动物学报 48(2) :233～239, 2002A cta Zoologica S i nica
异源四倍体鲫鲤的受精细胞学
(湖南师范大学生命科学学院, 长沙410081)
3
李建中 张轩杰 刘少军 冯 浩 刘 筠33
摘 要 异源四倍体鲫鲤的成熟卵子处于第二次减数分裂中期, 精子通过受精孔进入卵内。精子入卵以后, 受精孔立即被受精塞堵住。受精后8min , 受精卵出现明显的精子星光, 同时进入第二次减数分裂后期, 即将排出第二极体; 13min 时, 精子头部开始膨胀, 趋向核化; 18min 时, 雌雄原核均已形成, 并向胚盘中央靠近; 23
min 时, 雌、雄原核开始接触; 33min 时, 雌、雄原核完全融合成为一个合子核; 38min 时, 受精卵开始第一次
卵裂, 53min 后分裂形成两个子核。该研究证明异源四倍体鲫鲤和大多数二倍体鱼一样, 具有正常的受精细胞学程序, 受精方式为单精受精。
关键词 异源四倍体鲫鲤 二倍体卵子 二倍体精子 单精受精
人工诱导四倍体鱼的研究是鱼类育种领域中的重大课题。国内外学者采用物理或化学的方法, 在
虹鳟(Chourrout , 1982; Refstie , 1981) 、罗非鱼(Myers , 1986) 、鳙鱼(洪云汉, 1990) 、水晶彩) ) ×鲫(桂建芳等, 1991) 和白鲫(♀红鲫(♂
(陈敏容等, 1997) 等鱼类获得了同源四倍体或异
用Smith ’s 液固定受精卵, 酒精脱水, 二甲苯透明, 石蜡包埋, 连续切片厚度为6～8μm , 苏木精2伊红染色, 中性树胶封片, Olympus 显微镜观察并摄影。
2 结 果
211 受精前
源四倍体, 但到目前为止, 成功获得两性可育且能自然繁殖的人工诱导四倍体鱼的研究甚是少见。湖南师范大学生命科学学院与湘阴县东湖渔场合作,
利用生物学的方法, 在红鲫(Carassi us aurat us red variety , ♀) 与湘江野鲤(Cypri nus carpio , ) 的杂交三代(F 3) 中发现了异源四倍体鱼(刘♂
筠, 1993) , 该四倍体鱼雌雄可育并且能自然繁殖,
成熟的异源四倍体鲫鲤的二倍体卵子和精子都比一般鲤鲫鱼产生的单倍体配子要大。其中卵子直径约为116～118mm , 精子头部直径约为214μm (Liu et al . , 2001) 。卵膜上的受精孔呈典型的漏
斗状(图版Ⅰ:1) , 外部口径较大, 直径约为716～1513μm , 能同时容纳多个精子; 但精孔管道细长而狭小, 直径约为310～315μm , 可以限制大量精子涌入卵中。即将受精的异源四倍体鲫鲤的卵子处于第二次减数分裂中期, 受精孔敞开, 精孔细胞消失。
212 精子入卵和卵激动反应(受精至受精后8min)
已形成了一个新的稳定的四倍体鱼种群(刘少军等, 1999; Liu et al . , 2001; 李建中等, 2001) 。为了进一步了解异源四倍体鲫鲤能自然繁殖的生物学基础, 我们对异源四倍体鲫鲤自交的受精细胞学进行了研究。
1 材料与方法
1998年4月在湘阴县东湖渔场进行异源四倍
异源四倍体鲫鲤的精子是通过受精孔进入卵内的。通常情况下, 只有一个精子进入受精孔。但在受精后1min 的受精卵切片上, 作者也观察到有4
个精子依次进入受精孔管道的现象(图版Ⅰ:1) 。精子入卵以后, 受精孔立即被一个像瓶塞一样的受精塞堵住(图版Ⅰ:2, 3) 。受精后3min , 受精
体鲫鲤的人工繁殖时取得实验所用材料。在18～21℃的水温下, 采用干法人工授精。先取刚受精的
卵, 然后在受精后1、2、3和8min , 到以后每隔5min 分别取材固定, 直到受精后68min 结束。采
2000210208收稿, 2001204228修回 3国家“863”计划项目(国科生字1996182) 33通讯作者 E 2mail :liuyun @hunnu.edu. cn
第一作者简介 李建中, 男, 32岁, 博士研究生, 讲师。研究方向:鱼类生殖生理。
卵仍处于第二次减数分裂中期(图版Ⅰ:5) 。此
时, 精子头部已离开受精孔, 游过新月池, 到达卵质边缘(图版Ⅰ:4) 。精子入卵后, 卵即被激动。卵子激动的最初表现是卵体显著收缩, 放射膜离开卵体向外隆起形成受精膜(图版Ⅰ:5) ; 在受精膜与卵体之间出现围卵腔(图版Ⅰ:2) , 皮质小泡破裂释放到围卵腔中; 细胞质向动物极流动, 形成胚盘。213 雌、雄原核形成(受精后8～18min)
受精后8min , 精子深入卵质, 在动物极卵质边缘形成精子星光(图版Ⅰ:8) 。同时, 受精卵进入第二次减数分裂晚期(图版Ⅰ:6) , 准备排出第二极体(图版Ⅰ:7) 。受精后13min , 精子头部开始膨胀, 趋向核化(图版Ⅱ:1) ; 受精后18min , 精子头部完全核化形成雄原核(图版Ⅱ:2) , 此时已由一个星光发展为成对的子星光, 或称
双星光。精子星光的形成与发展以及精子头部的核化, 是异源四倍体鲫鲤正常受精的标志。在雄原核和双星光出现的同时, 卵子已完成第二次减数分裂, 排出了第二极体, 留在卵子里的那部分遗传物质形成雌原核(图版Ⅱ:3) , 雌原核周围未见星光发出。214 雌、雄原核靠近、接触及融合(受精后18～33min)
