Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus).

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.

Smad4 mediates Bmf involvement in sheep granulosa cell apoptosis.

Bcl-2-modifying factor (Bmf) functions to mediate follicular atresia and oocyte growth in mice. It has been proven that TGF-ß can induce Bmf expression via the Smad4 pathway in a variety of cells, and then induce cell apoptosis. Based on this, we hypothesized that Smad4 and Bmf may play important roles in the apoptosis of granulosa cells (GCs) in domestic animals. This study used small-tailed Han sheep follicular GCs cultured in vitro as a model system, and overexpression or interference experiments, to explore the biological roles of Bmf and reveal the preliminary regulatory mechanisms between Smad4 and Bmf in the process of GCs' apoptosis. We found that the proliferation rate of sheep GCs was significantly increased after the knockdown of Bmf, whereas overexpressing Bmf increased the apoptosis rate of GCs, results also verified by the expression patterns of PCNA, Bcl-2, and Bax genes. After the Smad4 knockdown, the apoptosis rate of GCs was increased, while the mRNA and protein expression of Bmf was significantly up-regulated. A rescue experiment verified that the Bmf knockdown could alleviate GCs' apoptosis induced by Smad4 knockdown. In conclusion, our study not only elucidated an important role for Bmf in the apoptosis of sheep GCs but also revealed a new regulatory pathway between Smad4 and Bmf in this process.

Newcastle disease virus V protein interacts with hnRNP H1 to promote viral replication.

The interactions between host cellular proteins and viral proteins are important for successful infection by viruses. Previous studies from our group have identified various host cellular proteins that can interact with the Newcastle disease virus V protein (Chu et al., 2018a), but their function in NDV replication has not been fully determined. The present study reports that heterogenous nuclear ribonucleoprotein H1 (hnRNP H1) can interact with NDV V protein in yeast. The immunofluorescence results showed that hnRNP H1 and V protein could colocalize in the cytoplasm of a chicken embryo fibroblast cell line (DF-1 cells). Co-immunoprecipitation assays further verified the interaction of these two proteins. The effects of overexpression and knockdown of hnRNP H1 on NDV replication were evaluated in DF-1 cells through real time quantitative PCR (RT-qPCR) and plaque assays. The regulation of V protein on hnRNP H1 expression was also examined. The results indicated that overexpression of hnRNP H1 facilitated NDV replication, while knockdown of hnRNP H1 decreased NDV replication. It was also shown that V protein could regulate hnRNP H1 expression at the protein level instead of the transcription level. The effect of V protein and hnRNP H1 on the DF-1 cell cycle was also tested and the results revealed that V protein may regulate cell proliferation by controlling the expression of hnRNP H1. Taken together, these results suggest that NDV V protein could promote viral replication by interacting with hnRNP H1.

Knockdown of phosphatase and tensin homolog (PTEN) inhibits fatty acid oxidation and reduces very low density lipoprotein assembly and secretion in calf hepatocytes.

Dairy cows with fatty liver exhibit hepatic lipid accumulation and disturbances in fatty acid oxidation and lipid transport. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, regulates intrahepatic fatty acid oxidation and lipid transport in mice. Whether PTEN play a role in fatty acid oxidation and very low density lipoprotein (VLDL) assembly in calf hepatocytes are unknown. Hepatocytes isolated from 3 healthy female Holstein calves (1 d old, 30-40 kg) were infected with empty adenovirus with green fluorescent protein for 48 h (Ad-GFP group) or infected with PTEN knockdown adenovirus for 48 h (Ad-shPTEN group), or cultured in RPMI-1640 without Ad-shPTEN or Ad-GFP (control group). Compared with the Ad-GFP group, PTEN knockdown decreased mRNA and protein abundance and the activity of fatty acid oxidation-related molecules, including acyl-coA synthetase long-chain 1, carnitine palmitoyltransferase 1, carnitine palmitoyltransferase 2, and 3-hydroxy acyl-coA dehydrogenase. Furthermore, PTEN knockdown decreased mRNA and protein abundance of VLDL assembly-related molecules, including apolipoprotein B100, apolipoprotein E, microsomal triglyceride transfer protein, and low density lipoprotein receptor. Importantly, PTEN knockdown promoted triglyceride accumulation in hepatocytes and reduced the VLDL content in culture medium. A subsequent study was conducted on the following 4 groups: cells infected with Ad-GFP for 48 h and then treated with 2% BSA for another 24 h (Ad-GFP + BSA); cells infected with Ad-GFP for 48 h and then treated with 1.2 mM free fatty acids (FFA) and 2% BSA for another 24 h (Ad-GFP + 1.2 mM FFA); cells infected with Ad-shPTEN for 48 h and then treated with 2% BSA for another 24 h (Ad-shPTEN + BSA); cells infected with Ad-shPTEN for 48 h and then treated with 1.2 mM FFA and 2% BSA for another 24 h (Ad-shPTEN + 1.2 mM FFA). Compared with Ad-GFP + BSA, the abundances of PTEN and of fatty acid oxidation- and VLDL assembly-related proteins were lower in the Ad-GFP + 1.2 mM FFA group. Importantly, PTEN knockdown heightened the increase in triglyceride accumulation of hepatocytes and the decrease in VLDL content in culture medium induced by FFA. Overall, these in vitro data indicate that FFA inhibits PTEN expression, leading to triglyceride accumulation and the inhibition of VLDL assembly in calf hepatocytes. These findings suggest that PTEN may be a potential therapeutic target for FFA-induced hepatic steatosis in dairy cows.

