小鼠体外受精和精子冷冻的研究
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摘要
为建立一套小鼠体外受精体系，本研究对不同受精浓度、精子的浮游处理、注射激素后卵母细胞的不同收集时间进行比较；对CD-1、B6DF1、FVB、C57BL／6、DBA小鼠的卵母细胞进行直接、去卵丘细胞、切割透明带法处理后，再分别进行体外受精、体外培养和胚胎移植试验。结果表明，小鼠体外受精的精子适宜浓度为5-7×10~5个／ml。C57BL／6小鼠精子浮游处理有利于提高体外受精率。卵母细胞收集的最佳时间为注射hCG后15h。CD-1和B6DF1小鼠体外受精率及受孕率均高于近交系FVB、C57BL／6、DBA小鼠的受精率和受孕率。五种品系小鼠卵母细胞三种不同处理试验，受孕率均差异不显著；去卵丘细胞卵体外受精率(10-23％)均显著低于卵丘卵母细胞复合体的受精率(34-60％)和切割透明带卵的受精率(41-65％)，而C57BL／6和DBA小鼠切割透明带卵的体外受精率还显著高于卵丘卵母细胞复合体的受精率。因此，C57BL／6和DBA小鼠理想的鲜精体外受精方法为切割透明带法，而CD-1、B6DF1、FVB小鼠切割透明带卵的受精率与卵丘卵母细胞复合体的受精率均差异不显著，故此三种品系小鼠则直接用卵丘卵母细胞复合体进行鲜精体外受精法简单有效。
为探索、建立一套小鼠精子冷冻方法，本研究通过CPS和RS318两种冷冻液冷冻小鼠精子试验；进行干、湿解冻法和解冻时采用离心法和稀释法处理冷冻液试验；并以RS318为冷冻液，对B6DF1、CD-1、DBA、C57BL／6小鼠精子分别进行快速冷冻和缓速冷冻，将解冻的精子和鲜精分别与同品系小鼠的卵丘卵母细胞复合体进行体外受精，快速冷冻精子解冻后还与去卵丘细胞卵、切割透明带卵受精，然后进行体外培养、胚胎移植试验。根据体外受精率和受孕率对小鼠精子冷冻方法进行筛选。结果，两种冷冻液试验和干、湿解冻法试验及离心法、稀释法处理冷冻液试验的受精率均差异不显著。B6DF1、CD-1、C57BL／6小鼠快速冷冻精子试验(与卵丘卵母细胞复合体)的受精率(35％、34％、17％)显著低于鲜精的(60％、59％、34％)、缓速冷冻精子试验的受精率(62％、58％、30％)，后两者受精率均差异不显著；此三种品系小鼠快速与缓速冷冻精子体外受精试验的受孕率均
显著低于鲜精试验的受孕率。
DBA小鼠快速冷冻精子体外受精试验的受精率和受
孕率也低于缓速冷冻精子
、鲜精体外受精试验的受精率和受孕率
，但三种精子处
。四种品系小鼠卵母细胞切割透明带后与解
冻精子的体外受精率(43一49%)显著高于去卵丘细胞卵的受精率(7一19%)，并高于卵
丘卵母细胞复合体的受精率(17一35%)。因此缓速冷冻法是小鼠精子冷冻的适宜方
法，干解冻法和稀释法处理冷冻液可简单有效地解冻精子，切割透明带法可提高
卵与解冻精子的受精率。
To establish a suit of method on the in vitro fertilization (IVF) of mouse, the different concentration and swimming up of mouse spermatozoa and the different time of the oocytes collected were compared. Five strains of mouse (CD-I, B6DF1, FVB, C57BL/6 and DBA) oocytes were removed cumulus cells, dissected partial zona-pellucida, surrounded by cumulus cells. The techniques of IVF, culture and embryos transfer were used. Results showed that the feasible concentration of mouse spermatozoa was 5-7 X 105/ml. Swimming up of mouse spermatozoa in C57BL/6 can improve the fertilization rate. The best time of the oocytes collected was 15h after injected hCG The higher rates of IVF and pregnancy were CD-land B6DF1. There was no significantly difference of the pregnancy rate in three disposal ways of oocytes in each of five strains of mouse. The rates of IVF of the denuded and zona-intact oocytes (10-23%)were evidently lower than that of oocytes surrounded by cumulus cells (34-60%). We found the positive effect of parti
al zona-pellucida dissection(PZD) on the insemination rate (41-65%), above all the strains of C57BL/6 and DBA. So this is the best method of IVF of C57BL/6 and DBA while the oocytes surrounded by cumulus cells of CD-1,B6DF1,FVB are directly used to IVF in the tests of fresh spermatozoa.
to explore and establish a suit of method of cryopreserved mouse spermatozoa, two kind of cryopreservation protection solutions (CPS and RS318) and two thawed methods and two methods to dispose RS318 were studied. Four strains (B6DF1,CD-1,DBA, C57BL/6) of mouse spermatozoa were cooled at a high rate and low rate when RS318 was used .Then frozen-thawed spermatozoa and oocytes surrounded by cumulus cells were used for IVF under the control of unfrozen sperm. Four strains of mouse oocytes removed cumulus cells and of PZD were inseminated with sperm cooled at a high rate. The techniques of culture and embryos transfer were used. The rate of IVF and pregnancy was used to select good method of cryopreservation of mouse
spermatozoa. Results, there was no remarkable difference in the rate of IVF of the test of CPS and RS318 or two thawed methods or two methods to dispose RS318. The rates of IVF(35%,34%,17%)of B6DFl,CD-l,C57BL/6 mouse of tests of spermatozoa cooled rapidjy were remarkably lower than that of fresh spermatozoa(60%,59%,34%)or that of spermatozoa cooled slowly(62%,58%,30%)and the rates of pregnancy in the spermatozoa cooled tests were remarkably lower than them in fresh spermatozoa except for DBA. The fertilization rates of PZD oocytes (43-49%) in the tests of spermatozoa cooled rapidly were significantly higher than that of the zona-intact oocytes(7-19%) and higher than that of oocytes surrounded by cumulus cells (17-34%). So mouse spermatozoa cooled slowly is a feasible method. And dry thawed and dilution methods are used. PZD oocytes can improve the rate of IVF with frozen-thawed spermatozoa.
