猪卵巢卵母细胞体外成熟与体外受精的研究
摘要
本研究利用屠宰场采集的母猪卵巢，用机械法从卵巢表面直径为3～6mm的卵泡中分离紧密包裹三层以上卵丘细胞的卵丘—卵母细胞复合体(COC)，用于猪卵巢卵母钼胞体外成熟、体外受精及早期胚胎(体外受精卵)体外培养的研究。主要结果如下：
1．卵母细胞的体外成熟 采用微滴培养，卵丘—卵母细胞复合体体外培养42～44h后(38.8℃，5％CaO_2，100％相对湿度)，卵丘细胞扩展，部分卵母细胞排出第一极体。mTCM199+15％NCS(v／v)和NCSU23+10％PFF(v／v)为基础培养液用于卵母细胞体外成熟培养，在添加15iu／ml的PMSG和HCG时，卵母细胞体外培养成熟率分别为41.2±3.98％和38.28±4.02％；在添加20iu／mlPMSG和HCG时，卵母细胞体外培养成熟率为44.8±5.34％和39.12±1.25％，在添加相同浓度的激素时mTCM199+5％NCS(v／v)和NCSU23+10％PFF(v／v)对卵母细胞成熟率的影响差异不显著(P>0.05)。mTCM199+15iu／ml PMSG—15iu／ml HCG添加浓度为0，10％，15％，20％的血清(NCS)体外培养卵母细胞，卵母细胞成熟率分别为0，37.78±1.92％，43.33±2.72％，43.33±3.85％，添加组与不添加组之间差异极显著(P<0.01)：添加10％与20％组之间差异显著(P<0.05)；添加15％与20％组之间差异不显著(P>0.05)；添加10％与15％组之间差异显著(P<0.05)。在mTCM199+15％NCS(v／v)中共同添加0iu／ml、10iu／ml、15iu／ml、20iu／ml的相同剂量PMSG和HCG进行体外培养时，卵母细胞的成熟率分别为0，35.56±1.92％，41.2±3.98％，46.27±7.36％，添加组与未添加组之间差异极显著(P<0.01)，添加15iu／ml组与20iu／ml组之间的差异不显著(P>0.05)，添加15iu／ml组与添加10iu／ml组之间差异不显著(P>0.05)，添加20iu／ml组与添加10iu／ml组之间差异显著(P<0.05)。添加一定浓度的血清、激素有利于提高卵母细胞体外成熟率。
2．体外受精 将鲜精(或常温保存精液)在mTBM+100ug／ml肝素中运用“上游法”获能40～60min，然后将获能看的精子与成熟卵母细胞在mTBM-50
u g／。1肝素中共孵育6h （3．4aC，5％CO。，100％相对湿度）。受精后 24～28h有部
分受精卵排出第二极体，部分受精卵发生卵裂，总卵裂率为盯．9弧（“～50h观
察统计）。
3．早期胚胎的体外培养 体外受精后，三种培养体系被用于受精卵
的体外培养：1．TCM199＋15％NCS，11．输卵管上皮细胞＋TCM199＋15％NCS共培养系
统，fll．颗粒／卵丘细胞＋TCM199＋15％＼CS共培养系统，培养条件为38．S”C，
5 ％ CO。，100％相对湿度。三种培养体系中早期胚胎发育率分别为
38．49士6．59％；37．19IS．19％，37．85土4．25％（48～50h观察统计），三种培养体系
对早期胚胎发育率的影响差异不显著（P＞0．05）。在1中，胚胎发育到4细胞后便
不再向前发育，在＊和＊中，部分胚胎能越过 4细胞期发育到 8～16细胞期及至
桑檀胚期。桑堪胚发育率分别为（，12．84土6．04％，4．76土6．73％），11组与1组
间差异极显著中＜O．N），H组与＊I组间差异显著沪＜O．“），HI组与I组间无显
著差异（P＞0．05）．
总之，在本实验条件下，猪卵巢卵母细胞在体外条件下能够发育成熟，成熟
卵母细胞与体外获能的精子能进行体外受精发生卵裂，发育成胚胎；通过共培养
体系培养，早期胚胎能突破4细胞发育阻滞，发育到桑堪胚。
Porcine ovaries were obtained from Changsha Wulipai abattoir.
