Ca2+，Mg2+，K+及胰蛋白酶和胰蛋白酶抑制剂对中国对虾卵子激活的影响

对虾成熟卵子的激活都是由海水引起的,海水的刺激引起成熟的卵母细胞发生皮层反应,完成减数分裂,这一过程同有无精子的作用无关(Clark et al.,1977,1980; Lynn et al.,1991; Pillai et al.,1987)。在对锐脊单肢虾(Sicyonia ingentis)的卵子激活研究中发现,其成熟卵子无论受精与否在接触海水以后均具有相同的激活过程,受精卵和未受精卵的皮层反应及减数分裂的恢复没有区别,而海水中刺激卵子发生皮层反应直接因子是Mg2+而不是Ca2+。在无Mg2+人工海水中,锐脊单肢虾的卵子不能激活;而在无Ca2+人工海水中,卵子则可正常激活(Pillai et al.,1987; Lindsay et al.,1992a)。Mg2+对锯额长臂虾(Palaemon serratus)卵子减数分裂的形响的研究表明,锯额长臂虾卵子减数分裂的恢复必须有Mg2+参与,而Ca2+则是非必须的;胞外Mg2+可以诱导卵子的皮层反应,并使卵膜外的微绒毛消失;胞外Mg2+的增加可使卵子内部游离Ca2+浓度发生变化,从而诱导卵子的激活(Goudeau et al.,1986,1991,1996)。 根据前人的报道,胰蛋白酶和胰蛋白抑制剂对对虾精子的顶体反应、卵子的激活都具有明显的影响(Lynn et al.,1987; Griffin et al.,1990; Lindsay et al.,1992b; Chen et al.,1994)。Lynn等(1987)报道褐对虾(Penaeus aztecus)的卵胶前体(jelly procusor)含有约70%-75%的蛋白质及25%-30%的多糖,胰蛋白酶对于人工分离的卵胶前体具有明显的降解作用,胰蛋白酶抑制剂可以抑制卵子在海水中的激活,Griffin等(1990)的研究发现,锐脊单肢虾的卵子内含有类胰蛋白酶的物质,解剖性成熟雌虾获得的卵子置入海水摇匀,上清液-卵水(egg water)可以诱导取自雌虾纳精囊内的精子发生顶体反应,胰蛋白酶也可以诱导对虾精子的顶体反应,而胰蛋白酶抑制剂则完全可以抑制对虾精子在人工海水中发生顶体反应时顶体丝的形成。Lindsay等(1992b)的研究也得出了类似的结果,并对卵水和胰蛋白酶不同浓度及不同作用时间下对对虾精子顶体反应的诱导作用进行比较研究。Chen等(1994)对锐脊单肢虾精子的提取物进行生化分析发现,精子内部也有类胰蛋白酶的物质,并通过免疫电镜技术将其进行定位,精子的提取物可以降解分离的卵黄膜。 激光扫描共聚焦显微镜(Laser scanning confocal microscope)在海洋动物的受精及发育生物学的研究中已有一些报道(Sardet et al.,1992; Stricker et al.,1992; Shen et al.,1993; Jaffe, et al.,1994; Malinda, et al.,1994)。而在对虾的受精及发育生物学方面应用还比较少,只有锐脊单肢虾的卵子激活及早期发育的研究上有一些报道(Hertzler et al., 1992, 1994: Lindsay et al.,1992a)。 海水中的离子成分是影响卵子激活的关键因子,而胰蛋白酶和胰蛋白酶抑制剂对对虾受精也有着很大影响,这两方面的工作在国内、外中国对虾的受精生物学研究中均未见报道。 作者对上述两类理化因子对中国对虾卵子激活的影响进行研究,并使用激光扫描共聚焦显微镜对中国对虾卵子受胰蛋白酶和胰蛋白酶抑制剂的作用而发生的形态变化进行研究,初步分析了造成异常卵裂的原因。     

