A visualized and quercetin-optimized three-dimensional culture model of mouse ovaries derived from fetal gonads

Abstract

The in vitro culture of ovarian tissue is emerging as a popular technology to study female reproductive medicine. However, standard in vitro culture conditions usually increase the level of reactive oxygen species (ROS), hindering ovarian development. Here, we establish an in vitro visualized mouse ovarian explant 3D culture model with the GFP-BVSC reporter system and obtain the early follicle pool from fetal female gonads. This model recapitulates in vivo ovarian characteristics and allows non-invasive monitoring of ovarian development. Importantly, supplementation with quercetin, a plant-derived natural antioxidant, increases the tissue area and total follicle count in cultured ovaries by protecting mitochondria and reducing ROS, thus more closely mimicking in vivo growth conditions. Finally, this visualized and optimized ovarian explant culture platform has been proven to be effective in modelling female ovarian diseases, such as the fetal reproductive aberrations of female offspring affected by gestational diabetes mellitus (GDM). Overall, our work extends the understanding of ovarian biology and creates an efficient and simplified platform for the morphological monitoring of ovarian development, as well as for drug screening and the clinical treatment of ovarian hypofunction.

1. Introduction

On the basis of advancements in three-dimensional (3D) culture systems and increasing knowledge of ovary biology, many attempts have been made to establish ovarian organoid or ovarian explant culture models from fetal and adult ovaries, as well as pluripotent stem cells [1–8]. Although this technology is still in its early stages, it can mimic the ovarian microenvironment and support follicle maturation, driving progress in reproductive and regenerative medicine [9]. Remarkably, Morohaku et al. [4] generated a complete ovarian explant model starting from mouse fetal female gonads and successfully obtained fertile pups. However, compared with that of germinal vesicle (GV) oocytes in vivo, the rate of embryonic development up to the pup stage remains to be further enhanced. Moreover, in vitro conditions inevitably lead to an increase in reactive oxygen species (ROS) generation from cellular metabolism or external factors [10,11]. Excessive ROS can trigger a cascade of abnormal effects in the ovary, including apoptosis, inflammation, and mitochondrial damage, which collectively contribute to impaired ovarian development [12,13]. Therefore, the real challenge is to reduce the impact of harmful exogenous factors on ovarian cells and simulate the growth system in vivo as much as possible. These findings will ultimately facilitate more precise studies of ovarian biology and disease pathology.

Ovarian dysfunction is a main cause of female infertility [14]. Approximately 1% of women under the age of 40 years experience primary ovarian insufficiency (POI), marked by a significant reduction in oocyte count and vitality, along with irregular menstrual cycles [15]. Germ cells in the ovary initiate meiosis around embryonic day 14.5 (E14.5) and then arrest at the diplotene stage of the first meiotic division from E17.5 to postnatal day 3 (PND3) in mice [16]. At this stage, primordial follicles begin to assemble, consisting of an arrested oocyte and a single layer of flattened granulosa cells [17]. Then, a cohort of primordial follicles are irreversibly activated and initiate foll