Embryonic factors mediate the maternal age-induced programming of offspring postnatal behavior in mice†.

Advanced maternal age is associated with adverse pregnancy and offspring outcomes, including neurodevelopmental disorders. While age-related oocyte and embryonic abnormalities may underlie this association, the aged maternal uterine environment also plays an important role in offspring development and survival. The aim of this study was to evaluate the contribution of maternal age-related embryonic and uterine factors on pregnancy and offspring behavior, by using a model of reciprocal embryo transfer between old and young female mice. Pregnancies were obtained by transferring embryos collected from either old (9-14 months) or young (3-4 months) C57BL/6J female mice to either young or old recipients. The results showed that embryos from old and young donors have comparable developmental potential when transferred to young recipients, whereas no pregnancies were obtained by transferring embryos of young females to old recipients. Moreover, the offspring conceived by aged females displayed altered ultrasonic vocalization and learning skills compared to the progeny of young females, even though they were both prenatally and postnatally fostered by young recipients. These results indicate that maternal factors mostly determine the occurrence of age-related pregnancy complications, whereas the long-term effects of maternal aging on the offspring's behavior could be already established at pre-implantation stages and depend on embryonic factors.

Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos.

Studies of mitochondrial dynamics have identified an intriguing link between energy supply balance and mitochondrial architecture. This suggests that inappropriate culture conditions might inhibit mitochondrial functions, and affect embryonic development. Therefore, this study was conducted to determine whether in vitro culture (IVC) might affect mitochondrial function, distribution, organization (by Mitotracker Green), gene expression on RNA level (by qPCR), and protein expression and localization (by western blot and immunostaining) involved in regulation of mitochondrial functions. Mitochondria in 2-cell IVC embryos were less numerous compare to IN VIVO while the localization and distribution do not differ between the groups. Mitochondria of in vivo blastocysts formed elongated network along the cells, while in IVC were fragmented, rounded, and aggregated mainly in the perinuclear region. Additionally, mitochondria of IN VIVO embryos moved back and forth along their long axis on radial tracks, while in IVC blastocysts were much less active. mtDNA copy number in IVC blastocysts (92,336.65 ± 5860.04) was significantly lower than that of IN VIVO (169,103.92 ± 16,322.41; P < 0.02) as well as lower protein expressions responsible for mitochondrial fusion was observed in IVC blastocysts. Results indicate that in vitro culture affect on perturbations in mitochondrial number and function, which is associated with decreased developmental competence of in vitro produced mouse embryos.

Canine Smell Preferences-Do Dogs Have Their Favorite Scents?

The available evidence on dogs' scent preferences is quite limited. The purpose of this study was to verify the canine response to selected odors that may also be preferred by humans. The experiment was performed using 14 adult dogs (10 female and 4 male) of different breeds, body size, and age (1-14 years). During the experiment, dogs were exposed to 33 odor samples: a neutral sample containing pure dipropylene glycol (control) and 32 samples containing dipropylene glycol and fragrance oils. The dog was brought to the experimental area by its handler, who then stopped at the entrance, unleashed the dog, and remained in the starting position. The dog freely explored the area for 30 s. All dog movements and behavior were recorded and analyzed. The methodology of observing the dogs freely exploring the experimental area allowed us to determine the smells that were the most attractive to them (food, beaver clothing). Our study shows that dogs interacted more frequently with the scents of blueberries, blackberries, mint, rose, lavender, and linalol.

Delayed parenthood and its influence on offspring health: what have we learned from the mouse model†.

Delayed parenthood is constantly increasing worldwide due to various socio-economic factors. In the last decade, a growing number of epidemiological studies have suggested a link between advanced parental age and an increased risk of diseases in the offspring. Also, poor reproductive outcome has been described in pregnancies conceived by aged parents. Similarly, animal studies showed that aging negatively affects gametes, early embryonic development, pregnancy progression, and the postnatal phenotype of resulting offspring. However, how and to what extent parental age is a risk factor for the health of future generations is still a subject of debate. Notwithstanding the limitation of an animal model, the mouse model represents a useful tool to understand not only the influence of parental age on offspring phenotype but also the biological mechanisms underlying the poor reproductive outcome and the occurrence of diseases in the descendants. The present review aims at i) providing an overview of the current knowledge from mouse model about the risks associated with conception at advanced age (e.g. neurodevelopmental and metabolic disorders), ii) highlighting the candidate biological mechanisms underlying this phenomenon, and iii) discussing on how murine-derived data can be relevant to humans.

Embryo structure reorganisation reduces the probability of apoptosis in preimplantation mouse embryos.

Programmed cell death plays a key role in mammalian development because the morphological events of an organism's formation are dependent on apoptosis. In the mouse development, the first apoptotic waves occur physiologically at the blastocyst stage. Cell number and the mean nucleus to cytoplasm (N/C) ratio increase exponentially throughout subsequent embryo cleavages, while cell volume concurrently decreases from the zygote to blastocyst stage. In this study we tested the hypothesis that reorganisation of the embryo structure by manipulating cell number, the N/C ratio and the cell volume of 2-cell embryos may result in the earlier and more frequent occurrence of apoptosis. The results indicate that doubling ('Aggregates' group) or halving ('Embryos 1/2' group) the initial cell number and modifying embryo volume, ploidy ('Embryos 4n' group) and the N/C ratio ('Embryos 2/1' group) reduce the probability of apoptosis in the resulting embryos. There was a higher probability of apoptosis in the inner cell mass of the blastocyst, but apoptotic cells were never observed at the morula stage in any of the experimental groups. Thus, manipulation of cell number, embryo volume, the N/C ratio and ploidy cause subtle changes in the occurrence of apoptosis, although these are mostly dependent on embryo stage and cell lineage (trophectoderm or inner cell mass), which have the greatest effect on the probability of apoptosis.

Preconceptional Resveratrol Supplementation Partially Counteracts Age-Related Reproductive Complications in C57BL/6J Female Mice.

Aging is associated with a drastic decline in fertility/fecundity and with an increased risk of pregnancy complications. Resveratrol (RES), a natural polyphenolic compound, has shown anti-oxidant and anti-inflammatory activities in both human and animal models, thus representing a potential therapeutic and prophylactic anti-aging supplement. Here, we investigated whether preconceptional resveratrol supplementation improved reproductive outcomes in mid-aged (8-month-old) and old (12-month-old) C57BL/6J female mice. Female siblings were cohoused and assigned to either RES or vehicle supplementation to drinking water for 10 consecutive weeks. Subsequently, females were mated with non-supplemented males and their pregnancy outcomes were monitored. RES improved mating success in old, but not in mid-aged females, and prevented the occurrence of delivery complications in the latter. These results indicate that preconceptional RES supplementation could partially improve age-related reproductive complications, but it was not sufficient to restore fecundity in female mice at a very advanced age.

Blastomere removal affects homeostatic control leading to obesity in male mice.

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise - compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

Reproductive biotechnology and critically endangered species: Merging in vitro gametogenesis with inner cell mass transfer.

A fifth of mammalian species face the risk of extinction. A variety of stresses, and lack of sufficient resources and political endorsement, mean thousands of further extinctions in the coming years. Once a species has declined to a mere few individuals, in situ efforts seem insufficient to prevent its extinction. Here we propose a roadmap to overcome some of the current roadblocks and facilitate rejuvenation of such critically endangered species. We suggest combining two advanced assisted reproductive technologies to accomplish this task. The first is the generation of gametes from induced pluripotent stem cells, already demonstrated in mice. The second is to 'trick' the immunological system of abundant species' surrogate mothers into believing it carries conceptus of its own species. This can be achieved by transferring the inner cell mass (ICM) of the endangered species into a trophoblastic vesicle derived from the foster mother's species. Such synthesis of reproductive biotechnologies, in association with in situ habitat conservation and societal changes, holds the potential to restore diversity and accelerate the production of animals in the most endangered species on Earth.

Birth Cohorts in Highly Contaminated Sites: A Tool for Monitoring the Relationships Between Environmental Pollutants and Children's Health.

