Hypothyroidism through the reproductive cycle alters the DNA methylation level of mammary differentiation-associated genes

GARCÍA, D.; HAPON, M.B.; CARON, R.; CAMPO VERDE ARBOCCO, F.

Congreso; XL Reunión Cientifica Anual de la Sociedad de Biología de Cuyo; 2022

Sociedad de Biología de Cuyo

Mammary epithelial functional differentiation involves an abrupt turnover in the cell´s transcriptome. These changes arise from an epigenomic modification that confers lineage differentiation. The epigenomic imprinting depends on reproductive hormones from gestationto involution. In previous work, we proved that hypothyroidism (hypoT) disrupts the mammary transcriptome through lactation. In recent studies, we demonstrated that hypoT through the reproductive cycle alters the long-term mammary cell ability to respond to the hormonalfluctuation of cycle. In this study, we analyzed the impact of hypoT through a reproductive cycle in the mammary epigenome, focused on the methylation state of differentiation-associated genes. In this work, we investigated the mammary glands of hypothyroid Sprague Dawley hypothyroid (HypoT) and euthyroid (Ctrl) rats that have undergone a complete cycle of gestation, lactation, and involution. On day 28 after weaning, we dissected the inguinal mammary glands and isolated genomic DNA using CTAB reagent. We evaluated the DNA methylation state using MSRE and designed the bioinformatic assay with online ensemble database for gene sequence analysis and both beacon designer and methyl primer express software for primer designer and CpG island analysis. Next, we analyzed the methylation level by Real-Time PCR of GATA-3, STAT6, TET2, ELF5 y STAT5. The digested/non-digested ratio allows us to estimate the methylation level of eachsequence. The results show that hypoT through the reproductive cycle, long-term alters the methylation state of STAT6, STAT5, and TET2. This suggests both, an impediment in lineage commitment and in the mammary ability to respond to the prolactin pathway in successivelactation. Particularly, the increase in STAT5 promoter methylation suggests a partial loss in the cellular ability to synthesize milk compounds. These results, added to previous work, prove that hypoT through the reproductive cycle alters the mammary differentiation and in consequence its long-term ability to respond to hormonal stimulus.