Intestinal transcriptomes in Kazakh sheep with different haplotypes after experimental Echinococcus granulosus infection.

Cystic echinococcosis (CE) is a chronic zoonosis caused by infection with the larval stage of the cestode Echinococcus granulosus. As the intermediate host, sheep are highly susceptible to this disease. Our previous studies have shown that sheep with haplotype MHC Mva Ibc-Sac IIab-Hin1I ab were resistant to CE infection, while their counterparts without this haplotype were not. In order to reveal the molecular mechanism of resistance in Kazakh sheep, after selecting the differential miRNA in our previous study, herein, transcriptome analyses were conducted to detect the differential expression genes in the intestinal tissue of Kazakh sheep with resistant and non-resistant MHC haplotypes, after peroral infection with E. granulosus eggs. A total of 3835 differentially expressed genes were identified between the two groups, with 2229 upregulated and 1606 downregulated. Further function analysis showed that the most significant genes were related to both innate immune response and adaptive response participating in the defense against E. granulosus infection and the metabolic changes associated with it. The results suggest that genes related to lectin receptors, NK cells activation, chemokines, and tumor necrosis factor, may play important roles in the response of intestinal tissue to E. granulosus.

TITLE: Transcriptomes intestinaux chez des moutons kazakhs de différents haplotypes après une infection expérimentale avec Echinococcus granulosus. ABSTRACT: L'échinococcose kystique (EK) est une zoonose chronique qui est causée par une infection au stade larvaire du cestode Echinococcus granulosus. En tant qu'hôte intermédiaire, les moutons sont très sensibles à cette maladie. Nos études précédentes ont montré que les moutons avec l'haplotype MHC Mva Ibc-Sac IIab-Hin1I ab étaient résistants à l'EK, alors que leurs homologues sans cet haplotype ne l'étaient pas. Afin de révéler le mécanisme moléculaire de la résistance chez le mouton kazakh, après avoir sélectionné le miARN différentiel dans notre étude précédente, des analyses de transcriptome ont été menées dans ce travail pour détecter les gènes d'expression différentielle dans le tissu intestinal de mouton kazakh avec des haplotypes MHC résistants et non résistants, après une infection pérorale par des œufs d'E. granulosus. Un total de 3835 gènes différentiellement exprimés ont été identifiés entre les deux groupes, avec 2229 régulés à la hausse et 1606 à la baisse. Une analyse fonctionnelle plus poussée a montré que les gènes les plus significatifs étaient liés à la fois à la réponse immunitaire innée et à la réponse adaptative participant à la défense contre l'infection à E. granulosus et aux changements métaboliques qui y étaient associés. Les résultats suggèrent que les gènes liés aux récepteurs de la lectine, à l'activation des cellules NK, aux chimiokines et au facteur de nécrose tumorale peuvent jouer un rôle important dans la réponse du tissu intestinal à E. granulosus.

iTRAQ-based comparative proteomic analysis in different developmental stages of Echinococcus granulosus.

Cystic echinococcosis, caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis. The lifecycle of the E. granulosus parasite includes three consecutive stages that require specific gene regulation or protein expression to survive environmental shifts between definitive hosts and intermediate hosts. The aim of the present study is to screen and analyze the stage differential antigens to be considered for vaccine development against E. granulosus. By using the iTRAQ (isobaric tags for relative and absolute quantification) method, the differentially expressed proteins were selected from the three consecutive developmental stages of E. granulosus: oncosphere, adult tapeworms, and protoscolex. Through a bioinformatics analysis including Clusters of Orthologous Groups (COG), Gene Ontology (GO), and pathway metabolic annotation, we identified some proteins of interest from each stage. The results showed that a large number of differentially expressed proteins (375: oncosphere vs. adult, 346: oncosphere vs. protoscolex, and 391: adult vs. protoscolex) were identified from the three main lifecycle stages. Analysis of the differential protein pathways showed that these differential proteins are mainly enriched in metabolic pathways, Huntington's diseases, Alzheimer's diseases, and ribosome metabolic pathways. Interestingly, among these differential proteins, expression levels of paramyosin, HSP60, HSP70, HSP90, cathepsin L1, cathepsin D, casein kinase, and calmodulin were significantly higher in the oncosphere than in the adult or protoscolex (p < 0.05). We hope our findings will help to identify potential targets for diagnosis or for therapeutic and prophylactic intervention.

TITLE: Analyse protéomique comparative basée sur iTRAQ de différents stades de développement d'Echinococcus granulosus. ABSTRACT: L'échinococcose kystique, causée par une infection au stade larvaire du cestode Echinococcus granulosus, est une zoonose chronique. Le cycle de vie du parasite E. granulosus comprend trois étapes consécutives qui nécessitent une régulation génétique ou une expression de protéines spécifiques pour survivre aux changements environnementaux entre les hôtes définitifs et les hôtes intermédiaires. Le but de la présente étude est de cribler et d'analyser les antigènes différentiels de stade à considérer pour le développement de vaccins contre E. granulosus. En utilisant la méthode iTRAQ (étiquettes isobares pour la quantification relative et absolue), les protéines différentiellement exprimées ont été sélectionnées parmi les trois stades de développement consécutifs d'E. granulosus : l'oncosphère, les ténias adultes et le protoscolex. Grâce à une analyse bioinformatique comprenant les grappes de groupes orthologues (COG), l'ontologie des gènes (GO) et l'annotation des voies métaboliques, nous avons identifié certaines protéines d'intérêt à chaque étape. Les résultats ont montré qu'un grand nombre de protéines exprimées différentiellement (375 : oncosphère vs adulte, 346 : oncosphère vs protoscolex et 391 : adulte vs protoscolex) sont identifiées pour les trois étapes principales du cycle de vie. L'analyse des voies différentielles des protéines a montré que ces protéines différentielles sont principalement enrichies dans les voies métaboliques, la maladie de Huntington, la maladie d'Alzheimer et les voies métaboliques des ribosomes. Fait intéressant, parmi ces protéines différentielles, les niveaux d'expression des protéines paramyosine, HSP60, HSP70, HSP90, cathepsine L1, cathepsine D, caséine kinase et calmoduline étaient significativement plus élevés dans l'oncosphère que chez l'adulte ou le protoscolex (p < 0,05). Nous espérons que nos résultats pourront identifier des cibles potentielles pour un diagnostic ou une intervention thérapeutique ou prophylactique.

