斜带石斑鱼(♀)×鞍带石斑鱼(♂)受精细胞学的组织切片观察

人工诱导雌核发育二倍化的关键是掌握极体排出和卵裂的时机,掌握该规律最直接的方式即进行受精细胞学的研究。以鞍带石斑鱼(Epinephelus lanceolatus)为父本,斜带石斑鱼(Epinephelus coioides)为母本,通过人工授精获得受精卵,收集受精后不同时刻的卵子,经Smith′s液固定、石蜡包埋、切片、苏木精-伊红染色和显微组织观察。结果表明:受精后第30~60 s精子入卵;第3 min精子星光出现;第5 min卵子排出第二极体;第7~15 min雌雄原核相互靠近,并最终融合;第15 min以后合子核开始分裂。鞍带石斑鱼精子进入斜带石斑鱼卵子的时间与多数硬骨鱼类比较没有显著差别;但是,第二极体排出及第一次卵裂的时间较早。研究展示了斜带石斑鱼与鞍带石斑鱼杂交的受精细胞学的基本发育过程,并为后续的人工诱导石斑鱼雌核发育研究提供了理论依据。     

点带石斑鱼的亲鱼培育、产卵受精和胚胎发育

在水泥池人工养殖条件下,对人工诱导性逆转的“功能性雄性”、天然雄性及天然雌性点带石斑鱼(Epinephelus malabaricus)亲鱼进行强化培育,自然产卵受精。2002年繁殖季节,共获3cm以上的点带石斑鱼苗145万尾。亲鱼培育、产卵受精和胚胎发育的程序：(1)经过强化培育的亲鱼,自然产卵受精,受精率平均81％,最高97％;(2)成熟的“人工变性雄鱼”的精子头部呈球型,精子头径约3．7—6．1gm,尾长12．4～44．5μm;在水温26℃、盐度3％、pH7．9条件下,精子游动时间最长超过58min,精液中90％精子在31min停止游动,与天然雄亲鱼的精子无异,可作为生产雄亲鱼的来源;(3)在东山岛,产卵期由4月初持续到11月中旬,产卵高潮集中在4月底至5月底、9月底至10月初。在24h内,自然产卵通常从16：30持续到次日1：30;(4)产卵适宜水温为23．5—28．6℃,最适产卵水温为25．5—26．5℃;(5)水温26℃时,受精卵在盐度2．82％～2．96％,的海水中呈悬浮状态,盐度2．82％以下时下沉,盐度2．96％以上时上浮;(6)在水温24．9—27．6℃、盐度3％—3．3％、pH7．6—8．2的情况下,胚胎发育时间为21h。     

雌核发育兰州鲇胚胎发育和形态特征的研究

该研究通过紫外线(UV)灭活的黄颡鱼(Pelteobagrus fulvidraco)精子利用冷休克方法诱导兰州鲇(Silurus lanzhouensis)雌核发育二倍体(G_(2n))、单倍体(N)、杂交二倍体(H_(2n)),以及同源精子诱导的普通二倍体(2N)的胚胎发育时序和发育生物学特征进行了观察比较。结果显示,(1)受精率为2NH_(2n)NG_(2n),孵化率为2NG_(2n)N,畸形率为NG_(2n)2N。(2)在水温23 °C下,普通二倍体48 h孵化出膜,雌核发育二倍体51 h孵化出膜,杂交二倍体在神经胚期(26 h 20 min)停止发育,单倍体在出膜前期(43 h)停止发育。(3)各组胚胎发育形态上的差别主要表现在杂交二倍体未形成正常胚体,单倍体表现出典型的单倍体综合征,雌核发育二倍体初孵仔鱼在形态学上与普通二倍体无明显差异。该研究为兰州鲇雌核发育技术提供了技术支撑,同时也为单倍体、杂交和雌核发育胚胎的细胞生物学研究提供了理论依据。     

人工诱导泥鳅雌核发育的细胞学研究和诱导参数优化

为培育泥鳅(Misgurnus anguillicaudatus)全雌品种,研究通过细胞荧光染色后进行细胞学观察确定人工诱导泥鳅雌核发育的热休克起始时间,并进一步利用核型和流式细胞分析等方法对人工诱导雌核发育泥鳅进行鉴定。结果显示,二倍体泥鳅受到灭活的鲤(Cyprinus carpio)精子刺激后,第二极体与卵核分开的时间在人工授精后3—5min;卵子在人工授精后3.5min后再热休克2min的诱导孵化率达到10.26%。通过分析野生型泥鳅胚胎、人工诱导雌核发育泥鳅胚胎、泥鳅×鲤杂交胚胎和单倍体泥鳅胚胎的发育,发现部分雌核发育的胚胎可以正常发育,而杂交胚胎和单倍体胚胎会出现明显的发育障碍。核型和流式细胞检测分析表明利用本研究获得的热休克参数进行处理可得到二倍体雌核发育子代,二倍率达64.71%。研究从细胞学层面获得泥鳅雌核发育的诱导参数,可为利用人工诱导雌核发育开展全雌泥鳅育种提供指导。     

