-observable-adverse-effect level (NOAEL) in the F0 and F1 offspring for all tested GBCAs.

Return to fertility, toxicology, and transgenerational impact of treatment with WIN 18,446, a potential male contraceptive, in mice

ObjectivesWe examined the return to fertility and transgenerational impact of treatment with WIN 18,446, an experimental male contraceptive, in mice. Study designWe paired male mice treated with WIN 18,446 for 4 weeks to suppress spermatogenesis, followed by a 9-week recovery, and mated them with normal females to assess fertility. F1 generation mice were subsequently mated to ascertain any transgenerational impact of treatment on fertility. Testes were examined histologically. ResultsWIN 18,446–treated mice and their progeny produced normally sized litters (6.5 pups per litter after treatment and 7.3 pups per litter from the progeny). However, testes histology revealed rare residual intratesticular foci of mineralization after treatment. ConclusionsFertility normalizes after WIN 18,446 treatment, and progeny also have normal fertility.

Reproductive and neurobehavioral effects of dinotefuran in an F1 -generation toxicity study in mice.

Few studies were found for neurobehavioral toxicity of dinotefuran in mammals. This study was designed to evaluate the reproductive and neurobehavioral effects of dinotefuran exposure in mice. Dinotefuran was given in the diet to provide levels of 0% (control), 0.015%, 0.03%, and 0.06% from 5 weeks of age of the F0 generation to 11 weeks of age of the F1 generation in mice. Selected reproductive and neurobehavioral parameters were measured. Movement time increased with a significant dose-related trend, and the related variables of rearing time decreased in significant dose-related trends in adult males in the F0 generation. Litter size and weight increased in significant dose-related trends, and sex ratio decreased in a significant dose-related trend. The average body weight of offspring increased in a significant dose-related trend on postnatal day (PND) 21 in both sexes. In the olfactory orientation on PND 14 in female offspring, the time required lengthened in a significant dose-related trend. In male offspring, total distance and the average speed decreased in significant dose-related trends, and the average time of rearing, number of defecations, and frequencies of mice with urination increased in a significant dose-related trend. In female offspring, the related variables of rearing increased in significant dose-related trends. In spontaneous behavior of males, the parallel lines during the control and treatment groups indicated a significant distance in the number of horizontal activities. The dose levels of dinotefuran in the present study produced several adverse effects on reproductive and neurobehavioral parameters in mice.

Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

Effects of gestational inflammation on age-related cognitive decline and hippocampal Gdnf-GFRα1 levels in F1 and F2 generations of CD-1 Mice

BackgroundIt has been reported that age-associated cognitive decline (AACD) accelerated by maternal lipopolysaccharide (LPS) insult during late pregnancy can be transmitted to the second generation in a sex-specificity manner. In turn, recent studies indicated that glial cell line‐derived neurotrophic factor (GDNF) and its cognate receptor (GFRα1) are critical for normal cognitive function. Based on this evidence, we aimed to explore whether Gdnf-GFRα1 expression contributes to cognitive decline in the F1 and F2 generations of mouse dams exposed to lipopolysaccharide (LPS) during late gestation, and to evaluate also the potential interference effect of pro-inflammatory cytokines.MethodsDuring gestational days 15–17, pregnant CD-1 mice (8–10 weeks old) received a daily intraperitoneal injection of LPS (50 μg/kg) or saline (control). In utero LPS-exposed F1 generation mice were selectively mated to produce F2 generation mice. In F1 and F2 mice aged 3 and 15 months, the Morris water maze (MWM) was used to evaluated the spatial learning and memory ability, the western blotting and RT-PCR were used for analyses of hippocampal Gdnf and GFRα1 expression, and ELISA was used to analyse IL-1β, IL-6 and TNF-α levels in serum.ResultsMiddle-aged F1 offspring from LPS-treated mothers exhibited longer swimming latency and distance during the learning phase, lower percentage swimming time and distance in targe quadrant during memory phase, and lower hippocampal levels of Gdnf and GFRα1 gene products compared to age-matched controls. Similarly, the middle-aged F2 offspring from the Parents-LPS group had longer swimming latency and distance in the learning phase, and lower percentage swimming time and distance in memory phase than the F2-CON group. Moreover, the 3-month-old Parents-LPS and 15-month-old Parents- and Father-LPS groups had lower GDNF and GFRα1 protein and mRNAs levels compared to the age-matched F2-CON group. Furthermore, hippocampal levels of Gdnf and GFRα1 were correlated with impaired cognitive performance in the Morris water maze after controlling for circulating pro-inflammatory cytokine levels.ConclusionsOur findings indicate that accelerated AACD by maternal LPS exposure can be transmitted across at least two generations through declined Gdnf and GFRα1 expression, mainly via paternal linage.

