Ovary and ovulation: In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

Abstract

A simplified culture system was developed for the in-vitro maturation of early preantral mouse ovarian follicles. The follicles were cultured singly in 20 microliters droplets under oil in medium supplemented with recombinant follicle stimulating hormone (r-FSH) at 37 degrees C and 5% CO2 in air. The follicles grew and became attached to the bottom of the dish, progressively lost their spherical structure by outgrowth of the granulosa cells through the basal membrane and developed follicles with antral-like cavities. The normal three-dimensional follicular structure was lost but all components, i.e. theca, granulosa and oocyte, remained functional, as was proven by the oestradiol, inhibin and progesterone secretion patterns. Follicle survival exceeded 80% and histological analysis proved the absence of atresia and cell death in granulosa cells up to day 16. Oocytes of 55 (+/-4) microns diameter on the day of isolation reached 74 (+/-3) microns by day 16 of culture. The optimal moment for inducing the final meiotic maturation with human chorionic gonadotrophin was investigated: the highest absolute numbers of metaphase II oocytes were obtained on days 12 and 14 (39 and 41%). The fertilizing potential of the in-vitro matured oocytes was comparable to in-vivo matured controls. A 50% hatched-blastocyst development rate was observed.

In vitro follicular growth affects oocyte imprinting establishment in mice

“…Follicles were placed individually into 10 ml drops of culture medium, under pre-equilibrated mineral oil (Sigma-Aldrich) at 371C in an atmosphere of 5% CO 2 in air, using a system similar to the one previously described. 8 Culture medium consisted of a minimal essential medium (Life Technologies) supplemented with 5% heat inactivated FCS, 10 mg/ml transferrin (Boehringer Mannheim, France), 5 mg/ml insulin (Boehringer Mannheim) and 100 mIU/ml r-FSH (Serono, France). Healthy oocyte -cumulus complexes consisting of compact granulosa cells with regular borders surrounding the entire mass were used in these experiments.…”

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

“…Washed pre-antral follicles were cultured in a medium described by Cortvrindt et al (1996) with some modifications. The culture medium consisted of a-minimum essential medium (Invitrogen) supplemented with 5% heat-inactivated FCS (Invitrogen), 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenium (ITS), 50 mIU/ml penicillin, 50 mg/ml streptomycin, and 2 mg/ml FSH (Folltropin-V, BIONICHE Animal Health Canada Inc. Belleville, Ontario, Canada).…”

“…On day 12 of culture, ovulation was induced by adding 5 ng/ml recombinant human epidermal growth factor (Invitrogen) and 1.5 IU/ml human chorionic gonadotropin (Chorulon, Intervet Canada Corp., Kirkland, Quebec, Canada) into pre-antral follicle culture droplets (Cortvrindt et al 1996, Liu et al 2001. After 14-16 h, oocyte release was observed (and aided, if required), and the presence and mucification of the cumulus were recorded using an inverted microscope.…”

Carbohydrate metabolism by murine ovarian follicles and oocytes grown in vitro

“…Follicle diameter was recorded and the degree of theca coverage (Cortvrindt et al 1996) was graded as either grade 1 (O50%) or 2 (!50%). The apposition between the oocyte and the surrounding granulosa cells was also noted (Cortvrindt et al 1996) and was characterised as the degree of close contact or adhesion between an oocyte and its immediate granulosa neighbours.…”

“…The process of folliculogenesis involves many intricate and timely developmental events, which must be replicated in the laboratory. Avascular preantral follicles survive relatively well in vitro and culture of preantral follicles is possible in several species, including mouse (Nayudu & Osborn 1992, Boland et al 1993, Cortvrindt et al 1996, Rose et al 1999, sheep (Newton et al 1999, Picton et al 2003 and human (Roy & Treacy 1993, Picton & Gosden 2000. However, after long-term culture, oocyte viability is reduced and this is especially a problem in large animals and humans.…”