Gonadotropin receptors of Labeo rohita: Cloning and characterization of full-length cDNAs and their expression analysis during annual reproductive cycle.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), secreted from pituitary, stimulate gonadal function by binding to their cognate receptors FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR). Rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the regulation of expression of gonadotropins and their receptors gene during different phases of annual reproductive cycle has not been investigated. We envisaged the critical role of these molecules during seasonal gonadal development in this carp species. We cloned full- length cDNAs of fshra and lhcgrba from rohu testis using RACE (Rapid amplification of cDNA ends) and analyzed their expression along with fsh and lh by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu fshra and lhcgrba cDNA encodes 670 and 716 amino acids respectively, and in adult fish, they were widely expressed in brain, pituitary, gonad, liver, kidney, head kidney, heart, muscle, gill, fin, eye and intestine. In male, both fsh and fshra transcripts showed high level of expression during spermatogenesis, however, in female, expression level was found to be higher in the fully grown oocyte stages. The expression of rohu lh and lhcgrba mRNA increased with increment of gonadosomatic index and showed highest level during spermiation stage in male and fully matured oocyte stage in female. These results together may suggest the involvement of fshra and lhcgrba in regulating function of seasonal gonadal development in rohu.

The ß-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

We previously characterized the ß-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The ß-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of ß-actin gene expression across eukaryotes. Furthermore, the isolated ß-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

Gene structure and identification of minimal promoter of Pou2 expressed in spermatogonial cells of rohu carp, Labeo rohita.

Mammalian Pou5f1 is a known transcriptional regulator involving maintenance of embryonic and spermatogonial stem cells. Little is known about teleost Pou2, an ortholog of mammalian Pou5f1. Evidences of discrepancy in expression pattern between fish species were documented. To better understand, we have cloned and characterized Pou2 gene of farmed rohu carp, Labeo rohita. It contained five exons with an open reading frame of 1419 bp long, translatable to 472 aa. A bipartite DNA binding domain termed POU domain, comprising of POU-specific and POU-homeo sub-domains, was identified. Rohu Pou2 is highly conserved with zebrafish counterpart, as evidenced by 92% overall sequence identity of deduced protein. The POU domain remained highly conserved (showing more than 90% identities) within fish species. Even though there is a divergence between Pou2 and Pou5f1, the common POU-specific domain remained conserved throughout eukaryotes indicating their possible involvements in common trans-activation pathway(s) between mammals and non-mammals. In support, we have provided evidence that Pou2 is indeed abundantly expressed in proliferating rohu spermatogonial cells and hence participates in stem cell maintenance. Its mRNA accumulation in the ovary supported about its maternal transmission with possible regulatory roles during embryogenesis. The 5'-flanking region (~2.7 kb) of rohu Pou2 was sequenced and computational analysis detected several putative regulatory elements. These elements have been conserved among fish species analysed. Luciferase assay identified a mammalian-type 'TATA-less promoter' capable of driving Pou2 gene transcription. These findings will help for future studies in elucidating participatory role of fish Pou2 in male germ cell development.

Identification and characterization of differentially expressed transcripts in the gills of freshwater prawn (Macrobrachium rosenbergii) under salt stress.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important species. It is a euryhaline shrimp, surviving in wide-range salinity conditions. A change in gene expression has been suggested as an important component for stress management. To better understand the osmoregulatory mechanisms mediated by the gill, a subtractive and suppressive hybridization (SSH) tool was used to identify expressed transcripts linked to adaptations in saline water. A total of 117 transcripts represented potentially expressed under salinity conditions. BLAST analysis identified 22% as known genes, 9% as uncharacterized showing homologous to unannotated ESTs, and 69% as unknown sequences. All the identified known genes representing broad spectrum of biological pathways were particularly linked to stress tolerance including salinity tolerance. Expression analysis of 10 known genes and 7 unknown/uncharacterized genes suggested their upregulation in the gills of prawn exposed to saline water as compared to control indicating that these are likely to be associated with salinity acclimation. Rapid amplification of cDNA ends (RACE) was used for obtaining full-length cDNA of MRSW-40 clone that was highly upregulated during salt exposure. The sequenced ESTs presented here will have potential implications for future understanding about salinity acclimation and/or tolerance of the prawn.

