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.     

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.     

Identification,isolation, and properties of a plasma membrane protein involved in the adhesion of boar sperm to the porcine zona pellucida

Boar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt: J Cell Biol 105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (AP_z) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. AP_z does not exhibit enzymatic properties thought possibly to be involved in sperm-zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and anliproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p-aminoben-zamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm-egg binding. When plasma membranes or fractions containing AP_z that bind to dextran sulfate agarose were chromatographed on L-fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti-AP_z antibody, Anti-AP_z was absorbed by fractions that did not contain proacrosin. These data indicate that AP_z is not proacrosin. Since anti-AP_z monovalcnt antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing AP_z completely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view AP, to be the major and perhaps only plasma membrane protein involved in the adhesion of capacitated boar sperm to eggs prior to the acrosome reaction.     

Applying near-infrared reflectance spectroscopy to predict carbon,nitrogen, phosphorus,and organic-bound cadmium in lake picoplankton

This study describes the 0.1–3 m particle size fraction in a Precambrian Shield lake (37-ha Lake 382 in the Experimental Lakes Area, northwestern Ontario) receiving experimental additions of cadmium to determine fate and effects of low cadmium loading. This size fraction is important in binding cadmium in water. The study examined the feasibility of using near-infrared reflectance spectrophotometry (NIRS) for quantifying carbon, nitrogen, and phosphorus in this size fraction in 20-fold concentrated water samples from the lake and from a limnocorral experiment exploring the effect of fertilization on sedimentation of cadmium from the water column. NIRS was also used for detecting and characterizing organic matter in this size fraction associated with cadmium. Aliquots (1.5 ml) of the concentrated samples were applied to pre-ashed Whatman GF/C glass fibre filters. The filters containing 40–150 g carbon, 1–21 g nitrogen, 1–10 g phosphorus, and 0.21–2.21 ng cadmium, were scanned by NIRS, then analyzed by traditional methods for carbon, nitrogen, and phosphorus. Cadmium was determined in the concentrated samples by atomic absorption spectrophotometry. Coefficients of determination,r ², between chemically-measured and NIRS-predicted values were 0.921 for carbon, 0.852 for nitrogen, 0.869 for phosphorus, and 0.752 for cadmium. Several lines of evidence suggested that the organic material associated with cadmium was predominantly algae <3 m. NIRS is useful for measuring organic matter in this size fraction and is potentially useful for characterizing organic matter that binds metals.     

Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa

Apoptosis is a type of cell death characterized by the activation of a family of cysteine-proteases called caspases. We made a comparative study to determine the presence of several caspases and other regulators of apoptosis in rat, mouse, and hamster spermatozoa. Our results showed that the three species have both active and inactive caspases-8 and -3, the proapoptotic protein BID, p53, and the endogenous caspase inhibitor cIAP-1. However, we did not find evidence for the presence of active caspase-9. The acrosome reaction (i.e., the exocytic process of sperm acrosome) and sperm viability were not affected by the presence of a general caspase inhibitor. On the other hand, valinomycin, which promotes caspase-dependent cell death in somatic cells, induced caspase-independent cell death in spermatozoa. TRAIL, a ligand whose receptor induces apoptosis in malignant cells, did not have any effect in the viability of mouse spermatozoa, despise the presence of its receptor in rat and mouse, but not in hamster spermatozoa. Therefore, our results strongly suggest that rodent spermatozoa have some components of the apoptotic pathway. However, the role of caspases in mammalian spermatozoa appears to be unrelated to sperm survival or to the acrosome reaction under physiological conditions.     

Chromatin transitions in the fertilizing sperm nucleus of the sea urchin,strongylocentrotus purpuratus

Electron microscopic analysis of fertilization in the sea urchin, Strongylocentrotus purpuratus, has been carried out in an effort to establish the sequence of events involving dispersion of the paternal chromatin. Subsequent to loss of the nuclear envelope the condensed sperm chromatin begins to disperse under the influence of egg cytoplasmic factors. However, this process does not proceed at a uniform rate as is observed in other species examined to date. Portions of the paternal genome rapidly transform into dispersed chromatin while other adjacent regions disperse at a reduced rate. This variation in the time sequence of dissociation of the paternally derived chromosomes results in a reticulum of electron lucent and electron dense chromatin within the developing male pronucleus. As the paternally derived chromatin is dispersing and migrating centrad, membranous vesicles of maternal origin become aligned along the peripheral aspect of the chromatin. Deposition of a continuous bilaminar nuclear envelope around the dispersing sperm chromatin results in the formation of the definitive male pronucleus. At the time the male pronucleus is formed the paternally derived chromosomes have not completely dispersed and are visualized as a reticulum of condensed and dispersed chromatin. These results indicate that not all the paternally derived chromatin is modified in the same manner during pronuclear development.     

