Patterns of growth, oestradiol and progesterone released by in vitro cultured mouse ovarian follicles indicate consecutive selective events during follicle development

Abstract

This study investigated the relationship between individual follicle growth, steroid release and follicular morphology to provide basic information about critical stages in follicle development. Preantral mouse follicles secreted significant and constant amounts of progesterone that were not related to oestradiol production but did appear to be related to thecal organization. Oestradiol release was variable among follicles of equivalent diameter, but marked increases in oestradiol concentrations were measured in follicles of 300 and 400 microns diameter. Over 4 days of culture, the proportion of follicles growing beyond the threshold diameter of 300 microns was lower in small (140-160 microns) than it was in large preantral follicles (170-210 microns). Retardation of growth below this diameter was associated with significantly decreased steroid concentrations. In follicles growing beyond the threshold diameter, antrum formation progressed, but full Graafian stage was not attained. Among large follicles, variability in oestradiol release could not be associated with obvious histological differences. In contrast, structural disturbances were associated with reduced steroid production and growth abnormalities. Therefore, it is concluded that the preantral follicles selected for culture comprise distinct follicle populations with respect to their developmental potential in vitro, and that follicle development is controlled by endogenous processes involving oestradiol at distinct stages of follicle development.

In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence

“…These results confirm the important role of FSH, but also stress the fact that low FSH concentrations are able to support follicle survival, antral cavity development and granulosa cell differentiation, yet allowing the oocyte to resume meiosis (more than 93%) and being fertilizable. Furthermore, follicles in the NA cultures reached large antral stages (mean diameter: 424±18 μm), with a recognizable antral-like cavity at diameters ≥350 μm; similar to those reported under other non-attachment conditions (~400 μm and~300 μm, respectively) [15,20,22,28,38,39]. This becomes significant since initial follicle diameters in previous reports differ from ours (150-200 μm; with a length of 5-6 days of culture versus 110-130 μm; with a length of 9 days of culture, respectively).…”

“…Different spherical follicle culture systems have used strategies to prevent the attachment of follicles to the culture wells, with the aim to provide a more natural environment to the growing oocyte [15,18,20,22,24,25,28,[38][39][40][41]. The present study evaluates the impact of culturing follicles in a non-attachment system by comparing gene expression levels in oocytes and their surrounding cumulus cells to those found in a previously characterized attachment follicle culture system and to in-vivo conditions.…”

A Reproducible Two-Step Culture System for Isolated Primary Mouse Ovarian Follicles as Single Functional Units1

“…To keep the three-dimensional structure intact during the first step, follicles were detached mechani- cally should attachment of the thecal cells (fibroblast-like cells) occur. Mechanical detachment is easy and avoids the use of more sophisticated techniques to inhibit attachment, such as membrane inserts [25] and coated wells [26]. Detachment kept thecal cells surrounding the follicle, perhaps providing essential biophysical factors to support the basal membrane and, thereby, ensuring functional integrity to the follicle mass [27].…”

Luteinizing Hormone Has a Stage-Limited Effect on Preantral Follicle Development In Vitro1

“…Intact preantral mouse follicles with a minimum size of 150 m and between 2 to 3 granulosa cell layers [7,9,10] can be routinely cultured to the preovulatory stage in a virtually LH-free mixture containing FSH and 5% serum [4] in 3-5 days, depending on follicle size at the beginning of culture. In contrast, preantral follicles from 140 m down to 85 m in diameter (between 1 to 2 layers of granulosa cells) were shown in this study to additionally require LH together with an increase in serum to support rapid growth and antral development.…”

“…The follicle culture protocol was adapted from Fehrenbach et al [7] and Vitt et al [4]. For culture, follicles were transferred from the holding medium into the culture medium, which consisted of ␣ minimum essential medium (␣MEM) supplemented with 3.5 g/ml insulin (I 1882; Sigma, St. Louis, MO), which was a lower amount than had been used in the previous studies; 1 M L-glutamine (Gibco 15039; Gibco BRL, Karlsruhe, Germany); 10 g/ml transferrin (Sigma T 5391); 50 g/ml L-ascorbic acid (A 4544, Sigma); and, depending on the experiment, either 5%, 7.5%, or 10% mouse serum.…”