The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations () according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid () significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of folic acid to culture medium improved in vitro development of pig PA embryos.

Inositol 1,4,5-trisphosphate (IP₃) plays an important role in the release of Cα²+ from intracellular stores into the cytoplasm in a variety of cell types. IP₃ translocation dynamics have been studied in response to many types of cell signals. However, the dynamics of cytosolic IP₃ in salivary acinar cells are unclear. A green fluorescent protein (GFP)-tagged pleckstrin homology domain (PHD) was constructed and introduced into a phospholipase C δ1 (PLC δ1) transgenic mouse, and then the salivary acinar cells were isolated. GFP-PHD was heterogeneously localized at the plasma membrane and intracellular organelles in submandibular gland and parotid gland cells. Application of trypsin, a G protein-coupled receptor activator, to the two types of cells caused an increase in GFP fluorescence in the cell cytoplasm. The observed time course of trypsin-evoked IP₃movement in acinar cells was independent of cell polarity, and the fluorescent label showed an immediate increase throughout the cells. These results suggest that GFP-PHD in many tissues of transgenic mice, including non-cultured primary cells, can be used as a model for examination of IP₃intracellular dynamics.

This study was carried out to investigate the effect of morphology of oocytes, kinds of media, cysteine and myo-inositol supplementation on IVM rate of porcine oocytes. Cumulus- enclosed oocytes were incubated in maturation NCSU-23 and TCM-199 medium with supplementation with 3, 5, 10, 20 mM myo-inositol and 0.05, 0.1, 0.5, 1.0 mM cysteine. 1. When classified by morphology, excellent, good and fair of cumulus-enclosed oocytes were incubated for 48 hrs and the IVM rate were , respectively. The rate were greater in oocytes with excellent cumulus cells than those without cumulus cells. 2. The IVM rate of oocytes cultured in TCM-199 and NCSU- 23 medium supplementation or non-supplementation with 1.0 mM myo-inositol were , respectively. Supplementation with myo-inositol significantly increased the IVM rate of oocytes. 3. The IVM rate of oocytes cultured in NCSU-23 medium supplementation of 3, 5, 10, 20 mM myo-inositol for 48 hrs were , respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM myo-inositol were significantly increased compared to control (). 4. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 media supplement with 0.3, 0.5, 1.0, 2.0 mM myo-inositol were , respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM cysteine were significantly increased compared to control ().

본 연구는 미성숙돼지 난모세포의 체외 성숙에 있어서 myo-inositol의 영향을 알아보기 위하여 실시하였다. 미성숙 돼지 난모세포을 myo-inositol을 포함하는 또는 포함하지 않은 체외 성숙배지에서 44시간 체외 성숙을 유도하였을 때 성숙율은 myo-inositol을 첨가한 성숙배지에서 성숙시킨 실험구에 유의하게 높았다(P<0.05). Myo-inositol 에 의한 성숙율 향상에 있어서 난구세포의 영향을 알아보기 위하여 난구세포의 치밀도에 따라 분류하여 미성숙 난모세포을 myo-inositol을 포함하는 체외 성숙배지에서 배양하였을 때 난구세포가 치밀한 미성숙 난모세포에서가 더 높은 성숙율을 나타내었다(P<0.05). 그러나 난구세포가 치밀하지 않은 미성숙 난모세포도 myo-inositol을 포함하는 성숙배지에서 배양하면 성숙율은 대조구에 비하여 유의하게 높았다(P<0.05). 이러한 결과는 돼지 난모세포의 체외 성숙배지에 myo-inositol의 첨가는 체외 성숙율을 향상시킬 수 있음을 나타내고 있다.

흰쥐의 뇌로부터 읽는구조 전체를 포함하는 1.8kbp 크기인 IP_3K를 암호화 하는 IP_3KcDNA 유전자속의 NotⅠ부위 GCGGCCGC를 GCAGCCGC로 site-directed mutagenesis 하여 얻은 변이 IP_3KcDNA를 pSP72·Not2 운반체의 EcoR I 부위에 클로닝하여 증폭시키고, 유전자 재조합 과정을 거쳐서 포유동물의 발현 운반체인 pZIP·NeoSV(X)의 NotⅠ부위에 IP_3KcDNA를 서브클론하였다. 이것을 CCL39 hamster lung fibroblasts 세포에 transfection 하여 발현시키고, anti-IP_3K antibody를 사용하여 Western blot 법으로 효소량과 활성도를 각각 측정한 결과, 효소량은 5배, 효소의 활성도는 무려 16배로서 기대하였던 것보다 훨씬 발현효율이 높았다. 또한 흰쥐의 각 조직속에 IP_3K의 분포와 생화학적인 특성이 논의되었다.

본 연구에서는 혼합지방과 쇠기름, 옥수수기름, 들기름을 각각 15%(w/w) 수준으로 Sprague Dawley종 수컷 주에게 섭취시키면서 식이와 동시에 화학적 발암원인 DMH를 투여하여 식이지방의 조성이 대장점막 및 인지질과 phosphatidyl inositol의 지방산 조성에 미치는 영향을 살펴보았다. 대장점막과 인지질 및 PI의 지방산 조성은 식이지방의 지방산 조성에 의해 영향을 받았다. 대장점막 인지질의 C_18:2 분포는 식이중의 linoleic acid함량에 비례하는 경향을 보여 CO>BF>PO>BT군의 순서로 유의성있게 낮아졌으나 C_20:4 분포는 C_18:2 분포와는 다르게 CO군과 BT군이 같은 수준으로 높았으며 PO군이 가장 낮아 식이의 linoleic acid 함량에 꼭 비례하지는 않았다. DMH를 투여한 경우에는 PO군을 제외한 세 군에서 C_20:4 함량이 증가하였다(P<0.05). PI의 지방산 조성도 인지질과 비슷한 양상을 보여 C_18:2 분포는 CO군에서 가장 높았으며 C_20:4는 BT군이 대조군과 DMH처리군 모두에서 PO군 보다 유의성있게 높았다.

