J Vet Sci.  2023 Jan;24(1):e4. 10.4142/jvs.22223.

Optimized study of an in vitro 3D culture of preantral follicles in mice

• Ren H
• Zhang Y
• Zhang Y
• Qiu Y
• Chang Q
• Yu X
• Pei X

• KMID: 2539437
• DOI: http://doi.org/10.4142/jvs.22223

Abstract

Background
in vitro culture of preantral follicles is a promising technology for fertility preservation.
Objectives
This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation.
Methods
The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E 2 ) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization.
Results
The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E 2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro.
Conclusions
The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBSfree system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

Keyword

Preantral follicles; 3D culture; ultra-low attachment plate; sodium alginate