Introduction: A diminishing ovarian follicular reserve is normal for women throughout their functional reproductive life; however, the cause for this decline is unknown. Constant remodeling of extracellular matrix (ECM) mechanical properties initiates signaling cascades via mechanotransduction that significantly influence ovarian follicle development and could potentially dictate the exhaustion of the remnant follicle pool. A three-dimensional cell culture system was implemented to examine the effects of ECM stiffness on ovarian follicle development by modulating ovarian hydrogel concentrations. Our findings indicate that increasing ECM stiffness may prematurely trigger follicle activation causing a decrease in the immature follicle population.
Materials and Methods: Derivation of Ovarian Hydrogel: An ovarian microenvironment was simulated using tissue-specific hydrogels derived from decellularized porcine ovaries. The ovaries were treated with several chemical reagents including triton X-100, sodium deoxycholate, and peracetic acid to remove genetic material. Decellularized tissues were lyophilized and ground into fine particles for pepsin and HCl digestion. The pH of the ECM digest was neutralized using NaOH and buffered with PBS. Physical crosslinking of hydrogels occurred at 37°C within 15-20 minutes.
In vitro Ovarian Culture: Two concentrations (2 mg/mL and 5 mg/mL) of ovarian hydrogels were used to test differing ECM stiffness on follicle development. Newborn mCherry mouse ovaries were microdissected and cultured for 7 days on top of the hydrogels in Waymouth’s MB 7521 media. After day 7 culture, the ovaries were imaged using confocal microscopy and quantified using Volocity software to determine the total number of viable oocytes. The ovaries were fixed and serial sectioned for histological analysis using a periodic-acid Schiff (PAS) stain.
Decellularized Ovarian Tissue and Hydrogel Characterization: Decellularized ovarian tissue was characterized using native ovarian tissue as a baseline. Histological analyses were performed using DAPI and H&E staining. Collagen and glycosaminoglycan (GAG) presence was quantified using hydroxyproline and GAG assays. Residual DNA content was measured by agarose gel electrophoresis and PicoGreen dsDNA assays. Ovarian hydrogel viscoelastic properties were assessed through rheology testing (time, frequency, and strain sweeps).
Results and Discussion:
Rheology testing of the two ovarian hydrogel concentrations confirmed that the 5 mg/mL peak storage (G’) and loss (G’’) moduli were approximately double the 2 mg/mL gel concentration. The increase in viscoelasticity resulted in a decrease in the total number of oocytes between the two gel concentrations (2 mg/ml ~1850 oocytes; 5 mg/mL ~1300 oocytes) suggesting that modulating ECM stiffness has a significant impact on follicle viability. As a control, ovaries from day 7 wild-type mice were microdissected for oocyte quantification (~1900 oocytes) and showing similar viability to the 2 mg/mL hydrogel ovary culture. PAS stained sections confirmed oocyte presence and morphology.
Conclusion: The three dimensional culture was aided by the ability to effectively tune mechanical properties of the ovarian hydrogels with varying concentrations. It was evident that stiffening the hydrogel substrate directly correlated to a diminishing oocyte population. Future studies will focus on modulating ECM stiffness with known cross-linking reagents to further analyze the effects of localized mechanical stimuli on follicle development.