Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

Differential expression of fertility genes boule and dazl in Chinese sturgeon (Acipenser sinensis), a basal fish.

The gene family DAZ (deleted in Azoospermia), including boule, dazl and DAZ, performs highly conserved functions in germ cell development and fertility across animal phyla. Differential expression patterns have been demonstrated for the family members in invertebrates and vertebrates including fish. Here, we report the identification of boule and dazl and their expression at both RNA and protein levels in developing and mature gonads of Chinese sturgeon (Acipenser sinensis). Firstly, the isolation of the boule and dazl genes in Chinese sturgeon and the observation of the two genes in coelacanth suggest that dazl originated after the divergence of bony fish from cartilaginous fish but before the emergence of the Actinistia. Quantitative real-time PCR and western blot analyses reveal that boule and dazl RNA and proteins are restricted to the testis and ovary. In situ hybridization and fluorescent immunohistochemistry show that the bisexual mitotic and meiotic germ cell expression of dazl RNA and protein is conserved in vertebrates, while Chinese sturgeon boule RNA and protein exhibit mitotic and meiotic expression in the testis, and also likely display mitotic and meiotic expression in female. Moreover, we directly demonstrate for the first time that sturgeon Balbiani body/mitochondrial cloud disperses in the cytoplasm of early developing oocytes and co-localizes with Dazl to some extent. Finally, urbilaterian boule may also have an ancestral function in oogenesis. Taken together, these results provide useful information on the evolution of DAZ family genes, expression patterns and functions in animal reproduction.

Oocyte-specific H2A variant H2af1o is required for cell synchrony before midblastula transition in early zebrafish embryos.

Oocyte-specific histone variants have been expected to play significant roles in early embryonic development, but the exact evidence and the biological function have remained unclear. Here, we present evidence that H2af1o, an oocyte-specific H2A variant, is required for cell synchrony before midblastula transition in early zebrafish embryos. The H2A variant is oocyte specific, peaks in mature eggs, and is supplied to early embryos. We constructed a series of deletion plasmids of the zebrafish h2af1o tagged with EGFP and determined the main key function regions including nuclear localization signal of N-terminal 25 amino acids and nucleosome binding region of 110-122 amino acid sequence in the C-terminus by microinjecting them into one-cell-stage zebrafish embryos. In comparison with ubiquitous H2A.X, the H2af1o was revealed to confer a more open structure than canonical H2A in the nucleosomes. Furthermore, we conducted the h2af1o-specific morpholino knockdown analysis in early embryos of zebrafish and revealed its biological function for maintaining cell synchrony division because the H2af1o deficiency disturbed cell synchrony in early cleavages before midblastula transition. Therefore, our current findings provided the first case to understand the biological function of maternal oocyte-specific histone variants in vertebrates.

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.

Feedback regulation of 17ß-estradiol on two kisspeptin genes in the Dabry's sturgeon (Acipenser dabryanus).

In tetrapods, kisspeptins are a group of peptides that play essential roles in the regulation of the Gonadotropin-releasing hormone secretion, and may participate in the feedback regulation of sex steroids as well. In this study, two kiss paralogs, designated as dskiss1 and dskiss2 were identified in Acipenser dabryanus. The full-length cDNA sequences of dskiss1 and dskiss2 are 1265 and 744 base pairs (bp), encoding 130 and 146 amino acids, respectively. Multiple sequence alignment indicated that both Kiss1 and Kiss2 decapeptides were highly conserved among vertebrates. Besides, Kiss1 of Dabry's sturgeon shared closer evolutionary relationship with the holostean species spotted gar (Lepisosteus oculatus), while Kiss2 of Acipenser dabryanus was conservatively grouped with the early sarcopterygian coelacanth (Latimeria chalumnae) in the phylogenetic analysis. Tissue distribution analysis showed that dskiss1 transcribed exclusively in the brain, whereas dskiss2 exhibited wider tissue distribution including brain, testis and ovary. Furthermore, male Dabry's sturgeons were intraperitoneally injected with 17ß-estradiol (E2) and the effect of E2 on hypothalamus kiss and its receptors kissr mRNA levels was evaluated by relative real-time PCR. The transcription levels of dskiss2 and dskissr1 were significantly increased by E2 injection (P < .05). However, the mRNA levels of dskiss1 and dskissr2 were not changed in E2-treated group compared to the control group. These results indicate that E2 exerts positive feedback effects through dskiss2/dskissr1 in male Dabry's sturgeon.

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis.

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon.

C1q-like factor, a target of miR-430, regulates primordial germ cell development in early embryos of Carassius auratus.

C1q-like is a significant maternal factor of TNF/C1q super-family, and the abundant protein has been observed in both mature eggs of Carassius auratus and Carassius auratus gibelio, but its biological function in early embryo development has remained unclear. In this study, we firstly revealed a high level of maternal C1q-like transcript existence only in mature eggs of Carassius auratus, whereas no any maternal C1q-like transcript was observed in that of Carassius auratus gibelio. During embryonic development, the C1q-like zygotic expression begins around cardiopalmus stage in embryos of both Carassius auratus and Carassius auratus gibelio. Then, we examined the biological role of C1q-like by morpholino-mediated knockdown in early embryo development. Knockdown of CaOC1q resulted in a significant reduction of primordial germ cells (PGCs) in Carassius auratus, as shown by whole mount in situ hybridization with vasa-specific RNA probe, fluorescence immunostaining of vasa protein, and GFP imaging of the GFP-nanos1-3'UTR mRNA reporter. In vitro and in vivo evidence indicated that a microRNA, miR-430 could repress the C1q-like expression and PGC development. These data suggest that C1q-like should be a direct target of miR-430 and play an essential role in PGC development of Carassius auratus.