Expression profile of HSP genes during different seasons in goats (Capra hircus).

The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P < 0.05) in all age groups during peak summer season as compared with peak winter season in both tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P < 0.05) during summer season as compared with winter season in tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.

First evidence of molecular characterization of rohu carp Sox2 gene being expressed in proliferating spermatogonial cells.

Because little is known about the function of Sox2 (Sry-related box-2) in teleosts, the objective of this study was to clone and characterize Sox2 complementary DNA (cDNA) from the testis of Indian major carp, Labeo rohita (rohu). The full-length cDNA contained an open reading frame of 936 nucleotides bearing the typical structural features. Phylogenetically, Sox2 of L rohita was most closely related to freshwater counterparts than marine water. The sequence information of cDNA and genomic DNA together revealed that the Sox2 gene is encoded by an uninterrupted exon. Furthermore, comparative mRNA expression profile in various organs including proliferating spermatogonial stem cells (SSCs) suggested about the participatory role of Sox2 during fish male germ cell development and maintenance of stem cells. In support, we have also provided evidence that Sox2 protein is indeed present in rohu SSCs by Western blot analysis. The evolutionarily conserved high-mobility group box domain indicated its possible involvement in common networking pathways for stem cell maintenance and pluripotency between mammals and nonmammals. Our findings could be the first step toward the use of Sox2 as a potential biomarker for proliferating SSCs and understanding the transcriptional regulatory network involved during male germ cell development and maintenance in fish species.

First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection.

Toll-like receptor 22 (TLR22) is present in teleost but not in mammals. Among Indian farmed carps, Catla catla is relatively more resistant than Labeo rohita to Argulus siamensis lice infection. TLR22 is believed to be associated with innate immunity against ectoparasite infection. To investigate the TLR22 mediated immunity against argulosis, we have cloned and characterized TLR22 genes of L. rohita (rTLR22) and C. catla (cTLR22). The full-length cDNAs of rTLR22 and cTLR22 contained an open reading frame of 2838 and 2841 nucleotides, respectively; bearing the typical structural features. Phylogenetically rTLR22/cTLR22 was most closely related to Cyprinus carpio (common carp) counterpart, having highest sequence identity of 86.0%. The TIR domain remained highly conserved with 90% identity within freshwater fishes. The sequence information of cDNA and genomic DNA together revealed that the rTLR22/cTLR22 genes are encoded by uninterrupted exons. The co-habitation challenge study with A. siamensis infection confirmed that C. catla is comparatively more resistant than L. rohita. Further, comparative mRNA expression profile in immune relevant tissues also suggested about the participatory role of TLR22 during lice infection. However, TLR22 might not solely be involved in conferring relative resistance among carp species against argulosis.

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis).

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.

Cloning of cDNA and prediction of peptide structure of Plzf expressed in the spermatogonial cells of Labeo rohita.

The promyelocytic leukemia zinc finger (Plzf) gene containing an evolutionary conserved BTB (bric-a-brac/tramtrack/broad complex) domain plays a key role in self-renewal of mammalian spermatogonial stem cells (SSCs) via recruiting transcriptional co-repressors. Little is known about the function of Plzf in vertebrate, especially in fish species. To gain better understanding of its role in fishes, we have cloned Plzf from the testis of Labeo rohita (rohu), a commercially important freshwater carp. The full-length cDNA contains an open reading frame (ORF) of 2004bp translatable to 667 amino acids (aa) containing a conserved N-terminal BTB domain and C-terminal C(2)H(2)-zinc finger motifs. L. rohita Plzf, which is phylogenetically related to Danio rerio counterpart, abundantly expressed in spermatogonial stem cells (SSCs). A three-dimensional (3D) model of BTB domain of Plzf protein was constructed by homology modeling approach. Molecular docking on this 3D structure established a homo-dimer between two BTB domains creating a charged pocket containing conserved aa residues: L33, C34, D35 and R49. Thus, Plzf of SSC is structurally and possibly functionally conserved. The conserved aa residues in the cleft resulting from Plzf BTB self-association are likely to be the binding platform for interaction with recruited co-repressor peptides. The identified Plzf could be the first step towards exploring its role in rohu SSC behavior.