Cumulus-oocyte complexes (COC) with more than three layer' s compact cumulu cells were mechanically isolate& from 3~-6 mm sized ovarian surface follicles. The COC were cultured in vitro to study the porcine ovarian oocyte' s in vitro maturation (IVM), in vitro fertilization (lyE) and in vitro culture (IVC) of the early embryos. The results were presented as follows:
1. Oocyte' s in vitro maturation In the experiment, COCs were cultured in 300 microliters droplets (38. 8~C, 5%C02, 100% humidity). After cultured 42-44h, the cumulu cells expansion, some oocytes extrusion the first polar body (PBI). mTCM199-'-lO%NCS(v/v) and NCSU23-4-l0%PFF(v/v) were used to study their effects on oocyte' s IVM. The results show that, when supplemented with l5iu/ml PMSG and HCG the maturation rate were 41.2±3.%98 and 38.28±4.02% (P>0.03); When supplemented with 2Oiu/ml PMSG and HCG the maturation rate were 44. 8±5. 34% and 39. 12±l.25%(P>0. 05). When supplemented 0, 10%, 13%, 20%(v/v) NCS(New-born cattle serum) into mTCM199-15iu1"ml PMSG--lSiu ml HCG, the maturation rates were 0,37.78±1.92%, 43. 33±2.72% 43 33±3.85%, maturation rates were remarkbabiy different (P<0.0l) between the groups with lO%-----20%(vlv) serum and the group without supplemented serum, the difference of the maturation rate among 10% and 20% or 15% and 10% were significant (P<0. 05), the difference of the maturation rate among 15% and 20% was not significant (P>0. 05) . When supplemented 0, lOiu/ml, lSiu/ml, 2Oiu/ml PMSG and L-JCG into mTCM199~-l5%NCS, the maturation rates were 0, 35.56±1.92%, 41.2±3.98%, 46.27±7 36% the maturation rates between 0 (group) and l0iui'mb- 20iu11m1 (groups) were remarklablv different (P<0. 01); between l5iu/ml and 2Oiu ml or lSiu,"'mi and lOiu/ml were not significantly differen? (p>O.OS> between 2Oiu"ml (group) and lOiu/ml (group) were
3
significant different(p<0. 05). The results showed that add serum~ PMSG and HCG can improve oocyte' s maturation rate in vitro.
2. In vitro fertilization Fresh sperm were capacitated in medium mTBM+100 ~ g/ml Heparin with the "swim-up" method. After capacitated 40- 60 mm. the sperm and the maturation oocytes were incubated in mTBM+50 ii g/ml Heparin (39. 40C, 5%C02, 100% humidity) for Gb. After 24'-28h some fertifized eggs extrusion the second polar body (PB II) and some fertilized eggs were clevaged. The clevage rate was 37. 95%(48-50h).
3. Early embryo' s in vitro culture After in vitro fertilization, the fertilized eggs were cultured in three systems. I .TCM199+l5%NCS, II. Porcine oviduct epithelial cell monolayer (POEC)+TCM199-r15%NCS, III. granule/cumulus cell monolayer(G/C>TCM199+l5%NCS. The culture condition was 38. 80C, 5%C02, 100% humidity. Their development rates were 38. 49±6. 59% 37 19±5.19%,37.85±4.25%(48 -~ 50h), there were no significant different (P>0. 05) between them. In group I, the embryos were blocked at 4 cell stage, in group II and Ill some embryos can surmount 4 cell stage block and developed to 8-~l6 cell stage and morula. The morula rates were 0 12 84± 6. 04%, 4. 76 ±6. 73%. The difference of the morula rates among group II and I was remarkably (P0.05).
