Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of Clq/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of Clq family with a Clq domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific Clq-like factor, CaOClq-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOClq-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOClq-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization. (C) 2004 Elsevier Inc. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells. (C) 2008 Elsevier B.V. All rights reserved.

Heterodimerization of integrin Mac-1 subunits studied by single-molecule imaging

Heterodimerization of integrin Mac-1 (alpha(M) beta(2)) Subunits plays important role on regulating leukocytes adhesion to extracellular matrix or endothelial cells. Here, using total internal reflection microscopy, we investigated the heterodimerization of integrin Mac-1 subunits at the single-molecule level in live cells. Individual alpha(M) subunit fused to the enhanced yellow fluorescent protein (eYFP) was imaged at the basal plasma membrane of live Chinese hamster ovary (CHO) cells. Through analysis of mean square displacement (MSD), diffusion coefficient, the size of restricted domain and fraction of molecules undergoing restricted diffusion, we found that as compared with the diffusion in the absence of beta(2) subunit, the diffusion of single-molecule of alpha(M)-YFP was suppressed significantly in the presence of beta(2) subunit. Thus, based on the oligomerization-induced trapping model, we suggested that in the presence of beta(2) subunit, the am subunit may form heterodimer with it. (C) 2008 Elsevier Inc. All rights reserved.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

在异倍性水平多胚苗水稻的特征特性研究

通过无融合生殖方式固定水稻杂种优势是水稻育种中极具诱惑力的研究内容之一。自从1979年以来，我国水稻无融合生殖充种研究主要集中在筛选和鉴定水稻多胚苗材料。根据前人的研究结果，具有无融合生殖特性的植物大多数为多倍体。本试验以多胚苗水稻APIV及其衍生系为研究材料，在二倍体[APIV_((2))]、同源三倍体[APIV_((3))]和同源四倍体[APIV_((4))]水平，对其多个世代中的特征特性，其中包括形态学特征，遗传学和胚胎学特性进行了研究，旨在探讨在多倍性水平筛选水稻无融合生殖种质的可能性，获得如下主要研究结果： 1.在二倍体APIV_((2))的多个世代中，只发现单胚苗、双胚苗和三胚苗植株，多胚苗频率比较低(4.67～5.14%)，多胚苗性状的表达在一定程度上受到环境因素的影响，并且，多胚苗植株的成活率比较低(11.70～17.39%)，大部分多胚苗植株在三叶期之前死亡，这很可能与其胚乳的营养供应有关。根据APIV_((2))的多胚苗性状在多个自交世代和杂交世代中的表达特点，多胚苗性状不是显性性状，也不是隐性性状，而是一种比较特殊的数量性状。 2.胚胎学的研究结果表明，在APIV_((2))的2857个子房的胚囊中没有观察到与不定胚生殖有关的特异生殖现象;APIV_((2))在发生受精之前的胚囊构型包括正常蓼型胚囊（76.5%）、退化型胚囊（16.0%）和变异型胚囊（7.5%），在变异型胚囊中包括双卵卵器胚囊（86.7%）和三卵卵器胚囊（13.3%）;APIV_((2))的双受精与前人在正常二倍体水稻中所观察到的结果大致相符;在不同季节的颖花和同一季节同一稻穗的不同颖花内多卵和多胚苗频率存在着明显差异。 3.在同源四倍体水稻的诱导中对种芽进行预处理，促使其胚芽鞘明显伸长后再进行诱导处理是诱导成功的关键技术。在同源四倍体APIV_((4))的同一稻穗中强势颖花的多卵频率要明显地高于弱势颖花的多卵频率。在APIV_((4))去雄后的颖花中意外地观察到了单胚和双胚现象。同源四倍体水稻APIV_((4))的有性生殖能力明显变弱，在花药内正常花粉粒少而败育花粉粒多;在受精前正常胚囊数少而退化胚囊数比较多（36.0%）;花粉管进入胚囊的时间比较晚;双受精频率低而单受精频率高。 4.异倍性水稻间具有一定的可交配性，但其结实率比较低（0.20～-1.64%），通过常规杂交方法所获得的同源三倍体成活植株的频率更低（0.07%）。在湖南湘潭的秋季条件下同源三倍体水稻植株雄性完全败育，但有部分稻穗能结实（0.59%～7.71%)，由此可获得饱满种子和不饱满种子。 5.通过子房培养可以获得异倍性水稻间杂种植株。在APIV_((4))/APIV_((2))杂交组合的子房培养中出现了一株双胚苗;同源三倍体成活植株的获得率仍然比较低（0.78%），但比通过常规杂交法首先获得种子，进而获得同源三倍体成活植株的效率要倍出11.14倍。根据试验结果，提出了6个问题进行讨论。认为在杂交后代中有可能筛选到多胚苗发生频率更高的单株;多胚苗性状是比较复杂的数量性状;同源四倍体水稻的有性生殖能力明显变弱;通过合理配组和复重授粉可以提高异倍性水稻间的杂并结实率;通过子房培养可以明显提高获得同源三倍体成活植株的效率;在多倍体水稻中筛选无融合生殖种质在比二倍体水稻中筛选可能更容易成功。

