RESUMEN
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.
Asunto(s)
Técnicas de Cultivo , Técnicas de Maduración In Vitro de los Oocitos , Nacimiento Vivo , Oocitos/fisiología , Folículo Ovárico/fisiología , Animales , Femenino , Fertilización In Vitro , Masculino , Ratones , Ratones EndogámicosRESUMEN
OBJECTIVE: To determine an optimum condition for vitrification of various animal species and to examine the developing capacity into pups of mouse oocytes in preantral follicles originating from cryopreserved ovaries. DESIGN: Experimental animal study. SETTING: Laboratory environment. ANIMAL(S): Normal (C57BL/6 x DBA2) F1 mice. INTERVENTION(S): Vitrification of animal ovaries using polyester sheets as a storage device; collection of oocyte-granulosa cell complexes by enzymatic treatment; and in vitro growth (IVG), in vitro maturation (IVM), and IVF, with embryo transplantation to pseudopregnant mice. MAIN OUTCOME MEASURE(S): Histological analysis of vitrified and warmed ovaries from several animal species; measurement of successful rates in IVG, IVM, and IVF of mouse oocytes in oocyte-granulosa cell complexes collected from the vitrified and warmed ovaries; and achievement of pup birth. RESULT(S): The vitrification method used was effective for storage of various animal ovaries. The oocytes enclosed in preantral follicles that were yielded from the vitrified-warmed ovaries preserved the capacity for developing into pups after IVG, IVM, and IVF. CONCLUSION(S): The findings indicated that ovarian cryopreservation by vitrification and subsequent IVG, IVM, and IVF are promising methods for restoring young cancer patients' fertility. The practical information presented here is applicable to human ovarian tissues.
Asunto(s)
Animales Recién Nacidos , Criopreservación , Fertilización In Vitro , Ratones/fisiología , Oocitos/fisiología , Folículo Ovárico , Ovario , Animales , Desarrollo Embrionario , Femenino , Técnicas In Vitro , Masculino , Ratones/embriología , Ratones Endogámicos , Ovario/anatomía & histología , Conejos , PorcinosRESUMEN
The mammalian zona pellucida is an extracellular matrix that occurs in growing oocytes, ovulated eggs and pre-implantation embryos, and is known to be involved in several important events during ovarian folliculogenesis and fertilization. Since the zona pellucida is formed at an early stage of oocyte growth, circulating antibodies against zona pellucida may impair ovarian function. In this article we discuss whether anti-zona antibodies cause ovarian dysfunction and infertility. The discussion is based on clinical examination and animal experiments including the following approaches: 1/ immunological method using solubilized human zona pellucida detected anti-zona antibody with a high frequency in infertile patients, especially premature ovarian failure syndrome; 2/ in vivo experiment using hamsters showed that some, but not all, animals experienced ovarian failure after immunization with hamster recombinant zona proteins; 3/ in vitro experiment using mouse isolated ovarian follicles showed significant inhibitory effects on follicular growth and oocyte development. We concluded that anti-zona antibody may be involved in causing ovarian failure.