小鼠SN、NSN卵体外成熟与核酸酶敏感性变化研究
摘要
完全生长的小鼠卵泡卵根据染色质构型可分为两种类型，即SN型和NSN型。SN型，即surrounded nucleolus，染色质高度凝集，集中围绕在核仁周围；NSN型，即non-surrounded nucleolus，染色质结构较松散，均匀分布于核中。又根据卵周围的卵丘细胞的有无将卵泡卵分成四种类型，即COC-NSN型(有卵丘细胞的NSN型卵)、COC-SN(有卵丘细胞的SN型卵)、DO-NSN(无卵丘细胞的NSN型卵)和DO-SN(无卵丘细胞的SN型卵)。本研究比较了各类型卵减数分裂过程中染色质构型、体外成熟能力以及Dnase Ⅰ和S1核酸酶敏感性变化。
1．卵泡卵用0.1μg／ml的Hoechst 33342短暂染核后在低强度汞灯光线下短时间内(低于15秒)观察可成功地将不同染色质结构的卵子分开，而且该操作程序不影响卵子的减数分裂能力(P＞0.05)。
2．完全生长的卵泡卵子中SN型比例为63.4％，NSN型为36.6％。在COC卵子中，SN型与NSN型的比例分别为72.5％，27.5％；DO卵子中，SN型与NSN型的比例分别为59.2％，40.8％。DO卵中NSN型卵子的比例显著高于COC卵中NSN型卵子的比例，SN型卵则相反(p＜0.01)。
3．各类型卵子体外减数分裂的能力存在极显著差异(p＜0.01)。生发泡破裂(GVBD)发育至第一次减数分裂中期(MI)能力，SN型卵高于NSN型，COC卵中的NSN型高于DO中的NSN型。排出第一极体(PBI)发育至第二次减数分裂中期(MII)的能力，SN型卵高于NSN型卵，COC卵中的NSN和SN型分别高于DO中的NSN型和SN型。
4．GV期NSN、SN型卵子DNase Ⅰ敏感性无显著差异(P＞0.05)，而在体外成熟培养后减数分裂的MI期S1核酸酶的敏感性NSN型卵显著高于SN型(P＜0.01)，在MII期两种类型卵子S1核酸酶的敏感性无显著差异(P＞0.05)。
总之，SN型卵与NSN型卵在染色质构型、GVBD能力、排出PBI能力与MI期S1核酸酶敏感性方面存在显著的差异。卵成熟前的生长过程中卵丘细胞的存在对卵减数分裂能力的获得性有重要作用。卵丘细胞与染色质构型间存在着协同作用。
According to chromatin organization, full growing mouse follicle oocytes were identified as SN-type, whose condensed chromatin surrounded nucleolus, or NSN-type, whose decondensed chromatin distributed in almost whole nuclear. Surrounded by cumulus, oocytes were also identified as COC-NSN or COC-SN, otherwise they were identified as DO-NSN or DO-SN. The objection of this study was to research the difference of each type oocytes in chromatin organization, mature competence and nuclease sensitivity in vitro.
Dyed by 0. 1μg/ml Hoechst 33342 for 10 minutes and exposed to lower irradiation shorter than 5 second oocytes could be successfully grouped without any meiosis competence losing.
In full growing follicle oocytes, COC-NSN, COC-SN, DO-NSN and DO-SN were respectively accounted for 8.6%, 22.7%, 28.0% and 40.7%. Total of SN-type oocytes was larger than that of NSN-type oocytes. Among DO oocytes percents of NSN-type oocytes, 40. 8%, was higher than that among COC ooctyes, 27. 5%, while percents of SN-type among DO oocytes, 59. 2%, was lower than that among COC oocytes, 72. 5%.
Ratio of oocytes undergoing GVBD and developing to MI stage in SN-type oocytes was higher than in NSN-type ooctyes while in COC-NSN oocytes was higher than in DO-NSN oocytes. Competence of oocytes' developing to MII after excluding FBI in SN-type oocytes was also higher than in NSN-type oocytes while in COC-NSN and in COC-SN oocytes were higher than in DO-NSN oocytes and in DO-SN oocytes respectively.
The DNase I sensitivity of each type oocytes in GV stage didn' t show significant difference. But NSN-type oocytes undergoing GVBD had more S1 nuclease sensitivity than SN-type oocytes in same stage while no significant difference between COC-type oocytes and DO-type oocytes. Little difference among oocytes in Mil stage in S1 nuclease sensitivity was found.
On the whole, significant difference exists between NSN-type and SN-type oocytes in chromatin organization, competence of undergoing GVBD or excluding PBI and in MI stage S1 nuclease sensitivity. Cumulus surround growing oocytes
are playing important roles on oocytes' obtaining meiosis competence, and are also in coordination with chromatin organization.
