Research Department: Cecil H. And Ida Green Center for Reproductive Biology Graduation Date: December 2018
Abstract: INTRODUCTION: In term and preterm (PTB), cervical changes precede the onset of labor. Defining the molecular changes that drive cervical remodelling during term leading to loss of structural integrity and tensile strength is necessary for risk detection and prematurity prevention. Previous studies from lab have demonstrated steroid hormones, progesterone and estrogen (E2) as key modulator of tissue mechanical function via collagen and elastic fibers. Many of E2ʼs actions via its receptor, ERα, occur in the context of activation of the signalling pathways controlled by the peptide hormone relaxin (Rln) through its receptor, Rxfp1 during term pregnancy. However, the molecular mechanisms by which cross-talk between E2 and Rln is achieved for optimal cervical ripening is unknown. We hypothesized that Rln signalling in the cervix alters or regulates a parturition-specific E2 signaling that may regulate genes encoding matricellular proteins (CCN genes) and ECM remodeling during cervical ripening. METHODS: We used cervical tissue from ovariectomized mice treated with E2+Rln or either hormone alone, or from pregnant mice with or without targeted mutations in Rxfp1. We generated high quality transcriptomic (RNA-seq) data sets in the ovx mice model to identify novel genes encoding matricellular proteins and proteases. Gene ontology (GO) analyses grouped genes into up and down regulated biological and molecular processes. RT-qPCR was performd eto analyse expression of CCN genes identified as differential regulated in the RNA-seq data sets in WT and Rxfp1 KO mice. The functional interplay between E2 and Rln was examined by performing western blot for various signalling molecules downstream of Rln-Rxfp1 signaling. Collagen fibres ultrastructure was visualized in d18, WT and Rxfp1 KO using Transmission Electron Microscopy (TEM). Trichrome and H&E staining were performed to visualize the collagen and overall cervix histology, respectively. RESULTS: RNA-seq analyses and RT-qPCR results identifies several CCN genes and few proteases stimulated by Rln. RT-qPCR results from WT and Rxfp1 KO identifies one of the CCN genes, Wisp1 and Adamts4 encoding for a protease as Rln-dependent during day 18 of mouse pregnancy. GO analyses identifies enrichment of genes involved in extracellular matrix organization, inflammatory response, regulation of cell proliferation and cell death associated with proposed functions of cervix. In support of these findings, insufficient cervical epithelial differentiation and ECM remodelling was observed in the microscopy images from day 18 Rxfp1 KO mice cervical sections. In contrast, the western blot results did not show any striking differences between the WT and Rxfp1 KO mice. DISCUSSION: This study on regulatory expression of CCN genes and Adamts activity, a protease might put potential insights into the successful ECM disruption required at term. These findings add up to our knowledge of diverse mechanism by which cervical ECM can be reorganized to increase compliance and facilitate cervical opening.
. What does research mean to you? Research is an opportunity for me to experience lecture concepts in a practical context. It taught me the importance of learning for understanding. you learn to learn, not to pass an exam and forget.
Tell us about your journey. Dr. Mahendroo handed me my entire research project description on one sheet of paper. “Simple,” I naively thought to myself, glancing at the deceptively brief summary. By the end of the meeting, however, I recieved a few dozen articles and a textbook to read. I realized that I still had a lot to learn before I would even be able to begin to understand. I had previously been working in the Texas Biomedical Devices Center at UTD. TXBDC was a large lab with dozens of undergraduate volunteers. When I started at Dr.Mahendrooʼs lab, I was not used to working so closely with a supervisor in such a small lab. It was the first time I had seen so many basic biology concepts and lab techniques work fully in practice. I soon adjusted my expectations that I would arrive at textbook-perfect data and images. My project focused on how estrogen and relaxin regulate selected proteins in the cervix to facilitate gestation and labor. The project wasnʼt simple at all, and I spent many days trying to yield viable data. In school, I knew how to find the right answer and regurgitate it. In research, everything I was creating was new, so I had to adjust experimental protocols to find the answer I sought. The Green Fellowship provided me with the opportunity to see years of biology lectures in practice and to hone my problem-solving skills, but I got caught up in small details. Sensing my frustration, my supervisor taught me a lesson that I made my motto: you learn to learn, not to pass an exam and forget. Most importantly, the Green Fellowship taught me how to deal with the frustrating setbacks that arise when conducting original research and the necessity of creative problem-solving. When my results were not what I expected, I treated them as opportunities to learn. These lessons in flexibility and resilience will follow me throughout my medical career, whether I do part-time research or encounter a challenging medical case.
Advice for Future Green Fellows
Do not expect to come up with a break through in a semester! For most of you this is your first immersive research experience so learn. Ask questions, understand every bit of what. You are doing and why. More than just your research itself, you will learn valuable lessons that you can use throughout your careers regardless of whether you choose to do research.