EPIGENETIC INVESTIGATION ON CYCLIC ONSET OF REPRODUCTIVE ACTIVITY IN DAIRY GOATS

At our latitude and under natural conditions, goats show autumn heats with calvings in spring. Individual variability in the onset of puberty and reproductive activity could hide a genetic and epigenetic variability that can allow a selection of goats more prone to deseasonalization. As a consequence, breeders could easily spread milk production throughout the year. The aim of the project was to analyze epigenetic profiles responsible for the reproductive seasonality in goat species. Considering the lack of information in literature for the species, we decided, first of all, to focus our effort on the characterization of the epigenetic profile (involving DNA methylation and microRNA) of two key organ in reproduction performances: hypothalamus and ovary. This basic research gave a first picture of the epigenome of goat species, representing a starting point for future researches in this field. In the three year of my PhD we gathered biological samples of interest for the project and we set up a pipeline for the epigenetic analysis (for DNA methylation and smallRNA) of the data produced. We saw different DNA methylation patterns across the goat genome between two organs, hypothalamus and ovary, and even a marked difference in the same organ (hypothalamus) between two physiological stages, before and after reaching puberty. In particular, the comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. We discovered interesting differences in the expression of microRNA in hypothalamus and ovary. We confirmed microRNAs already known on databases and we discovered some new, through structure homology with other related species. A selection of differentially expressed miRNAs was identified among hypothalamus, pituitary and ovary. In addition, 44, 55, and 69 miRNAs identified in pituitary, hypothalamus and ovary respectively, were specifically up-regulated in only one organ. Then we focused our efforts on the characterization of the epigenetic factors that could drive to puberty in the species. Genome wide DNA methylation analysis revealed 664 Differentially Methylated Genes (DMGs) among pubertal goats and their control. The crossing of DMGs and Differentially Expressed Genes (DEGs) among pubescent goats and their control, revealed new 8 genes, as potential marker for puberty onset. Among these, ATG16L1 (autophagy related 16 like 1), LRP5 (low-density lipoprotein receptor-related protein 5) and MERTK (proto-oncogene tyrosine-protein kinase MER) are suitable examples of puberty-related genes. According to False Discovery Rate threshold, it seems that miRNA population does not play a crucial role in the reaching of puberty in goat species in the organ analyzed. Furthermore, we conducted a SNP discovery on three key genes for photoperiod response (Melatonin Receptor 1 - MTNR1A, type 2 Deiodinase Gene - DIO2, Thyroid Stimulating Hormone beta subunit - TSHB), confirming and identifying new genetic polymorphisms. We found a novel non-synonymous polymorphism in the caprine DIO2 gene and we confirmed the high variability of the MTNR1A gene, discovering a new SNP bringing to a silent mutation. The first genome-wide comparison among organs of DNA methylation in Capra hircus has been generated. The DNA methylome in the hypothalamus and ovary of goat offers a valid base for studying the involvement of epigenetic modifications in goat reproduction performances. By confirming that the neuroendocrine control of female puberty entail mechanisms subjected to epigenetic regulation, our results provide insight into genes and pathways involved in the system used by the hypothalamus to control the initiation of mammalian puberty. As such, they are consistent with the concept that the pubertal process depends not only on genetic determinants, but also on developmentally regulated changes in epigenetic information.