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1.
Reprod Biol Endocrinol ; 9: 18, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21281523

ABSTRACT

BACKGROUND: BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. METHODS: For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. RESULTS: We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. CONCLUSIONS: Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover, our findings demonstrate, for the first time, that a correct balance of BMP signaling is needed for proper pre-implantation development of bovine embryos.


Subject(s)
Bone Morphogenetic Protein 4/physiology , Carrier Proteins/physiology , Animals , Autoantigens/biosynthesis , Blastocyst/drug effects , Blastocyst/metabolism , Bone Morphogenetic Protein 4/antagonists & inhibitors , Cattle , Embryonic Development/drug effects , Fertilization in Vitro , HSP70 Heat-Shock Proteins/biosynthesis , Parthenogenesis/physiology
2.
Mol Endocrinol ; 23(7): 1102-14, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19342445

ABSTRACT

The regulatory role of estrogen, bone morphogenetic protein-4 (BMP-4), and TGF-beta has a strong impact on hormone secretion, gene transcription, and cellular growth of prolactin (PRL)-producing cells. In contrast to TGF-beta, BMP-4 induces the secretion of PRL in GH3 cells. Therefore, we studied the mechanism of their transcriptional regulation. Both BMP-4 and TGF-beta inhibited the transcriptional activity of the estrogen receptor (ER). Estrogens had no effect on TGF-beta-specific Smad protein transcriptional activity but presented a stimulatory action on the transcriptional activity of the BMP-4-specific Smads. BMP-4/estrogen cross talk was observed both on PRL hormone secretion and on the PRL promoter. This cross talk was abolished by the expression of a dominant-negative form for Smad-1 and treatment with ICI 182780 but not by point mutagenesis of the estrogen response element site within the promoter, suggesting that Smad/ER interaction might be dependent on the ER and a Smad binding element. By serial deletions of the PRL promoter, we observed that indeed a region responsive to BMP-4 is located between -2000 and -1500 bp upstream of the transcriptional start site. Chromatin immunoprecipitation confirmed Smad-4 binding to this region, and by specific mutation and gel shift assay, a Smad binding element responsible site was characterized. These results demonstrate that the different transcriptional factors involved in the Smad/ER complexes regulate their transcriptional activity in differential ways and may account for the different regulatory roles of BMP-4, TGF-beta, and estrogens in PRL-producing cells.


Subject(s)
Bone Morphogenetic Protein 4/metabolism , Estrogens/pharmacology , Lactotrophs/metabolism , Prolactin/genetics , Promoter Regions, Genetic , Transforming Growth Factor beta/metabolism , Animals , Binding Sites , Bone Morphogenetic Protein 4/physiology , Cells, Cultured , Estrogens/metabolism , Lactotrophs/drug effects , Prolactin/metabolism , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/physiology , Protein Binding/physiology , Rats , Receptor Cross-Talk/drug effects , Receptor Cross-Talk/physiology , Receptors, Estrogen/metabolism , Receptors, Estrogen/physiology , Smad Proteins/metabolism , Smad Proteins/physiology , Transcriptional Activation/physiology , Transforming Growth Factor beta/physiology
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