受精后18min , 雌、雄原核形成后均向胚盘中央靠近(图版Ⅱ:4) ; 受精后23min , 雌、雄原核开始接触(图版Ⅱ:5) ; 受精后28min , 雌、雄原核进一步融合, 但两者的接合线还存在; 受精后33min , 雌、雄原核完全融合, 来自父母双方的二倍染色体组, 融合成为一个具有四倍染色体组的合子核(图版Ⅱ:6) 。此时核中染色体嗜碱性大为增强, 容易看到许多染成黑紫色的染色体。215 第一次卵裂(受精后33～53min)
雌、雄原核融合后经过短时间的准备, 便开始第一次卵裂。受精后38min , 合子核的核膜消失, 染色质凝聚形成染色体; 受精后43min , 受精卵进入第一次卵裂中期, 染色体整齐地排列在纺锤体的赤道板上(图版Ⅱ:7) ; 受精后48min , 在纺锤丝的牵引下, 染色体分为两部分, 各自移向纺锤体的两极, 最终染色体被拉至两端, 形成两子核。与此同时, 在两子核之间的细胞质也分别向两端收缩形成卵沟裂(图版Ⅱ:8) ; 受精后53min , 第一次卵裂结束。分裂形成的两子核并不停止活动, 紧接着进行第二次卵裂。
3 讨 论
受精孔是无顶体鱼类精子入卵的唯一通道(朱洗等, 1960; 宋慧春等, 1999) 。一般认为, 硬骨鱼类都是单精入卵、单精受精(刘筠等, 1981; 王瑞霞等, 1982; 黄永松, 1993; 尹洪滨等, 2000) ; 但也有人发现一些硬骨鱼类有多精入卵、多精受精的现象(叶玉珍等, 1994) 或多精入卵、单精受精的现象(许雁等, 1990) 。我们的研究发现, 异源四倍体鲫鲤只有一个精子由受精孔进入卵内形成雄原核, 并与雌原核结合, 形成合子核。这证明异源四倍体鲫鲤和大多数二倍体鱼一样, 具有正常的受精细胞学程序, 受精方式为单精入卵、单精受精。
我们在观察中还发现, 异源四倍体鲫鲤虽然有多个精子进入受精孔管道的现象(图版Ⅰ:1) , 但最终只有一个精子进入卵内。这表明异源四倍体鲫鲤有完善的阻止多精入卵的机制存在。我们的研究表明, 达到生理成熟的异源四倍体鲫鲤Ⅴ时相卵子的受精孔是畅通无阻的, 而精子入卵以后, 受精孔立即被一个像瓶塞一样的受精塞堵住。在受精后1min 的一个受精卵切片上(图版Ⅰ:2, 3) 可以看到, 由于精孔管的内孔被封闭, 受精孔的外口边缘和精孔管道完全被受精塞堵住, 已到达受精孔管道口准备入卵的两个精子因受到受精塞的阻止而不能继续入卵, 另外还有几个精子被受精塞堵在受精孔的外部。所以我们认为, 受精塞的形成是异源四倍体鲫鲤也是大多数硬骨鱼类阻止多精受精的一种主要的机制。形成受精塞的物质是从何而来? 又是如何产生? 有学者(尹洪滨等, 2000) 提出, 受精塞可能是精子入卵以后精孔细胞释放出来的一种物质所形成的, 而作者则认为受精塞的形成与精孔细胞无关。我们的观察证实, 成熟的异源四倍体鲫鲤卵子的受精孔是敞开的, 精孔细胞已经消失了, 这同刘筠等(1981) 对青鲂杂交受精的光学显微镜观察和王瑞霞等(1982) 对鲂鱼受精前后的扫描电镜观察结果是一致的。有学者(Brummett et al . , 1979) 认为, 受精塞是精子入卵后皮质小泡破裂释放的物质形成的, 我们同意这种观点, 但尚需进一步的研究证明。当然, 异源四倍体鲫鲤可能还有一些其它阻止多精受精的机制, 例如, 成熟卵子的卵膜上可能含有精子的受体, 当精子入卵后由于膜电位的变化, 使没有被精子附着的受体结构发生改变, 变为没有功能, 从而迅速阻止第二个精子入卵(Epel , 1978) 。另外, 异源四倍体鲫鲤的成熟卵子
的受精孔可能释放某些特殊的物质, 能使精子入卵以后附着在卵子表面的精子凝集并融化而失去受精的功能(Yanagimachi , 1957) , 从而阻止多精受精; 还有, 异源四倍体鲫鲤的精子入卵以后, 放射膜离开卵体向外隆起形成了受精膜, 受精膜的形成和举起等因素, 对阻止多精受精也有重要的作用(吴长功等, 2000) 。
异源四倍体鲫鲤虽然含有四套染色体(4n =200) , 但是由于它们的染色体是整倍体的, 所以异源四倍体鲫鲤能进行正常的减数分裂, 产生可育的二倍体配子(楼允东, 1984) , 这是异源四倍体鲫
鲤能自然繁殖的前提条件; 而异源四倍体鲫鲤具有正常的单精受精方式, 则是异源四倍体鲫鲤能自然繁殖的重要保障。正是因为异源四倍体鲫鲤的二倍体精子与二倍体卵子正常受精形成了四倍体的合子核, 保证了异源四倍体鲫鲤染色体数目的恒定性, 使异源四倍体鲫鲤能代代相传, 已形成了一个染色体数目为4n =200的四倍体鱼新种群。能自然繁殖的异源四倍体鲫鲤的获得, 不仅为探讨自然界鱼类多倍体的形成机制提供了重要的理论依据, 而且在生产上有重要的应用价值。
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外 文 摘 要(Abstract)
CYTOLOGICAL STU DY ON FERTIL IZATION OF ALLOTETRAPLOID
H YBRIDS OF RED CRUCIAN CARP (♀) ×COMMON CARP (♂) 3
L I Jian 2Zhong ZHAN G Xuan 2Jie L IU Shao 2J un FEN G Hao L IU Yun 33
(College of L if e Science , Hunan Normal U niversity , Changsha 410081, Chi na )
Serial sections of fertilized eggs were used to study the fertilization of the allotetraploid fish which was ) with common carp (Cypri nus developed with hybridizing red crucian carp (Carassi us aurat us red variety , ♀
