Expression pattern analysis of anti-Mullerian hormone in testis development of pearlscale angelfish (Centropyge vrolikii).

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.

Characterization of the transcription factor Sox3 regulating the gonadal development of pearlscale angelfish (Centropyge vrolikii).

As a member of the Sox gene family, Sox3 plays a vital role in gonadal development and gametogenesis. Nevertheless, the exact expression pattern of this gene in fish is still unknown. Here, we identified the Sox3 gene of Centropyge vrolikii, namely, Cv-Sox3. The Cv-Sox3 mRNA expression in the ovary and testis was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the mRNA expression level of Cv-Sox3 in the ovary in the resting stage was significantly higher than that in other tissues. The phylogenetic tree and alignment of multiple sequences were constructed to analyze the evolutionary relationships of Cv-Sox3. Cv-Sox3 was relatively conserved in the evolution of teleost fish, indicating the importance and similarity of its function. The in situ hybridization results demonstrate that Cv-Sox3 was present in the follicle cells and cytoplasm of oocytes in the ovary of different stages, and the positive signals occurred in germ cells of the testis. After interfering with Cv-Sox3, the growth rate of ovarian cells in culture became slow, and the expression of ovary-bias-related genes Cyp19a and Foxl2 significantly increased. Meanwhile, the expression of testis-bias-related genes Dmrt1, Sox9, Cyp11a, Amh, and Sox8 significantly decreased. These results suggest that Cv-Sox3 gene might be expressed in the germ cells of male and female gonads during gonadal development. This study provides a precise expression pattern of Cv-Sox3 and demonstrates that Cv-Sox3 might play a significant role in the reproductive regulation of C. vrolikii. In this study, Sox3 of C. vrolikii (Cv-Sox3) was cloned to understand the expression pattern in the gonadal development, which is expressed in germ cells, involved in the process of gonadal development. The results demonstrated that Cv-Sox3 may play a significant role in the reproductive regulation of C. vrolikii.

Genome-wide investigation of Dmrt gene family in large yellow croaker (Larimichthys crocea).

The Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM (Doublesex/Mab-3) domain. Previous researches indicate this gene family is involved in various physiological processes, especially in sex determination/differentiation and gonad development. Despite the vital roles of the Dmrt gene family in physiological processes, the comprehensive characterization and analysis of the dmrt genes in large yellow croaker (Larimichthys crocea), one of the most commercially important marine fish in China, have not been described. In this study, we performed the first genome-wide systematic analysis of L. crocea dmrt genes through the bioinformatics method. A total of seven members of the Dmrt gene family including Lcdmrt1, Lcdmrt2a, Lcdmrt2b, Lcdmrt3, Lcdmrt4, Lcdmrt5, and Lcdmrt6 were excavated based on the genome data of L. crocea. Further analysis revealed that the dmrt genes of L. crocea were distributed unevenly across four chromosomes. There were three dmrt genes (Lcdmrt1, Lcdmrt2a, and Lcdmrt3) on 3rd chromosome, one (Lcdmrt6) on 13th chromosome, one (Lcdmrt4) on 14th chromosome, two on (Lcdmrt5 and Lcdmrt2b) 17th chromosome. The gene structure analysis indicated that the number of introns of different dmrt genes of L. crocea had some differences: Lcdmrt1 had four introns, Lcdmrt2a, Lcdmrt2b, and Lcdmrt6 had two introns, Lcdmrt3, Lcdmrt4, and Lcdmrt5 had only one intron. The expression pattern analysis with published gonad transcriptome datasets and further confirmed by qRT-PCR revealed that these members of the Dmrt gene family except for Lcdmrt4 were all sexually dimorphic and preferred expressing in testis. Furthermore, the expression pattern analysis also revealed that the expression level of Lcdmrt1 and Lcdmrt6 was significantly higher than that of other members, suggesting that these two genes may play a more important role in testis. Overall, our studies provide a comprehensive insight into the Dmrt gene family members and a basis for the further study of their biological functions in L. crocea.

[A clinical trial of anti-CD4 CD8 monoclonal antibodies combined with cyclosporine A in treatment of severe aplastic anemia].

OBJECTIVE: To explore the safety and efficacy of a therapeutic regimen for treating severe aplastic anemia (SAA) in its early stage. METHODS: Two groups of SAA patients were enrolled in the present study. One was a treatment group including 21 patients being treated with anti-CD(4), CD(8) monoclonal antibodies as well as cyclosporine A. Another was a control group including 20 patients being treated with anti-lymphocyte globulin and cyclosporine A. RESULTS: The response rates in the two groups were more or less some, being 76.2% for the treatment group and 65.0% for the control group (P > 0.05), but the blood routine examination results showed quicker recovery in the treatment group than in the control group (P < 0.05) and the incidences of side effects related to therapy such as fever were less in the treatment group than in the control group (P < 0.01). CONCLUSIONS: The combination of anti-CD(4), CD(8) monoclonal antibodies and cyclosporine A is safe and efficient in treating SAA.