C.J. McCauley1,2, H. McDowell2,3, E. Rowell1, M. Laronda1,2,3,4 1Ann and Robert H. Lurie Children’s Hospital of Chicago, Division Of Pediatric Surgery, Chicago, IL, USA 2Ann and Robert H. Lurie Children’s Hospital of Chicago, Stanley Manne Children’s Research Institute, Chicago, IL, USA 3Northwestern University Feinberg School of Medicine, Department Of Pediatrics, Chicago, IL, USA 4Northwestern University Feinberg School of Medicine, Department Of Obstetrics And Gynecology, Chicago, IL, USA
Introduction:
Patients diagnosed with childhood cancer often undergo chemotherapeutic regimens that result in premature ovarian insufficiency (POI) and infertility. The only fertility preservation option for prepubertal female patients is ovarian tissue cryopreservation (OTC). OTC requires unilateral oophorectomy, specialized tissue processing, and cryopreservation for long-term storage. Ovarian tissue transplantation has been successful in restoring fertility in adults but has limitations in terms of eligible patient populations and duration of hormone restoration and fertility function. An alternative approach utilizing a bioengineered matrix that regulates follicle activation could provide a safe, long-term solution for fertility and hormone restoration. Interstitial cells and the matrisome (extracellular matrix proteins) are essential for ovarian hormone production, immune response, and vascularization in vivo; however, how this microenvironment influences the production of good quality eggs and which components are required within a transplantable system are unknown.
Methods:
Ovaries from reproductive-aged cows were utilized as a mono-ovulatory model of the human ovary. Ovaries were sliced in the sagittal and axial planes, decellularized, and proteins were isolated for analysis. Bottom-up shotgun proteomics was performed and Jess Western and immunohistochemistry were used to validate the presence and relative abundance of key proteins. Heat maps with a spatial resolution of 0.5 mm2 were created for matrisome proteins using overlapping data from sagittal and axial planes.
Results:
Proteomic analysis identified 1471 unique proteins, of which 218 (14.8%) represented matrisome proteins. ECM proteins from all six matrisome divisions were identified. 39 (17.9%) matrisome proteins were significantly differentially expressed by depth in the sagittal plane and 35 (16.1%) were differentially expressed in the axial plane. In the sagittal plane, stromal-expressed matrisome proteins including FRAS1, LAMB3, LAMC3, and SFRP4 were cortex-predominant and CST3, MGP, SERPINF2 were medulla-predominant. Key matrisome proteins including VTN, COL1, and FN1 were identified equally across compartments. Validation using Jess Western was performed for VTN, EMILIN1, COL4, and ZP3 and confirmed compartmental differences seen in the proteomic dataset. Matrisome protein heat maps were created to spatially map the ovary.
Conclusion:
We map the matrisome of ¼ of the bovine ovary to a spatial resolution of 0.5 mm2 and found 54 of 218 identified matrisome proteins to be differentially expressed by depth within the ovary. This novel information can be utilized to inform the inclusion of matrisome proteins within a bioengineered matrix to better recapitulate the ovarian microenvironment. Further, this work will inform ongoing research on the incorporation of stromal cells into a bioprosthetic and transplantable scaffold to improve follicle survival and transplant efficacy.