Industrial areas are characterized by the dispersion of environmental stressors that could possibly have long-term detrimental effects on both human health and the environment. Environmental contamination has been indicated to be one of the major risks for reproductive health. In this context, the effects of environmental pollution on pregnant women living in heavily polluted areas is of special interest. In fact, fetal development is a crucial phase due to the dynamic interaction between the maternal/external environments and the developing organs and tissues. Moreover, following Barker's postulate of the intrauterine origin of health and disease, the events occurring in this time window could affect future health. Birth cohorts provide the most suitable design for assessing the association between early-life and possible long-term health outcomes in highly contaminated sites. By providing an assessment of the early life environment throughout the collection of biological samples, birth cohorts offer the opportunity to study in-depth several possible confounders and outcomes by means of questionnaires and follow-ups based on clinical evaluations and bio-specimen samplings. The exposome comprises the totality of exposures from conception onwards; the birth cohort approach allows the integration of the exposures as a whole, including those related to socioeconomic status, with "omics" data from biological samples collected at birth and throughout life. In the characterization of the "fetal exposome," the placenta represents a highly informative and scarcely considered organ. For this purpose, the "Neonatal Environment and Health Outcomes" (NEHO) birth cohort has been established by enrolling pregnant women residing in contaminated sites and in surrounding areas.

Food toxicology: quantitative analysis of the research field literature.

Total-scale quantitative research literature analysis on the food toxicology scientific field has yet to be conducted. In this work, we identified and analysed food toxicology publications in the existing scientific literature. A literature search was performed with the online Web of Science database. Full records and cited references of the 73,099 identified manuscripts were imported into VOSviewer software for analysis. This research field has been growing steadily since the 1990s. Article to review ratio was 7.4:1. The publications were mainly related to toxicology, environmental sciences, food science and technology, pharmacology/pharmacy and biochemistry/molecular biology. The United States and China are major contributors to food toxicology research, followed by other European and Asian countries. The prolific authors have formed three major clusters within a citation network. Toxic or hazardous chemicals related to food with high citations included aflatoxin, dioxin, fumonisin, malondialdehyde, mycotoxin, ochratoxin, phthalate, and polychlorinated biphenyl.

Assessing the epigenetic risks of assisted reproductive technologies: a way forward.

Since the birth of the first baby conceived by in vitro fertilization (IVF), assisted reproductive technologies (ART) have been constantly evolving to accomodate needs of a growing number of infertile couples. Rapidly developing ART procedures are directly applied for human infertility treatment without prior long-term safety evaluation. Although the majority of ART babies are healthy at birth, a comprehensive assessment of the long-term risks associated with ART is still lacking. An increased risk of epigenetic errors has been associated with the use of ART, which may contribute to the onset of civilization disease later in adolescence/adulthood and/or in subsequent generations. Therefore, our investigations should not focus on (or be limited to) the occurrence of a few very rare imprinting disorders in ART children, which might be associated with parental age and/or the use of ART, but on the possibly increased disease susceptibilities later in life and their potential transmission to the subsequent generations. Retrospective studies do not offer exhaustive information on long-term consequences of ART. Animal models are useful tools to study long-term effects including transgenerational ones and the epigenetic risk of a given ART procedure, which could then be translated to the human context. The final goal is the establishment of common guidelines for assessing the epigenetic risk of ART in humans, which will contribute to two key objectives of the Horizon2020 programme, i.e. to improve our understanding of the causes and mechanisms underlying health and disease, and to improve our ability to monitor health and prevent/manage disease.

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies.

Pregnancy at Advanced Maternal Age Affects Behavior and Hippocampal Gene Expression in Mouse Offspring.

There is growing evidence that advanced maternal age is a risk factor for neurological and neuropsychiatric disorders in offspring. However, it remains unclear whether the altered brain programming induced by advanced maternal age is mediated by pre- or postnatal factors. Here, a mouse model was used to investigate whether pregnancy at advanced age may provoke behavioral and brain gene expression changes in offspring. Swiss Albino mice conceived by 3-month-old males and either 15-18-month-old (n = 11) or 3-month-old control females (n = 5), were delivered by cesarean section, fostered after birth by 3-month-old dams and subjected to a battery of behavioral tests. Furthermore, genome-wide mRNA expression was analyzed in the hippocampi of 4-month-old males offspring using microarrays. Offspring conceived by old mothers exhibited increased ultrasound vocalization activity during separation from the foster mother, increased anxiety-like behaviors in adult life, and altered patterns of hippocampal gene expression, compared to controls. These effects were not reversed by the postnatal maternal care provided by the young foster mothers, suggesting that the altered brain programming is already established at birth, consistent with prenatal effects related to maternal aging.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.