miR-1306 induces cell apoptosis by targeting BMPR1B gene in the ovine granulosa cells.

Bone morphogenetic protein receptor type-1B (BMPR1B) is one of the major gene for sheep prolificacy. However, few studies investigated its regulatory region. In this study, we reported that miR-1306 is a direct inhibitor of BMPR1B gene in the ovine granulosa cells (ovine GCs). We detected a miRNA response element of miR-1306 in the 3' untranslated region of the ovine BMPR1B gene. Luciferase assay showed that the ovine BMPR1B gene is a direct target of miR-1306. qPCR and western blotting revealed that miR-1306 reduces the expression of BMPR1B mRNA and protein in the ovine granulosa cells. Furthermore, miR-1306 promoted cell apoptosis by suppressing BMPR1B expression in the ovine granulosa cells. Overall, our results suggest that miR-1306 is an epigenetic regulator of BMPR1B, and may serve as a potential target to improve the fecundity of sheep.

Identification of novel genes associated with litter size of indigenous sheep population in Xinjiang, China using specific-locus amplified fragment sequencing technology.

BACKGROUND: There are abundant sheep breed resources in the Xinjiang region of China attributing to its diverse ecological system, which include several high-litter size sheep populations. Previous studies have confirmed that the major high prolificacy gene cannot be used to detect high litter size. Our research team found a resource group in Pishan County, southern Xinjiang. It showed high fertility with an average litter size of two to four in one birth, excellent breast development, and a high survival rate of lambs. In the present study, we used this resource as an ideal sample for studying the genetic mechanisms of high prolificacy in sheep. METHODS: Indigenous sheep populations from Xinjiang, with different litter sizes, were selected for the research, and specific-locus amplified fragment sequencing (SLAF-seq) technology was used to comprehensively screen single nucleotide polymorphisms (SNPs) from the whole genome that may cause differences in litter size. Novel genes associated with litter size of sheep were detected using genome-wide association studies (GWAS), providing new clues revealing the regulation mechanism of sheep fecundity. Candidate genes related to ovulation and litter size were selected for verification using Kompetitive Allele Specific polymerase chain reaction (KASP) cluster analysis. RESULTS: We identified 685,300 SNPs using the SLAF-seq technique for subsequent genome-wide analysis. Subsequently, 155 SNPs were detected at the genome-wide level. Fourteen genes related to sheep reproduction were notated: COIL, SLK, FSHR, Plxna3, Ddx24, CXCL12, Pla2g7, ATP5F1A, KERA, GUCY1A1, LOC101107541, LOC101107119, LOC101107809, and BRAF. Based on literature reports, 30 loci of seven genes and candidate genes (CXCL12, FSHR, SLK, GUCY1A1, COIL, LOC101107541, and LOC101107119) related to ovulation and litter size were selected for verification using KASP cluster analysis. Among them, nine loci of three genes were successfully genotyped. Three loci of FSHR (GenBank ID: 443299, g. 75320741G>A site), GUCY1A1 (GenBank ID: 101110000, g. 43266624C>T site), and COIL (GenBank ID: 101123134, g. 7321466C>G site) were found to be significantly or extremely significantly associated with litter size. These three loci are expected to be used as molecular markers to determine differences in litter size in sheep.

Transcriptome profiling analysis reveals key genes of different coat color in sheep skin.

BACKGROUND: To investigate the molecular mechanisms determining the coat color of native breed sheep in Xinjiang. METHODS: Bashibai sheep, Yemule white sheep and Tulufan black sheep were selected. Illumina HiSeq X Ten sequencing technology was used to detect the genes responsible for the white, light brown, black and cyan gray coat colors in sheep. Sequence analysis and functional gene annotation analysis were performed to analyze the results. The signal pathways and differentially expressed genes related to sheep hair color production regulation were screened and finally verified by real-time polymerase chain reaction. RESULTS: Functional annotation by Kyoto Encyclopedia of Genes and Genomes analysis revealed significant differences in enrichment of immunity-related pathways as well as melanogenesis synthetic and tyrosine metabolism pathways. Our results showed that the DCT, TYR, TYRP1, PMEL, SLC45A2 and MLANA six genes may be associated with the regulation of coat color development and provide a theoretical basis for selecting natural coat colors of sheep.

Smad4 Feedback Enhances BMPR1B Transcription in Ovine Granulosa Cells.

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5' RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region -405 to -200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5' regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

Oviduct-specific expression of tissue plasminogen activator in laying hens.

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.

Oviduct-specific expression of tissue plasminogen activator in laying hens

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.