title: Gastrulation in rabbit blastocysts depends on insulin and insulin-like-growth-factor 1.
abstract: Insulin and insulin-like-growth-factor 1 (IGF1) are components of the uterine secretions. As potent growth factors they influence early embryo development. The underlying molecular mechanisms are largely unknown. Here we report on the effects of insulin and IGF1 on early gastrulation in rabbit blastocysts. We have studied blastocysts grown in vivo in metabolically healthy rabbits, in rabbits with type 1 diabetes and in vitro in the presence or absence of insulin or IGF1. Embryonic disc morphology and expression of Brachyury, Wnt3a and Wnt4 were analysed by qPCR and IHC. Pre-gastrulated blastocysts (stage 0/1) cultured with insulin or IGF1 showed a significantly higher capacity to form the posterior mesoderm and primitive streak (stage 2 and 3) than blastocysts cultured without growth factors. In gastrulating blastocysts the levels of the mesoderm-specific transcription factor Brachyury and the Wnt signalling molecules Wnt3a and Wnt4 showed a stage-specific expression pattern with Brachyury transcripts increasing from stage 0/1 to 3. Wnt4 protein was found spread over the whole embryoblast. Insulin induced Wnt3a, Wnt4 and Brachyury expression in a temporal- and stage-specific pattern. Only blastocysts cultured with insulin reached the Wnt3a, Wnt4 and Brachyury expression levels of stage 2 in vivo blastocysts, indicating that insulin is required for Wnt3a, Wnt4 and Brachyury expression during gastrulation. Insulin-induced Wnt3a and Wnt4 expression preceded Brachyury. Wnt3a-induced expression could be depleted by MEK1 inhibition (PD98059). Involvement of insulin in embryonic Wnt3a expression was further shown in vivo with Wnt3a expression being notably down regulated in stage 2 blastocysts from rabbits with type 1 diabetes. Blastocysts grown in diabetic rabbits are retarded in development, a finding which supports our current results that insulin is highly likely required for early mesoderm formation in rabbit blastocysts by inducing a distinct spatiotemporal expression profile of Wnt3a, Wnt4 and Brachyury.
authors.affiliation: Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, 06108 Halle (Saale), Germany. rene.thieme@medizin.uni-halle.de
authors.forename: René
authors.initials: R
authors.lastname: Thieme
authors.forename: Nicole
authors.initials: N
authors.lastname: Ramin
authors.forename: Sünje
authors.initials: S
authors.lastname: Fischer
authors.forename: Bernd
authors.initials: B
authors.lastname: Püschel
authors.forename: Bernd
authors.initials: B
authors.lastname: Fischer
authors.forename: Anne Navarrete
authors.initials: AN
authors.lastname: Santos
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D003921:Diabetes Mellitus, Experimental / Q000150:complications; D046149:Embryo Culture Techniques; D005260:Female; D047109:Fetal Development; D005317:Fetal Growth Retardation / Q000209:etiology; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism; D054262:Gastrulation; D015870:Gene Expression; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D008648:Mesoderm / Q000502:physiology; D011817:Rabbits; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism; D060528:Wnt4 Protein / Q000235:genetics / Q000378:metabolism; D000098908:Brachyury Protein
chemical_list: D005326:Fetal Proteins; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D020825:T-Box Domain Proteins; D060509:Wnt3A Protein; D060528:Wnt4 Protein; D000098908:Brachyury Protein