Porcine circovirus type 2 upregulates endothelial-derived IL-8 production in porcine iliac artery endothelial cells via the RIG-I/MDA-5/MAVS/JNK signaling pathway.

BACKGROUND: Dysfunction of endothelial cells and vascular system is one of the most important pathological changes of porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2). PCV2-infected endothelial cells can upregulate the production of endothelial-derived IL-8, which can inhibit the maturation of dendritic cells. Endothelial-derived IL-8 has different structural and biological characteristics compared with monocyte-derived IL-8. However, the mechanism of endothelial-derived IL-8 production is still unclear. RESULTS: Key molecules of RIG-I-like signaling pathway RIG-I, MDA-5, MAVS and a key molecule of JNK signaling pathway c-Jun in PCV2-infected porcine iliac artery endothelial cells (PIECs) were upregulated significantly detected with quantitative PCR, Western blot and fluorescence confocal microscopy, while no significant changes were found in NF-κB signaling pathway. Meanwhile, the expression of endothelial-derived IL-8 was downregulated after RIG-I, MDA-5, or MAVS genes in PIECs were knocked down and PIECs were treated by JNK inhibitor. CONCLUSIONS: PCV2 can activate RIG-I/MDA-5/MAVS/JNK signaling pathway to induce the production of endothelial-derived IL-8 in PIECs, which provides an insight into the further study of endothelial dysfunction and vascular system disorder caused by PCV2.

Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells.

Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.

Molecular characterization of GTP binding protein overexpressed in skeletal muscle (GEM) and its role in promoting adipogenesis in goat intramuscular preadipocytes.

GTP binding protein overexpressed in skeletal muscle (GEM) is an important gene with many functions, such as regulating the rearrangement of cytoskeleton and the activity of voltage-dependent calcium channel, and GEM was regarded as a candidate gene for obesity. However, little investigation has been carried out to explore whether GEM affected the intramuscular fat (IMF) deposition of goat. To explore the role of GEM gene in goat, this gene was cloned and its tissue and temporal expression profile were detected. Effect of GEM on adipogenesis was examined by losing function of GEM in vitro. Thereafter, several lipid metabolism-related genes were examined, including CCAAT/enhancing-binding protein α (C/EBPα), CCAAT/enhancing-binding protein ß (C/EBPß), lipoprotein lipase (LPL), preadipocyte factor 1 (Pref-1), peroxisome proliferator activated receptor γ (PPARγ) and sterol regulatory element binding protein 1 (SREBP1). We found that the goat GEM gene consisted of 936 bp, which encoded a protein of 311 amino acids. The expression of GEM was higher in spleen, lung and large intestine and it appeared sharp in the interim stage of differentiation. Furthermore, GEM knockdown blocked adipogenesis and the expression of C/EBPα, C/EBPß, LPL, PPARγ and SREBP1. These results indicated that GEM might promote lipid accumulation and adipogenesis.

Improvement of developmental competence of cloned male pig embryos by short hairpin ribonucleic acid (shRNA) vector-based but not small interfering RNA (siRNA)-mediated RNA interference (RNAi) of Xist expression.