西太平洋沿海石斑鱼属鱼类DNA条形码及分子系统进化研究

研究测定了分布于西太平洋沿海的35种石斑鱼属鱼类共142个个体线粒体基因COⅠ及核基因TMO-4C4标记序列, 基于最大似然法与贝叶斯法构建分析了石斑鱼类分子系统进化关系。同时, 探讨了COⅠ基因作为DNA条形码在石斑鱼属鱼类物种鉴定中的有效性问题。结果显示: 35种石斑鱼属鱼类COⅠ同源序列为636 bp, 编码212个氨基酸, TMO-4C4同源序列为486 bp, 编码162个氨基酸, 在COⅠ基因中, 种间遗传距离在0.030—0.202, 平均遗传距离为0.143, 物种间遗传距离均大于Hebert 推荐的物种鉴定最小种间遗传距离0.020(2%)。种内遗传距离0.000—0.008, 平均遗传距离为0.003, 种间平均遗传距离是种内平均遗传距离的48倍, 显著大于种内平均遗传距离, 表明COⅠ基因可作为石斑鱼属物种鉴定的有效条形码基因。基于COⅠ及TMO-4C4构建的系统进化树上, 35种石斑鱼主要形成2个主要类群, 类群Ⅰ由细点石斑鱼(Epinephelus cyanopodus)和蓝棕石斑鱼(Epinephelus multinotatus)等22种石斑鱼聚成, 类群Ⅱ由吊桥石斑鱼(Epinephelus morrhua)和小点石斑鱼(Epinephelus epistictus)等13种石斑鱼组成。部分存在同种异名争议的种类如云纹石斑鱼(Epinephelus moara)/褐石斑鱼(Epinephelus bruneus)和斜带石斑鱼(Epinephelus coioides)/马拉巴石斑鱼(Epinephelus malabaricus)经COⅠ及TMO-4C4序列差异及进化分析, 均存在较大的遗传分化。结果支持近期的分类研究, 云纹石斑鱼/褐石斑鱼和斜带石斑鱼/马拉巴石斑鱼均为独立的物种, 并非同种异名的分类。     

埋植17α-甲基睾酮诱导点带石斑鱼性转化技术

论述了埋植17α-甲基睾酮诱导点带石斑鱼(Epinephelus malabaricus)性转化的方法,包括药物的处理、用量、手术操作等．3龄的雌性点带石斑鱼经过2次埋植外源激素手术转化为功能性雄鱼,139d后58％可挤出精液,301d后100％可挤出精液．功能性雄鱼用于人工繁殖,亲鱼自然产卵。1999～2000年2年内共孵出仔鱼7103万尾．所得仔鱼用于鱼苗培育,得到发育正常的幼鱼．认为埋植外源激素诱导点带石斑鱼完成性转化的方法简便可行,能满足规模化人工繁殖的需要．     

赤点石斑鱼促甲状腺激素TSHβ启动子的克隆及其在斑马鱼胚胎原始性腺和垂体中定位表达

促甲状腺激素(Thyroid-stimulating hormone,TSH)具有调节生长,发育,代谢和生殖的功能,但是在低等脊椎动物中的功能研究较少。斜带石斑鱼(Epinephelus coioides)促甲状腺激素TSHβ除在垂体中表达外还在性腺中表达,进一步发现TSHβ也在赤点石斑鱼(Epinephelus akaara)性腺中表达。且在人工诱导的赤点石斑鱼中,随着雄性化的转变,TSHβ转录水平升高。为研究石斑鱼TSHβ特殊表达方式的调控机制,采用基因组步移技术扩增获得赤点石斑鱼TSHβ5′端序列共5112bp。将1864bp核心片段顺向克隆到pBK-TOL2载体中,pTSHβ-TOL2重组质粒与转座酶mRNA共注射斑马鱼一细胞期受精卵。Western blot结果显示,在受精后10h的尾芽胚中可检测到GFP的表达,胚体晚期GFP表达量最高。荧光显微镜追踪观察发现,受精后12-30hpf(Hours post fertilization)观察到GFP在原始生殖细胞(PGC)区域表达;36hpf-7dpf观察到GFP除在PGC区域表达外还在垂体中表达。由此可见,赤点石斑鱼TSHβ5′端1864bp的序列具有特异定位的启动子活性。       