1．卵泡卵用0.1μg／ml的Hoechst 33342短暂染核后在低强度汞灯光线下短时间内(低于15秒)观察可成功地将不同染色质结构的卵子分开，而且该操作程序不影响卵子的减数分裂能力(P＞0.05)。
2．完全生长的卵泡卵子中SN型比例为63.4％，NSN型为36.6％。在COC卵子中，SN型与NSN型的比例分别为72.5％，27.5％；DO卵子中，SN型与NSN型的比例分别为59.2％，40.8％。DO卵中NSN型卵子的比例显著高于COC卵中NSN型卵子的比例，SN型卵则相反(p＜0.01)。
3．各类型卵子体外减数分裂的能力存在极显著差异(p＜0.01)。生发泡破裂(GVBD)发育至第一次减数分裂中期(MI)能力，SN型卵高于NSN型，COC卵中的NSN型高于DO中的NSN型。排出第一极体(PBI)发育至第二次减数分裂中期(MII)的能力，SN型卵高于NSN型卵，COC卵中的NSN和SN型分别高于DO中的NSN型和SN型。
4．GV期NSN、SN型卵子DNase Ⅰ敏感性无显著差异(P＞0.05)，而在体外成熟培养后减数分裂的MI期S1核酸酶的敏感性NSN型卵显著高于SN型(P＜0.01)，在MII期两种类型卵子S1核酸酶的敏感性无显著差异(P＞0.05)。
总之，SN型卵与NSN型卵在染色质构型、GVBD能力、排出PBI能力与MI期S1核酸酶敏感性方面存在显著的差异。卵成熟前的生长过程中卵丘细胞的存在对卵减数分裂能力的获得性有重要作用。卵丘细胞与染色质构型间存在着协同作用。
According to chromatin organization, full growing mouse follicle oocytes were identified as SN-type, whose condensed chromatin surrounded nucleolus, or NSN-type, whose decondensed chromatin distributed in almost whole nuclear. Surrounded by cumulus, oocytes were also identified as COC-NSN or COC-SN, otherwise they were identified as DO-NSN or DO-SN. The objection of this study was to research the difference of each type oocytes in chromatin organization, mature competence and nuclease sensitivity in vitro.
Dyed by 0. 1μg/ml Hoechst 33342 for 10 minutes and exposed to lower irradiation shorter than 5 second oocytes could be successfully grouped without any meiosis competence losing.
In full growing follicle oocytes, COC-NSN, COC-SN, DO-NSN and DO-SN were respectively accounted for 8.6%, 22.7%, 28.0% and 40.7%. Total of SN-type oocytes was larger than that of NSN-type oocytes. Among DO oocytes percents of NSN-type oocytes, 40. 8%, was higher than that among COC ooctyes, 27. 5%, while percents of SN-type among DO oocytes, 59. 2%, was lower than that among COC oocytes, 72. 5%.
Ratio of oocytes undergoing GVBD and developing to MI stage in SN-type oocytes was higher than in NSN-type ooctyes while in COC-NSN oocytes was higher than in DO-NSN oocytes. Competence of oocytes' developing to MII after excluding FBI in SN-type oocytes was also higher than in NSN-type oocytes while in COC-NSN and in COC-SN oocytes were higher than in DO-NSN oocytes and in DO-SN oocytes respectively.
The DNase I sensitivity of each type oocytes in GV stage didn' t show significant difference. But NSN-type oocytes undergoing GVBD had more S1 nuclease sensitivity than SN-type oocytes in same stage while no significant difference between COC-type oocytes and DO-type oocytes. Little difference among oocytes in Mil stage in S1 nuclease sensitivity was found.
On the whole, significant difference exists between NSN-type and SN-type oocytes in chromatin organization, competence of undergoing GVBD or excluding PBI and in MI stage S1 nuclease sensitivity. Cumulus surround growing oocytes
are playing important roles on oocytes' obtaining meiosis competence, and are also in coordination with chromatin organization.
