The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue

Abstract

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.

Keywords: Alginate; Follicle; Mechanobiology; Microfluidic; Microtissue; Ovulation.

Fig. 1

Design and materials of biomimetic ovarian microtissue. (A) A schematic illustration of mouse ovary that consists of two mechanically distinct tissue layers: the more rigid cortex and the softer medulla. (B) A schematic illustration of the in vitro engineered biomimetic ovarian microtissue. (C) The storage (G′, representing elastic effect) and loss (G″, representing viscous effect) moduli of materials for making the microtissue shell. Two different harder hydrogels, 2% alginate (Alg(2)) and 2% alginate with oxidization (O-alg(2)), were used for making the shell (cortex). The moduli of O-alg(2) decrease by >16 times after ~ 7 days incubation in culture medium at 37 °C. (D) The two moduli of materials for making the microtissue core. Two different softer hydrogels, 0.5% alginate (Alg(0.5)) and 0.5% collagen (Col(0.5)), were used for making the core (medulla).

Fig. 2

Non-planar microfluidic flow-focusing device for encapsulating early secondary preantral follicle in core-shell microcapsules to produce the biomimetic ovarian microtissue. (A) A schematic view of the microchannel system (top) together with a zoom-in view of the nonplanar design of the flow-focusing junction (bottom) where W1×H1 = 200×200 µm, W2×H2 = 80×300 µm, and W3×H3 = 200×400 µm. (B) Typical image of the boxed region in panel A showing the dispatching and flow-focusing areas. (C) Typical image of the boxed region in panel A showing the entrance of the extraction channel. (D) Typical image of the boxed region in panel A showing the exit of the extraction channel. I-1, I-2, I-3, I-4, and I-5 are the inlets of core, shell, mineral oil emulsion, dispatching, and extraction flows, respectively. O-1 and O-2 are outlets for the aqueous (containing microtissues) and oil emulsion flows, respectively.

Fig. 3

In vitro development of early secondary preantral follicles of deer mice under miniaturized 3D culture in microtissue. (A) Typical micrographs showing the development to antral stage over 9 days of an early secondary preantral follicles in the collagen core of biomimetic ovarian microtissue with an alginate (non-oxidized) shell. The defining feature of an antral follicle is that it contains a cumulus-oocyte complex (COC) inside a fluid-filled antral cavity (the area enclosed in dashed line). A schematic illustration of the anatomic features of COC and follicles at various stages together with an enlarged view of the antral follicle on day 9 for better visualizing the COC and antral cavity is shown in Fig. S4. (B) Quantitative data (pooled, the number of early secondary preantral follicles n ≥ 40) showing the effect of the core (0.5% collagen or alginate) and shell (2% oxidized or nozn-oxidized alginate) materials for making the ovarian microtissue on the development of early secondary preantral follicle into antral stage together with that from 2D culture. Col(0.5): 0.5% collagen. Alg(2): 2% alginate. O-alg(2): 2% alginate with oxidization. Alg(0.5): 0.5% alginate. Scale bar: 100 µm.

Fig. 4

In vitro ovulation of antral follicles obtained by culturing early secondary preantral follicles in the collagen core of biomimetic ovarian microtissues with an alginate shell. (A) Quantitative data (pooled, the number of antral follicles n ≥ 6) showing the effect of luteinizing hormone (LH) and epidermal growth factor (EGF) on ovulation. (B) A typical micrograph of in vitro ovulation showing a cumulus-oocyte complex (COC) was released leaving behind a corpus luteum-like tissue complex in a biomimetic microtissue cultured in the presence of LH and EGF (LH⁺EGF⁺). (C) A typical micrograph of in vitro ovulation from a biomimetic microtissue cultured in the absence of LH and EGF (LH⁻EGF⁻). (D) A typical phase contrast image showing the oocyte morphology. (E) A typical fluorescence image showing the nucleus of the oocyte in panel D. Scale bars: 100 µm in panels B-C and 50 µm in panels D-E.