Representative cDNA Library from Isolated Rice Sperm Cells

利用Percoll密度梯度离心和多次滤膜过滤分离到适于分子生物学研究的水稻 (OryzasativaL .cv .Guichao2 )生活精细胞。借助RT_PCR和SMART技术构建了水稻精细胞的cDNA文库。初级文库的大小为 1.5 8× 10 6 pfu ,插入片段平均大小为 980bp。 10 3个随机挑选的克隆与DIG标记的水稻幼苗根、叶及二细胞时期花粉、成熟花粉、精细胞和授粉 5～ 7d的子房cDNA探针杂交表明 ,除少数克隆外 ,它们在上述器官中的表达情况很相似。 10个随机挑选的克隆用来测序及分析 ,有 4个克隆含有完整的开放阅读框。 1个克隆与水稻Polyubiquitin (Rubq1)mRNA高度同源。Northern杂交表明它在二细胞时期花粉、成熟花粉中的表达显著少于在根、叶和授粉子房中的表达。另一个克隆的开放阅读框编码与拟南芥 (Arabidopsisthaliana (L .)Heynh .)AtRAD17同源的蛋白。这是第一次正式报道高等植物精细胞cDNA文库的构建 ,也是第一次从高等植物精细胞中分离到其表达的基因。

F-Actin Visualization in Generative and Sperm Cells of Living Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Molecular Cloning and Preliminary Study of a Gene Differentially Expressed in Rice Sperm Cells

A gene with complete sequence of 1 176 bp containing an open reading frame (ORF) of 281 amino acids, RSG6 (accession number in GenBank: AF442490) was cloned from rice (Oryza sativa L.) sperm cell cDNA library by using sperm cell higher expression clone BF475207 as probe. It is the first gene isolated directly from sperm cells of higher plants. The sequences of RSG6 gene and its deduced amino acid did not reveal remarkable similarity to that of any known ones in GenBank. Southern hybridization suggested that there may be introns. in this gene. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) showed that the RSG6 gene was expressed in all nice organs or cell types examined (including roots, leaves, two-cell stage pollen, mature pollen, pollinated ovary and sperm cells), but the level of expression in sperm cells was particularly higher than that in all the other organs or cells. The RSG6 gene was cloned into a vector pQE30 and expressed in Escherichia coli M15 (pREP4). High specific antibody with high titer was obtained from rabbits.

Mechanism of action of Wuzi Yanzong pill in the treatment of oligoasthenozoospermia in rats determined via serum metabolomics

Objective:To investigate the mechanism of action of Wuzi Yanzong pill(WYP)in rats with oligoasthenozoospermia(OAZ)via metabolomics and to provide a possible basis for improving this WYP-based treatment.Methods:A rat model of OAZ was established by treating male SpragueeDawley rats with glucosides from Tripterygium wilfordii Hook.F.Seventy-two rats were randomly divided into six groups:control,L-carnitine(positive control),model,and low-,medium-,and high-dose WYP groups.Rats in the experimental groups were treated with WYP for 4 weeks.At the end of the treatment period,sperm cell quality(density,motility,and viability)was assessed using a semen analysis system,mitochondrial membrane potential(MMP)was assessed using flow cytometry,and testicular injury was assessed using hematoxylin and eosin staining to validate the therapeutic effect of WYP in OAZ.Further,serum metabolomics-based analysis was performed using high-performance liquid chromatography-mass spectrometry to identify differential metabolic pathways and possible mechanisms of action of WYP in OAZ treatment.Results:A rat model of OAZ was considered successfully-established after comparing the quality of spermatozoa in the model group to that in the control group.WYP-M and WYP-H treatments significantly improved sperm cell density,motility,and viability compared with those in the model group(all P<.05).Compared with the model group,both WYP-M and WYP-H treatments increased MMP values(P=.006 and P=.021 respectively),while there was no significant difference in the L-carnitine group.L-carnitine and WYP administration reversed damage to the testes to varying degrees compared with that in the model group.Further,44 differential metabolites and four metabolic pathways,especially autophagy pathway,related to OAZ were identified via metabolomics.Conclusions:WYP improves sperm cell quality and MMP in OAZ primarily via autophagy regulation.These findings can be employed to improve the efficacy of WYP in humans.