) . The cytological behavior during the fertilization of the allotetraploid hybrid was depicted in carpio L. , ♂
detail in this paper. The fertilized eggs of the allotetraploid fish were fixed in Smith ’s solution and dehydrated by alcohol. Then the tissue sections were stained with hematoxylin and eosin. The thickness of the tissue sections was from 6to 8micrometers. Before the s perm entering the egg , the mature eggs of the allotetraploid fish remained at the metaphase of the secondary maturation division. At this stage , a funnel 2like depression could be seen nearby the animal pole on the chorion and at the bottom of the depression was located the micropylar canal where no micropylar cell was observed. The diameter of the outer opening of the micropylar canal was always bigger than the inner one. The number of diploid sperm penetrating into the egg was limited because of the narrow micropylar canal. In the fertilized eggs , as many as four sperms entering the micropyle were observed. The micropyle was blocked by the fertilization plug as soon as the sperm entered the egg. When the water temperature was between 18℃and 20℃, the spermaster could be clearly seen and the second polar body was extruded about 8minutes after insemination which showed that the egg was developing into the anaphase of the secondary maturation division. At 13minutes after insemination , the s perm nucleus became vesicular nuclear. At 18minutes after insemination , the male pronucleus was observed and the female pronucleus was formed at the same time. At 23minutes after insemination , the male pronucleus and the female pronucleus
3The research was supported by the Chinese 863High Tecnology Program (No. 1996182) 33Corresponding author E 2mail :liuyun @hunnu.edu. cn
moved toward the center of the blastodisk , then fused with each other. At about 33minutes after insemination , the zygote nucleus was formed. At 38minutes after insemination , the first cleavage was observed and the first cleavage furrow of the blastodisk appeared at 53minutes. From all the results , we concluded that the ) ×common carp (♂) underwent the normal fertilization process allotetraploid hybrids of red crucian carp (♀and the fertilization type was monospermism.