海水双壳类受精生物学研究进展

1 研究概况,在海水双壳类中开展的大量受精生物学研究表明,绝大部分的海水双壳类为卵生型,雌雄配子被排放到海水中完成受精。排放的卵子处于第一次减数分裂前期(生发泡期)或中期。精子入卵后,受精卵恢复减数分裂,先后排放出两个极体,形成雌雄原核并最终启动卵裂。     

黄海太平洋鲱受精前后卵膜的动态变化

本文使用透射电镜，细胞化学和扫描电镜等方法，在亚微水平上对黄海太平洋鲱鱼（ＣｌｕｐｅａｐａｌａｓｉＣｖｖ，＆Ｖｏｌ．）卵黄膜和受精膜的结构以及皮层小泡在卵黄膜转化为受精膜过程中的作用，受精卵新质膜的形成及其细胞化学变化等进行了研究。观察到，太平洋鲱鱼卵黄膜由５层组成，皮层小泡参与了受精膜的形成，在卵黄膜转化为受精膜的过程中，５层都发生不同程度的形态和糖蛋白及ＰＡＳ阳性物质的变化。另外发现太平洋鲱有３种皮层小泡存在。在皮层反应中，第一种皮层小泡在受精后３ｍｉｎ内迅速破裂，未受精卵尚含有ＰＴＡ染色的小颗粒从小泡中泌出，其界膜形成了质膜的一部份。受精后，此质膜与皮层小泡形成的质膜相同排列构成卵子新的质膜。另外，还发现第一小泡顶部，与卵子质膜相接触的卵子表面有富含糖蛋白的泡状结构。卵子质膜表面糖蛋白及ＰＡＳ阳性物质，随时间推延而不断发生变化     

栉孔扇贝♀×华贵栉孔扇贝♂受精及早期胚胎发育过程的细胞学荧光显微观察

为探讨贝类杂交优势 ,利用HOECHST3 3 2 5 8对已固定样品进行染色的方法 ,连续观察了栉孔扇贝♀×华贵栉孔扇贝♂受精的细胞生物学过程 ,初步证明了这 2种远缘扇贝种类之间进行杂交的可行性。结果表明 ,栉孔扇贝的成熟未受精卵子处于第一次减数分裂中前期 ,部分来自华贵栉孔扇贝的异源精子可顺利进入卵子并激发后者完成 2次减数分裂 ,排出第一及第二极体。成熟的雌雄原核形成后完成融合过程 ,形成合子核 ,受精过程结束。大部分杂交受精卵及杂种早期胚胎可以正常发育 ,但其发育进程较种内近交对照组有明显的差别 ,具体表现在延缓性和不同步性 2个方面。     

三疣梭子蟹(Portunus trituberculatus)卵子发生、激活与早期卵裂的细胞学观察

采用连续切片和光镜技术,对三疣梭子蟹卵子发生、激活和早期卵裂进行了细胞学观察.结果表明,在发育及再发育的卵巢中,三疣梭子蟹卵子发生主要经历了卵原细胞的增殖和初级卵母细胞的分化、生长、成熟这两个阶段.依据卵子发生过程中雌性生殖细胞形态的变化,这两个阶段又可细分为卵原细胞期、卵黄发生前卵母细胞期、小生长期、大生长期、近成熟卵母细胞期和成熟卵母细胞期等6期.成熟卵母细胞处于第一次减数分裂中期,这可作为卵子成熟的形态学标志.第一次减数分裂中期纺缍体长轴的旋转可以发生于卵巢中,与精子及海水的刺激无关.初级卵母细胞达到形态和生理上都成熟的时间窗口很窄.精子入卵引起卵子的激活和减数分裂的完成.23℃下离体培养时,第一和第二极体分别在卵产出后约40min和2h释放.雄原核的形成早于雌原核.雌、雄原核在卵产出后约5h通过联合形成合子核.卵产出后约6h,受精卵处于第一次核分裂后期,但卵裂沟直到卵产出后13h才能观察到,此时核已完成了2次分裂,形成合胞体胚胎.     

