A prominent cause of premature ovarian insufficiency (POI) is gonadotoxic cancer therapies, which deplete the ovarian reserve of follicles, oocytes, and hormone-producing cells. Current fertility preservation methods include the removal and cryopreservation of ovarian tissue prior to gonadotoxic treatment. This cryopreserved tissue can be transplanted back and has been found to restore fertility in 20-40% of cases and restore ovarian hormone production for 2 months to 12 years. Our goal is to understand the physical properties and biochemical components of the ovarian microenvironment and their role in maintaining the ovarian reserve to restore fertility and ovarian hormones for patients using a bioprosthetic ovary. The extracellular matrix (ECM) is a network of proteins providing physical and biochemical support to organs. This thesis characterizes spatial maps of the porcine matrisome (ECM and ECM-associated proteins) identifying undiscovered proteins and potential modulators of follicle activation, mapping of the physical properties of ovaries, and the development of tools to dissect the mechanistic relationship between the matrisome and follicle activation.First, we mapped the composition of the matrisome of porcine ovaries through the cortical compartment, where quiescent follicles reside and the medullary compartment, where the larger follicles grow and mature. To do this we sliced the ovaries, uniformly in two anatomical planes, enriched for matrisome proteins and performed bottom-up shotgun proteomic analyses. We identified 42 matrisome proteins that were significantly differentially expressed across depths, and 11 matrisome proteins that have not been identified in previous ovarian protein analyses. We validated these data for nine proteins and confirmed compartmental differences with a second processing method. Here we describe a processing and proteomic analysis pipeline that revealed spatial differences and matrisome protein candidates that may influence folliculogenesis. Second, using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla, with a rigidity gradient showing a gradual decrease in rigidity across compartments. To determine if this difference in rigidity was maintained in isolated matrisome proteins from bovine ovarian compartments, we cast, and 3D printed hydrogels created from decellularized bovine ovarian cortex and medulla slices. The 3D printed scaffolds from the cortex were more rigid than those derived from the medulla. To expand our bioengineering toolbox that will aide in the investigation of how biochemical and physical cues may affect folliculogenesis, we sought to confirm the concentration of matrisome proteins in bovine ovarian compartments. The matrisome proteins, COL1, FN, EMILIN1 and AGRN were more abundant in the bovine ovarian cortex than the medulla. Whereas VTN was more abundant in the medulla than the cortex and COL4 was present in similar amounts within both compartments. Finally, we removed proteins of interest, EMILIN1 and AGRN, from decellularized bovine ovarian cortex materials and confirmed that this specifically depleted these proteins without affecting the rigidity of cast or 3D printed hydrogels. Culturing human mesenchymal stem cells (hMSCs) on EMILIN1 depleted materials we saw no change in proliferation or cell survival. However, we saw a significant difference in gene expression of candidate genes downstream of TGFβ that was reversed upon supplementation with EMILIN1. Taken together our results indicate the existence of a rigidity gradient in the bovine ovary, that this rigidity gradient is maintained in resulting engineered materials strongly implicating a role for matrisome proteins in contributing to the physical properties of the bovine ovary. By establishing additional engineering tools, we will continue to explore mechanisms behind matrisome-follicle interactions. This work defines the physical and biochemical properties of the ovarian microenvironment across compartments and develops tools to further investigate the role of specific proteins in modulating follicle quiescence, follicle activation, and the progression of folliculogenesis. Additionally, these results feed our underlying knowledge and provides new tools for investigating mechanistic relationships towards creating an effective next generation bioprosthetic ovary by controlling primordial follicle activation to improves bioprosthetic longevity.

Creator

• Henning, Nathaniel

DOI

• https://doi.org/10.21985/n2-sbne-ce50

Subject

• Biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:16071
• etdadmin_upload_902584

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright