Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thalictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (L.) Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

Mode of pollen tube growth in pistils of Ticodendron incognitum (Ticodendraceae,Fagales) and the evolution of chalazogamy

Ticodendron incognitum is the sole species of the Ticodendraceae, which was established as a new family in the Fagales less than 20 years ago. Considering the diverse modes of pollen tube growth observed in other Fagales, we investigated the growth of pollen tubes in the pistil of Ticodendron. At the time of pollination, T. incognitum had four immature ovules in a bilocular ovary, thus exhibiting delayed fertilization, as in other Fagales. During the period when fertilization was delayed, pollen tube growth in the pistil was intermittent, consisting of five steps associated with development of the ovules and embryo sacs. Four cessation sites occurred: in the style, in the tissue of the upper part of the ovary, inside and outside of the funicle and at the chalaza. A single pollen tube eventually reaches a mature embryo sac through the chalaza in one of the four ovules. While both delayed fertilization and intermittent pollen tube growth play a role in male and female gametophyte selection, as in other Fagales, the five‐step process of pollen tube growth through the chalaza (i.e. chalazogamy) is characteristic of lineages of the Casuarinaceae, Ticodendraceae and Betulaceae (the latter with the loss of one step). © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 621–631.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thalictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thafictroides （L.） Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

Ultrastructure of the Mature Egg and Fertilization in the Fern Ceratopteris thafictroides

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (L.) Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.     

不测土条件下半定量施肥原理和模型评述

1　不测土条件下半定量推荐施肥的需求矿质营养学说是施肥最基本原理 ,它的诞生标志着人类主动施肥时期的开始。从早期的定性施肥阶段 ,发展到今天的定量施肥阶段 ,如何确定施肥量一直是施肥技术的核心和难点。测土是定量施肥或精确施肥的前提。然而 ,测土本身存在各种偏差或精度、测土需要费用 ,本文仅对代表性的不测土施肥模型及其原理进行评述。即使有了土壤有效养分的测定结果 ,如何将其转化为一季内土壤提供的养分量一直是个理论难题 ,特别是Ｎ素土壤供应量更为难测或难以估算。因此 ,如何在使用定量施肥模型的同时 ,而又将土壤养分因…     

不同施肥处理下蔬菜塑料大棚土壤微生物活性及功能多样性

利用土壤酶学方法、微生物培养方法及Biolog微生物自动分析系统,分析了不同施肥处理下塑料大棚种植黄瓜与番茄的土壤中微生物活性及功能多样性.结果表明,与传统施肥相比,配方施肥下塑料大棚黄瓜土壤脱氢酶活性提高了36.5%,番茄土壤脱氢酶活性则提高了66.5%,且达到了显著水平.配方施肥下塑料大棚黄瓜与番茄土壤可培养放线菌数量分别比传统施肥处理增加了30.0%和72.2%,且都达到了显著水平.Biolog结果显示,在土壤微生物培养过程中,配方施肥下塑料大棚番茄土壤微生物群落AWCD始终大于无肥处理及传统施肥处理.与传统施肥相比,配方施肥下塑料大棚黄瓜土壤微生物培养96 h的AWCD增加了1.9%,番茄土壤微生物培养96h的AWCD则增加了68.5%,且达到了显著水平.配方施肥下塑料大棚番茄土壤微生物Shannon指数、Simpson指数及McIntosh指数都大于传统施肥处理,并且McIntosh指数达到了显著水平.PCA分析表明,配方施肥下蔬菜塑料大棚土壤微生物群落碳源利用能力与传统施肥处理明显不同.     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻（Oryza sativa）受精过程中雌雄性细胞融合时的形态特征及时间进程,确定合子期,为花粉管通道转基因技术的实施提供理论依据。结果表明：授粉后,花粉随即萌发,花粉管进入羽毛状柱头分支结构的细胞间隙,继续生长于花柱至子房顶部的引导组织的细胞间隙中,而后进入子房,在子房壁与外珠被之间的缝隙中向珠孔方向生长,花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞,释放精子。精子释放前,两极核移向卵细胞的合点端：两精子释放于卵细胞与中央细胞的间隙后,先后脱去细胞质,然后分别移向卵核和极核,移向卵核的精核快于移向极核的精核：精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下：授粉后,花粉在柱头萌发：花粉萌发至花粉管进入珠孔大约需要0．5小时：授粉后0．54,时左右,花粉管进入一个助细胞,释放精子：授粉后0．5—2．5小时,精卵融合形成合子：授粉后约10．0小时,合子第1次分裂,合子期为授粉后2．5-10．04,时：授粉后1．0-3．04,时,精核与两极核融合：授粉后约5．0小时,初生胚乳核分裂。’     

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial,the brushtail possum (Trichosurus vulpecula)

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E. coli) strain JM109, M15, SG13009, or BL21 codon plus. Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3. Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3. Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence. Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only. The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials.     

