The immune-stromal interplay of the cycling, pregnant and aged female reproductive tract (Flow cytometry)

ABSTRACT: We investigated whether the magnitude of the female reproductive tract (FRT) organ-wise cyclic inflammatory and remodeling responses naturally occurring during the estrus cycle of young mice lead to a memory of past environmental stimuli and chronic inflammation and fibrosis development in old age. Additionally, we investigated how inflammatory and remodeling responses change in pregnancy. We used single-cell RNA-sequencing (scRNA-seq) to profile how the female reproductive tract is remodeled during the estrus cycle, decidualization, and ageing (Arrayexpress, accession number E-MTAB-11491). Additionally, to explore in vivo spatial transcriptional landscapes, we used the 10x Visium system to profile 27 sections of young ovaries in diestrus, 33 sections of acyclic old ovaries, 27 sections of young uterus in diestrus and 65 sections of old uteruses in three to five biological replicates. H&E staining was used to confirm that decidualization occurred in impregnated mice as well as to generate pathology reports and asses immune infiltration/inflammation of FRT organs for young (3 months of age, n=3) and aged mice (18 months, n=9). To assess fibrosis development Picrosirius red staining was used to label collagen I and III fibers in 3, 12, 15, 18 and 21 month-old mice. Additionally, to uncouple the effect of aging and cycling on fibrosis development, a chemical protocol was used to prematurely terminate cycling in the FRT of young mice. Administration of 4-vinylcyclohexene diepoxide (VCD) causes the loss of ovarian small follicles in mice and rats, rapidly leading to acyclicity. VCD was administrated to two-month old mice, and their acyclicity was confirmed by five months of age. These mice were then further aged for six months, and sacrificed at 11 months old, in parallel with sham-injection control mice. Their uteri and oviducts were collected to assess the development of fibrosis using Picrosirius red staining. RNA in situ hybridization was used to validate stromal to epithelial cell abundance ratio in uterus of young mice (3 months) and decrease in follicular cells in aged ovary by comparing 3 months and 18 months old mice. Flow cytometry was used to validate immune cell infiltration in vagina during estrus cycle and change in myeloid cells abundance in uterus and vagina in aging (3 months vs 18 months).

ORGANISM(S): Mus musculus (mouse)

SUBMITTER:  

PROVIDER: S-BSST864 | biostudies-other |

REPOSITORIES: biostudies-other