Transgenerational effects of developmental neurotoxicity induced by exposure to a no-observed-adverse-effect level (NOAEL) of neonicotinoid pesticide clothianidin.

Neonicotinoid pesticides (NNs) transfer rapidly from mother to offspring, which exhibit neurobehavioral effects. However, no studies have investigated NNs' transgenerational effects. We exposed F0 generation mice (mothers) to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) during gestation and lactation, and examined the adult neurobehavioral effects of three generations of offspring (F1, F2, F3). F1 had lower birth weight, decreased locomotor activity, and increased anxiety-like behavior. In F2, body weight was affected, and there was a decreasing trend in locomotor activity and an increasing trend in anxiety-like behavior. In F3, locomotor activity tended to increase. Thus, even when only the mothers were exposed, the effects of CLOs were still observed in F1, F2, and F3 but the effects became smaller.

Thyroid hormone elicits intergenerational epigenetic effects on adult social behavior and fetal brain expression of autism susceptibility genes.

Genetic mutations identified in genome-wide association studies can only explain a small percentage of the cases of complex, highly heritable human conditions, including neurological and neurodevelopmental disorders. This suggests that intergenerational epigenetic effects, possibly triggered by environmental circumstances, may contribute to their etiology. We previously described altered DNA methylation signatures in the sperm of mice that experienced developmental overexposure to thyroid hormones as a result of a genetic defect in hormone clearance (DIO3 deficiency). Here we studied fetal brain gene expression and adult social behavior in genetically normal F2 generation descendants of overexposed mice. The brain of F2 generation E13.5 fetuses exhibited abnormal expression of genes associated with autism in humans, including Auts2, Disc1, Ldlr, Per2, Shank3, Oxtr, Igf1, Foxg1, Cd38, Grid2, Nrxn3, and Reln. These abnormal gene expression profiles differed depending on the sex of the exposed ancestor. In the three-chamber social box test, adult F2 generation males manifested significantly decreased interest in social interaction and social novelty, as revealed by decrease total time, distance traveled and time immobile in the area of interaction with novel strangers. F1 generation mice, compared to appropriate controls also exhibited altered profiles in fetal brain gene expression, although these profiles were substantially different to those in the F2 generation. Likewise adult F1 generation mice showed some abnormalities in social behavior that were sexually dimorphic and milder than those in F2 generation mice. Our results indicate that developmental overexposure to thyroid hormone causes intergenerational epigenetic effects impacting social behavior and the expression of autism-related genes during early brain development. Our results open the possibility that altered thyroid hormone states, by eliciting changes in the epigenetic information of the germ line, contribute to the susceptibility and the missing-but heriTables-etiology of complex neurodevelopmental conditions characterized by social deficits, including autism and schizophrenia.

Effects of prenatal and lactational exposure to iodoacetic acid on the F1 generation of mice†.

