Embryonic expression patterns of Hox genes in the oligochaete annelid Tubifex tubifex.

We have cloned and characterized the expression of seven Hox genes (designated Ttu-lab, Ttu-Dfd, Ttu-Scr1, Ttu-Scr2, Ttu-Lox5, Ttu-Lox4 and Ttu-Lox2) from the oligochaete annelid Tubifex tubifex. RT-PCR analyses show that except for Ttu-Lox4 and Ttu-Lox2 which begin expression as early as cleavage stages, Tubifex Hox genes are expressed during stages 13-18 when embryos undergo germ band formation, segmentation and body elongation. In terms of combination of tissues (or organs) exhibiting positive cells, the Tubifex Hox genes examined in this study are classified into three groups. Ttu-lab, Ttu-Scr1 and Ttu-Lox5 are expressed only in the ventral nerve cord; Ttu-Scr2 and Ttu-Lox4 are expressed not only in the ventral nerve cord but also in distinct lateral segmental tissues; and Ttu-Dfd and Ttu-Lox2 are expressed not only in the segmental ectoderm along the length of the AP body axis but also in the prostomium. Anterior expression boundaries of Ttu-lab, Ttu-Scr1, Ttu-Lox5 and Ttu-Lox4 are at segments 3, 4, 5, and 9, respectively. Anterior expression boundary of Ttu-Scr2 is at segment 2, and Ttu-Dfd and Ttu-Lox2 are expressed even at the anteriormost portion, the prostomium. These observations suggest that as in other annelids, so-called "spatial colinearity" of anterior expression boundaries of Hox genes has been conserved in the oligochaetes. It is also evident that there are some oligochaete Hox genes which violate the spatial colinearity rule.

Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish.

BACKGROUND: Telomeres are located at ends of eukaryotic chromosomes and can affect proper chromosomal positioning. During spermatogenesis, the appropriate dynamics and behavior of chromosomes is crucial to generate haploid cells through meiosis. Here, we describe telomere distribution patterns during spermatogenesis in zebrafish, especially during meiotic prophase I, using fluorescence in situ hybridization. This was combined with synaptonemal complex protein 3 immunostaining, which allows the staging of spermatocytes. RESULTS: During spermatogonial proliferation and the preleptotene stage, telomeres were dispersed throughout the nucleus. During the leptotene stage, telomeres temporarily moved to one pole of the nucleus at which γ-tubulin was located, forming the telomere bouquet. The cluster lasted until the onset of zygotene where it coincided with terminal synapsis initiation. They then spread around the periphery of the nucleus during the zygotene to pachytene stages. During postmeiotic stages, telomeres in spermatids and sperm were again dispersed throughout the nuclei. Application of this procedure in meiotic mutants confirmed that meiotic telomere clustering is independent of axial element formation of the synaptonemal complex. CONCLUSIONS: These data clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish. This procedure could be used to screen for mutants that have primary defects in telomere clustering.

Production of zebrafish offspring from cultured spermatogonial stem cells.

Germ-line stem cells have the potential to be a very powerful tool for modifying the genetic information of individual animals. As a first step to use spermatogonial stem cells (SSCs) to enable genetic modification, we here describe effective long-term culture conditions for propagating zebrafish SSCs and for the production of offspring from these cultured SSCs after their differentiation into sperm in transplanted testicular cell aggregates. Dissociated testicular cells were cultured in specific medium with some modified supplements, including several mammalian growth factors. The spermatogonia actively proliferated and retained the expression of exogenous green fluorescent protein under the control of vas and sox17 promoters and also of promyelocytic leukemia zinc finger (Plzf), a marker of undifferentiated spermatogonia, after 1 month in culture. This is a longer period than the entire natural spermatogenic cycle (from SSCs to sperm). The use of subcutaneously grafted aggregates of these cultured spermatogonia and freshly dissociated testicular cells showed that these SSCs could undergo self-renewal and differentiation into sperm. Artificial insemination of these grafted aggregates successfully produced offspring. This culture method will facilitate the identification of new factors for the maintenance of SSCs and enable the future enrichment of genetically modified SSCs that will produce offspring in zebrafish.

Artificial fertilization by intracytoplasmic sperm injection in a teleost fish, the medaka (Oryzias latipes).

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.

Structural components of the synaptonemal complex, SYCP1 and SYCP3, in the medaka fish Oryzias latipes.

The synaptonemal complex (SC) is a meiosis-specific structure essential for synapsis of homologous chromosomes. For the first time in any non-mammalian vertebrates, we have isolated cDNA clones encoding two structural components of the SC, SYCP1 and SYCP3, in the medaka, and investigated their protein expression during gametogenesis. As in the case of mammals, medaka SYCP1 and SYCP3 are expressed solely in meiotically dividing cells. In the diplotene stage, SYCP1 is diminished at desynaptic regions of chromosomes and completely lost on the chromosomes at later stages. SYCP3 is localized along the arm and centromeric regions of chromosomes at metaphase I, and its existence on the whole chromosomes persists up to anaphase I, a situation different from that reported in the mouse, in which SYCP3 is confined to the centromeric regions but lost on the arm regions at metaphase I. Thus, the expression patterns of SC components are different in mammals and fish despite the resemblance in morphological structure of the SC, suggesting divergence in the function of the SC in regulation of meiosis-specific chromosomal behavior. Since the antibody against medaka SYCP3 is cross-reactive to other fishes, it should be generally useful for a meiosis-specific marker in fish germ cells.