Common carp pentraxin gene: Evidence for its role in ovarian differentiation and growth.

Pentraxins (PTX), belong to an evolutionarily conserved family, containing a PTX protein domain, having role in acute immunological responses and fertility in higher vertebrates. However, information regarding the action of ptx on reproduction is extremely limited in fish. To study this, ptx cDNA was cloned for downstream analysis. Tissue distribution and ontogeny expression analysis indicated the prevalence of ptx in ovary. Varied phase-wise expression during carp ovarian cycle and elevated ptx expression after human chorionic gonadotropin induction, in vitro and in vivo, indicated probable regulation of gonadotropin. In situ hybridization and immunohistochemistry revealed the presence of ptx transcript and protein in the follicular layer of stage-III/IV oocytes indicating a role in ovarian growth. To assess the functional significance of ptx, transient silencing was performed using follicular primary cell culture, in vitro and in common carp, in vivo, through ovary-targeted injection of PEI-siRNA. Transient silencing of ptx-siRNA reduced the expression of various genes/factors related to oogenesis such as transcription factors, several steroidogenic enzymes, and esrs genes. These alterations in expression suggested a plausible role for ptx in ovarian steroidogenesis either, directly or indirectly, which is evident from the changes in the serum estradiol-17ß (E2) and 17α,20ß-dihydroxyprogesterone levels. Furthermore, downregulation of aromatase activity was also noticed after transient silencing. Increased ptx expression after E2 induced sex reversal to juvenile carp showed the correlative role of ptx during ovarian differentiation and development. Taken together, these findings suggest that ptx exerts an important role during ovarian growth, maturation and/or recrudescence of common carp.

Identification, expression profiling and localization of thoc in common carp ovary: Influence of thoc3-siRNA transient silencing.

THO complex is a multisubunit family with a function in transcription and mRNA export. In the present study, transcripts of THO complex (thoc) were identified in developing ovary of common carp and their role during ovarian development and growth has been characterized for the first time in a teleost using expression profiling and transient siRNA silencing. Thoc expression revealed a spatiotemporal pattern in the gonads with high levels at 120 days post-hatch, with moderately high levels thereafter. In situ hybridization and immunohistochemical localization revealed the presence of thoc3 in follicular layer of stage-III/IV oocytes. High levels of thoc3, thoc5, and thoc7 genes in the follicular layer suggest a possible role in ovarian growth. Reduced levels of serum estradiol-17ß and 17α, 20ß-dihydroxypregn-4-en-3-one after thoc3 transient silencing indicated differential action on steroidogenic enzyme, transcription factor, and growth factor genes. Furthermore, transient silencing of thoc3, in vivo and in vitro, downregulated ad4bp/sf1, amh, cyp19a1a, foxl2, hsd3b, hsd11b1, hsd20b, hsd17b1, rspo1, and vtg. Incidentally, gdf9 and igf1 were upregulated, while no change was seen in esr1/2, nanos, and vasa. These observations imply that thoc3 seems to regulate ovarian function including steroidogenesis, either directly or indirectly.

Gonadal transcriptome analysis of the common carp, Cyprinus carpio: Identification of differentially expressed genes and SSRs.

Common carp (Cyprinus carpio) is a world-wide freshwater fish of eutrophic waters. C. carpio, have various reproductive traits, including early sexual maturity, that may make them excellent, large, realistic, aquaculture model species. In the present work, de novo assembly of gonadal (testicular and ovarian) transcriptomes from juvenile common carp was performed to identify genes involved in gonadal development. A total of 81,757 and 43,257 transcripts with average lengths of 769 and 856 bp, were obtained from the immature testicular and ovarian transcriptomes, respectively. About 84,367 unigenes were constructed after removing redundancy involving representation of transcripts in both gonadal transcriptomes. Gene ontology (39,171 unigenes), clusters of orthologous group's analysis (6651 unigenes) and Kyoto encyclopedia of genes, and genomes automatic annotation server analysis (4783 unigenes) were performed to identify potential genes along with their functions. Furthermore, 18,342 (testis) and 8693 (ovary) simple sequence repeats were identified. About 298 differentially expressed genes were identified, of which 171 and 127 genes were up-regulated in testis and ovary, respectively. Quantitative real-time reverse transcription PCR was performed to validate differential expression of selected genes in testis and ovary. Nearly 809 genes related to reproduction were identified, sex-wise expression pattern of genes related to steroid synthesis, endocrine regulation, germ cell maintenance and others factors related to gonadal differentiation was observed, and expression analysis of nanos, ad4bp/sf-1, and gdf9 was performed. The present study identified certain important genes/factors involved in the gonadal development of C. carpio which may provide insights into the understanding of sex-differentiation and gonadal development processes.

Enterotoxic effects of Aeromonas hydrophila infection in the catfish, Clarias gariepinus: Biochemical, histological and proteome analyses.

Aeromonas hydrophila is considered as a potential risk to fish populations in the aquaculture industry and could also pose a serious threat to humans. In this study, the impact of A. hydrophila infection in the air-breathing catfish, Clarias gariepinus was analyzed using a multidimensional approach. Aeromonas hydrophila (1 × 107 cells) was injected into C. gariepinus intraperitoneally and maintained at an ambient temperature and photoperiod with periodical monitoring for morphological changes. After 7 days post-infection, tissue samples of the gills, liver, intestine, and kidney were subjected to biochemical, histological, transmission electron microscope (TEM) and proteomic analyses. Observed results indicated distinct morphological changes with the significant increase of ROS and oxidative stress enzymes (CAT and SOD) in tissues of the infected group when compared to the control. Histological analysis in infected fish revealed the presence of pyknotic nuclei, early stages of necrosis in the liver, degradation of renal tubules and widened sinusoidal space in kidneys along with enlargement of the epithelial region in the intestine. TEM analysis of the infected intestine showed degeneration of villi and the presence of multinucleated erythrocytes. Two-dimensional proteomic and mass spectrometry analysis of intestine and liver displayed up-regulation of several immune regulatory proteins such as proteasome subunit 3 protein, prolactin and intermediated filament protein; and down-regulation of proteins including actin, serine/arginine-rich splicing factor and carbonic anhydrase. Taken together, these results suggest that the identified proteins may have a role in immune regulation against A. hydrophila infection in C. gariepinus and support further investigations of host-pathogen interactions.