6—DMAP诱导太平洋牡蛎三倍体——抑制受精卵第二极体释放↑（*）

于１９９６～１９９７年，用６－ＤＭＡＰ抑制太平洋牡蛎受精卵第二极体的释放，诱导产生三倍体，最高诱导率为９３．８％。该实验组胚胎孵化率为８１．０％，Ｄ形幼虫畸形率为１７．６％。实验选用Ｌ９（３４）设计，进行三因素三水平的正交试验：６－ＤＭＡＰ浓度，设３００、４５０和６００μｍｏｌ／Ｌ等三水平；诱导时机（观察静止状态的受精卵，以受精卵出现第一极体的百分率指示），设１０％、３０％和５０％等三水平；诱导持续时间，设１０、１５和２０ｍｉｎ等三水平，试验设三次重复。解剖法获取精卵，人工授精。染色体倍性检查采用染色体计数法。根据正交试验直观分析结果，得出诱导太平洋牡蛎三倍体各因素的最优水平组合：当３０％受精卵出现第一极体时，将受精卵浸泡在含６－ＤＭＡＰ４５０μｍｏｌ／Ｌ的海水中１０ｍｉｎ；三因素的主次顺序：６－ＤＭＡＰ浓度→诱导时机→诱导持续时间。６－ＤＭＡＰ的浓度和诱导时机对三倍体诱导率的影响显著；但诱导持续时间影响不显著。部分试验组得到４．２％～１３．６％的四倍体和１０％左右的非整倍体，这种情况可能与受精不同步抑制第一极体释放而导致的染色体多极分离有关。     

6_二甲基氨基嘌呤诱导栉孔扇贝三倍体

用６－二甲基氨基嘌呤（６－ＤＭＡＰ）处理以抑制栉孔扇贝第二极体释放产生三倍体的适宜诱导参数进行了试验研究，结合ＤＡＰＩ染色和荧光显微镜观察，分析了开始处理前和处理结束时的受精卵染色体核相组成与三倍体诱导率和幼虫成活率的关系，对三倍体处理组和对照组幼虫的生长进行了观察，结果表明，获得充分成熟的卵子，起始处理时间以第一极体占４０％，药物浓度６０ｍｇ／Ｌ，持续处理时间１５ｍｉｎ及产卵至处理结束保持水温２     

6—二甲在氨基嘌呤诱导太平洋牡蛎三倍体—抑制受精卵第一极体释放

于１９９６－１９９７年，用６－二甲基氨基嘌呤抑制受精卵第一体体的释放，诱导太平洋牡蛎产生三倍体。选用Ｌ１６（４＾５）设计，进行三因素四水平的正交试验，６－ＤＭＡＰ肖度，设１５０，３００，４５０和６００μｍｏｌ／Ｌ；诱导时机，设１０，１５，２０和２５ｍｉｎ诱导持续时间，设１０，１５，２０和２５ｍｉｎ。试验平行重复二次。     

太平洋牡蛎三倍体的繁殖潜力和遗传学研究

在太平洋牡蛎三倍体的繁殖潜力和遗传学研究工作，三倍体精子所含ＤＮＡ经流式细胞光度术测定，在１．５℃存在一个单一峰值，尽管染色体联会程度在雌体内和雌体间存在很大不同，三倍体卵内联会染色体中，三价染色体占优势（为显性）一些卵经完全联合形成１０个三价染色体，大部则由１１－１３个三价染色体，二价染色体及单价染色体组成，用双倍体（Ｄ）和三倍体（Ｔ）亲本牡蛎交配，形成四种杂交体：ＤＤ，ＤＴ，ＴＤ及ＴＴ（雌性在     