) , Diploid oocyte , Diploid ) ×common carp (♂K ey w ords Allotetraploid hybrids of red crucian carp (♀
sperm , Monospermism
李建中等:异源四倍体鲫鲤的受精细胞学图版Ⅰ
) L I Jian 2Zhong et al . :Cytological study on fertilization of allotetraploid hybrids of red crucian carp (♀
) ×common carp (♂Plate
Ⅰ
11受精后1min , 4个精子进入受精孔(1min after insemination , four sperms entered the micropyle ) ×1000
21受精后1min , 受精孔被受精塞堵住, 白箭头示受精塞, 黑箭头示围卵腔(1min after insemination , the micropyle was blocked by fertilization plug , white arrow showing fertilization plug , black arrow showing perivitelline space. ) ×40031图2放大(magnification of picture 2) ×1000
41受精后3min , 到达卵质边缘的精子, 白箭头示新月池, 黑箭头示精子(3min after insemination , a sperm arrived at the edge of ooplasm , white arrow showing crescentic area , black arrow showing a sperm ) ×1000
51受精后3min , 受精卵处于第二次成熟分裂中期, 白箭头示受精膜(3min after insemination , the fertilized egg remained at the metaphase of secondary maturation division , white arrow showing fertilization membrace ) ×1000
61受精后8min , 受精卵进入第二次成熟分裂后期, 白箭头示留在卵内将来形成雌原核的遗传物质(8min after insemination , the egg was developing into the anaphase of secondary maturation division , white arrow showing the genetic material which would become female pronucleus ) ×1000
71受精后8min , 受精卵即将排出第二极体(8min after insemination , the second polar body was being extruded ) ×100081受精后8min , 精子星光形成(8min after insemination , the spermaster appeared ) ×1000
李建中等:异源四倍体鲫鲤的受精细胞学研究图版Ⅱ
) L I Jian 2Zhong et al . :Cytological study on fertilization of allotetraploid hybrids of red crucian carp (♀
) ×common carp (♂Plate
Ⅱ
11受精后13min , 精子头部开始核化(13min after insemination , the sperm nucleus became vesicular nuclear ) ×1000
21受精后18min , 雄原核形成, 白箭头示雄原核(18min after insemination , male pronucleus formed , white arrow showing male pronucleus ) ×1000
31受精后18min , 雌原核形成, 白箭头示雌原核(18min after insemination , female pronucleus formed , white arrow showing female pronucleus )
×1000
41受精后18min , 雌、雄原核彼此靠近(18min after insemination , female pronucleus and male pronucleus approached each other ) ×100051受精后23min , 雌、雄原核开始接触(23min after insemination , female pronucleus and male pronucleus came into contact ) ×1000
61受精后33min , 雌、雄原核完全融合成为合子核, 白箭头示合子核(33min after insemination , female pronucleus and male pronucleus fused into a zygotic nucleus , white arrow showing a zygotic nucleus ) ×1000
71受精后43min , 受精卵进入第一次卵裂中期(43min after insemination , the first cleavage entered into metaphase ) ×500
81受精后53min , 白箭头示两个子核, 黑箭头示分裂沟(53min after insemination , white arrows showing the daughter cell nucleus , black arrow showing the cleavage furrow ) ×300