title: Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice.
abstract: Although the ovary has a large store of germ cells, most of them do not reach mature stages. If a culture system could be developed from early growing follicles to mature oocytes, it would be useful for biological research as well as for reproductive medicine. This study was conducted to establish a multistep culture system from isolated early growing follicles to mature oocytes using a mouse model. Early growing follicles with diameters of 60-95 μm corresponding to primary and early secondary follicles were isolated from 6-day-old mice and classified into three groups by diameter. These follicles contained oocytes with diameters of ~45 μm and one or a few layered granulosa cells on the basal lamina. Embedding in collagen gel was followed by first-step culture. After 9-day culture, the growing follicles were transferred onto collagen-coated membrane in the second step. At day 17 of the culture series, the oocyte-granulosa cell complexes were subjected to in vitro maturation. Around 90% of the oocytes in follicles surviving at day 17 resumed second meiosis (metaphase II oocytes: 49.0-58.7%), regardless of the size when the follicle culture started. To assess developmental competence to live birth, the eggs were used for IVF and implantation in pseudopregnant mice. We successfully obtained two live offspring that produced next generations after puberty. We thus conclude that the culture system reported here was able to induce the growth of small follicles and the resultant mature oocytes were able to develop into normal mice.
authors.affiliation: Department of Obstetrics and Gynecology, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 663-8501, Japan.
authors.forename: Nahoko
authors.initials: N
authors.lastname: Mochida
authors.forename: Akiko
authors.initials: A
authors.lastname: Akatani-Hasegawa
authors.forename: Kayo
authors.initials: K
authors.lastname: Saka
authors.forename: Mai
authors.initials: M
authors.lastname: Ogino
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hosoda
authors.forename: Ryu
authors.initials: R
authors.lastname: Wada
authors.forename: Hideaki
authors.initials: H
authors.lastname: Sawai
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shibahara
mesh_terms: D000818:Animals; D046508:Culture Techniques; D005260:Female; D005307:Fertilization in Vitro; D059471:In Vitro Oocyte Maturation Techniques; D050498:Live Birth; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009865:Oocytes / Q000502:physiology*; D006080:Ovarian Follicle / Q000502:physiology*