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.     

The response of soil CO2 flux to changes in atmospheric CO2, nitrogen supply and plant diversity

We measured soil CO₂ flux over 19 sampling periods that spanned two growing seasons in a grassland Free Air Carbon dioxide Enrichment (FACE) experiment that factorially manipulated three major anthropogenic global changes: atmospheric carbon dioxide (CO₂) concentration, nitrogen (N) supply, and plant species richness. On average, over two growing seasons, elevated atmospheric CO₂ and N fertilization increased soil CO₂ flux by 0.57 µmol m^?2 s^?1 (13% increase) and 0.37 µmol m^?2 s^?1 (8% increase) above average control soil CO₂ flux, respectively. Decreases in planted diversity from 16 to 9, 4 and 1 species decreased soil CO₂ flux by 0.23, 0.41 and 1.09 µmol m^?2 s^?1 (5%, 8% and 21% decreases), respectively. There were no statistically significant pairwise interactions among the three treatments. During 19 sampling periods that spanned two growing seasons, elevated atmospheric CO₂ increased soil CO₂ flux most when soil moisture was low and soils were warm. Effects on soil CO₂ flux due to fertilization with N and decreases in diversity were greatest at the times of the year when soils were warm, although there were no significant correlations between these effects and soil moisture. Of the treatments, only the N and diversity treatments were correlated over time; neither were correlated with the CO₂ effect. Models of soil CO₂ flux will need to incorporate ecosystem CO₂ and N availability, as well as ecosystem plant diversity, and incorporate different environmental factors when determining the magnitude of the CO₂, N and diversity effects on soil CO₂ flux.     

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.     

体外受精-胚胎移植中完全受精失败的原因分析

受精是生命起源至关重要的一个步骤。在辅助生殖的过程中,完全受精失败发生具有其复杂性和不可预见性。受精失败常伴随着一些胞间调控机制异常,其中,可能阻滞在与精子穿越卵冠丘复合体、精子-透明带结合/穿透、精子-卵膜结合、卵子激活、精子去浓缩或原核形成等任一阶段。通过卵胞浆内单精子注射可以避免大部分受精失败现象,但某些患者仍无法成功受精,即使采用辅助人工激活也无法完全避免其发生。对于在辅助生殖过程中完全受精失败患者,结合其卵子成熟情况、精子质量及相关检测结果,在后续周期调整临床方案可有效避免受精失败的再次发生。     

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.     

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.     

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.     

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.     

杂交石竹授粉受精及其胚胎发育过程的观察

以‘杂交石竹’为试验材料,利用荧光显微镜观察其授粉后花粉萌发、花粉管生长情况,采用石蜡切片法对其受精及胚胎发育过程进行观察研究。结果表明:(1)授粉后1h母本柱头上少量花粉开始萌发;授粉后4h大量花粉萌发,花粉管生长至柱头中部有胼胝质出现;授粉后6h花粉管生长至子房组织并有少量与胚珠结合;授粉后15h柱头中出现大量胼胝质,花粉管与胚珠结合数增多;授粉后24h胚珠周围出现多条花粉管,其中1条花粉管进入胚珠,部分进入胚囊的花粉管卷曲盘绕生长并产生胼胝质;精细胞与极核的融合主要发生在授粉后17~48h,与卵细胞融合主要于授粉后1~3d。(2)杂交石竹胚发育经过原胚、球形胚、棒状形胚、心形胚、鱼雷形胚和子叶形胚阶段。(3)杂交障碍表现为:只有游离的胚乳核而无胚发育的胚囊、合子未分裂、两极核未融合、球形胚败育。研究表明,杂交石竹存在受精前和受精后障碍,这是导致其结实率低的主要原因。     