Xist is an X-linked ribonucleic acid (RNA) gene responsible for the cis induction of X chromosome inactivation (XCI). In cloned mammalian embryos, Xist is ectopically activated at the morula to blastocyst stage on the X chromosome that is supposed to be active, thus resulting in abnormal XCI. Suppression of erroneous Xist expression by injecting small interfering RNA (siRNA) remarkably increased the developmental efficiency of cloned male mouse embryos by approximately 10-fold. However, injection of anti-Xist siRNA resulted in only a slight increase in the developmental ability of injected cloned male pig embryos because the blocking effect of the injected siRNA was not maintained beyond the morula stage, which is 5 days post-activation. To develop a more effective approach for suppressing the ectopic expression of Xist in cloned pig embryos, we compared the silencing effect of short hairpin RNA (shRNA) and siRNA on Xist expression and the effects of these two Xist knockdown methods on the developmental competence of cloned male pig embryos. Results indicated that an shRNA-based RNA interference (RNAi) has a longer blocking effect on Xist expression than an siRNA-mediated RNAi. Injection of anti-Xist shRNA plasmid into two-cell-stage cloned male pig embryos effectively suppressed Xist expression, rescued XCI at the blastocyst stage, and improved the in vitro developmental ability of injected cloned embryos. These positive effects, however, were not observed in cloned male pig embryos injected with anti-Xist siRNA. This study demonstrates that vector-based rather than siRNA-mediated RNAi of Xist expression can be employed to improve pig cloning efficiency.

The interleukin-6 regulates the function of monocytes/macrophages (MO/MФ) via the interleukin-6 receptor ß in ayu (Plecoglossus altivelis).

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor ß (PaIL-6Rß) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rß knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rß knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.

The effect of nutrient restriction and syndecan-4 or glypican-1 knockdown on the differentiation of turkey pectoralis major satellite cells differing in age and growth selection.

Skeletal muscle growth is mediated by the proliferation and differentiation of satellite cells, whose activity is affected by both nutrition and the expression of syndecan-4 and glypican-1. Previous research has not addressed if there is an interactive effect of nutrition with the expression of syndecan-4 and glypican-1. Thus, the objective of the current study was to determine if the response of satellite cells to nutrient restriction was altered by syndecan-4 or glypican-1 knockdown and if age and growth selection are factors. Satellite cells were isolated from pectoralis major muscle of 1-day, 7-wk, and 16-wk-old turkeys selected for increased 16-wk body weight (F line) and the randombred control (RBC2) line from which the F line was selected. Syndecan-4 or glypican-1 expression was knocked down in both lines using small interfering RNAs along with nutrient restriction of 0 or 20% of the standard cell culture medium either applied during proliferation with subsequent normal differentiation medium (RN) or during differentiation with preceding normal proliferation medium (NR). For both lines, nutrient restriction and syndecan-4 or glypican-1 knockdown had an independent and additive effect on satellite cell differentiation at 72 h of differentiation except for 1 d satellite cells. The 1 d satellite cell differentiation was increased by RN treatment, but when combined with syndecan-4 or glypican-1 knockdown, the increase in differentiation was negated. At 48 h of differentiation, syndecan-4 knockdown in 7 and 16 wk satellite cells and glypican-1 knockdown in 7 wk cells cancelled the effect of the RN treatment, but enhanced the effect of NR treatment at 24 h of differentiation. Growth selection had little effect on the interaction between nutrient restriction and syndecan-4 or glypican-1 knockdown. Taken together, these data demonstrate that the satellite cell response to nutrition is dependent on the expression of syndecan-4 and glypican-1 in an age-dependent manner with growth selection having little impact.

Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos.

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.

14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection.

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in 2006 in China and caused great economic losses for the swine industry because of the lack of an effective vaccine. 14-3-3 proteins are generating significant interest as potential drug targets by allowing the targeting of specific pathways to elicit therapeutic effects in human diseases. In a previous study, 14-3-3s were identified to interact with non-structural protein 2 (NSP2) of PRRSV. In the present study, the specific subtype 14-3-3ε was confirmed to interact with NSP2 and play a role in the replication of the HP-PRRSV TA-12 strain. Knockdown of 14-3-3ε in Marc-145 cells and porcine alveolar macrophages (PAMs) caused a significant decrease in TA-12 replication, while stable overexpression of 14-3-3ε caused a significant increase in the replication of TA-12 and low pathogenic PRRSV (LP-PRRSV) CH-1R. The 14-3-3 inhibitor difopein also decreased TA-12 and CH-1R replication in Marc-145 cells and PAMs. These findings are consistent with 14-3-3ε acting as a proviral factor and suggest that 14-3-3ε siRNA and difopein are therapeutic candidates against PRRSV infection.