In sumftiary, under the present culture conditions, the porcine ovarian oocvtes can matured in vitro, fertilized in vitro and developed to early embryos. When the early embryos co-cultured with monolayer porcine oviduct epithelial cells or granual/cumulus cells some of them can surmount the 4 cell stage block and developed to morula
1．卵母细胞的体外成熟 采用微滴培养，卵丘—卵母细胞复合体体外培养42～44h后(38.8℃，5％CaO_2，100％相对湿度)，卵丘细胞扩展，部分卵母细胞排出第一极体。mTCM199+15％NCS(v／v)和NCSU23+10％PFF(v／v)为基础培养液用于卵母细胞体外成熟培养，在添加15iu／ml的PMSG和HCG时，卵母细胞体外培养成熟率分别为41.2±3.98％和38.28±4.02％；在添加20iu／mlPMSG和HCG时，卵母细胞体外培养成熟率为44.8±5.34％和39.12±1.25％，在添加相同浓度的激素时mTCM199+5％NCS(v／v)和NCSU23+10％PFF(v／v)对卵母细胞成熟率的影响差异不显著(P>0.05)。mTCM199+15iu／ml PMSG—15iu／ml HCG添加浓度为0，10％，15％，20％的血清(NCS)体外培养卵母细胞，卵母细胞成熟率分别为0，37.78±1.92％，43.33±2.72％，43.33±3.85％，添加组与不添加组之间差异极显著(P<0.01)：添加10％与20％组之间差异显著(P<0.05)；添加15％与20％组之间差异不显著(P>0.05)；添加10％与15％组之间差异显著(P<0.05)。在mTCM199+15％NCS(v／v)中共同添加0iu／ml、10iu／ml、15iu／ml、20iu／ml的相同剂量PMSG和HCG进行体外培养时，卵母细胞的成熟率分别为0，35.56±1.92％，41.2±3.98％，46.27±7.36％，添加组与未添加组之间差异极显著(P<0.01)，添加15iu／ml组与20iu／ml组之间的差异不显著(P>0.05)，添加15iu／ml组与添加10iu／ml组之间差异不显著(P>0.05)，添加20iu／ml组与添加10iu／ml组之间差异显著(P<0.05)。添加一定浓度的血清、激素有利于提高卵母细胞体外成熟率。
2．体外受精 将鲜精(或常温保存精液)在mTBM+100ug／ml肝素中运用“上游法”获能40～60min，然后将获能看的精子与成熟卵母细胞在mTBM-50
u g／。1肝素中共孵育6h （3．4aC，5％CO。，100％相对湿度）。受精后 24～28h有部
分受精卵排出第二极体，部分受精卵发生卵裂，总卵裂率为盯．9弧（“～50h观
察统计）。
3．早期胚胎的体外培养 体外受精后，三种培养体系被用于受精卵
的体外培养：1．TCM199＋15％NCS，11．输卵管上皮细胞＋TCM199＋15％NCS共培养系
统，fll．颗粒／卵丘细胞＋TCM199＋15％＼CS共培养系统，培养条件为38．S”C，
5 ％ CO。，100％相对湿度。三种培养体系中早期胚胎发育率分别为
38．49士6．59％；37．19IS．19％，37．85土4．25％（48～50h观察统计），三种培养体系
对早期胚胎发育率的影响差异不显著（P＞0．05）。在1中，胚胎发育到4细胞后便
不再向前发育，在＊和＊中，部分胚胎能越过 4细胞期发育到 8～16细胞期及至
桑檀胚期。桑堪胚发育率分别为（，12．84土6．04％，4．76土6．73％），11组与1组
间差异极显著中＜O．N），H组与＊I组间差异显著沪＜O．“），HI组与I组间无显
著差异（P＞0．05）．
总之，在本实验条件下，猪卵巢卵母细胞在体外条件下能够发育成熟，成熟
卵母细胞与体外获能的精子能进行体外受精发生卵裂，发育成胚胎；通过共培养
体系培养，早期胚胎能突破4细胞发育阻滞，发育到桑堪胚。
Porcine ovaries were obtained from Changsha Wulipai abattoir.
Cumulus-oocyte complexes (COC) with more than three layer' s compact cumulu cells were mechanically isolate& from 3~-6 mm sized ovarian surface follicles. The COC were cultured in vitro to study the porcine ovarian oocyte' s in vitro maturation (IVM), in vitro fertilization (lyE) and in vitro culture (IVC) of the early embryos. The results were presented as follows:
1. Oocyte' s in vitro maturation In the experiment, COCs were cultured in 300 microliters droplets (38. 8~C, 5%C02, 100% humidity). After cultured 42-44h, the cumulu cells expansion, some oocytes extrusion the first polar body (PBI). mTCM199-'-lO%NCS(v/v) and NCSU23-4-l0%PFF(v/v) were used to study their effects on oocyte' s IVM. The results show that, when supplemented with l5iu/ml PMSG and HCG the maturation rate were 41.2±3.%98 and 38.28±4.02% (P>0.03); When supplemented with 2Oiu/ml PMSG and HCG the maturation rate were 44. 8±5. 34% and 39. 12±l.25%(P>0. 05). When supplemented 0, 10%, 13%, 20%(v/v) NCS(New-born cattle serum) into mTCM199-15iu1"ml PMSG--lSiu ml HCG, the maturation rates were 0,37.78±1.92%, 43. 33±2.72% 43 33±3.85%, maturation rates were remarkbabiy different (P<0.0l) between the groups with lO%-----20%(vlv) serum and the group without supplemented serum, the difference of the maturation rate among 10% and 20% or 15% and 10% were significant (P<0. 05), the difference of the maturation rate among 15% and 20% was not significant (P>0. 05) . When supplemented 0, lOiu/ml, lSiu/ml, 2Oiu/ml PMSG and L-JCG into mTCM199~-l5%NCS, the maturation rates were 0, 35.56±1.92%, 41.2±3.98%, 46.27±7 36% the maturation rates between 0 (group) and l0iui'mb- 20iu11m1 (groups) were remarklablv different (P<0. 01); between l5iu/ml and 2Oiu ml or lSiu,"'mi and lOiu/ml were not significantly differen? (p>O.OS> between 2Oiu"ml (group) and lOiu/ml (group) were
3
significant different(p<0. 05). The results showed that add serum~ PMSG and HCG can improve oocyte' s maturation rate in vitro.
