Hum. Reprod. Advance Access published online on October 14, 2009
Human Reproduction, doi:10.1093/humrep/dep351
A new culture technique that allows in vitro meiotic prophase development of fetal human oocytes
1 Departament de Biologia Cel·lular i Genètica Mèdica, Universitat Autònoma de Barcelona, Bellaterra Campus, Cerdanyola del Vallès., Barcelona, Spain 2 Departamento de Farmacología y Fisiología, Facultad de Medicina de la, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México 3 Molecular Biology Laboratory of Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, NY, USA 4 Departament de Ginecologia I Obstetricia, Hospital de la Vall d' Hebron, Barcelona, Spain
5 Correspondence address. Tel: +34 935811175; Fax: +34 935811025; E-mail: montserrat.garcia.caldes{at}uab.es
BACKGROUND: Little is known about the mechanisms that regulate meiosis in the human female fetus as a result of the technical difficulties in obtaining samples. Currently, there is no technique for human fetal oocyte culture that permits the maintenance of fetal ovarian tissue in vitro which allows the progression of meiosis in a reproducible and standardized way.
METHODS: Meiotic progression was analyzed following pairing-synapsis and recombination progress. A total of 7119 oocytes were studied and analyzed. The proteins used to evaluate meiotic progression were: REC8, SYCP1, SYCP3 and MLH1, studied by immunofluorescence. Four different sample disaggregating methods were used, two enzymatic (trypsin and collagenase + hyaluronidase) and two mechanical (puncture and ovarian fragments). Two different culture media were used, control media and stem cell factor (SCF)-supplemented media. The oocytes were studied at initial time T0, and then at T7, T14 and T21 days after culture.
RESULTS: The mechanical methods increased the total number of oocytes found at the different times of culture and decreased the number of degenerated oocytes. Independently of the disaggregation method used, oocytes cultured with SCF-supplemented media showed a higher proportion of viable oocytes and fewer degenerated cells at all culture timepoints. No evidence of abnormal homologous chromosome synapsis was observed. Meiotic recombination was only observed in oocytes mechanically disaggregated and cultured with supplemented media.
CONCLUSIONS: The oocytes obtained by mechanical disaggregating methods and cultured with SCF-supplemented media are able to follow pairing-synapsis and recombination, comparable to oocytes in vivo. The culture conditions described herein confirm the methodology as a standardized and reproducible method.
Key words: meiosis/recombination/oocytes/culture/stem cell factor
Submitted on July 13, 2009; resubmitted on September 1, 2009; accepted on September 8, 2009.