Ovarian follicle culture: advances and challenges for human and nonhuman primates

Abstract

The removal and cryostorage of ovarian cortical biopsies is now offered as a fertility preservation option for young women. The only available option to restore fertility using this tissue is by transplantation, which may not be possible for all patients. The full potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development from the most immature stages to maturation. The techniques of in vitro growth (IVG) combined with in vitro maturation (IVM) are being developed with human tissue, but comparing different systems has been difficult because of the scarcity of tissue so nonhuman primates are being used as model systems. There are many challenges to developing a complete culture system that would support human oocyte development, and this review outlines the approaches being taken by several groups using tissue from women and nonhuman primate models to support each of the stages of oocyte development.

Figure 1

a) Digrammatic representation of follicle growth from the non-proliferating pool of primordial follicles. Primordial follicles are continuously activated into the growing population where they become primary follicles consisting of an oocye arrested at the dictyate stage of prophaseI of meiosis (yellow) surrounded by granulosa cells (green). Primary follicles undergo oocyte growth and granulosa cell proliferation and differentiation (purple) when they form an antral cavity. Antral follicles continue to grow and granulosa cells differentiate into two sub-populations of cells 1) cumulus surrounding the oocyte (blue) and 2) mural lining the wall of the follicle (orange). Exact timings for this developmental sequence to occur in humans are not known but estimations suggest several months, however, it is not known whether the growth profile is continuous or whether there are “resting” phases through follicle development. b) Simplified version of the PI3K pathway The factors initiating this process are largely unknown but a body of evidence is emerging to show that the phosphatidylinositol-3’-kinase (PI3K-AKT) signalling pathway is a major regulator of early follicle/oocyte development and that components of this pathway are involved in controlling the rate of activation from the non-growing population of follicles. The phosphatase PTEN converts PIP3 to PIP2, which negatively regulates PI3K activity. Signaling mediated by PI3Ks converge at PDK1. PDK1 phosphorylates Akt and activates it. Akt can phosphorylate and inactivate tuberous sclerosis complex 2 (TSC2, or tuberin), which leads to the activation of mTOR complex (mTORC1). mTORC1 can phosphorylate (activate) S6K1. S6K1 subsequently phosphorylates and activates rpS6, which enhances protein translation that is needed for cell growth. mTORC1 can be inhibited pharmacologically with Rapamycin and stimulated by leucine. The manipulation of this pathway could have important clinical applications in the field of fertility preservation.

Figure 2. Two-Step Culture System To Obtain Growing Human Follicles

A: An outline of the preparation of human ovarian cortical biopsies to initiate development of primordial follicles (Step 1) and after 6 days in serum free medium dissect out multi laminar growing follicles and grow these individually (16). Photomicrograph of freshly prepared micro-cortex before culture (B) and after 6 days of culture (C) followed by dissection to isolate multilaminar follicles with theca cells attached (D). Isolated follicles can be cultured individually as outlined in A and antral formation occurs during a further 4 day culture period.

Figure 3

Left panel depicts representative histological images of isolated rhesus macaque primordial follicles after encapsulation in alignate at Day 0 (A and B), cultured for 6 days without encapsulation (C and D), or cultured for 6 days while encapsulated in 0.5% (E and F) or 2% (G and H) alginate. Scale bar = 50 μm; asterisks denote oocytes. From reference (42) Right panel represents morphology and histology of isolated human primordial follicles encapsulated in alginate before in vitro culture (A), after 7 days in vitro (B), a cryopreserved follicle after 7 days in vitro (C), and a follicle isolated from cryopreserved-thawed ovarian cortex after 7 days in culture (D). Images are at 40X magnification. Semi-thin sections of a cryopreserved isolated follicle (E, 1000X) and a follicle isolated from frozen-thawed ovarian cortex (F, 400X) after 7 days of culture encapsulated in alginate. From reference (53).

Figure 4

Representative images illustrating in vitro growth of nonhuman primate and human preantral follicles enclosed in a 3D matrix to the small antral stage. The day of culture and the follicular diameter is noted under each picture. Top panel represents growth of a rhesus macaque secondary follicle (left) to a small antral follicle (right); scale bars = 250 μm; from M. Zelinski. Middle panel depicts growth of a baboon multilayer follicle (left; scale bar = 100 μm) to a small antral follicle (scale bar = 50 μm); from reference (31). Bottom panel shows a human secondary follicle that grew to a small antral follicle (scale bars = 100 μm); from reference (29).