大百合属间杂交亲和性、离体培养及耐热生理研究

本实验以大百合和百合东方杂种系“索蚌”为材料，对大百合、百合杂交的亲和性、大百合离体培养及其耐热性进行了研究，以期为大百合与百合杂交育种及相应的耐热百合材料的筛选、种质保存、新品种快繁及栽培应用提供理论依据。 　　以大百合为母本，百合为父本，对属间杂交授粉后花粉管的行为进行观察，结果表明：大百合与百合属间杂交授粉后，百合的花粉在大百合的花柱内的伸长过程中，出现少部分花粉管末端分叉、膨胀或变细，胼胝质大量不规则沉淀，及部分花粉管在伸长过程中受阻等不亲和现象，但大部分花粉仍能够正常萌发，穿过花柱道，进入子房，到达胚珠，且能够观测到早期的胚。虽然杂交亲和性与花粉管的行为有关，但杂交的成功与否还受到受精后诸多因素的影响，还需要从胚胎学和遗传学方面进一步探讨。 　　以大百合的鳞片、叶柄和子房为外植体，进行离体培养，结果表明：大百合的鳞片和叶柄外植体均可成功地诱导小鳞茎，叶柄相对更容易。鳞茎诱导小鳞茎的最佳培养基为MS＋NAA0.5－1.0mg/ml +BA2.5mg/ml +KT2.5mg/ml +蔗糖3%＋琼脂0.7%，28周后，每个外植体平均可以分化4-11个小鳞茎;叶柄诱导小鳞茎的最佳培养基为MS＋NAA1.0－2.0mg/ +BA2.5-3.0mg/ml +KT2.5-3.0mg/ml +蔗糖3%＋琼脂0.7%，26周后，每个外植体平均可以分化3-9个小鳞茎。同时也发现，用鳞茎作为外植体，污染率较高。在大百合的子房离体培养实验中发现：BA和KT 是影响大百合子房分化途径的关键因素，其浓度分别为0.1-1.0mg/L、2.0-4.0 mg/L和高于4.0mg/L时，外植体分别分化为愈伤组织、芽和叶。外植体分化的基本培养基以N6、B5为佳。愈伤组织诱导小鳞茎的最佳培养基为MS+0.1-0.5mg/L NAA +2.5mg/L BA+2.5mg/L KT +10%蔗糖+0.7%琼脂。在1/2MS +3%的蔗糖+0.7%琼脂+1%活性炭的生根培养基上，生根率为100%。炼苗一周后移栽，长势良好。 　　对长至5－6片真叶的大百合植株在不同高温（30℃、35℃和40℃）下，分别进行4h、10h及24h(热胁迫10h，然后在22℃对照温度下缓苗14h)的热胁迫处理，测定了不同处理下，植株的净光合速率（Pn），实际光化学效率（φPS2），最大光化学效率（Fv/Fm）和叶片的相对电导率，游离脯氨酸含量，可溶性蛋白含量，以及叶片中超氧化物歧化酶（SOD）和过氧化氢酶(CAT)的活性。结果表明：大百合对30℃的高温胁迫有较好的适应能力，表现为可溶性蛋白、游离脯氨酸等渗透调节物质的积累，抗氧化酶活性的提高，以及缓苗后细胞膜的自我修复和光合能力的恢复;随着胁迫温度的升高（35℃、40℃）和胁迫时间的延长（4h、10h），大百合一方面对高温胁迫做出了积极的响应，另一方面，光系统的光合能力，细胞膜的稳定性，抗氧化酶的活性，也受到了一定程度的伤害，在缓苗后,细胞膜的稳定性、细胞的渗透势、抗氧化酶的活性等都在一定程度上得到恢复。 　　

