担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cryobiol.2013.07.004

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CAMBRIDGE UNIV PRESS  

Medium that contains 17 beta-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte-granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17 beta-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 mu m (n = 191). Either steroid was necessary for maintainance of the organization of oocyte-granulosa cell complexes over the 14-day culture period. In the 17 beta-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22-24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17 beta-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17 beta-estradiol, can maintain the viability of bovine oocyte-granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose.

DOI： 10.1017/S0967199411000268

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 mu m in diameter) containing growing oocytes (approximately 60 mu m in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (P<0.05). Higher percentages of granulosa cell-enclosed oocytes were recovered from the follicles cultured in BPL-supplemented media with 0 and 25 ng/ml FSH, and the oocytes grew to 90 mu m or more in diameter. In FCS- and BFF-supplemented media, FSH increased the numbers of degenerating follicles. Next, vitrified-warmed secondary follicles were cultured in BPL-supplemented medium. One third of the follicles showed no degenerative signs, and the oocytes increased in diameter to 88.8 +/- 3.1 mu m after 4 weeks of culture. These results suggest that a BPL-supplemented medium supports oocyte growth in bovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.

DOI： 10.1262/jrd.10-047H

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The objective of the present study was to elucidate the involvement of FOXO3 in the activation of bovine primordial follicles. In immunohistochemistry, FOXO3 was detected in all of the oocytes in primordial and primary follicles. The FOXO3 decreased after treatment with FOXO3 small interfering RNAs (siRNAs). Ovarian tissues containing dominantly primordial follicles were treated with FOXO3 siRNAs and then xenografted to severe combined immune deficiency (SCID) mice. Two months after xenografting, some primordial follicles developed to the secondary and tertiary stages, and the total percentage of these developing follicles (secondary and tertiary follicles: 18 ± 7%) was higher than in the control grafts treated with control siRNA (7 ± 1%). It is thought that bovine primordial follicle activation is regulated by the FOXO3-dependent mechanism and that knockdown of FOXO3 induces the release of primordial follicles from FOXO3 suppression, initiating their growth. © 2011 by the Society for Reproduction and Development.

DOI： 10.1262/jrd.11-013H

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記述言語：英語   出版者・発行元：John Wiley and Sons Ltd  

Mammalian ovaries contain mixed populations of follicles at different developmental stages. A combination of vitrification and growth culture of ovarian follicles could provide the desired number of mature eggs from a preserved small amount of ovarian tissues. Secondary and primordial follicles from porcine and bovine ovaries were vitrified in solutions containing ethylene glycol, dimethyl sulfoxide and different concentrations of sucrose, and assessed via histological examination, viability staining, xenografting to immunodeficient mice, and in vitro culturing. Histological examination revealed the damage to oocytes and the damage to follicle components separately. The effects of sucrose in vitrification solutions on the follicles were different depending on the developmental stage of the follicle, oocyte size, cell type in the follicle, and species. Viability staining with fluorescein diacetate was useful to assess the damage to oocytes in secondary follicles. In the xenografts, vitrified bovine primordial and secondary follicles developed to the antral stage, and vitrified porcine primordial follicles developed to the secondary stage. Furthermore, bovine secondary follicles formed antrum-like structures in culture. These results suggest that histological examination and viability staining are valuable for assessing the direct effects of vitrification and warming conditions on follicles and oocytes, while xenografting and in vitro culturing can be useful for evaluating the developmental ability of vitrified follicles and oocytes. © Japan Society for Reproductive Medicine 2011.

DOI： 10.1007/s12522-011-0094-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE INC  

The objective was to cryopreserve porcine primordial follicles by vitrification and to assess the development of these follicles in xenografts. Ovarian tissues containing primordial follicles were collected from neonatal (15-d-old) piglets. They were vitrified in modified tissue culture medium (TCM)-199 containing 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, 20% (v/v) fetal calf serum, and 0, 0.25, or 0.5 M sucrose. After 1 wk of storage in liquid nitrogen (LN(2)), the tissues were warmed, and the morphology of follicles and oocytes was examined histologically. After vitrification in sucrose-free medium, there were 50 +/- 2 (mean +/- SEM; n = 10) follicles per tissue, in contrast with 108 +/- 10 (n = 10) in fresh tissues. Losses were attributed to puncturing oocytes during the vitrification-warming process, as oocytes were apparently normal after treatment of the sucrose-free vitrification solution without plunging into LN(2). When tissues were vitrified in sucrose-supplemented medium, loss of oocytes decreased (P < 0.05). However, the number of abnormal oocytes having nuclear shrinkage was increased (P < 0.05) by the addition of 0.5 M sucrose; this occurred in a small number of oocytes treated With sucrose-supplemented vitrification Solutions without vitrification. After 2 mo of xenografting, of vitrified-warmed tissues in SCID (severe combined immune deficiency) mice, primordial follicles developed to the secondary stage (accompanied by oocyte growth), whereas there was development to the antral stage in xenografts of fresh tissues. In conclusion, primordial follicles from neonatal pigs maintained their developmental ability after vitrification and warming, although their developmental rate was slower than that of the fresh control in xenografts. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2009.01.024

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記述言語：日本語   会議種別：口頭発表（招待・特別）  

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