Effects of Multiglycoside of Tripterygium Wilfordii(GTW)on Spermatogenic Cells and Enzymatic Activities in Male Rats

This study was undertaken to observe consecutively the morphology of testis andepididymis and the changes of enzymatic activities of AKP, ACP,3β-HSD andLDH-C4 in male rats treated with GTW 30 to 80 days.In addition,male antifertility effect and its possible reversibility were also observed.The results showed that GTW is apotential testicular toxicant in the animals.It can cause damage of the sperm cells withclose relation to the treated time and dosage.Sloughing of sperm cells and manymultinucleated giant cells in the seminiferous tubules were found on day 40 and 50 afterthe treatment.The lubules were hyperatrophic and the sperm cells were almost absent atthe end of the study(80days).No obvious morphological alteration was observed in theopididymal epithelial tissue.But changes of quality and number of the spermatozoa inepididymides were found prior to those of lestes.Reduced number and abnormal caudasperm were observed 30 days after the treatment.The ACP activity of Serloli cells increased slightly,whereas foe activities of ACP and LDH-C4 decreased gradually as thetreated lime prolonged.The 3β-HSD Of Leydik.cells was changed or subtle by m-Phaseor treatment and dramatically decreased at the end of treatment.The infertility caused byGTW was reversible 8 weeks after cessation of the treatment.

Ultrastructure Features of Sperm Cell in Amaryllis vittata

Ultrastructure of sperm cells in pollen tube of Amaryllis vittata Ait. has been investigated in details by electron microscopy ,with particular emphasis on the organization and distribution of microtubules.The two newly formed sperm cells are arranged in tandem and sometimes in transverse at the right angle to the long axis of the pollen tube.Thevegetative nucleus is ahead and closely associated with the two sperm cells in all examined pollen tubes. The microtubules are distributed in the region between the common cell wall and the proximity of the sperm nucleus,they are singles and dispersed with mainly orientation of longitudinal and oblique,forming a loose bucket-like structure as a whole.In late stage of development,all of the microtubule array longitudinally and enclose the sperm nucleus.This configuration is very similar to the basket-like structure of microtubule in the generative cell. These results show that the organization and distribution of microtubules in the sperm cells are dynamic during cell development.

Selection of High-quality Sperms by the Nanotechnological Method of Magnetic Activation in Brazilian Cervids

Cervids show a high degree of abnormalities in their sperm cells.Thus,this study aimed to select high-quality spermatozoa using magnetic-activated sperm sorting(MASS)compared to density gradient centrifugation(DGC)by assessing the post-selection cell quality.Semen from six Mazama deer was collected by electroejaculation after chemical restraint.The semen was analyzed in four samples:Fresh,DGC,SEMgood-non-apoptotic fraction,and SEMpoor-apoptotic fraction.The material was analyzed for motility and vigor(light microscopy),concentration(Neubauer chamber),semen morphology(phase contrast),and supravital staining test(eosin/nigrosine).The DGC method used 20 x 106 cells in 90%and 45%percoll®gradient.The MASS used 10 x 106 cells with 20μl of iron nanoparticles attached to Annexin V and filtration in a magnetic separation column.Both processing methods(DGC and MASS)were effective in producing high-quality sperm samples,with a marked reduction in abnormalities from 41.83±10.25(fresh)to 14.83±3.17(DGC)and 12±3.01(SEMgood),with 80.3%±2.06 livings cells.These findings suggest that this nanotechnological method,using nanoparticles,effectively produces high-quality semen samples in cervids for use in assisted reproduction.

