Seed money for reproductive biology research

Applicants: Carlos Guerrero Bosagna (EBC, Uppsala University, main applicant), Ylva Sjunnesson (KV, SLU, co-applicant).

Project summary

SLU has currently the only research laboratory for in vitro production (IVP) of bovine and porcine embryos in Sweden. The use of in vitro models has been a successful tool for studying the impact of different factors on early development (from oocyte maturation until the blastocyst stage) with several finished and ongoing studies that investigate the impact of different factors on the outcome in the offspring. The method includes collection of immature oocytes of cows and sows from slaughterhouse ovaries followed by maturation, fertilization and culture in vitro.

This methodology may greatly impact epigenetic research to understand epigenomic programming in mammalian embryos. It is well-know that in mammals an epigenetic reprograming period occurs, which starts with global demethylation immediately after embryo fertilization followed by re-methylation triggered at the time of blastocyst formation. However, this has not been investigated in detail in cows or pigs.

Embryos will be produced by IVP and then frozen. Once a suitable number of embryos are collected from both cattle and pigs at different developmental stages, we will proceed to apply Genotype-by-Sequencing (GBS) and Methylated-DNA-Immunoprecipitation (MeDIP) to construct a library based on these DNA samples. The establishment of epigenetic analyses of early embryos from cows and pigs in Uppsala will be of great importance to Swedish research and the close cooperation between the laboratory units at SLU and Uppsala University will promote a platform for high quality research, with the potential to open new exciting research avenues in the field of developmental biology.

Applicants: Fábio Pértille (UU) and Parisa Norouzitallab (SLU).

Project summary

Recent research has shown that environmental stimuli can affect exposed individuals and also their unexposed descendants. The phenomena, known as transgenerational epigenetic inheritance (TEI), is thought to be mediated by environmentally-altered epigenetic marks in the gametes that are transmitted across generations. TEI has been reported in a variety of vertebrates including lab rodents, fish, quails, ducks and chickens, as well as in invertebrates. Dr. Norouzitallab has shown that exposure of Artemia SPP. to biotic or abiotic environmental insults can lead to an increased robustness of the animals, to the same or a different stressor, in their 3 subsequent (unexposed) generations. This includes the exposure to trace elements, zinc and Cadmium, both of which are documented for being assimilated to the tissue and affect reproduction in marine zooplanktons. In the present study, we will take advantage of the Artemia model system to investigate the methylomic and genomic transgenerational consequences of dividing a founder population into two and exposing one of them to an environmental stress. The experiment will continue until a significant impact on animals’ reproduction is observed. The experimental treatments will be conducted at the Artemia lab at the Swedish University of Agricultural Sciences (SLU), while the genomic and epigenomic analyses will take place at the Environmental Epigenetics lab at Uppsala University.

Applicants: John Lees, Uppsala University and Jane Morrell, SLU.

Project summary

How biological information is transferred from parent to offspring has been a long-lasting central question in biology. Although genomic DNA is, of course, the primary means by which biological information is transferred across generations, it has become clear that epigenetic agents within germinal cells can impart information across generations. Recent evidence in C. elegans implicates the mitochondria as a novel but important transgenerational messenger of maternal stress, primarily through variation in oocyte mitochondrial DNA (mtDNA) copy number. Given that mtDNA copy number and integrity is correlated with male fertility parameters, the idea that maternally inherited mtDNA may maintain a memory of the mother’s environment poses a number of interesting and unanswered questions regarding male fertility. With this project, we will investigate the potential for the maternal metabolic environment to influence the mitochondria of her offspring. In particular, we will investigate the influence of maternal metabolic stress in chickens upon sperm mitochondria in sons, identifying fertility-relevant markers including mtDNA copy number as well as mtDNA methylation.

Main applicant: Ehsan Pashay Ahi, post doc at Uppsala University, Department of Organismal Biology, Comparative Physiology section.

Co-applicant: Johannes Pohl, PhD student at the Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Environmental Toxicology Section.

Project description

Human and veterinary pharmaceutical pollution is emerging as an environmental problem. Many pharmaceuticals pass through the patient in either metabolized or intact form and are excreted in feces and urine. The pharmaceutical compounds eventually reach the aquatic environment, as they are not readily removed by conventional sewage treatment plant (STP) technologies.

Subsequent effects in non-target aquatic biota exposed to pharmaceuticals in the aquatic environment is a concern. For instance, fish embryo development, reproduction, and behavior have been shown to be affected by pharmaceuticals. New sewage treatment methods have been developed and implemented in STPs to improve pharmaceutical removal.

One of these methods involves sewage ozonation. Ozonation is very efficient for removing pharmaceuticals. However, potentially toxic ozonation by-products (OBPs) can be formed as the parent compound is only partially degraded. One example is the anticonvulsant medication carbamazepine, which becomes more toxic in developing zebrafish (Danio rerio) embryos after ozonation. The increased toxicity is manifested in the embryonal cardiovascular system (reduced heart rate and induction of e.g. pericardial edemas).