Sex control by Zfy siRNA in the dairy cattle.

Zinc-finger Y is located in the short arm of the Y-chromosome and is a highly conserved gene that plays an important role in spermatogenesis. The objective of this study was to investigate the influence of silencing the Zfy gene during spermatogenesis on Y-sperm formation and offspring sex determination in Bos taurus cattle. Three recombinant expression vectors pLL3.7/a, pLL3.7/b and pLL3.7/c were evaluated and only pLL3.7/a effectively silenced the Zfy gene. The pLL3.7/a recombinant expression vector was injected into bull testes, using three injections. Semen was collected and preserved by extending and freezing. The frozen semen was subsequently used in artificial insemination of cows during a breeding season in accordance with the production plan on the farm where the experiment was conducted. Results showed that, after exposure to pLL3.7/a, sperm motility decreased (P < 0.01), but the sperm density was similar (p > 0.05) to the non-treated control semen. Injection of pLL3.7/a resulted in 72.0% female offspring, and was greater than the 49.4% female calves in the control (P ＜ 0.01), Results from this research suggests that the Zfy gene plays a role in the process of Y-sperm formation, and Zfy siRNA is a potential useful approach to control sex of offspring in cattle.

RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis.

The objective of this study was to explore a novel method to alter the sex-ratio balance of mouse offspring by silencing the paralogous genes Zfx/Zfy (Zinc finger X/Y-chromosomal transcription factor gene) during spermatogenesis. Four recombined vectors PRZ1, PRZ2, PRZ3, and PRZ4 (RNAi-Ready-pSIREN-RetroQ-ZsGreen) were constructed for interrupting the Zfx gene. Additionally, a recombined vector Psilencer/Zfy-shRNA was constructed for interrupting the Zfy gene. Male mice were randomly divided into 8 groups, with 20 animals per group. Five groups of mice were injected with PRZ1, PRZ2, PRZ3, PRZ4, and Psilencer/Zfy-shRNA vectors, respectively. The three control groups were injected with an equal volume of physiological saline, empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector, and empty Psilencer/Zfy-shRNA vector, respectively. All groups were injected every 7 days for a total of four injections. Fourteen days after the fourth injection, 10 male mice from each group were mated individually with 10 females. Testicular tissue of 10 male mice in each group was collected, and the expression level of Zfx/Zfy mRNA was determined by qRT-PCR. Results showed that, compared with the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and the physiological saline group, expression of Zfx mRNA decreased significantly after injection of PRZ1 (p < 0.01), PRZ3 (p < 0.01), and PRZ4 (p < 0.01), and 78.75 ± 7.50% of the offspring were male in PRZ4 group, significantly higher than the offspring derived from the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and physiological saline group (p < 0.01). In the PRZ1 group, the expression of Zfx mRNA was also significantly lower (p < 0.01), but the male rate of offspring was not different (p > 0.05). Conversely, the expression of Zfy mRNA decreased significantly after injection of Psilencer/Zfy-shRNA (p < 0.01) and 31.00 ± 11.00% of the offspring were male, significantly lower than in the physiological saline group (p < 0.01). In conclusion, our findings show that RNAi-mediated disruption of Zfx/Zfy in mouse testis affected X/Y spermatogenesis. Additionally, results suggest that the paralogous genes Zfx/Zfy play an important role in the process of X and Y sperm development. The individual interference of Zfx/Zfy may predict the outcome of X and Y haploid sperms. Presented herein is an advanced method developed to control mouse X/Y spermatogenesis and sex ratio of offspring.

MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach.

Cystic echinococcosis (CE), caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis, to which sheep are highly susceptible. Previously, we found that Kazakh sheep with different MHC haplotypes differed in CE infection. Sheep with haplotype MHCMvaIbc-SacIIab-Hin1Iab were resistant to CE infection, while their counterparts without this haplotype were not. MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at the post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. To identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant and non-resistant MHC haplotypes after peroral infection with E. granulosus eggs. A total of 351 known and 186 novel miRNAs were detected in the resistant group, against 353 known and 129 novel miRNAs in the non-resistant group. Among these miRNAs, 83 known miRNAs were significantly differentially expressed, including 75 up-regulated and 8 down-regulated miRNAs. Among these known microRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b, and miR-27a showed a significantly higher expression in CE-resistant sheep compared to the CE-non-resistant library, with the FC > 3. Functional analysis showed that they were NF-kB pathway-responsive miRNAs, which are involved in the inflammation process. The results suggest that these microRNAs may play important roles in the response of intestinal tissue to E. granulosus.