Cytological Mechanism of Cytoplasmic Inheritance in Pinus tabulaeformis: Ⅰ. Ultrastructural Aspects and Nucleoids of the Male Gametes

The cytological mechanism of plastid and mitochondrion inheritance in Pinus is an interesting research topic with only a limited number of published articles. The results indicate that the sperms of Pinus tabulaeformis Carr. contain abundant plastids, mitochondria and organelle DNA. These data provide reliable cytological evidence of paternal plastid and mitochondrion inheritance in Pinus . The results are in line with the confirmed general rule of paternal plastid inheritance in Pinaceae. But whether mitochondria in sperm cells can be transmitted into the embryos is an issue needs further developmental studies. Another important finding is that contrary to earlier classification of the male gamete of Pinaceae into the male nuclei type, the results reveal that male gametes in Pinus tabulaeformis are actually cells. However, the sperm cells are only surrounded by plasma membranes without cell walls. The larger leading sperm cell in a pollen tube section is long in shape, with a large amount of cytoplasm; while the second sperm cell is smaller, round in shape and contains less cytoplasm. Whether this feature of the male gamete type could be considered as a representative characteristic of the family is discussed and further conclusions await more experimental evidences from studies on plants from different species.

Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.

Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without （untreated control） 3-morpholinosydnonimine （SIN-l）, in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition （MPT）, externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-l, the sperm viability was significantly reduced compared to untreated control （P〈 0.001）. Furthermore, the MPT was induced （P〈 0.01） and increment in DNA oxidation （P 〈 0.01）, DNA fragmentation （P 〈 0.01）, tyrosine nitration （P 〈 0.0001） and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control （P 〈 0.001）, this process was observed in 〈10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.

Osmoregulation determines sperm cell geometry and integrity for double fertilization in flowering plants

Distinct from the motile flagellated sperm of animals and early land plants,the non-motile sperm cells of flowering plants are carried in the pollen grain to the female pistil.After pollination,a pair of sperm cells are delivered into the embryo sac by pollen tube growth and rupture.Unlike other walled plant cells with an equilibrium between internal turgor pressure and mechanical constraints of the cell walls,sperm cells wrapped inside the cytoplasm of a pollen vegetative cell have only thin and discontinuous cell walls.The sperm cells are uniquely ellipsoid in shape,although it is unclear how they maintain this shape within the pollen tubes and after release.In this study,we found that genetic disruption of three endomembrane-associated cation/H+exchangers specifically causes sperm cells to become spheroidal in hydrated pollens of Arabidopsis.Moreover,the released mutant sperm cells are vulnerable and rupture before double fertilization,leading to failed seed set,which can be partially rescued by depletion of the sperm-expressed vacuolar water channel.These results suggest a critical role of cell-autonomous osmoregulation in adjusting the sperm cell shape for successful double fertilization in flowering plants.

Small RNAs in pollen

In plants, each pollen mother cell undergoes two rounds of cell divisions to form a mature pollen grain, which contains a vegetative cell(VC) and two sperm cells(SC). As a companion cell, the VC carries the SCs to an ovule by germinating a pollen tube. In-depth sequencing analyses of mature pollen showed that micro RNAs(mi RNAs) and short interfering RNAs(si RNAs) are present in both the VC and SCs. Additionally, epigenetically-regulated transposable elements(TEs) are reactivated in the VC and these TE m RNAs are further processed into 21-nt epigenetically reactivated si RNA(easiR NA) in SCs, which prevent 24-nt si RNA accumulation and sequester mi RNA loading. Small RNAs are thought to move from the VC to SCs, where they regulate gene expression and reinforce TE silencing. Here, we summarize current knowledge of the biogenesis and function of mi RNAs, si RNAs, and easi RNAs in pollen, emphasizing how these different small RNAs coordinately contribute to sperm cell formation and TE silencing.

Gamete Recognition in Higher Plants:An Abstruse but Charming Mystery

Although much effort has been made to uncover the mechanism underlying double fertilization, little knowledge has been acquired for understanding the molecular base of gamete recognition, mainly because of technical limitations. Still, progress has been made in terms of the mechanism, including the identification of candidate molecules that are involved in gamete recognition in angiosperms. New cues for gamete recognition have been found by the successful separation of the gametes and construction of gamete-specific cDNA libraries in several species, and the application of molecular approaches for studying this process by mutations. Thus, the topic is considered an abstruse but charming mystery.