2015 Fiscal Year Research-status Report
| Project/Area Number |
26870839
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| Research Institution | National Agriculture and Food Research Organization |
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Principal Investigator |
ソムファイ タマス 国立研究開発法人農業・食品産業技術総合研究機構, 畜産研究部門 家畜育種繁殖研究領域, 主任研究員 (90547720)
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| Project Period (FY) |
2014-04-01 – 2017-03-31
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| Keywords | ガラス化保存 / ブタ / 未成熟卵子 / レドックス状態 / 成熟培地 / 胚発生 / アポトーシス / 遺伝発現 |
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| Outline of Annual Research Achievements |
We analyzed the integrity of gap junctions (GJ) in vitrified immature oocytes by immune- staining and laser scanning confocal microscopy. Vitrification reduces the abundance of GJs in the perivitelline space by approximately 30%. However, cumulus expansion during IVM was identical between vitrified and non-vitrified oocytes. The results suggests, that despite of the significant loss in GJs during vitrification/warming, the majority (70%) of GJs remain intact, which is enough to facilitate cumulus expansion during IVM. Hence, vitrification by the current method may not dramatically reduce GJ communication between the cumulus and the oocyte. This result is supported by our previous results showing similar GSH and ATP levels in vitrified and non-vitrified oocytes. Microscopic analysis of oocytes revealed that cryoprotectants causes sub-lethal damages in nucleolus and microfilaments which, however, recover during subsequent IVM culture. Also, we found similar mean RNA levels in vitrified and non-vitrified oocytes after sampling over 2000 oocytes in a total of 3 replications. Hence, vitrification by the current method may not cause major RNA degradation. Based on last year`s results, apoptotic process is suspected as the main factor for the reduced developmental competence of vitrified oocytes. Accordingly, the anti-apoptotic agent Resveratrol improved blastocyst development of vitrified oocytes (work in progress). We have performed additional replications for microarray analysis. Validation of the results for the 3 repliction by RT-qPCR is in progress.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We have optimized the vitrification system and analyzed the effects of vitrification on 1) nuclear progression and nuclear morphology, 2) metabolism (ATP levels), 3) redox status (glutathione levels), 4) apoptotic status and 5) gap junction integrity according to plan. Since vitrification damaged gap junctions, we added one more assay on cumulus expansion (which was not in the original plan). Because low RNA levels were detected in oocytes, we performed 2 additional replications for microarray to reduce false positive results. Therefore gene expression assay is slightly delayed. The results of the 3 microarray replications are now summarized and the validation of candidate genes by RT-qPCR is in progress.
Results show that vitrification does not alter metabolism and redox system in oocytes but triggers premature meiosis and apoptosis which suggests abnormal Ca2+ regulation. Also, vitrification causes gap junction damage; however, it is minor and did not affect cytoplasmic maturation and cumulus expansion. Total RNA levels are not affected by vitrification; however, several genes seem to be up and downregulated in vitrified oocytes and cumulus cells. Apoptosis seems to be a key factor contributing to reduced competence of vitrified oocytes. In accordance we have started testing anti-apoptotic agents such as Resveratrol on vitrified oocytes according to plan. Recent results show improved embryo development by Resveratrol. However, since this is a work in progress transfer of these embryos has not been performed yet. The results have been published according to plan.
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| Strategy for Future Research Activity |
Validation of the microarray results by RT-qPCR and application of the anti-apoptotic agents are in progress. Our first priority is to finish the validation of microarray results by RTqPCR. Optimization of IVM medium with anti-apoptotic agent Resveratrol will be finished within this year. Also, other anti-apoptotic and Ca2+ blocker agents (BAPTA-AM, Cyclosporine A) will be tested during IVM of vitrified oocytes to maximize blastocyst development. Finally, embryos produced from vitrified oocytes by optimized IVM/IVF will be transferred into recipients demonstrate their ability to develop to term. Regarding the publishing of results, at least 2 research papers will be submitted in this year; one describing nuclear and cytoplasmic changes on oocytes caused by vitrification and another describing altered gene expressions.
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| Causes of Carryover |
平成27年度に研究費の使用残額（177,917円）が生じた。これは、当該年度の研究費を効率的に使用した結果である。
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| Expenditure Plan for Carryover Budget |
上記理由により、発生した残額は平成28年度に繰り越し、同年度の研究計画に基づいて適正に使用する予定である。
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