In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

Abstract

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980-2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Keywords: follicle; follicle culture; follicular development; oocyte.

Figure 1

Key stages of follicle and oocyte development that must be recapitulated in vitro. During follicular development, primordial follicles are activated, and their surrounding squamous granulosa cells change to a cuboidal shape, making them primary follicles. Primary follicles grow to secondary follicles with a theca layer. Secondary follicles are gonadotropin dependent and can respond to FSH to grow into larger antral follicles and then preovulatory follicles. At the antral and preovulatory stage, the oocyte is surrounded by cumulus and mural granulosa cells. During oogenesis, immature oocytes with a germinal vesicle remain in prophase I arrest within the ovary. Oocytes resume meiosis at the time of ovulation and ultimately reach metaphase of meiosis II at which point they are called an egg and can be fertilized in the presence of sperm.

Figure 2

Distribution of species used in follicle culture studies examined. In studies that used more than one species, each species reported per study was accounted for in this analysis. Rodent studies are predominant, comprising 44.3% of the studies examined, followed, by humans at 15.9% and non-human primates at 14.7%. Agricultural species such as bovine (9.1%), caprine (4.6%), ovine (4.6%), and porcine (2.3%) are also used widely in follicle culture studies. Lesser used species are feline (2.3%), canine (1.1%), and marsupial (1.1%).

Figure 3

Strains of mouse used in follicle culture studies examined. Outbred strains such as CD1 were the most popular, followed by various F1 hybrid mice. Inbred strains such as FVB were used less often.

Figure 4

Distribution of the age of species used in follicle culture studies examined. Age was classified into three broad categories: prepubertal, reproductively young, and other (when age is not mentioned). If a citation mentioned the use of multiple age groups for follicle culture, each age group was counted independently.

Figure 5

Schematic overview of 2D and 3D culture systems. (A–C) 2D culture methods include droplet cultures (A), 2D substrate cultures with and without ECM coating (B), and membrane insert cultures with and without ECM coating (C). (D–F) 3D culture methods include suspension cultures (D) such as inverted droplet (i), magnetic levitation (ii), and roller culture (iii); encapsulated culture (E) between coatings of (i) ECM or(ii) alginate drops; and multistep culture (F). The multistep culture of McLaughlin et al. [83] is pictured as a representative technique. Cortical strips were first cultured in medium for 8 days (step 1). Intact follicles were dissected and cultured individually for an additional 8 days (step 2), and then COCs with mural and cumulus cells were isolated from the follicles and cultured on membranes for 4 more days (step 3). COCs with oocytes greater than 100 μm were selected for IVM (step 4).