海洋经济贝类多倍体研究的现状

综述了近年来国内外在海洋经济贝类多倍体育种方面的研究进展及其成果，指明６－ＤＭＡＰ是一种提高三倍体生产率的理想药物以及多倍体贝类的今后研究方向。     

雄烯二酮和甲基睾酮诱导雌性日本鳗鲡性发育的反馈调节作用

采用间隔１５ｄ多次埋植雄激素雄烯二酮（４－ａｎｄｒｏｓｔｅｎｅ－３，１７－ｄｉｎｅ，ＡＤＳＤ）或甲基睾酮（１７α－ｍｅｔｈｙｌ－ｔｅｓｔｏｓ－ｔｅｒｏｎｅ，ＭＴ）诱导雌性日本鳗鲡（Ａｎｇｕｉｌｌａ ｊａｐｏｎｉｃａ）性腺发育成熟；并着重研究埋植雄激素后，雌鳗血清中促性腺激素（ＧｔＨ）及脑和垂体中哺乳类促性腺激素释放激素（ｍＧｎＲＨ）含量的动态变化。一次或多次埋植ＡＤＳＤ后，１～５ｄ的血清ＧｔＨ含量上升，然后下降；并且，血清ＧｔＨ含量上升的幅度随埋植次数的增加而增加；对于ＭＴ处理组，只在埋植７次后第１５天测得血清ＧｔＨ水平显著高于对照组，但仍显著低于ＡＤＳＤ处理组。这表明埋植ＡＤＳＤ和ＭＴ可促进ＧｔＨ的分泌，但两者促进ＧｔＨ分泌的作用存在显著差异。埋植ＡＤＳＤ１ｄ后，脑和垂体中ｍＧｎＲＨ含明明显增加，第２天后和对     

矮小蛤的性别确定和多倍体的巨型性

通过雌核发育和用紫外线光照过和精子与卵受精及随后用ＣＢ处理和抑制ＰＢⅡ的释放都成功地诱导生产雌的和三倍体的矮小蛤。进一步处理结果显示矮小蛤可能有Ｙ显性化的ｘｘ－雌，ｘｙ－雄的性别确定，与二倍体相比，三倍体有一定的生殖力，雌性为５９％，雄性为８０％，三倍体雌性产生的卵的容量比二倍体雌性的卵容量大５３％（Ｐ〈０．００１）。     

太平洋牡蛎染色体G带和Ag－NORs研究

牡蛎科的种类染色体大多为２０条，其中又以Ｍ／ＳＭ染色体居多，仅从核型较难将它们区分，应用显带技术能反映染色体的“解剖学”特征，以提供更多的具鉴定性特征的信息［１］。牡蛎是我国重要的养殖贝类之一，目前尚无有关太平洋牡蛎（Ｃｒａｓｓｏｓｔｒｅａｇｉｇａｓ）带型研究的报道。本文对其Ｇ带和Ａｇ－ＮＯＲｓ带作了初步探索，为种间比较、染色体鉴定以及基因定位等基础理论研究提供参考。收稿日期：１９９８－０８－０３国家攀登计划Ｂ项目（专题编号：ＰＤ－Ｂ６－３－１）及国家高科技发展计划项目（８６３－８１９－０１－０…     

大西洋鲑性腺分化及热休克的影响

大西洋鲑性腺分化发生在孵出45－63天，约495－693度日。经热休克处理获得三倍体稚鱼，性腺在分化之前明显大于正常二倍体。雌性三倍体稚鱼性腺在细胞学分化时受抑，初级卵母细胞不能发育；雄性三倍体稚鱼性腺在细胞学分化阶段生长明显减慢，初级精母细胞尚能发育。     

泥蚶受精过程的细胞学荧光显微观察

采用荧光染色剂ＤＡＰＩ已固定的泥蚶成熟卵和受精卵进色染色的方法，在显微镜下观察泥蚶受精细胞学过程。通过ＤＡＰＩ的染色，在荧光显微镜下可清楚观察到，泥蚶的成熟未受精卵处于第一次成熟分裂的中期，精子入卵后分裂再次启动，释放出第一和第二极体，完成成熟分裂，部分受精卵的第一极体有极明显的分裂现象，精子入卵的部位是随机的，且自精子入卵至成熟分裂完成，入卵的精子在形态等方面将发生一系列变化，形成雄性原核。雌雄     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

刺参受精及早期胚胎发育过程的细胞学观察

采用HOECHST 33258染色荧光显微方法,对刺参成熟未受精卵以及受精过程中精子入卵、极体排放、雌雄原核的形成与结合、早期卵裂以及多精入卵等细胞学进行了研究。结果显示,刚产出的刺参成熟未受精卵呈圆形,核相处于第一次成熟分裂中期;在水温22~23 ℃、盐度29条件下进行受精,受精后12 min,完成第一次成熟分裂,释放第一极体;受精后20 min,大部分受精卵完成第二次成熟分裂,放出第二极体。受精后35 min,雌、雄原核开始在卵中央发生染色体联合;受精后80 min,部分受精卵完成第一次卵裂,受精后100 min,部分受精卵完成第二次卵裂。刺参在受精过程中存在极少数的多精入卵现象。     

