Research articleBirth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium
Introduction
Oocyte-handling techniques, including in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF), and in vitro culture (IVC) of embryos, constitute the foundation of reproductive technologies, such as production of transgenic animals, somatic cell cloned animals, and establishment of embryonic stem cells. These techniques have contributed to the field of medicine by creating animal models of human disease, and have led to the use of assisted reproductive technology (ART) in humans. Nonhuman primates are valuable in models for human disease and for ART, because they have many physiological, morphologic, and genetic similarities. Hence, optimization of oocyte-handling techniques in nonhuman primates can lead to important advances in human medicine. However, there are few reports regarding oocyte-handling techniques of nonhuman primates, because of their availability, age, size, viability, and management.
The common marmoset (Callithrix jacchus) is a nonendangered New World primate, which is native to Brazil and has been widely used as a model of human disease in the fields of neuroscience, reproductive biology, infectious disease, and behavioral research. Recently, the establishment of embryonic stem cells [1] and induced pluripotent stem cells [2] as well as the generation of a transgenic marmoset [3] and somatic cell cloned embryos [4], have been reported. The use of marmosets has many advantages for studying reproductive biology compared with other laboratory primates. In addition to their small body size and low feeding costs, they are the only anthropoid primates that routinely ovulate multiple oocytes per ovarian cycle, they have a short gestation period, and they reach sexual maturity at approximately 1 yr of age. Moreover, the ovarian cycle can be synchronized, and efficient protocols for ovarian stimulation have been developed. Therefore, common marmosets are ideal nonhuman primates for studying reproductive biology.
Although oocyte handling techniques in common marmosets have been developed [5] as have effective ovarian stimulation methods [6], [7], there are few reports regarding oocyte handling techniques in nonhuman primates. In the first IVM report of marmoset oocytes [8], alpha-minimum essential medium was used as preincubation medium. A subsequent report clearly demonstrated that in vitro-matured marmoset oocytes in Waymouth's medium supported advanced preimplantation embryonic development in vitro [9]. However, rates of development to the blastocyst stage of in vitro-matured and -fertilized embryos were very low. Therefore, improvement of IVM culture medium is required. Furthermore, a chemically defined medium for IVM in marmosets has apparently never been established or used to obtain offspring. A chemically defined medium is stable, with no lot-to-lot variation, as it is devoid of biologically derived materials (e.g., serum and extracts from yeast or plants). Hence, it is useful for analyzing the physical action of substrates, hormones, cytokines, and vitamins, on preimplantation oocytes and embryos. Moreover, studies of chemically defined medium in nonhuman primates have potential application to human ART, as a chemically defined medium prevents transmission of zoonotic infectious diseases.
Porcine oocyte medium (POM) was first reported by Yoshioka et al. in 2008 [10] and devised based on porcine zygote medium [11]. In that report, they successfully produced porcine blastocysts from germinal vesicle-stage oocytes that were matured, fertilized, and cultured in chemically defined medium designed to closely mimic in vivo environmental conditions in the porcine oviduct and to support normal development of porcine zygotes in vitro. As pigs have many physiological similarities to primates, perhaps a medium for IVM of porcine oocytes would be useful for IVM of marmoset oocytes. Although the components of most media for human oocytes were unclear because they are proprietary commercial products, components of POM are in the public domain. Furthermore, to our knowledge, none of the media used for human oocyte have been reported to be optimal for marmoset oocytes. Therefore, the objective of the present study was to investigate the maturation of marmoset oocytes in several IVM media, including a chemically defined medium.
Section snippets
Animals
All experiments were conducted with permission from the Institutional Animal Care and Use Committee of the Central Institute for Experimental Animals (CIEA) and were performed in accordance with CIEA guidelines.
For oocyte collection, ovaries were obtained from female marmosets that were killed because of disease, accidents, or the other experiments. All animals were purchased from a marmoset breeding company for experimental animals (CLEA Japan, Inc., Tokyo, Japan).
Oocyte collection and oocyte IVM
To collect germinal
Effect of pFF on IVM of marmoset oocytes
To improve the culture condition for IVM of marmoset oocytes, the effect of pFF on IVM was investigated. Twelve female marmosets were used. The IVM rates in oocytes matured to both the MI and MII stages were significantly higher for oocytes cultured in Waymouth-pFF medium than those cultured in Waymouth medium (48.5% vs. 36.4%, respectively, and 36.1% vs. 24.8%; P < 0.05; Table 1). Based on these results, POM was evaluated in the next experiment.
Differences in oocyte maturation and degradation rates between POM and Waymouth medium
The IVM rates for oocytes that developed to the
Discussion
The present study demonstrated that the IVM conditions for porcine oocyte were also effective for IVM of marmoset oocytes, and the marmoset offspring were successfully obtained from oocytes matured in chemically defined medium, apparently for the first time.
The pFF has been used in IVM medium for porcine oocytes [15], because factors secreted from follicular cells play a crucial role supporting oocyte maturation [16], [17]. Adding pFF to IVM medium increased maturation rates to the MI and MII
Acknowledgments
We thank Dr. Toshio Ito (Central Institute for Experimental Animals) for animal management, and Dr. Osamu Takayama for data analysis. This study was supported by the Strategic Research Program for Brain Science (SRPBS) and Grants-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to E.S. This study was also partly supported by PRESTO of the Japan Science and Technology Agency and the Global COE program for human
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