引文
1 Honglin liu, Fugaku Aoki. Transcriptional activity associated with meiotic competence in fully grown mouse GV oocytes. Zygote, 10:327-332
2 Fuente RDL, Eppig JJ, Transcriptional activity of the mouse oocyte genome: companion granulosa cells modulate transcription and chromatin remodeling. Dev Biol, 2001, 229:224-236
3 Debey P, Szllsi MS, Szllsi D et al. Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. Mol Reprod Dev, 1993, 36:59-74
4 Bouniol-Baly C, Hamraoui L, Guibert J et al. Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes. Biol Reprod, 1999, 60:580-587
5 Wickramasinghe D, Ebert KM, Albertini DF. Meiotic competence acquisition is associated with the appearance of M-Phase characteristics in growing mouse oocytes. Dev Biol, 1991,143:162-172
6 Zucotti M, Rossi PG, Martinez A et al., Meiotic and developmental competence of mouse antral oocytes. Biol. Reprod., 1998, 58:700-704
7 Worrad DM, Ram PT, Schultz RM, Regulation of gene expression in the mouse oocyte and early preimplantaion embryo: Developmental changes in Spland TATA binding protein, TBP. Development, 1994, 120:2347-2357
8 Parfenov V, Potchukalina G, Dudina L et al., Human antral follicles: Oocytes, nucleus and the karyosphere formation (electron microscopic and autoradiographic data). Gamete Res, 1989, 22:219-231
9 Mattson BA, Albertini DF. Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev, 1990, 25:374-383
10 Lefevre B, Gougeon A, Nome F. et al., IN vivo changes in oocyte germinal vesicle related to follicular quality and size at mid-follicular phase during stimulated cycles in the cynomolgus mondey. Reprod. Nutr. Dev. 1989, 29:523-532
11 Schramm RD, Tennier MT, Boatman DE et al., Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys. Biol. Repro., 1993, 48:349-356
12 Mandl AM, Preovulatory changes in the oocyte of the adult rat. Proc. R. Soc. 1962, London, 158:105-118
13 McGaughey RW, Montgomery DH, Richter JD. Germinal vesicle configurations and patterns of polypeptide synthesis of porcine oocytes from antral follicles of different sizes as related to their competency for spontaneous maturation. J. Exp. Zool., 1979, 209:239-254
14 Zucotti M, Piccinelli A, Rossi PG et al. Chromatin organization during mouse oocyte growth. Mol. Reprod. Dev., 1995, 41:479-485
15 Zucotti M, Rossi PG, Martinez A et al., Meiotic and developmental competence of mouse antral oocytes. Biol. Reprod., 1998, 58:700-704
16 陈大元，受精生物学——受精机制与生殖工程，科学出版社，2000年，第一版：146-164
17 Chesnel F, Eppig JJ, Induction of precocious germinal vesicle breakdown(GVB) by GVB-incompetent mouse oocytes: Possible role of MAP kinases rather than p34~(cdc2) kinase. Biol. Reprod., 1995, 52:895-902
18 Chesnel F, Eppig JJ, Synthesis and accumulation of p34~(cdc2) and cyclin Bin mouse oocytes during acquisition of competence to resume meiosis. Mol. Reprd. Dev., 1995,40:503-508
19 Chesnel F, Wigglesworth K, Eppig JJ, Acquisition of meiotic competence by denuded mouse oocytes: Participation of somatic-cell products and cAMP. Dev. Boil., 1994,161:285-295
20 Canipari R., Palombi F, Riminucci M et al., Early programming of maturation competence in mouse oogenesis. Dev. Biol. 1984, 102:519-524
21 http://rsb.info.nih.gov/nih-image/
22 Wyllie AH (1980). Glucocorticoid-induced thymocyte apoptosis is associated With engoeneous cndonuclease activation. Nature 284:555-556
23 Wiltshire T, Park C, Handel MA, Chromatin Configuration during Meiosis Ⅰ Prophase of spermatogenesis., Mol. Reprod. Dev. 1998, 49:90-8
24 Eppig JJ, Schultz RM, O Brien M et al. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev. Biol., 1994, 164:1-9
25 Eppig jj, Chesnel F, Hirao Y, Oocyte control of granulose cell development: How and why. Hum. Reprod. 12 Natl. Suppl. JBFS, 1997, 2:127-13
26 Bachvarova R, Gene expression during oogenesis and oocye development in mammals. Developmental Biology: A comprehensive Synthesis, 1985, 1:453-524
27 Wickramasinghe D, Albertini DF, Centrosome phosphorylation and the developmental
28 expression of meiotic competence in mouse oocyte. Dev. Biol., 1992, 152:60-72
29 Mitra J, Schultz RM, Regulation of the acquisition of meiotic competence in the mouse: Changes in the subcellular localization of cdc2, cyclin bl, cdc 25c and weel, and in the concentration of these proteins and their transcripts. J. Cell Sci. 1996, 109:2407-2415
29 De Vantery C, Stutz A, Vassalli JD et al., Acquisition of meiotic competence in growing mouse oocytes is controlled at both translational and posttranslational levels. Dev. Biol., 1997, 187:43-54
