Evolutionary trends in fossil gymnosperm pollen

The evolution of cordaite, conifer, and cycadophyte pollen types are discussed relative to germination polarity, external ornamentation, suture organization, saccuscorpus development, and sporoderm ultrastructure. Information is presented in which the development of the microgametophyte is considered together with an analysis of the reproductive biology of several taxa.     

The ultrastructure and reproductive significance of Lasiostrobus microspores

The normally inaperturate microspores of the Pennsylvanian cone Lasiostrobus are found to rarely possess trilete sutures, confirming the subequatorial position of the sacci. Sporoderm ultrastructure compares favourably with saccate pollen of some extant members of the Coniferales. The shedding of grains in the microspore stage suggests that microgametophyte evolution had passed the stage of prothallial cell production, and that Lasiostrobus is an advanced member of the Paleozoic gymnosperm complex.     

矮沙冬青小孢子发生和雄配子体发育的观察

对矮沙冬青（Ammopiptanthus nanus）小孢子发生及雄配子体发育过程进行了观察,结果表明：花约具4个花粉囊,花药壁发育为基本型,由表皮、药室内壁、中层（2—3层）和绒毡层组成,绒毡层为腺质型。小孢子母细胞减数分裂后胞质分裂为同时型,四分体为四面体型排列,成熟的花粉粒为二细胞型。在矮沙冬青小孢子发生及雄配子体发育过程中没有发现异常现象,认为矮沙冬青濒危不存在雄性生殖结构与发育过程异常的内在因素。     

Insect sperm motility

The flagellosperm of insects, although following a general ground plan, exhibit considerable variation in morphology and ultrastructure across taxa, consistent with a history of rapid and divergent evolution. Sperm competition, which occurs when sperm of two or more males compete for the fertilization of a female's ova, has been recognized as a significant driving force in the evolution of insect sperm structure. Despite a considerable volume of data on sperm morphology, little is known about the motility of insect sperm. Understanding insect sperm motility would help to refine models of sexual selection on insect sperm, and would throw light on the selective mechanisms that shape insect sperm structure and function. This review updates our present knowledge of the proximate and ultimate aspects of insect sperm motility.     

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.     

The tailored sperm cell

Sperm are ubiquitous and yet unique. Genes involved in sexual reproduction are more divergent than most genes expressed in non-reproductive tissues. It has been argued that sperm have been altered during evolution more than any somatic cell. Profound variations are found at the level of morphology, motility, search strategy for the egg, and the underlying signalling mechanisms. Sperm evolutionary adaptation may have arisen from sperm competition (sperm from rival males compete within the female’s body to fertilize eggs), cryptic female choice (the female’s ability to choose among different stored sperm), social cues tuning sperm quality or from the site of fertilization (internal vs. external fertilization), to name a few. Unquestionably, sperm represent an invaluable source for the exploration of biological diversity at the level of signalling, motility, and evolution. Despite the richness in sperm variations, only a few model systems for signalling and motility have been studied in detail. Using fast kinetic techniques, electrophysiological recordings, and optogenetics, the molecular players and the sequence of signalling events of sperm from a few marine invertebrates, mammals, and fish are being elucidated. Furthermore, recent technological advances allow studying sperm motility with unprecedented precision; these studies provide new insights into flagellar motility and navigation in three dimensions (3D). The scope of this review is to highlight variations in motile sperm across species, and discuss the great promise that 3D imaging techniques offer into unravelling sperm mysteries.     

Pollen mitosis in the slipper orchid Cypripedium fasciculatum

Pollen mitosis in the slipper orchid Cypripedium fasciculatum was studied using correlated methods of immunofluorescence and transmission electron microscopy. Unlike the more highly evolved orchids, the cypripedioid orchids shed pollen as monosulcate monads. Prior to pollen mitosis, the microspore nucleus migrates to a proximal position opposite the aperture, as is typical of monocotyledons. There is no distinct generative pole microtubule system (GPMS) like that recently reported in development of pollen polarity in the vandoid moth orchid Phalaenopsis. Instead, microtubules in early prophase are concentrated around the nucleus and extend into the cytoplasm toward the future generative pole. Once the nucleus has migrated to the continuous surface opposite the aperture, microtubules surround the nucleus evenly and show no tendency to be more concentrated in the generative domain. The mitotic spindle, which develops from the perinuclear microtubules, is asymmetrically placed in the microspore and is cone-shaped. The generative pole is broad and closely appressed to the continuous spore surface, while the vegetative pole is pointed and located in the interior of the microspore. As the chromosomes move poleward, microtubules proliferate in the interzone and a phragmoplast develops. The phragmoplast expands in a hemispherical path beyond the interzone following an array of microtubules that radiates from the generative nucleus. Data from this study indicate that evolution of pollen in orchids includes a shift in location of the generative cell from proximal to distal and the evolution of a GPMS, in addition in the well-known trend toward increased pollen aggregation and loss of exine.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

Effects of reactive oxygen species on sperm function

Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.     

