Cloning and expression of medaka dazl during ernbryogenesis and gametogenesis

The Deleted in azoospermia family consists of RNA-binding proteins Bottle, Daz, and Daz-like (Dazl) that are expressed in the germline. Here, we report the cloning and expression of the medakafish (Oryzias latipes) dazl gene (odazl). Interestingly, although the predicted medaka Dazl protein (oDazl) contains a RRM motif and a DAZ repeat characteristic of its mammalian homologs, it lacks 80 aa at the C-terminus. By RT-PCR, RNA in situ hybridization, Western blotting and fluorescent immunohistochemistry using a rabbit anti-DazI antibody (alpha Oazl), we analyzed the expression patterns of odazl and its protein. The odazl transcript persists throughout embryogenesis and delineates with primordial germ cells. In adults, the expression of odazl RNA and its protein is restricted to germ cells of both the testis and ovary. We observed differential expression of RNA and protein at critical stages of gametogenesis. In the testis, the odazl RNA is low at premeiotic stages, abundant at meiotic stages, but absent in postmeiotic stages; whereas the oDazl protein is rich in premeiotic stages, reduced at meiotic stages, becomes barely detectable or absent in postmeiotic round spermatids or sperm, respectively. This is in sharp mature spermatozoa. In the ovary, the odazl RNA contrast to the human situation where the Dazl transcript and protein are present in and protein persist throughout oogenesis and also show differential expression at premeiotic, meiotic and postmeiotic stages. Thus, the odazl or its protein is a marker for germ cells during embryogenesis and at critical stages of gametogenesis in both sexes of medaka. (c) 2006 Elsevier B.V. All rights reserved.

Bryodrilus fuscistriatus, a new enchytraeid species (Oligochaeta : Enchytraeidae) from northeastern China

Bryodrilus fuscistriatus, a new enchytraeid species from Mt. Changbaishan, Jilin Province, north-eastern China, is described. It is characterized by brown epidermal glands, 7 pairs of preclitellar nephridia, poorly-developed clitellar glands, spermatheca with 2 sessile globular diverticula, and a long sperm funnel with a very broad collar. It is similar to the Alaskan B. tunicatus Dozsa-Farkas & Christensen, 2002 in possessing two diverticula in the spermathecal ampulla and the origin of the dorsal vessel, and the Chinese B. longifistulatus and B. macrotheca Xie et al., 2000c in body size, long sperm funnel and undeveloped clitellar glands, but it differs from B. tunicatus by the presence of brown-striped epidermal gland cells in III-V, a poorly-elevated clitellum, the absence of copulatory glands in XIII-XIV, the regular outline of coelomocytes, and 7 pairs of preclitellar nephridia; from B. longifistulatus and B. macrotheca by the shape of spermatheca, the color of epidermal gland cells, the position of the first pair nephridia, and the origin of dorsal vessel.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Reproduction mode of an artificial allotetraploid carp (Pisces; Cyprinidae)

Mature eggs of allotetraploid carp were activated by inactive sperm or crossed with normal sperms of common carp (Cyprinus carpio), crucian carp (Carassius auratus), Chinese blunt snout bream (Megalobrama amblycephala), Hemiculter leucisculus and Pseudorasbora parva. Chromosome counts showed that all offspring of these crosses presented a mode number of 200 chromosomes (4n = 200), and their morphological traits are much like maternal. Microsatelite marker and RAPD patterns between allotetraploid maternal and its offspring, reproduced from different paternal species, were identical. Cytological, morphological and molecular evidences suggested that allotetraploid carp female nucleus would not fuse with any male nucleus and its reproduction mode might be gynogenesis and therefore their offspring are retaining their tetraploidy and give origin to clonal individuals.

Introduction of the human lactoferrin gene into grass carp (Ctenopharyngodon idellus) to increase resistance against GCH virus

Haemorrhage can be an epidemic and fatal condition in grass carp. It is known now that the Grass Carp Haemorrhage Virus (GCHV) triggers haemorrhage. Human lactoferrin (hLF) plays an important role in the non-specific immune system, making some organisms more resistant to some viruses. Sperm of grass carp was mixed with linearized pCAhLFc, which is a DNA construct containing an hLF cDNA and the promoter of common carp beta-actin gene, and then electroporated. Then, mature eggs were fertilized in vitro with the treated sperm cells. The fry were sampled and analyzed by polymerase chain reaction (PCR). Results indicated that the foreign gene had been transferred successfully into the cells of some fry. Under optimal electroporation conditions, the efficiency of gene transfer was as high as 46.8%. About 35.7% of treated 5-month-old grass carp contained foreign genes. Most transgenic fry demonstrated significant delays in onset of symptoms of haemerrhage after injection of GCHV, suggesting a significant positive relationship between hLF cDNA and levels of disease resistance (P < 0.01). Results suggest that transgenic grass carp could be bred for increased resistance to haemorrhage. (C) 2002 Elsevier Science B.V. All rights reserved.

The precocious Chinese mitten crab: Changes of gonad, survival rate, and life span in a freshwater lake

Changes of the gonad. survival rate, and life span of the precocious (i.e., sexually mature in their first October-November, at the age of 5 or 6 months) Chinese mitten crab (Eriocheir sinensis) were studied in a small, shallow, macrophytic, freshwater lake along the middle reaches of the Changjiang River, China. The gonosomatic index (GSI) reached peak during the next March when the female GSI was 12.32 +/- 1.75 SD% and the male GSI A as 4.24 +/- 0.19%. The sexual glands degraded from then on. In the first ten-day period of the following July, there was no complete ovum in the ovary. and the sperm became thin and lost adhesion. The population declined sharply from November to July, and the last one (a female) died in the middle of July, which indicated that the life span of the precocious crab was about 12 months (from larval hatching in June to death in July of next year). The survival curve might be expressed as Y = 1.09exp(-0.018x) (Y survival rate; x: days) for the precocious crabs stocked in experimental cages.

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp (Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp (Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.

Retroposon analysis of major cetacean lineages: The monophyly of toothed whales and the paraphyly of river dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram. and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28-33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

Gynogenetic silver crucian carp, Carassius auratus gibelio, is an intriguing model. system. In the present work, a systemic study has been initiated by introducing suppression subtractive hybridization technique into this model system to identify the differentially expressed genes in oocytes between gynogenetic silver crucian carp and its closely related gonochoristic color crucian carp. Five differential cDNA fragments were identified from the preliminary screening, and two of them are ZP3 homologues. Moreover, the full length ZP3 cDNAs were cloned from their oocyte cDNA libraries. The length of ZP3 cDNAs were 1378 bp for gyno-carp and 1367 bp for gono-carp, and they can be translated into proteins with 435 amino acids. Obvious differences are not only in the composition of amino acids, but also in the number of potential O-linked oligosaccharide sites. In addition, gyno-carp ZP3 amino acid sequence has an unexpected higher identity value with common carp (83.5%) than that with the closely related gono-carp (74.7%). The unique homology may be originated from the ancient hybridization. Northern blot analysis confirmed that expression of the ZP3 gene occurred exclusively in the oocytes. Because O-linked oligosaccharides on ZP3 have been demonstrated to play very important roles in fertilization, it is suggested that the extra O-linked glycosylation sites may be related to the unique sperm-egg recognition mechanism in gynogenesis.