2 Fuente RDL, Eppig JJ, Transcriptional activity of the mouse oocyte genome: companion granulosa cells modulate transcription and chromatin remodeling. Dev Biol, 2001, 229:224-236
3 Debey P, Szllsi MS, Szllsi D et al. Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. Mol Reprod Dev, 1993, 36:59-74
4 Bouniol-Baly C, Hamraoui L, Guibert J et al. Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes. Biol Reprod, 1999, 60:580-587
5 Wickramasinghe D, Ebert KM, Albertini DF. Meiotic competence acquisition is associated with the appearance of M-Phase characteristics in growing mouse oocytes. Dev Biol, 1991,143:162-172
6 Zucotti M, Rossi PG, Martinez A et al., Meiotic and developmental competence of mouse antral oocytes. Biol. Reprod., 1998, 58:700-704
7 Worrad DM, Ram PT, Schultz RM, Regulation of gene expression in the mouse oocyte and early preimplantaion embryo: Developmental changes in Spland TATA binding protein, TBP. Development, 1994, 120:2347-2357
8 Parfenov V, Potchukalina G, Dudina L et al., Human antral follicles: Oocytes, nucleus and the karyosphere formation (electron microscopic and autoradiographic data). Gamete Res, 1989, 22:219-231
9 Mattson BA, Albertini DF. Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev, 1990, 25:374-383
10 Lefevre B, Gougeon A, Nome F. et al., IN vivo changes in oocyte germinal vesicle related to follicular quality and size at mid-follicular phase during stimulated cycles in the cynomolgus mondey. Reprod. Nutr. Dev. 1989, 29:523-532
11 Schramm RD, Tennier MT, Boatman DE et al., Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys. Biol. Repro., 1993, 48:349-356
12 Mandl AM, Preovulatory changes in the oocyte of the adult rat. Proc. R. Soc. 1962, London, 158:105-118
13 McGaughey RW, Montgomery DH, Richter JD. Germinal vesicle configurations and patterns of polypeptide synthesis of porcine oocytes from antral follicles of different sizes as related to their competency for spontaneous maturation. J. Exp. Zool., 1979, 209:239-254
14 Zucotti M, Piccinelli A, Rossi PG et al. Chromatin organization during mouse oocyte growth. Mol. Reprod. Dev., 1995, 41:479-485
15 Zucotti M, Rossi PG, Martinez A et al., Meiotic and developmental competence of mouse antral oocytes. Biol. Reprod., 1998, 58:700-704
16 陈大元，受精生物学——受精机制与生殖工程，科学出版社，2000年，第一版：146-164
17 Chesnel F, Eppig JJ, Induction of precocious germinal vesicle breakdown(GVB) by GVB-incompetent mouse oocytes: Possible role of MAP kinases rather than p34~(cdc2) kinase. Biol. Reprod., 1995, 52:895-902
18 Chesnel F, Eppig JJ, Synthesis and accumulation of p34~(cdc2) and cyclin Bin mouse oocytes during acquisition of competence to resume meiosis. Mol. Reprd. Dev., 1995,40:503-508
19 Chesnel F, Wigglesworth K, Eppig JJ, Acquisition of meiotic competence by denuded mouse oocytes: Participation of somatic-cell products and cAMP. Dev. Boil., 1994,161:285-295
20 Canipari R., Palombi F, Riminucci M et al., Early programming of maturation competence in mouse oogenesis. Dev. Biol. 1984, 102:519-524
21 http://rsb.info.nih.gov/nih-image/
22 Wyllie AH (1980). Glucocorticoid-induced thymocyte apoptosis is associated With engoeneous cndonuclease activation. Nature 284:555-556
23 Wiltshire T, Park C, Handel MA, Chromatin Configuration during Meiosis Ⅰ Prophase of spermatogenesis., Mol. Reprod. Dev. 1998, 49:90-8
24 Eppig JJ, Schultz RM, O Brien M et al. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev. Biol., 1994, 164:1-9
25 Eppig jj, Chesnel F, Hirao Y, Oocyte control of granulose cell development: How and why. Hum. Reprod. 12 Natl. Suppl. JBFS, 1997, 2:127-13
26 Bachvarova R, Gene expression during oogenesis and oocye development in mammals. Developmental Biology: A comprehensive Synthesis, 1985, 1:453-524
27 Wickramasinghe D, Albertini DF, Centrosome phosphorylation and the developmental
28 expression of meiotic competence in mouse oocyte. Dev. Biol., 1992, 152:60-72
29 Mitra J, Schultz RM, Regulation of the acquisition of meiotic competence in the mouse: Changes in the subcellular localization of cdc2, cyclin bl, cdc 25c and weel, and in the concentration of these proteins and their transcripts. J. Cell Sci. 1996, 109:2407-2415
29 De Vantery C, Stutz A, Vassalli JD et al., Acquisition of meiotic competence in growing mouse oocytes is controlled at both translational and posttranslational levels. Dev. Biol., 1997, 187:43-54