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Transcriptional profiling of mouse uterus at pre-implantation stage under VEGF repression.

Ji Y, Lu X, Zhong Q, Liu P, An Y, Zhang Y, Zhang S, Jia R, Tesfamariam IG, Kahsay AG, Zhang L, Zhu W, Zheng Y - PLoS ONE (2013)

Bottom Line: Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells.Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development.Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.

ABSTRACT
Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in the pre-implantation stage. Results will contribute to further study the candidate genes and pathways in regulating implantation process and related diseases.

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VEGF repression effect on ovulation, pregnancy and implantation rate.(A) Reversible repression system of VEGF gene expression. (B) Relative VEGF mRNA levels by qPCR (n = 5). (C, D) Protein expression by Western blot analysis and the quantification (n = 3). (E) Embryo collection at E3.5 (n = 45). (F) Embryo dissection at E9.5 (n = 11). (G) Embryo transfer experiment (n = 22). Dox+ represented mouse chow with 200 mg/kg doxycycline. Dox− represented regular mouse chow. Data are presented as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001.
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pone-0057287-g001: VEGF repression effect on ovulation, pregnancy and implantation rate.(A) Reversible repression system of VEGF gene expression. (B) Relative VEGF mRNA levels by qPCR (n = 5). (C, D) Protein expression by Western blot analysis and the quantification (n = 3). (E) Embryo collection at E3.5 (n = 45). (F) Embryo dissection at E9.5 (n = 11). (G) Embryo transfer experiment (n = 22). Dox+ represented mouse chow with 200 mg/kg doxycycline. Dox− represented regular mouse chow. Data are presented as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001.

Mentions: Double transgenic mice VEGFtetO/tetO/β-actin-tetR-Krab (βAKtg/wt) were generated as described previously [18], [19]. In brief, four copies of tet operator (tetO) sequences were inserted into the promoter region of VEGF by gene targeting (VEGFtetO). The transgenic mice carrying universal expression of tetR-Krab fusion protein were generated by pronuclei DNA injection (β-actin-tetR-Krab). By crossing two lines, VEGF expression was under control of tetracycline (Figure 1A). When tetracycline is absent (Dox−), tetR-Krab fusion protein binds to VEGF promoter region and blocks VEGF expression. All the embryos die at E10.5. When tetracycline is administered in the food (Dox+), tetR-Krab fusion protein binds to tetracycline and falls off VEGF promoter VEGF expression goes back to the normal, following Mendel inheritance. These mice were phenotypically normal. The propagation of double transgenic mice in this experiment, therefore, was maintained on doxycycline food (200 mg/kg), a tetracycline analogue. Doxycycline was removed at the age of 3–4 weeks and total RNA was isolated from littermates of Dox+ and Dox− uteri after 6 weeks. All the mice (Dox+ and Dox−) used were littermates and analyzed at the age of 9–10 weeks. Double transgenic females (VEGFtetO/tetO/βAKtg/wt) were set for timed mating with VEGF males (VEGFtetO/tetO). The day of detecting vaginal plugs was designated as G0.5. Animals were euthanized and tissues were collected at G2.5. Uterine samples from two groups Dox+ and Dox− were collected for RNA isolation.


Transcriptional profiling of mouse uterus at pre-implantation stage under VEGF repression.

Ji Y, Lu X, Zhong Q, Liu P, An Y, Zhang Y, Zhang S, Jia R, Tesfamariam IG, Kahsay AG, Zhang L, Zhu W, Zheng Y - PLoS ONE (2013)

VEGF repression effect on ovulation, pregnancy and implantation rate.(A) Reversible repression system of VEGF gene expression. (B) Relative VEGF mRNA levels by qPCR (n = 5). (C, D) Protein expression by Western blot analysis and the quantification (n = 3). (E) Embryo collection at E3.5 (n = 45). (F) Embryo dissection at E9.5 (n = 11). (G) Embryo transfer experiment (n = 22). Dox+ represented mouse chow with 200 mg/kg doxycycline. Dox− represented regular mouse chow. Data are presented as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585347&req=5

pone-0057287-g001: VEGF repression effect on ovulation, pregnancy and implantation rate.(A) Reversible repression system of VEGF gene expression. (B) Relative VEGF mRNA levels by qPCR (n = 5). (C, D) Protein expression by Western blot analysis and the quantification (n = 3). (E) Embryo collection at E3.5 (n = 45). (F) Embryo dissection at E9.5 (n = 11). (G) Embryo transfer experiment (n = 22). Dox+ represented mouse chow with 200 mg/kg doxycycline. Dox− represented regular mouse chow. Data are presented as mean ± SEM. *P<0.05, **P<0.005, ***P<0.001.
Mentions: Double transgenic mice VEGFtetO/tetO/β-actin-tetR-Krab (βAKtg/wt) were generated as described previously [18], [19]. In brief, four copies of tet operator (tetO) sequences were inserted into the promoter region of VEGF by gene targeting (VEGFtetO). The transgenic mice carrying universal expression of tetR-Krab fusion protein were generated by pronuclei DNA injection (β-actin-tetR-Krab). By crossing two lines, VEGF expression was under control of tetracycline (Figure 1A). When tetracycline is absent (Dox−), tetR-Krab fusion protein binds to VEGF promoter region and blocks VEGF expression. All the embryos die at E10.5. When tetracycline is administered in the food (Dox+), tetR-Krab fusion protein binds to tetracycline and falls off VEGF promoter VEGF expression goes back to the normal, following Mendel inheritance. These mice were phenotypically normal. The propagation of double transgenic mice in this experiment, therefore, was maintained on doxycycline food (200 mg/kg), a tetracycline analogue. Doxycycline was removed at the age of 3–4 weeks and total RNA was isolated from littermates of Dox+ and Dox− uteri after 6 weeks. All the mice (Dox+ and Dox−) used were littermates and analyzed at the age of 9–10 weeks. Double transgenic females (VEGFtetO/tetO/βAKtg/wt) were set for timed mating with VEGF males (VEGFtetO/tetO). The day of detecting vaginal plugs was designated as G0.5. Animals were euthanized and tissues were collected at G2.5. Uterine samples from two groups Dox+ and Dox− were collected for RNA isolation.

Bottom Line: Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells.Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development.Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China.

ABSTRACT
Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in the pre-implantation stage. Results will contribute to further study the candidate genes and pathways in regulating implantation process and related diseases.

Show MeSH
Related in: MedlinePlus