斜带石斑鱼L-氨基酸氧化酶基因克隆及表达分析

鱼类的L-氨基酸氧化酶(L-amino acid oxidase, LAAO)具有广泛的抑菌杀虫效果, 为了解斜带石斑鱼(Epinephelus coioides)LAAO基因序列特征及其在刺激隐核虫(Cryptocaryon irritans)感染后的表达变化, 该试验克隆得到2个石斑鱼LAAO基因: EcLAAO-1和EcLAAO-2, 它们的ORF长度分别为1536和1569 bp, 编码511和522个氨基酸, 均含有氨基酸氧化酶(Amino_oxidase)结构域以及LAAO保守序列: DBM和GG motif。多重序列比对显示石斑鱼LAAO与其他鱼类LAAO具有较高的相似性。系统进化树分析表明, 斜带石斑鱼的LAAO与硬骨鱼类亲缘关系较近。实时荧光定量PCR检测结果显示EcLAAO-1和EcLAAO-2在石斑鱼各组织均有表达, 其中皮肤、鳃、胸腺、肝脏和肌肉中含量较丰富; 在感染刺激隐核虫后, 鳃和脾脏EcLAAO-1, EcLAAO-2表达量显著升高(P<0.05), 这些结果暗示了石斑鱼LAAO参与先天性免疫, 并在抗御刺激隐核虫感染中发挥重要作用。     

点带石斑鱼染色体显带技术及其描绘研究

点带石斑鱼(Epinephelus malabaricus)属于鲈形目, 科、石斑鱼亚科、石斑鱼属, 是中国东南沿海暖水性礁栖的名贵海产经济鱼类. 采用PHA活体注射结合秋水仙素培养, 取点带石斑鱼全肾, 低渗处理, 空气干燥制片法制作染色体标本, 利用Alu I 限制性内切酶介导的原位切口平移显带技术, 在点带石斑鱼有丝分裂中期染色体上诱导出带纹清晰、分散良好的多重带. 结果显示, 多数染色体显现出8-10条带纹, 最少的一对染色体也有4条带纹, 同源染色体带纹基本一致, 在每对染色体上的数目及其分布具明显特征性且相对稳定, 同时发现不同分裂相的同一号染色体上, 特征带纹鲜明一致, 带纹数目基本吻合, 具有可重复性和可操作性; 然后用人X和Y染色体文库特异DNA为探针, 对点带石斑鱼的有丝分裂中期分裂相染色体进行了描绘研究. 结果表明, 点带石斑鱼染色体组中测出了人X染色体特异DNA同源片段的3个保守同线群, 分别在点带石斑鱼的第7、第13和第22号同源染色体上, 它们的杂交信号最近边距着丝粒的百分比距离分别大约为62.3%、43.4%及44.4%; 人X染色质同源片段的大小约占点带石斑鱼基因组的4.63％. 但用人Y染色体DNA描绘点带石斑鱼染色体时, 没有检测出可见的同源片段. 研究结果可以为从低等脊椎动物到人类性染色体的进化过程提供一种新的研究思路.       

斜带石斑鱼IL-10基因的克隆及其原核表达

白介素10(Interleukin-10,IL-10)是一个重要的多效细胞因子,主要作用是介导炎症反应和调控一些免疫细胞的分化和增殖。利用同源克隆和RACE-PCR技术获得全长为1 104 bp的斜带石斑鱼(Epinephelus coioides)IL-10基因。该基因ORF为564 bp,编码187个氨基酸,预测蛋白分子量为21.7 k D,等电点为5.74。氨基酸同源性比对及进化分析结果显示,斜带石斑鱼IL-10氨基酸序列和吉富罗非鱼(O.niloticus)同源性最高,达72.25%。利用双酶切及质粒重组技术构建IL-10原核表达载体,并转化大肠杆菌BL21(DE3)中诱导原核表达。SDS-PAGE分析显示,融合蛋白分子量为37.5 k D,与预期相符;在IPTG为0.02mmol/L,37℃诱导3 h,蛋白包涵体的表达量最大。     

Disruption of normal meiosis in artificial inter-populational hybrid females of <Emphasis Type="Italic">Misgurnus</Emphasis> loach

Artificial cross between two genetically different populations of Japanese Misgurnus loach was made to examine the reproductive capacity of the artificial inter-populational hybrid females. Ploidy status and microsatellite genotypes of the eggs laid by these hybrids were inferred from those determined in progenies developed by normal fertilization with haploid loach sperm, induced gynogenesis with UV-irradiated goldfish sperm and/or hybridization with intact goldfish sperm. Some hybrid females laid unreduced diploid eggs genetically identical to the mother. However, these diploid eggs could not develop by spontaneous gynogenesis, but grow to triploid by incorporation of a sperm nucleus. Other hybrid females laid haploid eggs together with diploid eggs and/or various aneuploid and polyploid eggs. Thus, a disruption of normal meiosis occurred in inter-populational hybrid females. The results suggested that the two populations should be so distant as to give rise to atypical formation of unreduced and other unusual eggs in their hybrids.     