Water disinfection can generate water disinfection byproducts (DBPs). Iodoacetic acid (IAA) is one DBP, and it has been shown to be an ovarian toxicant in vitro and in vivo. However, it is unknown if prenatal and lactational exposure to IAA affects reproductive outcomes in female offspring. This study tested the hypothesis that prenatal and lactational exposure to IAA adversely affects reproductive parameters in F1 female offspring. Adult female CD-1 mice were dosed with water (control) or IAA (10, 100, and 500mg/L) in the drinking water for 35days and then mated with unexposed males. IAA exposure continued throughout gestation. Dams delivered naturally, and pups were continuously exposed to IAA through lactation until postnatal day (PND) 21. Female pups were euthanized on PND 21 and subjected to measurements of anogenital distance, ovarian weight, and vaginal opening. Ovaries were subjected to histological analysis. In addition, sera were collected to measure reproductive hormone levels. IAA exposure decreased vaginal opening rate, increased the absolute weight of the ovaries, increased anogenital index, and decreased the percentage of atretic follicles in female pups compared to control. IAA exposure caused a borderline decrease in the levels of progesterone and follicle-stimulating hormone (FSH) and increased levels of testosterone in female pups compared to control. Collectively, these data show that prenatal and lactational exposure to IAA in drinking water affects vaginal opening, anogenital index, the weight of the ovaries, the percentage of atretic follicles, and hormone levels in the F1 generation in mice.

Maternal Sodium p-Perfluorous Nonenoxybenzene Sulfonate Exposure Disturbed Lipid Metabolism and Induced an Imbalance in Tyrosine Metabolism in the F1 Generation of Mice.

The toxicity of perfluorinated compounds (PFCs) to mammals has recently received increasing attention. However, the effects of maternal sodium p-perfluorous nonenoxybenzene sulfonate (OBS) exposure during pregnancy and lactation on the liver function of dams (F0) and offspring (F1) mice are still unknown. The results demonstrated that maternal OBS treatment could not only induce lipid metabolism dysfunction but also disrupt amino acid metabolism in the liver of F0 and F1 generations. OBS had marked accumulation in the liver, and the serum and liver triglyceride (TG) levels increased in the F0 and F1 generations after maternal OBS exposure. Moreover, maternal OBS exposure changed the transcriptional levels of genes related to lipid metabolism (fatty acid (FA) synthesis, TG synthesis, and transport) and induced changes in the amino acid level in dams and 20-day-old mice offspring (F1-20 d). Additionally, the regulation of lipid metabolism by OBS was mainly dependent on the activation of peroxisome proliferator-activated receptor γ (PPARγ) and cluster of differentiation 36 (CD36). Interestingly, OBS could also disturb tyrosine (TYR) metabolism by increasing the TYR level and downregulating fumarate acetoacetate hydrolase (FAH). Together, these results indicated that the liver can be perceived as the major target tissue of OBS, which strongly affected metabolic function and ultimately led to an imbalance in the metabolism of lipids and TYR. In summary, maternal OBS exposure during pregnancy and lactation has toxic effects on the hepatic metabolism of dams and offspring, indicating that the toxic effects could obviously cross generations of mice, and we should pay more attention to understanding the health risk to both dams and offspring.

A Comparative Study of Cadmium Levels in Brain Tissue of Mice Observed Over Two Generations after Exposure During their Perinatal Period: Modulation by Quercetin

The perinatal period is very critical as the embryo or the new born is more susceptible to Cd toxicity. This study was done to measure Cd levels in brain tissue of F1 and F2 generation mice whose mothers were exposed to Cd during lactation or during the entire period of gestation and lactation and also to investigate whether quercetin could modulate this effect. Dams were exposed to cadmium during lactation and during the entire perinatal period. F1 and F2 generations were reared till 100 days of age. After being sacrificed, their brains were extracted, and cadmium levels were estimated using Atomic absorption spectrophotometer. It was found that Cd levels in brain tissue were significantly higher in the F1 generation when animals were exposed in lactation. There was slight increase in Cd in brain tissue of animals exposed during gestation as well as lactation, but the change was not statistically significant. Quercetin reduced the Cd levels significantly in a dose dependent manner in lactation group. In the other two groups it reduced the Cd levels even lower than the controls. This study shows that Cd is passed on to the next generation more efficiently when exposed during lactation. Lesser transmission is seen when exposure is during gestation followed by lactation. Quercetin effectively reduces Cd levels in brain tissue irrespective of the type of exposure.