Oligospermies sévères (< 5 millions/ml): facteurs pronostiques et évolution

Between March 1987 and January 1998, 1127 patients with an oligospermia < millions/ml were followed up for infertility problems. 876 of these patients were followed up at least one year (mean: 3.2 years). During this period, each patient performed at least two spermograms with an interval varying from 6 months to 3 years. We studied the evolution of the quality of sperm during follow up and tried to show the pronostic factors of a potential decline We found an impairment of all the sperm quality parameters from the outset of the sixth month. At the fourth year of follow up more than 2/3 of the patients had a significant impairment of sperm quality and none of them showed any improvement. The % of sperm motility appears to be the most important prognostic factor in predicting an impairment. Sperm motility <20% predicts a significant impairment in sperm quality in the following two years.     

Characteristics of sperm motility induced on the egg-jelly in the internal fertilization of the newt,Cynops pyrrhogaster

Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K(+)-channel antagonist, charybdotoxin (CTX), or a Ca(2+)-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

细胞运动、细胞迁移与细胞骨架研究进展

细胞定向运动与细胞骨架的动态循环密切相关。运动细胞在其伪足前沿依靠细胞骨架的不断聚合推动细胞膜的前进,在基部靠近细胞体部位通过细胞骨架的不断解聚收缩拖拉细胞体向前运动,细胞骨架的聚合与解聚通过伪足与支撑表面的吸附与解吸附而偶连。肌动蛋白组成的微丝骨架是大多数运动细胞的主要成分。外界刺激引起微丝细胞骨架动态变化的信号通路已逐步明了。线虫精子细胞的运动行为与阿米巴变形运动相似,但是在线虫精子细胞中没有肌动蛋白,而是以精子主要蛋白为基础形成细胞骨架驱动精子细胞的运动。与肌动蛋白不同,精子主要蛋白没有分子极性、ATP结合位点和马达蛋白。通过比较研究以上两种运动体系将有助于在分子水平上进一步阐明细胞运动的机理。     

Differentiation of generative and vegetative cells in angiosperm pollen

 Cellular differentiation of a generative and a vegetative cell is an important event during microspore and pollen development and is requisite for double fertilization in angiosperms. The generative cell produces two sperm cells, or male gametes, whereas the vegetative cell produces an elongated pollen tube, a gametophytic cell, to deliver the male gametes to the embryo sac. For typical differentiation of the gametic and gametophytic cells, cell polarity, including nuclear positioning, must be established prior to microspore mitosis and be maintained during mitosis. Microtubules are closely involved in the process of asymmetric cell division. On the other hand, alteration of the chromatin composition seems to be responsible for the differential gene expression between the generative and vegetative cells. Cytoplasmic regulatory molecules, which affect chromatin configuration, are postulated to be unequally distributed to the two cells at the asymmetric cell division. Thus, typical differentiation of the cells is accomplished by a cellular mechanism and a molecular mechanism, which might be independent of each other. These results are discussed in relation to one model that accounts for the different fates of generative and vegetative cells during sexual plant reproduction. Received: 3 September 1996 / Revision accepted: 23 September 1996     

莴苣花粉发育过程中ATP酶的分布

对莴苣花粉发育过程中ATPase的分布特征做了研究。四分体早期的小孢子细胞质中开始出现ATPase反应颗粒。之后,小孢子在发育过程中,花粉内壁聚集大量体积较大的ATPase反应颗粒,并一直保持到花粉即将成熟。在小孢子发育晚期,在花粉萌发孔处和小孢子大液泡中也特异性地聚集了较多ATPase颗粒。二胞花粉刚形成的生殖细胞表面呈现大量的ATPase反应颗粒,当生殖细胞脱离花粉内壁移入营养细胞,ATPase反应颗粒基本消失。生殖细胞分化过程中生殖细胞的ATPase反应颗粒逐渐低于营养细胞中的。在成熟花粉中,精细胞中的ATPase反应颗粒比营养细胞中的少,且主要集中在细胞核中。结果显示花粉发育过程中ATPase的特异分布与花粉发育的一些生物学事件密切相关。     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

Development-related changes of protein ubiquitination in pollen from male and female kiwifruit (Actinidia deliciosa)

Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non-functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early-uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.     

Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the "actoclampin" model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.