Stimulating effect of pyroglutamylglutamylprolineamide,a prostatic,TRH-related tripeptide,on mouse sperm capacitation and fertilizing ability in vitro

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin-releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome-intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome-intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide-treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley-Liss, Inc.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

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.     

Synthetic organic pH buffers can support fertilization of guinea pig eggs,but not as efficiently as bicarbonate buffer

Bicarbonate ions (HCO₃^?) in the medium are not absolutely essential for the fertilization of guinea pig eggs. However, fertilization takes place most efficiently in HCO₃^?-buffered medium. Capacitation, the acrosome reaction, and zona penetration by spermatozoa can occur in HCO₃^?-free media with synthetic organic buffers (i.e., MOPS, TES, HEPES, Tris, and TAPSO) but not as efficiently as in the HCO₃^?-buffered medium. It appears that HCO₃^? functions as more than just a pH-buffering molecule.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

Controlled ecdysteroid accumulation in eggs of the silkworm,Bombyx mori,by an imidazole compound (KK-42), and embryogenesis in these eggs

An imidazole compound (KK-42), a potent inhibitor of ecdysone synthesis, was applied to the female pharate adult of the silkworm, Bombyx mori, to control ecdysteroid accumulation in developing ovaries and mature eggs. KK-42 applied on day 2 or later completely suppressed an increase in ecdysteroid content in developing ovaries. The inhibitory action of KK-42 was restricted to vitellogenic follicles, i.e., those in which active ecdysteroid synthesis is occurring. Ecdysteroid content in the mature eggs of moths remained at the level accumulated in ovaries before KK-42 application. Thus, KK-42 was shown to be a novel agent to suppress the ecdysteroid accumulation in eggs. Eggs containing different amounts of ecdysteroids showed different levels of embryonic development. About 80% of the eggs which contained less than 10 ng free ecdysteroids/g eggs were not fertilized. More than 80% of the eggs containing less than 40 ng/g eggs of free ecdysteroids initiated embryogenesis but failed to hatch. Larvae hatched from almost all eggs which accumulated free ecdysteroids of more than 150 ng/g. Thus, maternal ecdysteroids appear to be required at different titers for fertilization, embryogenesis, and hatching of the silkworm larvae. © 1994 Wiley-Liss, Inc.     

SOB3, a human sperm protein involved in zona pellucida binding: Physiological and biochemical analysis,purification

LB5 antibody was selected from a monoclonal antibody (mAb) library directed against human sperm proteins. LB5 mAb detected the corresponding protein SOB3 in the neck region and the flagellum of most live ejaculated sperm while it labelled, in addition, the acrosome of about 10–20% of spermatozoa. The percentage of LB5 acrosome-stained sperm was significantly correlated with the percentages of either spontaneous or A23187-induced acrosome-reacted sperm. While SOB3 could not be detected in the testis, it appeared in spermatozoa from the corpus epididymis segment. LB5 mAb impaired neither sperm motion parameters, acrosomal reaction triggering, nor sperm binding to zona-free hamster oocytes. By contrast, LB5 Fab fragments (200 μg/ml) inhibited sperm binding to human zonae pellicidae by 35.7%. If sperm were induced to acrosome react with A23187 prior to LB5 treatment, the inhibitory effect shifted to 59.9%, while no significant effect was observed following A23187 incubation alone. Western blotting of human sperm and cauda epididymis extracts revealed two bands of 18 and 19 kDa. While no cross-reaction was observed with other tested organs, a similar 18-kDa band was revealed in erythocytes and one of 19 kDa in B-lymphocytes. No cross-reactivity could be evidenced in any animal sperm analyzed. SOB3 was first separated in a 17- to 20-kDa preparative electrophoresis fraction and finally purified by isoelectrofocusing according to its pI of 9.8. These results suggest that SOB3 is localized under the outer acrosomal membrane, that it participates in secondary sperm binding to the zona pellucida, and that it shares homologies with the immune system. Mol. Reprod. Dev. 49:286–297, 1998. © 1998 Wiley-Liss, Inc.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes,mature oocytes,or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15–30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed. © 1996 Wiley-Liss, Inc.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial,the brushtail possum,Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N-linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998. © 1998 Wiley-Liss, Inc.     

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…