Sperm-egg interaction in the mouse using live and glutaraldehyde-fixed eggs

A study of interaction between gametes in the mouse, using live and glutaraldehyde-fixed eggs, showed that, while in live eggs the binding is a two-step process (“early” and “late” binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca²⁺-dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm-egg interaction removed from all the other events of the fertilization process.     

Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro

To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67–68%) and formed normal foetuses (50–53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5–3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.     

A two-second delay confers first-male fertilization precedence within in vitro sperm competition experiments in Atlantic salmon

In vitro paired-male sperm competition experiments in Atlantic salmon Salmo salar for a single female's eggs revealed that 2 s delays in sperm release caused significant reductions in paternity, with second males achieving only 30% fertilization success (against an expected 50%). This first-male fertilization precedence supports previous work suggesting that sperm competition follows the principles of a race in Atlantic salmon, and suggests that any timing asymmetry in sperm release within natural competitive spawnings could have significant consequences for male fertilization success.     

番茄受精作用及其间隔期的研究

利用常规石蜡切片法研究了番茄受精作用的全过程,具体研究结果为:(1)授粉后2 h,花粉粒在柱头上萌发;约2~4 h,花粉管长入柱头,且末端膨大;约8 h后,生殖细胞进入分裂期;并于约两小时后,分裂为两个精细胞。(2)约14 h,花粉管进入子房腔;约18~24 h,花粉管进入胚囊,破坏一个助细胞,并在其珠孔端释放两个精子;随后被释放的精子移到卵细胞与次生核附近。(3)授粉后约30 h精核进入卵细胞;约34 h,精核与卵核融合,并在卵核内出现分散的雄性染色质,进而出现雄性核仁;44~50 h,雌、雄性核仁融合,形成合子;合子的休眠期为10 h左右。60 h之后,合子分裂形成二细胞原胚。(4)约26 h,另一个精子的精核与次生核核膜相贴伏,随后与之融合;约30~34 h,次生核内出现分散的雄性染色质,随之出现雄性核仁;约38~42 h,雌、雄性核仁融合,形成初生胚乳核。约44 h后,初生胚乳核进行有丝分裂,形成两个胚乳细胞。番茄胚乳发育属于细胞型。初生胚乳核无休眠期。(5)精子与次生核的融合比与卵核的融合快。(6)番茄的受精作用属于有丝分裂前配子融合类型。     

Predominance of outcrossing in Lymnaea stagnalis despite low apparent fitness costs of self-fertilization

We have quantified the natural mating system in eight populations of the simultaneously hermaphroditic aquatic snail Lymnaea stagnalis, and studied the ecological and genetic forces that may be directing mating system evolution in this species. We investigated whether the natural mating system can be explained by the availability of mates, by the differential survival of self- and cross-fertilized snails in nature, and by the effects of mating system on parental fecundity and early survival. The natural mating system of L. stagnalis was found to be predominantly cross-fertilizing. Density of snails in the populations had no relationship with the mating system, suggesting that outcrossing rates are not limited by mate availability at the population densities observed. Contrary to expectations for outcrossing species, we detected no evidence for inbreeding depression in survival in nature with inferential population genetic methods. Further, experimental manipulations of mating system in the laboratory revealed that self-fertilization had no effect on parental fecundity, and only minor effects on offspring survival. Predominance of cross-fertilization despite low apparent fitness costs of self-fertilization is at odds with the paradigm that high self-fertilization depression is necessary for maintenance of cross-fertilization in self-compatible hermaphrodites.     

Spermatozoon structure and motility in the anuran Lepidobatrachus laevis

Synthetic human gonadotropin releasing hormone (GnRH) injections were used for induction of spermatozoon release followed by cloacal lavage or mechanical stimulation of sperm release in Lepidobatrachus laevis . Light microscopic observations of Lepidobatrachus laevis spermatozoa indicated an acrosomal segment with a length of 4.1 μm delineated by an indentation, a nuclear region of 12.6 μm in length and a midpiece of 0.87 μm in length. The tail was 54.9 μm long by 1.35 μm wide with two lateral axial fibers and a central undulating membrane. At the electron microscopic level, the unusual tail had two complete axonemes that emanated from the distal centriole. The tail also contained two axial fibers 77 nm in diameter medial to the axonemes and was connected by an undulating membrane. An unusual accessory cell adherent to the head of the spermatozoon was noted in freshly obtained suspensions of spermatozoa. Spermatozoa with the accessory cell were motile and a subsequent loss of motility was correlated with the shedding of the accessory cell.     

