烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚.要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料.以前的研究表明,烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显.这种异型性的精细胞可能与倾向受精有关.烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞.用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞.用微量酶液可使两个精细胞分开.然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体.这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础.本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚。要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料。以前的研究表明, 烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显。这种异型性的精细胞可能与倾向受精有关。烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞。用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞。用微量酶液可使两个精细胞分开。然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体。这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础。本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件。     

五唇兰精细胞的分离

五唇兰(Doritis pulcherrima)具二胞花粉,授粉后1 d即有花粉开始萌发,授粉后5 d开始有生殖细胞完成有丝分裂形成一对精细胞。通过人工授粉使花粉管在子房内发育,再利用花粉管直接爆破,成功分离出五唇兰的精细胞。成对的2个精细胞在直径大小、荧光强弱上均显示出较大差异,预示2个精细胞具有不同的前途。     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000     

The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.)

 Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell. Received: 25 August 1997 / Revision accepted 16 March 1998     

A bias caused by ectopic development produces sexually dimorphic sperm in nematodes

Self-fertile hermaphrodites have evolved independently several times in the genus Caenorhabditis [1, 2]. These XX hermaphrodites make smaller sperm than males [3, 4], which they use to fertilize their own oocytes. Because larger sperm outcompete smaller sperm in nematodes [3-5], it had been assumed that this dimorphism evolved in response to sperm competition. However, we show that?it was instead caused by a developmental bias. When we transformed females of the species Caenorhabditis remanei into hermaphrodites [6], their sperm were significantly smaller than those of males. Because this species never makes hermaphrodites in the wild, this dimorphism cannot be due to selection. Instead, analyses of the related nematode Caenorhabditis elegans suggest that this dimorphism might reflect the development of sperm within the distinct physiological environment of the hermaphrodite gonad. These results reveal a new mechanism for some types of developmental bias-the effects of a novel physical location alter the development of ectopic cells in predictable ways.     

Double spermatogenesis in Chelicerata

Sperm dimorphism is a rare phenomenon in Chelicerata. Until now, it was known only from three species of the opilionid genus Siro (Sironidae, Cyphophthalmi). Fertilizing (eusperm) and nonfertilizing spermatozoa (parasperm) develop in the same cyst and are thus sister cells. The fine structure of the spermatozoa of two species has been examined and is compared here. In contrast to Siro rubens, S. duricorius spermatozoa lack an acrosomal complex. Both sperm types produce a transitional process, a more or less modified flagellum, which is later retracted. Hence, the spermatozoa are aflagellate. Eusperm and parasperm of all three species form highly ordered sperm balls that are stored in the deferent duct. Reviewing and adding new results about the sperm dimorphism in this arachnid taxon provides the basis for some considerations of another enigmatic morphological character found in Uropygi and Amblypygi, i.e., the tubular accessory genital glands that show holocrine extrusion. These glands are suggested to represent modified, infertile derivatives of the testis anlage. Their secretion is produced in a way reminiscent of a strongly degenerated spermatogenesis. Consequently, these products may be regarded as strongly degenerated germ cells representing a line of germ cell development, which has been separated very early in spermatogenesis from the usual line leading to fertilizing sperm cells. This further, although less evident, case of probable dichotomous germ cell development is discussed with respect to the controversial phylogenetic-systematic relationships between Uropygi (Thelyphonida and Schizomida), Amblypygi, and Araneae.     

Ultrastrukturelle Untersuchungen zur Morphologie und Genese der Spermien von Archaeogastropoda

The sperm cells ofPatella coerulea (Patellacea),Monodonta turbinata, andGibbula tumida (Trochacea) were investigated by means of transmission electron microscopy. They belong to the primitive type (sensu Franzén) and have more features in common with primitive Bivalvia sperms than with Neritacea. Their head contains an apical acrosome and a roundish nucleus followed by 4 or 5 mitochondria and a centriolar apparatus which consists of two centrioles, one of which bears a flagellum. The sperm cells ofMonodonta andGibbula are very similar to each other and differ mainly in size;Patella exhibits more differences (very small acrosome, subacrosomal space, variable number of spherical mitochondria (origin of spermic dimorphism ?). The development of the sperm cells shows no peculiarities.     

The evolution of male-female dimorphism: Older than sex?

This contribution considers the evolution of a dimorphism with respect to cell fusion characteristics in a population of primitive cells. These cells reproduce exclusively asexually. The evolution towards asymmetric fusion behaviour of cells is driven by selection promoting horizontal transfer of an endosymbiontic replicator. It is concluded that evolution of asymmetric cell fusion in this scenario is more likely than evolution of sexual differentiation in a sexually reproducing population. Pre-existing dimorphism with respect to cell fusion may thus have been the basis for the establishment of sexual differentiation at the level of gamete fusion, and this in turn is fundamental to the evolution of two different sexes, male and female.     

