The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

Abstract

The gonadotoxic effects of some cancers significantly increase the risk of developing infertility and cessation of ovary hormones (premature ovarian insufficiency, POI). Fertility preservation in the form of ovarian tissue cryopreservation (OTC) is offered to pediatric and adolescent cancer patients who cannot undergo oocyte retrieval and egg cryopreservation. The cryopreserved ovarian tissue can be transplanted back and has been found to restore fertility in 20 - 40% of transplants and restore hormone function for an average of 3 to 5 years. However, some individuals have primary or metastatic disease within their ovarian tissue and would not be able to transplant it back in its native form. Therefore, there is a need for additional methods for hormone and fertility restoration that would support a safe transplant with increased successful livebirths and long-term hormone restoration. To support this goal, we sought to understand the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds that would support isolated follicles. Using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla. 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 cast gels and 3D printed bioprosthetic ovary scaffolds from the cortex was still significantly more rigid than the medulla biomaterials. 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. 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.

Generation of Tailored Extracellular Matrix Hydrogels for the Study of In Vitro Folliculogenesis in Response to Matrisome-Dependent Biochemical Cues

“…There were no significant differences between the Young’s modulus of the two hydrogels CTX dECM and MED dECM. This was an unexpected finding as the ovarian cortex has previously been shown to be more rigid than the medulla [ 28 ]. However, the bovine cortical dECM has also been previously shown to be denser than the medulla.…”

“…Protein expression of EMILIN1 in the porcine ovary peaks within the cortex where PMFs reside [ 33 ]. Lower EMILIN1 abundance also coincides with an increase in ZP3, which is expressed after follicle activation [ 28 , 53 , 54 ]. ZP3 is a protein exclusively found in the ovary surrounding oocytes.…”

“…The utilization of bovine dECM is highly translatable for clinical purposes as bovine products have been FDA-approved and are often used for regenerative medicine and transplantations. Within the bovine ovary, the cortex is ≈8.5 times more rigid than the medulla and is comprised of different matrisome proteins [ 28 ]. The matrisome, the contingent of genes encoding proteins that comprise the ECM and ECM-related proteins, provides integral structural and physical support as well as necessary signaling cues that control tissue-specific cellular behavior [ 29 , 30 , 31 ].…”

Shear wave elastography to assess stiffness of the human ovary and other reproductive tissues across the reproductive lifespan in health and disease

“…In addition, ovarian collagen density directly correlates to increased tissue stiffness [ 24 ]. Increased cortical stiffness has also been demonstrated in an ex vivo bovine model [ 118 ]. However, an additional study of regional tissue stiffness using a mouse model, where the ovary is not distinctly compartmentalized into the cortex and medulla, found that the exterior region of the ovary was softer than the interior, as measured by atomic force microscopy [ 119 ].…”