鲫鲤杂种一代(F_1)自交二代(F_2)的受精细胞学研究

荷包红鲫 (♀ )×湘江野鲤 (♂ )杂交产生的杂种一代 ( F1)成熟卵的直径为1 .0～ 1 .2 5mm;为单受精孔卵 ,一般只允许单个精子入卵 ;成熟卵在受精前处于第二次减数分裂中期 ;鲫鲤杂种一代的雄性部分可育 ,能产生正常的精子 ;杂种一代的受精方式为单精受精 ,具有正常的受精细胞学程序 .受精 30 s后 ,精子通过受精孔进入卵质 ;5min后 ,精子产生明显星光 ;1 5min后 ,精子头部膨大核化 ,最后形成雄性原核 ,与此同时 ,雌性原核也开始形成 ;2 5min后 ,雌、雄原核向胚盘中央靠近 ,然后彼此接触 ,最后融合为合子核 ;40 min后 ,开始第一次卵裂 ,在一个受精卵第一次卵裂中期的切片上看到了一个三极纺锤体 .     

红鲫与湘江野鲤杂交的受精细胞学研究

红鲫成熟卵直径680—720μm;卵膜孔为精子入卵的唯一通道,包括前庭和精孔管两部分;精孔管内径约5μm。湘江野鲤精子头部直径约2.5μm。在通常情况下,红鲫卵为单精受精。尽管红鲫与湘江野鲤不同属,但杂交仍具有正常的受精细胞学程序。红鲫卵子处于第二次成熟分裂中期接受湘江野鲤精子入卵,精子入卵5min后,出现明显的精子星光;15min后,雄性原核及雌性原核形成;25min后,雌、雄性原核融合;30min后、开始卵裂,发现1个受精卵切片上有4个即将融合的原核,这可能是由于双精受精所致。     

人工复合三倍体鲤卵的受精的生物学研究

本文对人工复合三倍体鲤成熟卵的受精生物学进行了研究。结果表明,三倍体卵体壳具有凸凹不平的表面,与正常二倍体卵比较有明显差异。受精生物学观察发现有些卵为多精受精,进入卵的所有精子都能形成雄性原核,排出一个极体,但绝大部分受精卵在发育期间分裂不齐、畸形、直至死亡,只有具有三套完整染色体的卵子的雌核才能发育成为三倍体个体。三倍体鲤的成熟分裂不是完全的均等分裂,有些不等分裂的卵母胞核中只有两套染色体,或一     

异源四倍体鲫鲤的受精细胞学

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。精子入卵以来,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;23min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。     

异源四倍体鲫鲤的受精细胞学

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8 min,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;13 min时,精子头部开始膨胀,趋向核化;18 min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;33 min时,雌、雄原核完全融合成为一个合子核;38 min时,受精卵开始第一次卵裂,53 min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.     

人工复合三倍体鲤卵的受精生物学研究

本文对人工复合三倍体鲤成熟卵的受精生物学进行了研究。结果表明,三倍体卵外壳具有凸凹不平的表面,与正常二倍体卵比较有明显差异。受精生物学观察发现有些卵为多精受精,进入卵的所有精子都能形成雄性原核,排出一个极体,但绝大部分受精卵在发育期间分裂不齐、畸形、直至死亡,只有具有三套完整染色体的卵子的雌核才能发育成为三倍体个体。三倍体鲤的成熟分裂不是完全的均等分裂,有些不等分裂的卵母胞核中只有两套染色体,或一套染色体,甚至是一套半染色体。     

实验小鼠受精卵染色体的观察及制作法

哺乳动物精子穿人卵细胞后,精子头部膨 胀,进一步发育,出现雄性原核和雌性原核。受 精卵第一次成熟分裂时制作的染色体可以单独 分析雄性和雌性染色体．小鼠受精卵染色体制 作国外已有报道[2,3.87,国内尚未见报道。本文 根据Tarkowski的方法〔BI略加修改,能获得清晰 的小鼠受精卵染色体,可为胚胎发育、发育遗传 学和遗传毒理学等方面研究提供实验手段。     

中华鲟受精细胞学研究

中华鲟(Acipenser sinensis Gray)的成熟卵具有一层放射膜及二层卵黄膜。在动物极有9—15个受精孔。每一受精孔有一大的入口(12.7—13.9μm直径)及一细长的受精管道(1.2—1.3μm直径)。 进入受精孔的许多精子只能按序入卵。其中只有一个精子的头部膨大核化,最后形成雄性原核。同时活跃的卵子也形成雌性原核。雌、雄原核彼此接触,最后融合成合子核,随后分成两个子核。 中华鲟的受精方式为多精入卵,单精受精。     

