Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth

Abstract

Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and -dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. eIVFG is performed with alginate hydrogels, which are biologically inert, maintain cell-to-cell interactions between granulosa cells and the oocyte, and preserve follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.

Common mechanisms of physiological and pathological rupture events in biology: novel insights into mammalian ovulation and beyond

“…The role that these forces play and their potential for clinical and diagnostic use is better understood in the context of ICA and chorioamniotic membrane rupture than in ovulation. As a result of the similarities between rupture systems discussed herein, ex vivo methods that have been developed to study ICAs and chorioamniotic membrane rupture should be paired with existing ex vivo ovulation techniques to advance the field of ovulation biology ( Converse et al ., 2022 ; Skory et al ., 2015 ). The gaps identified in this review, specifically in the context of the biomechanical and contractile forces involved, should be explored further to enhance our understanding of the process of ovulation.…”

Generation of Tailored Extracellular Matrix Hydrogels for the Study of In Vitro Folliculogenesis in Response to Matrisome-Dependent Biochemical Cues

“…Animals were housed in a temperature- and light-controlled environment (12 h light/12 h dark) and provided with food and water ad libitum. Follicles were isolated from the ovary using previously published mechanical isolation methods [ 36 ]. High-quality single-layered primary follicles were selected for long-term follicle culture.…”

Age-associated increased stiffness of the ovarian microenvironment impairs follicle development and oocyte quality and rapidly alters follicle gene expression

“…Follicles were cultured and analyzed as previously described [20]. Briefly, ovaries from day 12 or 13 prepubertal CB6F1 and CD1 mice were placed in dissection media containing L15, 1% fetal bovine serum (FBS), and 0.5% penicillin-streptomycin (Pen-Strep) (Gibco, ThermoFisher).…”

“…Only follicles with an intact basement membrane and visible and healthy oocytes surrounded by two layers of granulosa cells were selected for culture. To encapsulate the follicles, the parafilm technique was performed [20]. Groups of 10 follicles were mixed with 7 µl of 0.5%, 2%, or 4% w/v alginate solution.…”