Limits...
Quantification of fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos.

Goossens K, Van Soom A, Van Zeveren A, Favoreel H, Peelman LJ - BMC Dev. Biol. (2009)

Bottom Line: In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1.The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation.Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. Karen.Goossens@UGent.be

ABSTRACT

Background: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.

Results: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).

Conclusion: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

Show MeSH

Related in: MedlinePlus

Results of the RT-PCR on oocytes, embryos, cumulus cells and tissue samples. The RT-PCR reactions were performed with primer pairs 1–3, surrounding the alternatively spliced regions and yielded PCR fragments of different lengths depending on the presence or absence of the alternatively splice region, and were compared to the expression of the control gene GAPD. MM: molecular marker, Oo: oocyte, 2C: in vitro 2-cell embryo, 8C: in vitro 8-cell embryo, M: in vitro morula, Bv: in vivo blastocyst, Bt: in vitro blastocyst, HB: in vitro hatched blastocyst, Cum: cumulus cells, K: kidney, Li: liver, Lu: Lung, Ud: udder, P: placenta, Sp: spleen, M: muscle, H: heart, Ut: uterus, Sk: skin, Ov: ovary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2648952&req=5

Figure 2: Results of the RT-PCR on oocytes, embryos, cumulus cells and tissue samples. The RT-PCR reactions were performed with primer pairs 1–3, surrounding the alternatively spliced regions and yielded PCR fragments of different lengths depending on the presence or absence of the alternatively splice region, and were compared to the expression of the control gene GAPD. MM: molecular marker, Oo: oocyte, 2C: in vitro 2-cell embryo, 8C: in vitro 8-cell embryo, M: in vitro morula, Bv: in vivo blastocyst, Bt: in vitro blastocyst, HB: in vitro hatched blastocyst, Cum: cumulus cells, K: kidney, Li: liver, Lu: Lung, Ud: udder, P: placenta, Sp: spleen, M: muscle, H: heart, Ut: uterus, Sk: skin, Ov: ovary.

Mentions: In a first step, conventional RT-PCR with primers surrounding the alternatively spliced regions (Figure 1 – Table 1; Primers 1–3) were used to get an indication of the presence or absence of each splice isoform in bovine oocytes, embryos, cumulus cells and adult tissues. The results (Figure 2) demonstrated that the EIIIB region was nearly absent in bovine oocytes and embryos whereas the EIIIA region and the full-length IIICS region were highly present. Cumulus cells on the other hand, had a different expression pattern with lower expression of the EIIIB region and near absence of the EIIIA and IIICS regions. In adult tissue samples, the EIIIA and EIIIB regions were mostly absent and the presence of multiple IIICS fragments, suggest the expression of more than one transcript isoform. The two main IIICS isoforms were the IIICS and the IIICS full-length isoforms while the intermediate IIICS forms were hardly expressed in the analysed samples.


Quantification of fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos.

Goossens K, Van Soom A, Van Zeveren A, Favoreel H, Peelman LJ - BMC Dev. Biol. (2009)

Results of the RT-PCR on oocytes, embryos, cumulus cells and tissue samples. The RT-PCR reactions were performed with primer pairs 1–3, surrounding the alternatively spliced regions and yielded PCR fragments of different lengths depending on the presence or absence of the alternatively splice region, and were compared to the expression of the control gene GAPD. MM: molecular marker, Oo: oocyte, 2C: in vitro 2-cell embryo, 8C: in vitro 8-cell embryo, M: in vitro morula, Bv: in vivo blastocyst, Bt: in vitro blastocyst, HB: in vitro hatched blastocyst, Cum: cumulus cells, K: kidney, Li: liver, Lu: Lung, Ud: udder, P: placenta, Sp: spleen, M: muscle, H: heart, Ut: uterus, Sk: skin, Ov: ovary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2648952&req=5

Figure 2: Results of the RT-PCR on oocytes, embryos, cumulus cells and tissue samples. The RT-PCR reactions were performed with primer pairs 1–3, surrounding the alternatively spliced regions and yielded PCR fragments of different lengths depending on the presence or absence of the alternatively splice region, and were compared to the expression of the control gene GAPD. MM: molecular marker, Oo: oocyte, 2C: in vitro 2-cell embryo, 8C: in vitro 8-cell embryo, M: in vitro morula, Bv: in vivo blastocyst, Bt: in vitro blastocyst, HB: in vitro hatched blastocyst, Cum: cumulus cells, K: kidney, Li: liver, Lu: Lung, Ud: udder, P: placenta, Sp: spleen, M: muscle, H: heart, Ut: uterus, Sk: skin, Ov: ovary.
Mentions: In a first step, conventional RT-PCR with primers surrounding the alternatively spliced regions (Figure 1 – Table 1; Primers 1–3) were used to get an indication of the presence or absence of each splice isoform in bovine oocytes, embryos, cumulus cells and adult tissues. The results (Figure 2) demonstrated that the EIIIB region was nearly absent in bovine oocytes and embryos whereas the EIIIA region and the full-length IIICS region were highly present. Cumulus cells on the other hand, had a different expression pattern with lower expression of the EIIIB region and near absence of the EIIIA and IIICS regions. In adult tissue samples, the EIIIA and EIIIB regions were mostly absent and the presence of multiple IIICS fragments, suggest the expression of more than one transcript isoform. The two main IIICS isoforms were the IIICS and the IIICS full-length isoforms while the intermediate IIICS forms were hardly expressed in the analysed samples.

Bottom Line: In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1.The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation.Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. Karen.Goossens@UGent.be

ABSTRACT

Background: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.

Results: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).

Conclusion: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

Show MeSH
Related in: MedlinePlus