In vitro development of ovine follicles from the preantral to antral stages depends on initial follicle diameter and promotes oocyte growth and transzonal projections formation

Abstract

Ovine follicle culture recapitulates several features of the preantral-to-antral transition, including morphological changes and granulosa cells proliferation and differentiation. Although the influence of initial follicle diameter on in vitro development has long been suspected, it had not been investigated systematically. To date, only a few studies in other species have compared the in vitro progression of preantral follicles from distinct size classes. In the present study, we finely characterized the effect of initial follicle diameter (ranging from 199 to 340 µm) on subsequent in vitro development, by incorporating this factor as a continuous variable in statistical analysis. Regardless of the presence of ovine FSH, increasing initial follicle diameter was associated with enhanced survival and antrum formation, and larger final follicle and oocyte diameters. Initial follicle diameter also impacted growth dynamics, with a positive effect during early culture (days 0–6) but a negative effect during late culture (days 13–20). The presence of leukemia inhibitory factor modulated some of these effects. Follicle culture sustained oocyte growth, albeit to a lesser extent than in vivo development. Among oocytes retrieved from in vitro grown follicles measuring 500–900 µm, 33% had reached 110 µm (excluding the zona pellucida), a threshold commonly associated with meiotic competence. Furthermore, using high-resolution confocal microscopy, we demonstrate for the first time that follicle culture supports the formation of actin-rich transzonal projections.