油橄榄授粉受精和胚胎发育过程观察

该研究以油橄榄“鄂植-8”为材料,应用压片荧光观察花粉在柱头和花柱中的萌发及生长情况,采用石蜡制片法观察油橄榄的胚珠结构特点和授粉受精过程。结果表明：油橄榄为1子房2心室4胚珠,珠心较发达,由多层细胞构成,属于厚珠心椭圆型胚珠,胚珠直生;花开时花粉粒落到柱头上立刻萌发,随后花粉管在花粉通道中生长,再后进入子房经子房内表面,大部分花粉管不能到达胚珠,仅少数沿胎座生长经珠柄进入珠孔,释放2个精子,2个精核分别进入卵细胞和极核,并与卵核及极核相互融合;观察到合子中雌、雄性核仁融合的过程。授粉受精各阶段经历的时间为花粉落到柱头上立刻萌发;授粉后6d左右,花粉管长入胚珠的珠孔,随后释放精子;10 d左右完成授粉受精,30 d左右形成心型胚,40 d左右胚发育几乎成熟。在进行子房石蜡切片中,共观察到612个完整子房切片,发育完整的子房为97.53％,完成授粉受精的胚珠为15.52％。油橄榄在自然授粉下,约85％的胚珠授粉受精过程不能完成,在一定程度上影响其坐果。该研究结果为油橄榄授粉受精、高效生产等提供了理论依据。     

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.     

不同缓控释肥对鲜食玉米产量、品质及氨挥发的影响

采用田间试验,设置不施氮对照(CK)、常规施肥(U)、增效尿素(DU)、包膜尿素(CU)、缓控释掺混尿素(CDU)共5个处理,研究了常规施肥(240 kg N·hm^-2)和不同缓控释肥料一次性减量施用(180 kg N·hm^-2)对鲜食玉米产量、品质与土壤无机氮变化和氨挥发的影响。结果表明: U处理氨挥发总量最高,追肥是产生氨挥发损失的重要因素;与U处理相比,DU、CU、CDU处理氨挥发减排78%～81%。收获后U处理80～100 cm土层硝态氮浓度最高,为51.6 mg·kg^-1,氮淋溶风险较高,而DU、CU、CDU处理同土层硝态氮含量均较低,降低了淋溶风险。与U处理相比,减氮25%的3个缓控释肥处理没有减产,并增加了籽粒维生素C、可溶性糖和蛋白质含量;缓控释肥处理之间,DU处理的氮肥农学效率和经济效益最高。综上,减量施用新型缓控释肥可以实现鲜食玉米稳产提质,显著降低氨挥发损失和硝态氮淋失风险。与成本较高的树脂包膜控释肥相比,双效抑制剂增效肥(DU)成本低、制作便捷,是鲜食玉米专用肥的较好选择。     

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.     

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.     

氮素指数施肥对杉木无性系苗生长及养分含量的影响

以1年生无性系杉木幼苗为材料,采用温室盆栽方法,设定对照、常规施肥和3个指数施肥共5个处理,施氮总量分别为每株0、0.5、0.5、1.0、2.0 g,施氮20次,间隔为10 d,施氮210 d后,测定杉木的苗高、地径、生物量、叶绿素荧光参数及养分含量.结果表明: 施肥显著促进了杉木无性系的苗高、地径和生物量的增长,以施氮量1.0 g·株^-1的指数施肥处理最佳,分别为59.0 cm、6.0 mm、52.99 g·株^-1;杉木无性系苗木叶绿素荧光参数特征均表现为指数施肥显著大于常规施肥,其中指数施肥的光化学淬灭系数、表观电子传递速率和PSⅡ的有效量子产量均随施氮总量的增加呈先增加后减小的趋势,在施氮量为1.0 g·株^-1时达到最佳,分别为0.89、35.79和0.71.杉木无性系苗木氮含量表现为叶>根>茎.与对照相比,常规施肥根、茎、叶的氮含量分别增加39.6%、16.6%和41.1%,指数施肥根、茎、叶的氮含量分别增加22.6%～81.4%、27.3%～152.6%和73.6%～135.5%.施氮量为1.0和2.0 g·株^-1的指数施肥处理杉木幼苗根、茎和叶氮含量均显著大于常规施肥,而不同器官磷含量和钾含量无显著差异.从苗木生长性状、叶绿素荧光参数特征、植株氮磷钾含量等综合考虑,在本试验条件下,施氮量为1.0 g·株^-1的指数施肥是杉木无系性苗木温室培育的适宜方法.