Oocytes grown in vitro are of low quality and yield few live births, thus limiting the ability to store or bank the ova of women wishing to preserve their fertility. We applied tissue engineering principles to the culture of immature mouse follicles by designing an alginate hydrogel matrix to maintain the oocyte's 3- dimensional (3D) architecture and cell-cell interactions in vitro. A 3D culture mimics the in vivo follicle environment, and hydrogel-encapsulated follicles develop mature oocytes within the capacity for fertilization similar to that of oocytes matured in vivo. Embryos derived from cultured oocytes fertilized in vitro and transferred to pseudopregnant female mice were viable, and both male and female offspring were fertile. Our results demonstrate that alginate hydrogel-based 3D in vitro culture of follicles permits normal growth and development of follicles and oocytes. This system creates new opportunities for discovery in follicle biology and establishes a core technology for human egg banks for preservation of fertility.

Cancer is now appreciated as not only a highly heterogenous pathology with respect to cell type and tissue origin but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes such as death, proliferation, differentiation and migration. Thus, the activities of molecular networks that execute metabolic or cytoskeletal processes, or regulate these by signal transduction, are altered in a complex manner by diverse genetic mutations in concert with the environmental context. A major challenge therefore is how to develop actionable understanding of this multivariate dysregulation, with respect both to how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments. While high-throughput experimental platform technologies ranging from genomic sequencing to transcriptomic, proteomic and metabolomic profiling are now commonly used for molecular-level characterization of tumor cells and surrounding tissues, the resulting data sets defy straightforward intuitive interpretation with respect to potential therapeutic targets or the effects of perturbation. In this review article, we will discuss how significant advances can be obtained by applying computational modeling approaches to elucidate the pathways most critically involved in tumor formation and progression, impact of particular mutations on pathway operation, consequences of altered cell behavior in tissue environments and effects of molecular therapeutics.

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (k(A)) was modeled for the two groups of animals, and consistent with the empirical data, the k(A) for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

The extracellular matrix (ECM) provides a three-dimensional structure that promotes and regulates cell adhesion and provides signals that direct the cellular processes leading to tissue development. In this report, synthetic matrices that present defined ECM components were employed to investigate these signaling effects on tissue formation using ovarian follicle maturation as a model system. In vitro systems for follicle culture are being developed to preserve fertility for women, and cultures were performed to test the hypothesis that the ECM regulates follicle maturation in a manner that is dependent on both the ECM identity and the stage of follicle development. Immature mouse follicles were cultured within alginate-based matrices that were modified with specific ECM components (e.g., laminin) or RGD peptides. The matrix maintains the in vivo like morphology of the follicle and provides an environment that supports follicle development. The ECM components signal the somatic cells of the follicle, affecting their growth and differentiation, and unexpectedly also affect the meiotic competence of the oocyte. These effects depend upon both the identity of the ECM components and the initial stage of the follicle, indicating that the ECM is a dynamic regulator of follicle development. The development of synthetic matrices that promote follicle maturation to produce meiotically competent oocytes may provide a mechanism to preserve fertility, or more generally, provide design principles for scaffold-based approaches to tissue engineering.

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4-6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.