唇鱼骨受精的细胞学研究

唇精孔器属深凹陷、短孔径型。精子在受精后2s到达精孔管、5s进入卵子。受精后8—15min,卵子进入第二次减数分裂后期。受精后10min,开始形成雄性原核。受精后20min,进入第二次减数分裂末期。受精后25min,雌性原核形成。受精后30—35min,雌性原核向雄性原核移动。受精后40min,雌雄原核接近。受精后50min,雌雄原核结合。受精后70min,受精卵进入第一次有丝分裂中期,受精后80min,进入第一次有丝分裂后期,受精后120min,进入末期。卵黄降解与其内部或外周小泡的泡状缺口紧密相关。雌雄原核结合是精子星光扩张、牵引和细胞质流动的共同结果。有多精入卵的现象。     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method

The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid-UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV-irradiated region during its formation. When the UV-irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV-irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.     

FERTILIZATION PROCESS IN THE HEART-URCHIN,CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE*

The observations of the fertilization process in the heart-urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.     

Selective identification of the paternal mitochondrion in living sea urchin eggs and embryos by chlorotetracycline

When sea urchin eggs are pretreated with fluorescent chelate probe chlorotetracycline (CTC) and then fertilized with unlabeled sperm, a small, brightly fluorescent particle resembling the mitochondrion of free-swimming sperm both in size and fluorescent staining characteristics appears in the egg cytoplasm. This particle first appears near the base of the insemination cone and, like the paternal mitochondrion identified in previous ultrastructural studies, remains closely associated with the male pronucleus during its microtubule-dependent migration toward the egg center. These similarities strongly suggest that the fluorescent particle observed in the cytoplasm of living, CTC-pretreated sea urchin eggs is, in fact, the mitochondrion of the fertilizing sperm.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。     

Reciprocal inheritance of centrosomes in the parthenogenetic hymenopteran Nasonia vitripennis

Background: In the majority of animals, the centrosome-the microtubule-organizing center of the cell-is assembled from components of both the sperm and the egg. How the males of the insect order Hymenoptera acquire centrosomes is a mystery, as they originate from virgin birth.Results: To address this issue, we observed centrosome, spindle and nuclear behavior in real time during early development in the parthenogenetic hymenopteran Nasonia vitripennis. Female meiosis was identical in unfertilized eggs. Centrosomes were assembled before the first mitotic division but were inherited differently in unfertilized and fertilized eggs. In both, large numbers of asters appeared at the cortex of the egg after completion of meiosis. In unfertilized eggs, the asters migrated inwards and two of them became stably associated with the female pronucleus and the remaining cytoplasmic asters rapidly disappeared. In fertilized eggs, the Nasonia sperm brought in paternally derived centrosomes, similar to Drosophila melanogaster. At pronuclear fusion, the diploid zygotic nucleus was associated only with paternally derived centrosomes. None of the cytoplasmic asters associated with the zygotic nucleus and, as in unfertilized eggs, they rapidly degenerated.Conclusions: Selection and migration of the female pronucleus is independent of the sperm and its aster. Unfertilized male eggs inherit maternal centrosomes whereas fertilized female eggs inherit paternal centrosomes. This is the first system described in which centrosomes are reciprocally inherited. The results suggest the existence of a previously undescribed mechanism for regulating centrosome number in the early embryo.     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastoeyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg.     

Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration

A male pronucleus migrates toward the center of an egg to reach the female pronucleus for zygote formation. This migration depends on microtubules growing from two centrosomes associated with the male pronucleus. Two mechanisms were previously proposed for this migration: a "pushing mechanism," which uses the pushing force resulting from microtubule polymerization, and a "pulling mechanism," which uses the length-dependent pulling force generated by minus-end-directed motors anchored throughout the cytoplasm. We combined two computer-assisted analyses to examine the relative contribution of these mechanisms to male pronuclear migration. Computer simulation revealed an intrinsic difference in migration behavior of the male pronucleus between the pushing and pulling mechanisms. In vivo measurements using image processing showed that the actual migration behavior in Caenorhabditis elegans confirms the pulling mechanism. A male pronucleus having a single centrosome migrated toward the single aster. We propose that the pulling mechanism is the primary mechanism for male pronuclear migration.     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.