锯缘青蟹皮层反应与受精膜形成研究

锯缘青蟹成熟卵子排出时具有两层卵黄膜，卵子排出后形成壳膜，与卵子附着有关。成熟卵子具有两种皮层颗粒，精子入卵后，皮层反应开始，两种皮层颗粒相继胞吐。首先由致密颗粒胞吐，形成一层很薄的膜性结构，接着环形颗粒大量胞吐，其内含物融合，并与外面薄层融合为受精膜。卵子质膜由于皮层颗粒胞吐作用形成镶嵌膜。     

对虾受精过程生理生化特征的研究进展

对虾受精细胞学目前已成为对虾生殖生物学的研究热点。近十几年来科研人员对对虾的受精过程、特点及影响条件,受精的生理生化特性及机理进行了广泛的研究,取得了很大进展。对单肢虾、褐对虾、白对虾、鹰爪虾、中国对虾等虾类受精细胞学的研究表明,对虾的受精过程包括精子的顶体反应、卵子的激活(卵子发生皮层反应形成孵化膜、减数分裂的恢复等)、精卵的结合(包括精子的入卵、雌雄原核的形成及融合等)。这些过程同其他动物的受精过程有些相似,但也有明显的不同,而且不同的对虾也有各自的特性。目前对锐脊单肢虾已研究得比较深入,而对其他虾类…     

毛蚶受精和早期卵裂过程核行为的荧光显微镜观察

利用Hoechst33258染色、荧光显微镜观察方法,对毛蚶受精和早期卵裂过程中核行为的细胞学变化进行了详细观察.结果表明,毛蚶成熟未受精卵呈卵圆形,卵径为52.909±2.092μm,核相处于第一次成熟分裂中期.在水温为26±1℃条件下进行受精,在受精后15~18、20~25 min,受精卵先后排出第一、第二极体,完成第一次和第二次成熟分裂;受精后30 min左右,雌、雄性原核形成;受精后35 min,雌、雄性原核各自形成染色体组,在卵子中央发生联合,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,形成2个大小不等的卵裂球;受精后55~60 min,受精卵完成第二次卵裂,形成1大3小4个卵裂球.另外,在研究中还发现了极少量的多精受精、多极分离等异常细胞学现象,对其成因及机制进行了分析和探讨.     

海蜇卵子卵径及其受精能力的初步探讨

研究观察了苗种生产过程中亲蜇产卵间隙卵巢中的卵子、自然产卵排放的卵子、受精卵的差异,结果发现其卵径依次增大,平均值依次为(74.57±1.05)、(84.22±1.13)、(92.03±1.39)μm,且非产卵时刻其卵巢中的卵子不具有受精能力,海蜇自然排放的卵子2h后亦失去受精能力。该结果表明,海蜇卵子产卵排放前须经历必要的生理转化过程才能成熟排放,具有受精能力。海蜇卵子受精后半径增大,表明海蜇卵子受精过程中亦会发生皮层反应,具体过程与机制需要进一步的研究。本文研究表明,海蜇人工授精不是可以随意进行的,应选择在海蜇自然产卵排精前后的时机及时进行。     

铜藻繁殖发育过程的研究

本研究观察了铜藻雌雄生殖托的形态、精子卵子的排放和受精卵的发育过程。研究发现,铜藻的雌雄性生殖托均为圆柱状,而雄性生殖托比雌性生殖托更加细长。生殖托发育成熟后会形成生殖孔,卵从生殖孔排出,靠粘液附在生殖托表面等待受精,且有8个核,被透明壁包围。而精子则呈放射状排列在生殖孔周围,排出的精子具有明显的鞭毛结构,游动到卵子周围完成受精过程。受精后的卵由8个核融为1个中央大核,受精卵经过多次细胞分裂后形成假根芽。然后,脱落在培养容器底部,发育成带有假根的幼孢子体。8周后生长成带有2～3个叶的幼苗,幼苗长达18 mm。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.