A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species

Injections of synthetic human gonadotropin releasing hormone (GnRH) into the dorsal pelvic area were used in an attempt to stimulate sperm release in isolated males of eight anuran species including Xenopus laevis, Rana pipiens and Lepidobatrachus laevis . Sperm were obtained within 1–5 h post injection either by mechanical stimulation or by cloacal lavage. Sperm suspensions varied from 8 μL to 7 mL and the cell densities ranged from 4 × 10⁵ to 4 × 10⁷ sperm/mL. The sperm obtained from seven species using GnRH-induced release were viable based on light microscopic observations of motility. In addition, sperm preparations fertilized eggs in vitro and produced normal tadpoles in the case of L. laevis and L. llanensis . This hormonal method of anuran sperm collection will provide a convenient non-injurious way to obtain anuran sperm for basic studies of reproduction and development.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

小鼠体外受精、胚胎培养及胚胎快速冷冻的研究

目的　为扩大胚胎来源并获取特定胚龄胚胎 ,建立小鼠冷冻胚胎库。方法　运用超数排卵、体外受精与胚胎培养及胚胎冷冻技术系统研究了小鼠受精卵的体内发育与运行规律。卵母细胞的体外成熟与受精、单细胞胚胎培养及胚胎快速冷冻。结果　 (1)注射hCG后 12～ 2 0h受精卵发育至原核期 ,4 2～ 4 8h为 2 细胞期 ,4 8～ 6 0h为 4 细胞期 ,6 0～ 6 8h为 8 细胞期 ,以上各期受精卵均处于输卵管中 ;75～ 78h为桑椹胚 ,78～ 80h为致密桑椹胚 ,90～ 92h为早期囊胚 ,92～ 96h为囊胚 ,以上各期均处于子宫角中。 (2 )培养液中添加促性腺激素 (FSH与hCG) ,能显著提高卵母细胞的体外受精率 ,添加FCS和激素组的体外受精率又显著高于单独添加激素组 ,FCS还能显著提高胚胎发育。 (3)在培养液中添加EDTA ,能有效克服小鼠胚胎的 2 细胞阻断 ,其 2 细胞胚的发育率达 10 0 % ,8 细胞胚发育率达 5 5 %以上 ;牛、羊上皮细胞培养液上清也能有效克服 2 细胞阻断。添加乳酸钠和丙酮酸钠可使 2细胞与 8细胞期胚的发育率显著提高。 (4)以D PBS +甘油 +蔗糖为冷冻液 ,以D PBS +蔗糖为稀释液 ,对小鼠胚胎进行快速冷冻 ,桑椹胚的存活率为 6 9 3% ,早期囊胚的存活率为 6 0 4 %。结论　研究为将生物技术应用于小鼠 ,扩大卵子和胚胎来源     

Carbohydrates and glycoproteins involved in bovine fertilization in vitro

In the present study, efforts were made towards identifying carbohydrates and glycoproteins involved in bovine in vitro fertilization (IVF). In vitro matured cumulus-oocyte complexes (COCs) were inseminated in the presence of a variety of carbohydrates and glycoproteins to determine which glycoconjugates act as competitive inhibitors of oocyte penetration. Among the carbohydrates and glycoproteins tested, D-mannose, fucoidan, dextran sulfate, and fibronectin were the most potent inhibitors of oocyte penetration (90% or more inhibition), while L-fucose and vitronectin inhibited the penetration rate to a lesser extent (around 50% inhibition). Other carbohydrates caused less than 40% inhibition (i.e., D-galactose, N-acetyl-D-galactosamine, D-fucose, and sialic acid) or were not effective as inhibitors of oocyte penetration (i.e., mannan, N-acetyl-D-glucosamine, dextran, and heparan sulfate). Heparin was the only carbohydrate that significantly increased the penetration rate. To exclude a possible toxic effect on spermatozoa, sperm motility was evaluated over time by means of computer-assisted sperm analysis in the presence of carbohydrates and/or glycoproteins that inhibited the penetration rate with 40% or more. L-fucose, dextran sulfate, and vitronectin did not significantly influence total and progressive sperm motility, whereas D-mannose, fucoidan, and fibronectin caused a significant, but slight reduction in both motility parameters. These results are indicative for the involvement of D-mannose, L-fucose, fucoidan, dextran sulfate, fibronectin, and vitronectin in bovine IVF.     

论有机肥料在农业生态系统中的地位和作用

人类为了生存,要从自然界取走一些物质和能量,并以不同的形式将物质和能量归还给自然即生态系统。要维持自然生态系统的动态平衡,“取走”和“归还”必须保持永续和相对