(305) Ovarian Phenotypes in Mice Expressing a Human MMP14 Transgene in Fibroblasts

Holly A. LaVoie¹;  Lauren E. James¹

1. Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC

Abstract Text:

The extracellular matrix (ECM) in the adult ovary is dynamic undergoing changes with the estrus/menstrual cycle to allow follicle growth, ovulation and corpus luteum formation. ECM protein turnover is regulated by several metalloproteinases (MMPs) and balanced by their inhibitors.  During a study of pregnant and postpartum maternal heart function, we investigated the phenotype of mice harboring a human MMP14 transgene under control of the murine Col1a2 gene promoter, which directs transgene expression mainly in fibroblasts (hMMP14 mice).  Serendipitously, we observed an increased incidence in grossly abnormal ovaries in the transgenic mice (12%) compared to wildtypes (1%). Ovaries were collected from wildtype and transgenic mice euthanized as age-matched virgins (in diestrus), at day 17 of pregnancy (late pregnancy) and at postpartum day 49. One ovary was fixed in paraformaldehyde, and the second ovary was snap frozen for molecular analyses. The ovarian abnormalities in hMMP14 mice included bloody cysts, a serous cystic adenoma, 2 teratomas, a fragmented ovary and unilaterally enlarged ovarian bursae with excess fluid.  Grossly abnormal wildtype FVB/NJ ovaries only exhibited teratomas.  We hypothesized that grossly normal hMMP14 mouse ovaries would exhibit molecular alterations capable of giving rise to abnormal ovarian phenotypes when compared to wildtype mice.  To address this, we performed quantitative protein analyses with grossly normal appearing diestrous ovaries from 5 wildtype and 5 hMMP14 virgin mice using DIA mass spectrometry. Numerous proteins were altered more than 1.5-fold in hMMP14 mouse ovaries compared to wildtypes. The top 19 significantly increased proteins were analyzed with the DAVID Bioinformatic Resource annotation tool. The prevalent pathways detected were ECM organization and degradation, neuronal growth, and interactions with zona pellucida. Select mRNAs from the differentially expressed proteins and mRNAs from the ECM pathway were evaluated by quantitative PCR.  We utilized Two-way ANOVAs to evaluate the main effects of genotype and reproductive status on mRNA levels.  We found a significant main effect of genotype for Col6a2, Flad1, Krt7, Mmp2 and Myh11.  We found a significant main effect of reproductive status for Col3a1, Ctgf, Lama1, Mmp2, Mmp9 and Tgfb1.  And interaction of genotype and reproductive status was found for Col1a1, Krt7 and Mmp2.   In conclusion, overexpression of human MMP14 in fibroblasts led to significant alterations in ovarian mRNAs for ECM proteins (Cola6a2, Mmp2), epithelial keratin (Krt7) and smooth muscle myosin (Myh11).  Alteration of the corresponding proteins might be responsible for the variety of aberrant ovarian phenotypes observed at the gross level.  Reproductive status (late pregnancy) showed the greatest alteration in the mRNAs encoding ECM-associated proteins which would correspond with functional changes in corpora lutea and the development of the next round of ovulatory follicles. 

Funding:   Magellan & SCHC Research awards, SC INBRE award (P20GM103499), USC ASPIRE-I & IDeA National Resource for Quantitative Proteomics (R24GM137786).