静水压诱导翘嘴鳜雌核发育的研究

为通过建立雌核发育纯系以实现快速优化种质资源, 文章以翘嘴鳜(Siniperca chuatsi)为研究对象, 探索出了一套静水压诱导雌核发育的方法: 将装有翘嘴鳜精子的培养皿置于摇床缓慢摇动, 在两只15 w的紫外灯管(培养皿与紫外灯管的垂直距离约17 cm)下照射18min, 然后与雌鱼卵子受精5min, 63 MPa静水压处理2min, 最后常规孵化。HRM分型技术检测其雌核发育群体的雌性率为100%。该方法简单快捷, 诱导孵化率(受精后72h的存活率)为7.56%, 比已有的冷休克诱导翘嘴鳜雌核发育的方法约提高3个百分点。研究结果可用于翘嘴鳜雌核发育品系的快速扩群, 也可用于翘嘴鳜抗逆相关品系的种质资源创制。     

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10 min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5 °C and 30 min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.     

中国南方海域4种石斑鱼的遗传多样性及分子系统发生关系的分析

对中国南方海域的青石斑鱼(Epinephelus awoara)、赤点石斑鱼(E.akaara)、橙点石斑鱼(E.bleekeri)及蜂巢石斑鱼(E.merra)进行了遗传多样性和分子系统发生关系的分析。对9个微卫星DNA标记的基因型测定结果进行计算,发现这4个地理分布重叠且形态相似的石斑鱼均具有较丰富的遗传多样性,其平均等位基因数、平均多态信息含量(PIC)、平均观测杂合度(Ho)及平均期望杂合度(He)的取值范围分别为(8.22±5.02)～(18.67±9.38)、(0.56±0.21)～(0.83±0.13)、(0.62±0.03)～(0.85±0.02)和(0.60±0.07)～(0.86±0.04)。经哈迪-温伯格平衡(HWE)检验,除橙点石斑鱼外,其他3种鱼的微卫星DNA位点都基本符合平衡。利用微卫星DNA标记和线粒体细胞色素b基因对这4种鱼重建的分子系统树均显示,青石斑鱼与赤点石斑鱼的亲缘关系较近。以微卫星DNA标记对所研究石斑鱼的全部个体进行贝叶斯聚类分析的结果表明,4个鱼物种之间的分化关系明确。     

Hybridization of tiger grouper (Epinephelus fuscoguttatus ♀) x giant grouper (Epinephelus lanceolatus ♂) using cryopreserved sperm

Using Ringer solution as extender, the present study examined the protective effect of dimethyl sulphoxide (Me₂SO; 8-12%, v/v) on the cryopreservation of giant grouper (Epinephelus lanceolatus) sperm. The cryopreserved sperm was then successfully applied in interspecific hybridization with tiger grouper (E. fuscoguttatus). Higher motility (90.56 ± 6.58%) and fertilization rate (69.61 ± 4.83%) was achieved in 10% Me₂SO with Ringer solution as extender (dilution ratio 1:1), which should no significant difference in comparison with fresh sperm (95.88 ± 1.64% and 73.10 ± 1.28%). There were no statistical differences in both fertilization and hatching rates between hybrid and non-hybrid tiger grouper by using cryopreserved sperm for fertilization, but malformation rate of the hybrid was higher than non-hybrid (17%) (P < 0.05). Survival rate of the hybrid was lower than that of the controls at 15 days post hatching (23% vs 48%). However, hybrids showed survival rate equal to the controls at the end of the 60-day study period. Hybridization of E. fuscoguttatus x E. lanceolatus was successfully achieved using cryopreserved sperm from giant grouper. The cryopreservation of giant grouper sperm and its application in hybridization provided a technical support for further grouper breeding work.     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

Flow cytometry for assessing the efficacy of interspecific gynogenesis induction in sturgeon

The efficacy of ploidy analysis for separating progeny of Siberian sturgeon Acipenser baerii after induced gynogenesis was demonstrated using sperm of a paternal species differing in ploidy level from the maternal species. Gynogenesis was induced in tetraploid A. baerii with UV‐C irradiated sperm from the diploid sterlet Acipenser ruthenus and vice‐versa. The success of sperm UV irradiation and diploidy restoration by heat‐shock was estimated based on the ploidy level of progeny, confirmed by microsatellite parentage assignment. Hatching rates of interspecific gynogenotes were comparable with rates reported for gynogenesis induction using sperm and eggs of the same species. Juvenile mortality was similar to that observed in the control hybrids. The efficiency and reliability of this method may foster its use for production of gynogenotes in aquaculture, potentially allowing interspecific gynogenesis to replace intraspecific.