不同方法制备的三倍体长牡蛎养殖效果的比较

比较了细胞松弛素B(CB)和6-二甲基氨基嘌呤(6-DMAP)通过抑制受精卵极体释放的方法批量诱导三倍体长牡蛎的养殖效果.长牡蛎卵子在25℃的海水中受精,20～30min后,开始用浓度为0.5mg@L-1的CB处理,持续18～22min,受精卵处理密度为4.0～4.5×107个@L-1,三倍体产率为65.2%～70.1%,面盘幼虫孵化率为12.3%～14.5%,诱导效率指数为0.09.6-DMAP的使用浓度为400～420μmol@L-1,受精卵处理密度为3.0～3.5×107个@L-1,授精水温、处理起始和持续时间等与CB方法相同,三倍体产率为58.7%～65.4%,面盘幼虫孵化率为52.1%～55.4%,诱导效率指数为0.32.两种方法的采苗率基本相同,采苗器为基质较硬的栉孔扇贝贝壳.海区养殖采用浮筏夹苗吊养技术,两种方法诱导的三倍体牡蛎养殖性状没有明显差别.通过比较CB和6-DMAP两种诱导方法及三倍体的养殖效果表明,后者具有更好的应用性.     

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Triploidy was induced in mud loach (Misgurnus mizolepis) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.     

微卫星标记在长牡蛎三倍体鉴定中的应用

为开发一种简单有效的倍性检测方法对长牡蛎三倍体诱导结果进行准确评估,本实验采用细胞松弛素B抑制第二极体释放诱导产生长牡蛎三倍体,选用7个微卫星位点扩增基因组DNA,通过亲子代基因分型进行倍性检测,检测结果采用流式细胞仪加以验证,以评估微卫星标记倍性检测的准确性。另外,本研究探讨了准确鉴定倍性所需的微卫星标记数量与微卫星—着丝粒重组率(y)之间的关系,以期为其他物种采用分子标记进行倍性检测提供理论依据。结果显示,细胞松弛素B诱导产生的115个长牡蛎子代经7个微卫星位点鉴定得到40个三倍体,与流式细胞仪检测结果一致,准确率达到100%,7个微卫星位点的(1-y)的乘积为0.005。随机挑选6个位点,也可鉴定出所有三倍体,(1-y)的乘积为0.005~0.042。研究表明,本研究中开发的微卫星标记可以简单高效地鉴定长牡蛎三倍体,微卫星位点的(1-y)的乘积小于0.005,倍性检测的准确率可达100%,采用微卫星标记进行倍性检测对于加速长牡蛎三倍体育种进程具有十分重要的意义。     

缢蛏受精和早期卵裂过程的细胞学变化观察

采用普通光镜、荧光显微镜和石蜡切片技术,对缢蛏受精和早期卵裂过程中的精子入卵、减数分裂、雌雄原核形成与结合、早期卵裂以及多精入卵等细胞学事件进行了显微观察。结果表明,缢蛏成熟未受精卵多呈圆球形或卵圆形,少数呈梨形,卵径为82～88μm,核相处于第一次成熟分裂中期;精子为典型的原生型,全长55～58μm,头部呈保龄球形,顶体前端的顶体杆呈特别细长的花丝状;在水温21～22℃、盐度10的条件下进行人工授精,精、卵混合后,精子迅速附着于卵子表面,启动卵子发育;受精后4～6 min,精子的头部已进入卵内并明显膨胀,卵子外形变圆,卵外附着的精子量明显减少;在受精后12～15 min、20～25 min,受精卵先后排出第一极体、第二极体,完成两次成熟分裂;第二次成熟分裂结束以后,精、卵核体积迅速膨胀,雄原核的膨胀早于雌原核,核膜重建,在受精后约30 min形成雌、雄原核;雌、雄原核均向卵子中央移动,雄原核旁边的精子星光清晰可见,随后二者在卵子中央以染色体联合的方式结合,联合核的染色体共同排列在纺锤体的赤道板上,形成第一次有丝分裂的中期分裂相;受精后40 min左右,受精卵进行第一次卵裂,染色体在纺锤丝的牵引下向两极移动,结果形成2个大小不等的卵裂球;受精后45～50 min,卵子进行第二次卵裂,核相变化与第一次卵裂相同,在与第一次卵裂垂直的纵轴方向上发生不等全裂,最终形成3小1大4个卵裂球;受精后60～70 min,胚胎进行第三次卵裂,仍为不等全裂,但自此次卵裂起已开始进行螺旋分裂。此外,对实验中发现的缢蛏极少量多精入卵、多极分离等异常细胞学现象进行了分析,并探讨了海洋贝类卵子阻止多精入卵发生的机制。     

