(322) In Vitro Growth of Oocytes is Available in the Inbred C57BL/6 Strain

Fertile oocytes—those capable of developing to term after fertilization and embryo transfer—have been successfully produced in vitro from ovarian oocytes at various stages in mice. In vitro growth of oocytes is an efficient approach for analyzing gene function during oogenesis and assessing essential maternal factors. Additionally, it has potential applications in assisted reproductive technologies for infertility treatment. Therefore, establishing a widely applicable method for in vitro growth of oocytes is essential. On the other hand, the inbred C57BL/6 (B6) mouse strain is recognized as the standard model and is commonly used for studying both forward and reverse genetics. However, the gametes of B6 mice are known to be highly sensitive to the in vitro culture environment and manipulation. For instance, the ability of blastocysts produced by in vitro fertilization to develop into living offspring is considerably lower in B6 mice compared to ICR, B6D2F1, and B6CBF1 mice. This presents a major challenge in utilizing in vitro systems for B6 mice, and addressing this issue is crucial. The aim of this study is to establish a method for generating offspring from ovarian oocytes of B6 mice using in vitro growth.
Ovaries were obtained from B6 and B6D2F1 neonatal mice and cultured in alpha-MEM supplemented with 10% FBS and 5 µM ICI. After six days of culture, ICI was removed from the medium, and the ovaries were then cultured for an additional nine days. On day 15 of culture, secondary follicles were isolated from the ovaries. However, follicles from the ovaries of B6 were smaller than those of B6D2F1 mice. To promote granulosa cell proliferation, dibutyryl cyclic AMP (dbcAMP) was added to the ovarian culture medium. As a result, the number and diameter of secondary follicles isolated from dbcAMP-treated B6 ovaries were significantly higher (32.7 ± 5.2/ovary, 103.0 ± 6.5 µm) than those from untreated B6 ovaries (12.3 ± 3.7/ovary, 88.4 ± 5.3 µm) and untreated B6D2F1 ovaries (25.3 ± 5.7/ovary, 99.3 ± 8.0 µm). These secondary follicles were cultured further. We routinely used alpha-MEM supplemented with 5% FBS, 2% PVP, and 0.1 IU/mL FSH as the follicle culture medium. Here, to investigate the optimal follicle culture conditions, we assessed the effects of oxygen concentration (7% vs. 20%) and the supplementation of dbcAMP and activin A. We found that dbcAMP but not activin A supplementation critically increased granulosa cells. Oocytes grown in vitro under 7% oxygen developed to the blastocyst stage after in vitro maturation and fertilization at higher rate compared with 20% oxygen. Using the most optimal culture condition, we successfully produced 2-cell stage embryos from secondary follicles (87/144). Finally, these 2-cell embryos were transferred into pseudopregnant mice, resulting in the birth of seven offspring using in vitro growth of ovarian oocytes. To our knowledge, this is the first report demonstrating the acquisition of full developmental competency during in vitro oogenesis in the B6 strain. The present study will further facilitate the use of in vitro growth of ovaries and/or follicles to understand gene function and maternal factors.