Study On In Vitro Maturation Of Mouse And Human Primary Follicles And Immature Oocytes

PurposeThe present study was designated to investigate the practical method of isolation of mouse primary follicles and to formulate cultural condition for its in vitro development & maturation, to investigate the maturational competence and time course of in vitro cultured mouse immature denuded oocytes, to set up a method testing the viability of cultured oocytes and embryos. The second purpose was to carry out a pilot study of in vitro development & maturation of human primary follicles and cumulus-oocyte complexes(CQCs). Methods 1. An enzymatic method was developed to collect intact follicles at primary or primordial stages from the ovaries at 370C, with the mixture of collagenase, deoxyribonuclease (DNase) and pronase. All the enzymetic separated follicles and mechanical collected follicles were cultured in Waymuoth medium MB 752/1 with bovine serum albumin (BSA) and insulin transferring and selenium (ITS) for comparative study of the two collective procedures. 2. Mechanical collected mouse primary follicles were cultured in Waymuoth medium MB 752/1 with fetal bovine serum (FBS) and follicle-stimulating hormone (FSH) or human menopausal gonadotropin (hMG) to mimic the physiological conditions of in vivo development. 3. Mechanical collected immature oocytes obtained from mouse ovary without gonadotropin stimulation were cultured with hMG and FSH for 24 or 48 hours for comparison. The cultured oocytes were evaluated for their fertilizing and developmental capacity through in vitro fertilization (IVF) and fluorescein diacetate (FDA). A preliminary pilot study has been carried out with human primary follicles and COCs in the formulated in vitro conditions to diserve their development & maturation. Results 1. The medium containing 3rng/mL collagenase IA and I OOIU/mL DNase with or without pronase obtained the better efficiency in collection, but their developmental competence was lower than those of mechanical collected follicles without enzymatic treatment. 2. About 15.94% mouse primary follicles have developed to VII stage in medium with I OOmIU/mL hMG; while about 28.57% mouse primary follicles to VIII stage with 300mIU/mL hMG. 3. No significant difference was observed on effect of hMG or FSH during 24h or 48h in vitro maturation for the mouse immature oocytes (P>O.05). However, the in vitro fertilization rates of two groups with hMG were lower than those of the two groups without hMG after cultured for 48h(P