Knockdown of ribosomal protein S3 causes preimplantation developmental arrest in mice.

Ribosomal protein S3 (RpS3), a member of the ribosome 40S subunit, has conventional ribosomal function and additional extraribosomal functions. The aim of the present study was to analyze the expression and localization of RpS3 and its function in early embryogenesis in mice. RpS3 mRNA and protein were expressed in multiple mouse tissues. In the ovary, RpS3 protein was ubiquitously and highly expressed in oocytes and granulosa cells. After ovulation and fertilization, RpS3 mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, RpS3 protein was localized in the cytoplasm of oocytes and preimplantation embryos. Moreover, knockdown of RpS3 in zygotes led to a significantly decreased rate of blastocyst formation. These results provide the first evidence for a novel function of RpS3 in regulating early embryonic development in mice.

Phenotypic characterization, virulence, and immunogenicity of Pseudomonas plecoglossicida rpoE knock-down strain.

Pseudomonas plecoglossicida, a temperature dependent bacterial pathogen in fish, expresses rpoE gene that is sensitive to temperature and probably critical for pathogen virulence and disease development. In this study, the rpoE silence strain rpoE-RNAi-1 was constructed by gene knock-down. The rpoE-RNAi-1 displayed significant changes in biofilm formation, swarming motility, adhesion and virulence. Meanwhile, vaccination of grouper with rpoE-RNAi-1 led to a relative percent survival (RPS) value of 85% after challenged with the wild-type P. plecoglossicida. qRT-PCR assays showed that vaccination with rpoE-RNAi-1 enhanced the expression of immune-related genes, including MHC-I, MHC-II, IgM, and IL-1ß, indicating that it was able to induce humoral and cell-mediated immune response in grouper. These results validated the possibility of rpoE as a potential target for constructing P. plecoglossicida live attenuated vaccine.

Neutrophil plays critical role during Edwardsiella piscicida immersion infection in zebrafish larvae.

Edwardsiella piscicida is a facultative intracellular pathogen that causes hemorrhagic septicemia and haemolytic ascites disease in aquaculture fish. During bacterial infection, macrophages and neutrophils are the first line of host innate immune system. However, the role of neutrophils in response to E. piscicida infection in vivo remains poorly understood. Here, through developing an immersion infection model in the 5 day-post fertilization (dpf) zebrafish larvae, we found that E. piscicida was mainly colonized in intestine, and resulted into significant pathological changes in paraffin sections. Moreover, a dynamic up-regulation of inflammatory cytokines (TNF-α, IL-1ß, GCSFb, CXCL8 and MMP9) was detected in zebrafish larvae during E. piscicida infection. Furthermore, a significant recruitment of neutrophils was observed during the E. piscicida infection in Tg(mpx:eGFP) zebrafish larvae. Thus, we utilized the CRISPR/Cas9 system to generate the neutrophil-knockdown (gcsfr-/- crispants) larvae, and found a comparative higher mortality and bacterial colonization in gcsfr-/- crispants, which reveals the critical role of fish neutrophils in bacterial clearance. Taken together, our results developed an effective E. piscicida immersion challenge model in zebrafish larvae to clarify the dynamic of bacterial infection in vivo, which would provide a better understanding of the action about innate immune cells during infection.

In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing.

IMPACT STATEMENT: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.

EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication.

Transmissible gastroenteritis coronavirus (TGEV) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. The TGEV membrane (M) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. To identify the cellular proteins that interact with the TGEV M protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified M-binding partners. Using the GST pull-down approach and a CO-IP assay, the M protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (EIF4A2), an essential component of the cellular translational machinery. Additionally, confocal microscopy revealed that EIF4A2 and M were colocalized in the cytoplasm. Furthermore, the function of EIF4A2 in intestinal cells during TGEV infection was examined. A knockdown of EIF4A2 by siRNA markedly decreased M protein proliferation and TGEV replication in target cells. Thus demonstrating that EIF4A2 plays a significant role in TGEV replication. The present study provides mechanistic insight into the interaction between the TGEV M protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for TGEV infection.

Identification and characterization of ERV transcripts in goat embryos.

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells.

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.