蓝猪耳精细胞的分离及两个精细胞群体的收集

蓝猪耳是二细胞型花粉,生殖细胞在花粉管中分裂形成两个精细胞。用体内-体外技术培养出花粉管后,将其置于爆破液中即可释放出花粉管内含物,其中包括两个精细胞和营养细胞。在显微镜下两个精细胞具二型性：体积较大的精细胞与花粉管的营养核相连,体积较小的精细胞只与大精细胞连接。两个精细胞之间的连接比较结实,需用微量酶液将两个精细胞分开。用显微操作仪就可分别挑选出两个精细胞群体,分别有上百个细胞。蓝猪耳精细胞的成功分离为利用蓝猪耳开展离体受精研究打下了良好的基础。这种单一纯化的精细胞群体的获得为用分子生物学方法区分两个精细胞的特异基因和蛋白质创造了条件。     

Metric variation and sexual dimorphism in the dentition of Ouranopithecus macedoniensis

The fossil sample attributed to the late Miocene hominoid taxon Ouranopithecus macedoniensis is characterized by a high degree of dental metric variation. As a result, some researchers support a multiple-species taxonomy for this sample. Other researchers do not think that the sample variation is too great to be accommodated within one species. This study examines variation and sexual dimorphism in mandibular canine and postcanine dental metrics of an Ouranopithecus sample. Bootstrapping (resampling with replacement) of extant hominoid dental metric data is performed to test the hypothesis that the coefficients of variation (CV) and the indices of sexual dimorphism (ISD) of the fossil sample are not significantly different from those of modern great apes. Variation and sexual dimorphism in Ouranopithecus M(1) dimensions were statistically different from those of all extant ape samples; however, most of the dental metrics of Ouranopithecus were neither more variable nor more sexually dimorphic than those of Gorilla and Pongo. Similarly high levels of mandibular molar variation are known to characterize other fossil hominoid species. The Ouranopithecus specimens are morphologically homogeneous and it is probable that all but one specimen included in this study are from a single population. It is unlikely that the sample includes specimens of two sympatric large-bodied hominoid species. For these reasons, a single-species hypothesis is not rejected for the Ouranopithecus macedoniensis material. Correlations between mandibular first molar tooth size dimorphism and body size dimorphism indicate that O. macedoniensis and other extinct hominoids were more sexually size dimorphic than any living great apes, which suggests that social behaviors and life history profiles of these species may have been different from those of living species.     

Sperm velocity and its relation to social status in Arctic charr (Salvelinus alpinus)

Sperm velocity has been shown to be a major determinant of fertilization success of external fertilizers. Yet, sperm velocity varies both within and between ejaculates and only a small number of fast sperm cells within an ejaculate are likely to have the potential of fertilizing the eggs. Having such fast cells should be of special importance during sperm competition, particularly for subordinate males that may release their sperm later or further away from eggs, than dominants. We examined ejaculates of dominant and subordinate male Arctic charr (Salvelinus alpinus), a species with sperm competition. Yet, rather than examining just average sperm velocity values, the aim was to examine whether the fastest fraction of sperm cell from dominant and subordinate males differed in velocity. While there was no difference in the average sperm velocity between dominant and subordinate males, analysis of the fastest swimming sperm cells show that subordinate males have significantly higher initial sperm velocity than dominant males within the 10, 5 and 1% fastest sperm cells. That is, the difference in sperm velocity between dominant and subordinate charr is most predominant among the fastest sperm cells. In sum, this study emphasizes the importance of studying the fastest sperm cells in the ejaculates, as status-dependent differences in sperm velocity may not be detected using average values.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Size variation in early human mandibles and molars from Klasies River,South Africa: Comparison with other middle and late Pleistocene assemblages and with modern humans

Previous studies of the Middle Stone Age human remains from Klasies River have concluded that they exhibited more sexual dimorphism than extant populations, but these claims have not been assessed statistically. We evaluate these claims by comparing size variation in the best‐represented elements at the site, namely the mandibular corpora and M₂s, to that in samples from three recent human populations using resampling methods. We also examine size variation in these same elements from seven additional middle and late Pleistocene sites: Skhūl, Dolní Věstonice, Sima de los Huesos, Arago, Krapina, Shanidar, and Vindija. Our results demonstrate that size variation in the Klasies assemblage was greater than in recent humans, consistent with arguments that the Klasies people were more dimorphic than living humans. Variation in the Skhūl, Dolní Věstonice, and Sima de los Huesos mandibular samples is also higher than in the recent human samples, indicating that the Klasies sample was not unusual among middle and late Pleistocene hominins. In contrast, the Neandertal samples (Krapina, Shanidar, and Vindija) do not evince relatively high mandibular and molar variation, which may indicate that the level of dimorphism in Neandertals was similar to that observed in extant humans. These results suggest that the reduced levels of dimorphism in Neandertals and living humans may have developed independently, though larger fossil samples are needed to test this hypothesis. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

The postmeiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the 3 weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization (dbf)), late spermatids (14-8dbf) and sperm (7-1dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than 2 weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of postmeiotic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e., smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.