2. In vitro fertilization Fresh sperm were capacitated in medium mTBM+100 ~ g/ml Heparin with the "swim-up" method. After capacitated 40- 60 mm. the sperm and the maturation oocytes were incubated in mTBM+50 ii g/ml Heparin (39. 40C, 5%C02, 100% humidity) for Gb. After 24'-28h some fertifized eggs extrusion the second polar body (PB II) and some fertilized eggs were clevaged. The clevage rate was 37. 95%(48-50h).
3. Early embryo' s in vitro culture After in vitro fertilization, the fertilized eggs were cultured in three systems. I .TCM199+l5%NCS, II. Porcine oviduct epithelial cell monolayer (POEC)+TCM199-r15%NCS, III. granule/cumulus cell monolayer(G/C>TCM199+l5%NCS. The culture condition was 38. 80C, 5%C02, 100% humidity. Their development rates were 38. 49±6. 59% 37 19±5.19%,37.85±4.25%(48 -~ 50h), there were no significant different (P>0. 05) between them. In group I, the embryos were blocked at 4 cell stage, in group II and Ill some embryos can surmount 4 cell stage block and developed to 8-~l6 cell stage and morula. The morula rates were 0 12 84± 6. 04%, 4. 76 ±6. 73%. The difference of the morula rates among group II and I was remarkably (P
In sumftiary, under the present culture conditions, the porcine ovarian oocvtes can matured in vitro, fertilized in vitro and developed to early embryos. When the early embryos co-cultured with monolayer porcine oviduct epithelial cells or granual/cumulus cells some of them can surmount the 4 cell stage block and developed to morula
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45. Romar R., Coy P, Campos L, et al, Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization, Anim Reprod Sci. 2001, 8(1-2):85～98
46. Hunter RHF, ichol R, Capacitation potential of the fallopian tube, a study involving surgical insemination and the subsequent incidence of polyspermy, Gamete Res. 1988, 1:255～266
47．北京农业大学，家畜寄生虫学，北京：北京农业出版社，1981
48．张元跃，陈斌，生物统计原理与方法，北京：中国科学技术出版社，1997
49. Mattioli M., et al, Effect of follicule somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation, Gamete Res, 1988(20): 177～183
50. Tsafriri A, et al, Control of the development of meiotic competence and of oocyte maturation in mammals. Fertilization of the human egg in vitro, 1983, 3～17
51．周虚，刘健，内分泌，旁分泌和自分泌对配子发生的调控，中国兽医学报．1997，17(3)：309～311
52. Dekel N, Beers WH, Development of the rat oocyte in vitro, inhibition and induction of maturation in the presence or absence of the cumulus oophorus, Dev Biol, 1980, 75:247～254
53. Richard FJ, Sirard MA, Effects of follicular cell on oocyte maturation. Biol Reprod, 1996, 54:22～28
54. Chen L, Wert SE, Hendrix EM, et al, Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass, Mol Reprod Dev, 1990, 26:236～247
55. Downs SM, Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro, Biol Reprod, 1989, 41:371～379
56. Hunter AG, Moor RM, Stage dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro, J Dairy Sci. 1987, 701646～1651
57．卢晟盛，卢克焕，不同血清种类及其浓度对牛卵母细胞体外受精的影响，广西农业大学学报，1994，13(1)：21～26
58．李朝军，范必勤，王余暖等，FSH，GEF，胰岛素促进卵母细胞体外成熟分裂恢复机制的研究，细胞生物学杂志，1997，19(1)：27～30
59．张涌，刘泽隆，李裕强，山羊卵泡卵母细胞体外成熟的研究，西北农业大学学报，1999，27(1)：14～18
