낮은 농도의 Hypoxanthine과 FSH가 미성숙난자의 체외성숙에 미치는 영향 = The Effect of Low Concentrated Hypoxanthine and FSH in 10% FBS Supplemented Medium on Immature Oocyte in vitro Maturatio

국문 초록 (Abstract)

목 적: Hypoxanthine (Hx)과 FSH가 미성숙난자의 배양에 미치는 영향을 관찰하기 위해 미성숙난자를 배양하여 GVBD, MII기 발생률을 비교 관찰하였다. 연구방법: 단순배양액인 BSAL-XI-HTF 배양액을 사용하여 (1) 0.3% BSA mBASAL-XI-HTF (0.3% BSA 배양액), (2) 0.1 IU/ml FSH를 첨가한 0.3% BSA mBASAL-XI-HTF (FSH 0.3% BSA 배양액), (3) 10% FBS mBASAL-XI-HTF (10% FBS 배양액), (4) 0.1 IU/ml FSH를 첨가한 10% FBS mBASAL-XI-HTF (FSH 10% FBS 배양액)의 4종류의 배양액을 만들었고, 각 종류의 배양액에서 생쥐 난구세포로 둘러싸인 미성숙난자의 성숙을 3, 6, 18시간별로 비교 관찰하였다. 각 배양액에 1 mM, 2 mM, 4 mM 농도의 미성숙난자성숙억제제인 Hx을 섞어 난자의 성숙억제양상을 관찰하였고 GVBD와 MII기 발생률을 비교 관찰하였다. 결 과: Hx을 첨가하지 않은 4종류의 배양액에서 미성숙난자의 자연성숙은 3시간 내에 대부분 GVBD가 발생하였고 MII기로의 발육은 6시간 이후 발생하였다. 18시간 후 각 군의 배양에서 모두 유사한 GVBD 발생률을 보였다. 1 mM, 2 mM, 4 mM Hx 농도의 0.3% BSA 배양액과 10% FBS 배양액에서 난자의 성숙억제양상을 비교해 보면 4 mM 농도의 Hx 배양액에서 18시간 동안 완전한 성숙억제를 보였다. 2 mM 농도의 Hx 배양액에서도 18시간 배양까지 억제를 보였으나 4 mM 농도의 Hx 배양액보다 작은 난자성숙억제양상을 보였다. 1 mM 농도의 Hx 배양액에서는 모두 난자성숙억제를 보이지 못했다. FSH를 첨가한 배양액에서는 2 mM 농도의 Hx 뿐만 아니라 1 mM 농도의 Hx 첨가에서도 초기 3시간까지 GVBD 성숙이 억제되었다. 또 같은 농도의 Hx을 함유하고 FSH를 첨가하지 않은 배양액에 비해 FSH를 첨가한 배양액에서 3시간, 6시간 동안 GVBD 발생을 더 억제하였다. 그러나 18시간 배양 후 난자성숙이 회복됨을 보였다. 이 결과로 FSH가 배양초기에 난자성숙을 억제하나 후기에는 성숙을 촉진함을 알 수 있었다. 18시간 후 MII기발육은 4 mM 농도의 Hx을 함유한 모든 배양액에서 낮은 발생률을 보였고, FSH를 함유한 10% FBS 배양액에 1 mM, 2 mM의 낮은 농도의 Hx을 첨가한 배양액은 다른 배양액에 비해 통계학적으로 높은 MII기 발생률을 보였다. 결 론: 본 실험에서 낮은 농도의 Hx과 FSH는 FBS를 함유한 배양액에서 미성숙난자의 초기배양 동안 성숙을 억제한 후 MII기 성숙을 촉진함을 알 수 있었다.

다국어 초록 (Multilingual Abstract)

Objective: We examined the effect of different culture media on oocyte maturation. Methods: Four groups of media, (1) 0.3% BSA mBASAL-XI-HTF, (2) 0.3% BSA mBASAL-XI-HTF with FSH, (3) 10% FBS mBASAL-XI-HTF and (4) 10% FBS mBASAL-XI-HTF with FSH were prepared. Mouse cumulus enclosed oocytes (CEOs) were incubated in each group of medium. Hypoxanthine (Hx) was mixed to each group of medium in increasing concentrations of 1 mM, 2 mM and 4 mM. CEOs were incubated and assessed for GVBD and MII development at 3, 6, 18 hours. Results: CEOs maturation to GVBD was seen in all four groups during 3 hours of culture, however MII stage of oocytes was seen after 6 hours. Complete arrest of GV stage in 4 mM Hx media without FSH and partial arrest in 2 mM Hx media without FSH were seen during 18 hours of culture but development was not suppressed in 1 mM Hx media without FSH. More prominent GVBD suppression was noted at early 3, 6 hours culture in 1 mM, 2 mM Hx media with FSH compared to media without FSH. But the suppression was recovered at 18 hours. This result suggests that low concentrated Hx and FSH supplemented media can suppress CEOs maturation during early culture period but recovery is resumed or even stimulated at late period. 1 mM, 2 mM Hx 10% FBS medium with FSH had significantly higher rates of MII development (71.7%, 66.7%) at 18 hours compared to other media. Conclusion: Our results show that low concentrated Hx and FSH supplemented 10% FBS media may stimulate MII development after an initial inhibitory effect.

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