为探索、建立一套小鼠精子冷冻方法，本研究通过CPS和RS318两种冷冻液冷冻小鼠精子试验；进行干、湿解冻法和解冻时采用离心法和稀释法处理冷冻液试验；并以RS318为冷冻液，对B6DF1、CD-1、DBA、C57BL／6小鼠精子分别进行快速冷冻和缓速冷冻，将解冻的精子和鲜精分别与同品系小鼠的卵丘卵母细胞复合体进行体外受精，快速冷冻精子解冻后还与去卵丘细胞卵、切割透明带卵受精，然后进行体外培养、胚胎移植试验。根据体外受精率和受孕率对小鼠精子冷冻方法进行筛选。结果，两种冷冻液试验和干、湿解冻法试验及离心法、稀释法处理冷冻液试验的受精率均差异不显著。B6DF1、CD-1、C57BL／6小鼠快速冷冻精子试验(与卵丘卵母细胞复合体)的受精率(35％、34％、17％)显著低于鲜精的(60％、59％、34％)、缓速冷冻精子试验的受精率(62％、58％、30％)，后两者受精率均差异不显著；此三种品系小鼠快速与缓速冷冻精子体外受精试验的受孕率均
显著低于鲜精试验的受孕率。
DBA小鼠快速冷冻精子体外受精试验的受精率和受
孕率也低于缓速冷冻精子
、鲜精体外受精试验的受精率和受孕率
，但三种精子处
。四种品系小鼠卵母细胞切割透明带后与解
冻精子的体外受精率(43一49%)显著高于去卵丘细胞卵的受精率(7一19%)，并高于卵
丘卵母细胞复合体的受精率(17一35%)。因此缓速冷冻法是小鼠精子冷冻的适宜方
法，干解冻法和稀释法处理冷冻液可简单有效地解冻精子，切割透明带法可提高
卵与解冻精子的受精率。
To establish a suit of method on the in vitro fertilization (IVF) of mouse, the different concentration and swimming up of mouse spermatozoa and the different time of the oocytes collected were compared. Five strains of mouse (CD-I, B6DF1, FVB, C57BL/6 and DBA) oocytes were removed cumulus cells, dissected partial zona-pellucida, surrounded by cumulus cells. The techniques of IVF, culture and embryos transfer were used. Results showed that the feasible concentration of mouse spermatozoa was 5-7 X 105/ml. Swimming up of mouse spermatozoa in C57BL/6 can improve the fertilization rate. The best time of the oocytes collected was 15h after injected hCG The higher rates of IVF and pregnancy were CD-land B6DF1. There was no significantly difference of the pregnancy rate in three disposal ways of oocytes in each of five strains of mouse. The rates of IVF of the denuded and zona-intact oocytes (10-23%)were evidently lower than that of oocytes surrounded by cumulus cells (34-60%). We found the positive effect of parti
al zona-pellucida dissection(PZD) on the insemination rate (41-65%), above all the strains of C57BL/6 and DBA. So this is the best method of IVF of C57BL/6 and DBA while the oocytes surrounded by cumulus cells of CD-1,B6DF1,FVB are directly used to IVF in the tests of fresh spermatozoa.
to explore and establish a suit of method of cryopreserved mouse spermatozoa, two kind of cryopreservation protection solutions (CPS and RS318) and two thawed methods and two methods to dispose RS318 were studied. Four strains (B6DF1,CD-1,DBA, C57BL/6) of mouse spermatozoa were cooled at a high rate and low rate when RS318 was used .Then frozen-thawed spermatozoa and oocytes surrounded by cumulus cells were used for IVF under the control of unfrozen sperm. Four strains of mouse oocytes removed cumulus cells and of PZD were inseminated with sperm cooled at a high rate. The techniques of culture and embryos transfer were used. The rate of IVF and pregnancy was used to select good method of cryopreservation of mouse
spermatozoa. Results, there was no remarkable difference in the rate of IVF of the test of CPS and RS318 or two thawed methods or two methods to dispose RS318. The rates of IVF(35%,34%,17%)of B6DFl,CD-l,C57BL/6 mouse of tests of spermatozoa cooled rapidjy were remarkably lower than that of fresh spermatozoa(60%,59%,34%)or that of spermatozoa cooled slowly(62%,58%,30%)and the rates of pregnancy in the spermatozoa cooled tests were remarkably lower than them in fresh spermatozoa except for DBA. The fertilization rates of PZD oocytes (43-49%) in the tests of spermatozoa cooled rapidly were significantly higher than that of the zona-intact oocytes(7-19%) and higher than that of oocytes surrounded by cumulus cells (17-34%). So mouse spermatozoa cooled slowly is a feasible method. And dry thawed and dilution methods are used. PZD oocytes can improve the rate of IVF with frozen-thawed spermatozoa.
引文
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2. Chang MC. Experimental studies of mammalian fertilization. ZOOL SCI, 1984,1:349-364.
3．陈大元主编．受精生物学．第一版．北京：科学出版社，2000，316-405．
4. Lannous DL, Blanchard Y. Nuclear maturity and morphology of hurnan spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure. J. REPROD FERTIL, 1988, 84: 551-556.
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