흰쥐로부터 뇌, 심장, 관장, 폐, 신장 및 흉선 등 12종류의 기관을 계통별로 해부하여 homogenates를 만들고 cytosol을 분획한 다음, 이것을 효소원으로 하여 inosltol phosphates 대사회로에서 핵심효소인 PLC, IP_3K 및 Ins(1, 4, 5)P_3 5-phosphatase의 활성도를 조직별로 비교 측정하였다. 흰쥐의 뇌에서 분리 정제된 53KDa의 IP_3K를 Balb/c 마우스에 면역 후 비장 세포를 얻어서 골수암세포인 myeloma cells (SP_2/Ag 0-14)에 세포융합, 스크리닝 및 클로닝과정을 거쳐서 anti IP_3K murine monoclonal antibodies를 만들었다. 이 과정에서 18개의 hybridoma clone이 얻어졌고 그 중 10개의 clone만이 nonspecific binding에 기인되는background가 낮았다. Ascitic fluid를 생산시킨 후 그 Ab를 Affi-gel 15로 정제하여 IgG의 subtype을 결정하였다. 이 항체들 중에서 IgG_1과 IgG_2b를 함께 사용하여 조직의 IP_3K에 대한 immuno 반응성을 검증한 결과 대체로 비슷한 비경쟁적 저해를 보였으며 뇌조직의 IP_3K가 예민한 반응을나타내었고, 심장조직에서는 현저히 activity가 낮았다. 흰쥐의 뇌로부터 16, 100배로 정제된IP_3K효소를 사용하여 얻어진 Km값은 1.8mM, 최대반응속도V_max 값은 5.41μ㏖/mln/㎖이었다. Western blot 결과 심장조직에서는 40KDa의 IP_3K만이 관찰되었으며, 뇌속에는 면역학적으로 서로 다른 3가지의 IP_3K(53, 51 및 40KDa)가 존재하였다. 그 중 53KDa의 단백질이 활성이 큰 주 효소이며, 1.8Kb의 IP_3K gene을 완전히 encoding하는 ^32P-labeled cDNA를 probe로 만들어 Northern blot법으로 IP_3K의 mRNA를 정량한 결과 성장단계별로 이들은 모두 transcriptional 수준으로 조절받고 있음을 밝혔다.

BACKGROUND Ca2+ oscillations during fertilization induce eggs activation and embryonic development in mammalian eggs.. The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is in charge of Ca2+ oscillations for the release of stored Ca2+ from the endoplasmic reticulum. The capacity of this oscillation is obtained during egg maturation and corresponds with an increase in the sensitivity of the IP3R1 and their localization in cytoplasm. Cluster formation of IP3R1 in the egg cortex is important to initiation of Ca2+ oscillations during egg and sperm fusion. In this study, we investigated that cell cycle–coupled redistribution of IP3R1 and Ca2+- oscillatory activity in mouse zygotes. MATERIALS AND METHODS Metaphase II arrested eggs were collected from ICR female mouse after super ovulation induction. At 14 hr post hCG, MII eggs were collected, and artificially activated in Ca2+ free CZB medium with 10 mM SrCl2 for 2 hrs. Pronuclear zygotes (PN) were collected from Strontium activated eggs at 8 hr post activation, and the first mitotic eggs were collected at 16~17 hr post activation. To identify cell cycle coupled IP3R1 redistribution, MII eggs, zygotes, and first mitotic eggs were collected, and fixed for immunostaining with anti-IP3R1antibody (CT-1) and observed on CLSM. Ca2+-oscillatory activity was monitored with fluorescence microscope mounted SimplePCI program (Hamamatsu) after injection of cRNA of mouse phospholipase C zeta (mPLCZ). RESULT IP3R1 were shown clusters, 1~2 um in diameter, in cortex of ovulated MII eggs with high Ca2+ oscillatory activity by mPLCZ injection. These eggs represent more than 6 spikes per 60 min. However, IP3R1 clusters were disappeared in PN eggs and these eggs showed very low Ca2+- oscillatory activity by mPLCZ. In mitosis I stage eggs, clusters of IP3R1 were appeared and Ca2+-oscillatory activity was reactivated slightly (2 spikes per 60 min). CONCLUSIOINS This study introduced the redistribution of IP3R1 clusters were occurred in egg activation according to cell cycle dependent manner. Also, functional modification of IP3R1 including protein phosphorylation was associated with cortical clustering of IP3R1 in cell cycle coupled Ca2+ oscillatory activity.

Inositol은 세포의증식 및 정보전달과정에 관여하는 phosphatidylinositol (PI)의 구성성분으로서 중요한 세포내 기능을 수행한다. P1는 세포내에서 특이적 인산화효소에 의하여 Pl4-phosphate(PIP), Pl4,5-phosphate()로 변환되며 PIP는 phospholipase C(PLC)에 의하여 세포내 second messengers인 1,2-diacylglycerol (DAG)와 inositol 1,4,5-triphos