60. Plancha CE. Albertini DF, Hormonal regulation of meiotic maturation in the hamster oocyte involves a cytokeleton mediated process, Biol Reprod. 1994, 51:852～864
61. Naito K, et al. Developmental ability of procine ova matured in porcine follicular fluid in vitro and fertilized in vitro, Theriogenology, 1989, 31:1049～1057
62．吴光明，秦鹏春，谭景和，张秋明，体外成熟猪卵母细胞的体外受精研究，东北农业大学学报，1996，27(2)：190～195
63．王建辰，章孝荣，动物生殖调控，合肥：安徽科学技术出版社，1998
64. Mccaffrey C, Successful co-culture of l-4-cell cattle ova to the morula or blastocyst stage, J Reprod Fert, 1991, 92:119～124
65．鄂征，细胞培养和分子细胞学技术，北京：北京出版社，1999
66．姜泊，细胞凋亡基础与临床，北京：北京人民军医出版社，1999
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43． Cran DG, Cheng WTK, The cortical reaction in pig oocytes in vivo an in vitro fertilization. Gamete Res, 1986, 13:241-251
44． Hunter RHF. Sperm egg ratios and putative molecular signals to modulate gamete interactions in polytocous mammals. Mol Reprod Dev. 1993, 35:324-327
45. Romar R., Coy P, Campos L, et al, Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization, Anim Reprod Sci. 2001, 8(1-2):85～98
46. Hunter RHF, ichol R, Capacitation potential of the fallopian tube, a study involving surgical insemination and the subsequent incidence of polyspermy, Gamete Res. 1988, 1:255～266
47．北京农业大学，家畜寄生虫学，北京：北京农业出版社，1981
48．张元跃，陈斌，生物统计原理与方法，北京：中国科学技术出版社，1997
49. Mattioli M., et al, Effect of follicule somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation, Gamete Res, 1988(20): 177～183
50. Tsafriri A, et al, Control of the development of meiotic competence and of oocyte maturation in mammals. Fertilization of the human egg in vitro, 1983, 3～17
51．周虚，刘健，内分泌，旁分泌和自分泌对配子发生的调控，中国兽医学报．1997，17(3)：309～311
52. Dekel N, Beers WH, Development of the rat oocyte in vitro, inhibition and induction of maturation in the presence or absence of the cumulus oophorus, Dev Biol, 1980, 75:247～254
53. Richard FJ, Sirard MA, Effects of follicular cell on oocyte maturation. Biol Reprod, 1996, 54:22～28
54. Chen L, Wert SE, Hendrix EM, et al, Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass, Mol Reprod Dev, 1990, 26:236～247
55. Downs SM, Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro, Biol Reprod, 1989, 41:371～379
56. Hunter AG, Moor RM, Stage dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro, J Dairy Sci. 1987, 701646～1651
57．卢晟盛，卢克焕，不同血清种类及其浓度对牛卵母细胞体外受精的影响，广西农业大学学报，1994，13(1)：21～26
58．李朝军，范必勤，王余暖等，FSH，GEF，胰岛素促进卵母细胞体外成熟分裂恢复机制的研究，细胞生物学杂志，1997，19(1)：27～30
59．张涌，刘泽隆，李裕强，山羊卵泡卵母细胞体外成熟的研究，西北农业大学学报，1999，27(1)：14～18
60. Plancha CE. Albertini DF, Hormonal regulation of meiotic maturation in the hamster oocyte involves a cytokeleton mediated process, Biol Reprod. 1994, 51:852～864
61. Naito K, et al. Developmental ability of procine ova matured in porcine follicular fluid in vitro and fertilized in vitro, Theriogenology, 1989, 31:1049～1057
62．吴光明，秦鹏春，谭景和，张秋明，体外成熟猪卵母细胞的体外受精研究，东北农业大学学报，1996，27(2)：190～195
63．王建辰，章孝荣，动物生殖调控，合肥：安徽科学技术出版社，1998
64. Mccaffrey C, Successful co-culture of l-4-cell cattle ova to the morula or blastocyst stage, J Reprod Fert, 1991, 92:119～124
65．鄂征，细胞培养和分子细胞学技术，北京：北京出版社，1999
66．姜泊，细胞凋亡基础与临床，北京：北京人民军医出版社，1999