Broad spectrum of CRISPR-induced edits in an embryonic lethal gene

Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype–phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.

Behavioral parameters evaluation after homeopathic Zincum metallicum treatment: a transgenerational study in mice

Background: ZINCUM METALLICUM (ZM) is a homeopathic medicine whose materia medica is defined by diverse behavioral and mental symptoms, including depression. Moreover, as a microelement, zinc itself is involved in several functions of Central Nervous System, including development and cell maturity during the intra-uterus life. Aims: Herein, the putative transgenerational effects of different homeopathic potencies of ZM upon behavioral parameters in P and F1 generations were evaluated. Methodology: Since mice and the tail suspension test (TST) are references for evaluating antidepressant agent activity, the TST together with the open field test (OPT) and the elevated plus maze test (EPM) were used to analyze offspring behavioral parameters. All animal procedures were in agreement with the Brazilian ethical research practices and were approved by the institutional ethical committee (CEUA-UNIP) under the protocol 156/2013. Four groups of seven females Balb/C mice were exposed to 0,1mL of ZM 5cH, 30cH, 200cH and lactose 5cH, diluted in 250mL of drinking water, during pregnancy and post partum period, in a total of 31 days. The flasks were coded before the remedies administration and all experimental procedures, including statistical analysis were done in blind. The parents were previously distributed in a Completely Randomized Design for the mates, according to the TST previous results. Mothers were re-evaluated for TST after weaning and mice of F1 generation were evaluated for TST, EPM and OPT when they reached two months old. According to the time of immobilization in TST, animals were classified as healthy (h), intermediate (i) and depressed (d) (&lt; 116; 117-180 and &gt;180 seconds of immobilization, respectively). Results: No significant changes were seen among the groups regarding to the number of newborns, sex proportions, TST, OPT and EPM behavioral parameters, besides the fact that the treatment with ZM 200cH was associated to the majority of healthy F1 mice (male: n=8: 7h+1i+0d; female: n=8: 8h+0i+0d), in relation to the number of delivery per group (Fisher test, p ≤ 0.01). Treatment with ZM 5cH, instead, produced reduced number of pups with no male mouse among F1 generation. We conclude that the treatment of pregnant females with ZM 200cH produced the best results in F1 generation regarding reproduction and behavioral parameters and the treatment with ZM 5cH reduced births in relation to control. The involved mechanisms have to be elucidated in the next steps of the study.

Prenatal exposure to a mixture of phthalates accelerates the age-related decline in reproductive capacity but may not affect direct biomarkers of ovarian aging in the F1 generation of female mice.

Phthalates are used in many consumer products, leading to daily human exposure. Although many studies focus on single phthalates, humans are exposed to mixtures of phthalates. Our laboratory created a phthalate mixture consisting of six different phthalates and found that it negatively affected female reproduction and accelerated some biomarkers of reproductive aging. However, it was unknown if prenatal exposure to the mixture accelerates the natural decline in reproductive capacity and ovarian aging in mice. Therefore, we tested the hypothesis that prenatal exposure to a phthalate mixture accelerates the age-related decline in reproductive capacity and biomarkers of ovarian aging in the F1 generation of mice. Pregnant CD-1 dams were orally dosed with control or phthalate mixture (20 µg/kg/day–200 mg/kg/day) daily from gestational day 10—birth. The F1 female pups were aged to 11–13 months, and then estrous cyclicity and breeding trials were conducted at 11 and 13 months. Ovaries were collected from the F1 females at 13 months to examine biomarkers of ovarian aging. Prenatal exposure to the phthalate mixture decreased the time the F1 females spent in proestrus and the ability of the F1 females to give birth at 11 and 13 months of age compared to control. In contrast, prenatal exposure to the mixture did not affect biomarkers of direct aging of the ovary in the F1 generation. Collectively, our data show that prenatal phthalate mixture exposure accelerates the natural age-related decline in reproductive capacity but may not affect some biomarkers of ovarian aging in the F1 generation.