Studies on aneuploid pearl oyster (Pinctada martensii Dunker) produced by crossing triploid females and a diploid male following the inhibition of PB1

Viable aneuploid embryos of pearl oyster, Pinctada martensii Dunker, were produced by inhibiting the first polar body (PB1) with cytochalasin B treatment in eggs from triploids fertilized with haploid sperm. During the period of growth measurement, aneuploid showed the slowest growth compared with diploid and triploid groups. The body size and weight measurement data showed that there were no differences between aneuploids (as a group) and diploids in body size and weight (P>0.10), but that they were significantly different from triploids (P<0.01). There were no differences between aneuploids within diploid condition (2n±n) and diploids in SL (P>0.1), but significant differences in BW were found (P<0.05). Aneuploids within triploid condition (3n±n) were significantly smaller than triploids in BW (P<0.05), but not different from triploids in SL (P>0.05), and almost identical to diploids in both (P>0.1). These dada indicated that some aneuploids might be associated with growth retardation. Karyotype analysis revealed that there were metacentric, submetacentric or subtelocentric chromosomes losses or gains; aneuploid pearl oysters with the same chromosome numbers had different chromosome composition. Aneuploids are valuable research materials for genetic analysis.     

人工诱导杂色鲍(Haliotis diversicolor)雌核发育受精细胞学观察

应用组织切片方法,比较观察了杂色鲍普通二倍体、人工诱导三倍体、人工诱导雌核发育单倍体和雌核发育二倍体在成熟分裂和第一次卵裂期间的细胞学变化,以探讨人工诱导杂色鲍雌核发育的细胞学机制。结果表明,精子经紫外线辐射进行遗传失活处理后,仍然能够进入卵子,并激动卵子发育,卵子能够正常完成两次减数分裂,与普通受精卵没有明显差别。精子入卵后形成精核,大多能够逐渐解凝、液化并膨大,最终形成形态正常的雄性原核,但在第一次卵裂早期退化成浓缩的染色质小体(DCB),并在胞质分裂时随机地分配到其中一个子细胞里的分裂沟附近。在人工诱导三倍体和雌核发育二倍体受精卵中,受精卵经细胞松弛素B处理后,染色单体在第二次成熟分裂期分离,但胞质分离受抑制,留在卵质中的两套染色体形成两个染色质团,并发育成两个雌性原核。     

Induced triploidy in the bay scallop, Argopecten irradians, and its effects on growth and gametogenesis

Triploidy was induced in the bay scallop, Argopecten irradians, by treating newly fertilized eggs with cytochalasin B. Treatment for 20 min duration 10 min after fertilization resulted in 66% triploidy of scallops in the 0.05 mg/l cytochalasin treatment group and 94% triploidy of scallops in the 0.1 mg/l cytochalasin treatment group, detected by measuring the relative DNA content of scallop granulocytes using flow cytometry. Individually labelled scallops of known ploidy were grown in the Damariscotta River estuary in south-central Maine, U.S.A. from May 24 to September 15, 1982. Mean adductor muscle weight in triploid scallops was 73% greater than that of diploid controls and mean total body tissue wet weight was 36% greater. Shell inflation was significantly greater in triploids, while shell height and length were unaffected. The indexes of weight and glycogen content of the adductor muscle were significantly greater in triploids at the end of the growing season. The majority of triploid scallops failed to ripen during the summer months, while diploid scallops ripened and spawned.