Murine MyaK, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary, Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos, Mol. Reprod. Dev. 55:372-378, 2000. (C) 2000 Wiley-Liss, Inc.

The reproductive strategies of an endemic Tibetan fish, Gymnocypris selincuoensis

The reproductive traits of Gymnocypris selincuoensis from Selincuo Lake and its tributaries were investigated in 1997 and 1998. The youngest mature male was age 7 with a standard length (SL) of 172.0 mm, and the youngest mature female was age 8 with a SL of 194.0 mm. The L(50)s Of SL and age at first maturity were respectively 250.32 mm and age 9 for males and 224.71 mm and age 8 for females. The gonadosomatic index (GSI) significantly changed with seasons for mature individuals but not for immature individuals. GSIs of mature females at stages IV and V of ovary development increased with SL and reached a maximum value at the SL range from 370 mm to 390 mm; the GSIs of mature males were negatively correlated with SL. The breeding season lasted from early April to early August. Egg size did not significantly change with SL but increased with the delay of spawning. The individual absolute fecundity varied from 1,341 to 28,002 eggs (mean 12,607+/-7,349), and the individual relative fecundity varied from 6.4 to 42.0 eggs.g(-1) (mean 25.5+/-9.7). The individual fecundity increased with total body weight; it also increased with SL for those of SL less than 370 mm. There was a rest of spawning for mature individuals.

牙鲆一株弹状病毒病原的分离与鉴定

从患病牙鲆中分离鉴定了一株弹状病毒(Paralichthys olivaceus rhabdovirus,PoRV)。用过滤除菌后的患病牙鲆组织匀浆液,接种不同的鱼类细胞,其中有7种鱼类细胞出现明显的病变在对病毒进行挑斑分离后,测定了PoRV的滴度,显示PoRV在敏感鱼类细胞(Grass Carp Ovary,GCO)中的滴度达到106.5TCID50/mL;绘制了PoRV生长曲线;经蔗糖密度梯度离心提纯PoRV,负染及宿主细胞超薄切片的电镜观察,显示PoRV大小约为60nm×200nm。测定了PoRV的

KHAWIA SAUROGOBII N. SP (CESTODA: CARYOPHYLLIDEA) FROM FRESHWATER FISH SAUROGOBIO SPP. (CYPRINIDAE) IN CHINA

Khawia saurogobii n. sp. is described from the intestine of the cyprinid fishes Saurogobio dabryi and Saurogobio dumerili in China. The new species belongs to the Lytocestidae and is placed in Khawia because of its afossate scolex, vitelline follicles in the cortical parenchyma, uterus not looping anterior to the cirrus sac, gonopores separate but close together in distinct genital atrium, external seminal vesicle absent, and postovarian follicles present. The new species differs from other congeneric species by the shape of the body markedly tapering posteriorly from the end of the anterior third, the shape of the scolex that is very short and markedly wider than the neck, spatulate without incisions, but shallow superficial grooves, vitelline follicles and testes starting immediately posterior to the scolex, and an ovary with long, posterior arms bent medially, thus giving the shape of an inverted A.

Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.