Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

<div><p>Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D <i>in vitro</i> culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) <i>in vitro</i> ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D <i>in vitro</i> ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.</p></div

Illustration of the HTP system.

<p>Images captured from 96-well plates are converted using MATLAB ® program to binary images. After comparing the area of fibrin degradation (shown as white circles) to control conditions, the program will then output results of follicle health evaluated by fibrin degradation: “√” indicates healthy follicles, “X” dead ones, and “?” requiring further evaluation.</p

Correlation of fibrin degradation and follicle health, evaluated by both manual calculation (M) and automated MATLAB ® program (A).

<p>(a) Representative images of a live follicle (top) and a dead follicle (bottom) in brightfield images and processed binary images. Scale bar = 800 μm. (b) Results from manual calculation (M) and automated program measurement (A) of fibrin degradation in GM+Apr condition for live follicles on D6. Data presented as mean ± SEM, with n = 9 in each case.</p

Summary of follicle growth and oocyte maturation under different conditions.

<p>†: %survival = (the number of survived follicles) ÷ (total number of follicles cultured)×100</p><p>%antrum = (the number of follicles with antrum) ÷ (the number of survived follicles)×100; %MII = (the number of MII oocytes) ÷ (total number of follicles matured) ×100; %GV = (the number of GV oocytes) ÷ (total number of follicles matured) ×100; %MI = (the number of MI oocytes) ÷ (total number of follicles matured) ×100; %DG = (the number of MII oocytes) ÷ (total number of follicles matured) ×100. Follicle diameter data was presented as mean ± SEM. Different letters (a, b, and c) within each column indicate statistical significance (p<0.05).</p><p>Summary of follicle growth and oocyte maturation under different conditions.</p

Aprotinin-controlled fibrin degradation.

<p>(a) Representative images of follicles encapsulated in FA-IPNs on different days of culture with higher magnification images of follicles in the lower right inserts. Comparison showed that optimized aprotinin concentration (0.025 to 0.05 TIU/mL) effectively delayed fibrin degradation till D8, a critical time for antrum formation. Arrow points to the fibrin degradation area surrounding the growing follicle. Arrow head (“<”) points to the encapsulated follicle in the FA-IPN. Scale bar = 500 μm. (b) Growth curves of follicles cultured in growth media with and without aprotinin. Data are shown as mean ± SEM, p < 0.05. n (total follicles, GM) = 286, n (total follicles, GM+Apr) = 258, n (total animals used) = 30, n (experiments) = 10 for each condition. (c) Representative images of <i>in vitro</i> follicle maturation (IVFM) results confirming oocyte quality from both GM (left) and GM+Apr (right) conditions. Data are shown as mean ± SEM, p < 0.05. MII rate (GM) = 78% ± 3%, MII rate (GM+Apr) = 81% ± 3%. Scale bar = 100 μm.</p

Quantitative measurements of fibrin degradation with different DXR treatments.

<p>(a) Manual calculation of fibrin degradation area; (b) Automated MATLAB® measurement of fibrin degradation. Data presented as mean ± SEM, n = 9, † n (GM+Apr+0.005nM DXR, dead, automated) = 3, *: p<0.05.</p

System validation with Doxorubicin (DXR).

<p>(a) Representative images of dose response of cultured follicles to DXR. Arrow points to the fibrin degradation area surrounding the growing follicle. Arrow head (“<”) points to the encapsulated follicle in the FA-IPN. Scale bar = 500 μm. (b) Kaplan-Meier cumulative survival curves for DXR treatments and control. Sample sizes for each condition [n (condition) = total follicles, animal used, experiments]: n (200 nM) = 69, 4, 3; n (10 nM) = 52, 3, 3; n (1 nM) = 75, 3, 3; n (0.1 nM) = 75, 3, 3; n (0.01 nM) = 80, 3, 3; n (0.005 nM) = 92, 4, 3. (c) Representative images of <i>in vitro</i> follicle maturation (IVFM) results from survived follicles in the 0.01 nM (left) and 0.005 nM (right) DXR treatment group. Scale bar = 100 μm. (d) The diameters of surviving follicles in control conditions (GM and GM+Apr) and 0.005 nM and 0.01 nM DXR treatment groups. Data presented as mean ± SEM.</p