In order to elucidate the molecular mechanisms underlying the increasing toxicity, a gene regulatory network (GRN) approach will be used. RNA collected from 48 hours post-fertilization zebrafish embryos exposed to tap water (control), carbamazepine and ozonated carbamazepine will be used for studies on expression patterns of candidate genes. The project work will then proceed to describe possible molecular pathways and deduce hierarchical maps of the GRNs explaining the observed embryotoxicity.

The GRN methodology enables a deeper understanding of molecular mechanisms giving rise to toxic responses in zebrafish embryos, and the outcomes of the project will be used in future studies.

Main applicant: Denise Laskowski, Dept. Clinical Sciences, SLU. 

Co-applicants: Theodora Kunovac Kallak, Dept. Women's and Children's Health, UU and Ylva Sjunnesson, Dept. Clinical Sciences, SLU.

Project description

The use of in vitro models has been a successful tool for studying the impact of different factors on early development (from oocyte maturation until the blastocyst staging), in order to predict the outcome in the offspring. Factors such as insulin, environmental toxins such as PCB126 (3,3',4,4',5-Pentachlorobiphenyl), PFOS (perfluorooctyl sulfonate) or cadmium are well studied substances with predictable and known effects on gene expression. Their known action on the gene level makes them valuable for setting up methods since such methods could be validated with already published data of known effects.

The connections between reproduction, toxicology and metabolism are a really interesting and emerging research field as fertility is decreasing in our modern society and the reasons for that are only partly explained. Contamination with different toxins could influence the reproduction of both humans and wildlife.

The in vitro embryo lab at Dept Clinical Sciences, SLU, has excellent expertise and equipment to produce bovine and porcine embryos for research purposes. Until now, descriptive and morphological studies have been performed at SLU, while gene expression analyses have been performed abroad. This project is a collaboration for performing gene expression analyses (quantitative real-time PCR for expression analysis of target genes, sequencing) in collaboration with Rudbeck Laboratory at Uppsala University.

Limitations for gene expression analyses in early embryos include the low number of cells per embryo (around 100-150 on day 8) and the time-consuming, high input of work of a skilled person to produce the embryos (in total 8 days with different steps to perform under several days) and the high costs for this type of analyses.

The aim of the project is to establish well working methods for gene analyses and result interpretation. RNA will be extracted at the lab at Dept Cinical Sciences, SLU, and further processed testing different methods at Rudbecks laboratory at UU. This would in the long run also facilitate the analysis of human embryos, which present a more limited resource for research as both numerical and ethical restrictions apply. In contrast to that, bovine or porcine oocytes for experiments are available at the slaughterhouse for large scale experiments.

Main applicant: Carolina Vogs, PhD, Department of Biomedical Science and Veterinary Public Health, Swedish University of Agricultural Science. 
Co-applicant: Joëlle Rüegg, Professor, Department of Organismal Biology, Environmental Toxicology, Uppsala University.

Project summary

Perfluorinated alkyl acids (PFAA) are broadly used in industrial products such as surfactants, fluorinated polymers, coatings or fire-resistant foams. They are extremely persistent and have globally been detected in the environment, animals, humans, and even in the human fetal brain.

This means that PFAA are able to cross the placenta and the blood-brain barrier and may impact on brain development during a very vulnerable window. Indeed, prenatal exposure to PFAA has been associated with neurodevelopmental disabilities along with effects on immunity, the cardio-metabolic, thyroid, renal system, and on puberty onset in children. Yet, mechanisms underlying developmental neurotoxicity of PFAA are poorly understood, partly because of the lack of suitable test models.

The zebrafish (Danio rerio) embryo model has emerged as an important alternative to mammalian models to study developmental neurotoxicity. Benefits are that effects on physiology, development and behavior can be assessed cost-efficiently and rapidly. Mechanisms of neurodevelopmental effects can be studied, because main parts of the brain develop until 120 hours post fertilization and the development of neuronal structures is highly conserved between mammals and zebrafish.

Our study aims at investigating whether the two dominating PFAA contaminants in the environment, PFOS and PFOA, potentially alter neurological processes during development. To this end, Carolina Vogs and Joëlle Rüegg will analyze early alterations on gene transcriptions in brains of PFAA exposed zebrafish embryos. We hypothesize that two different gene expression patterns will be identified because of their different distributions in the embryo; PFOS tends to accumulate much more in the embryo brain compared to PFOA, which is in agreement with reported accumulation in human fetal brains and opposite neurobehavioral effects in zebrafish embryo. A long-term goal with this study is to improve chemical hazard assessment of developmental neurotoxicity using the zebrafish embryo model to prevent harmful effects in the human population.