Components of the insulin-like growth factor system in in vivo - and in vitro-derived fetuses of cattle, and the association with growth and development.

This experiment was designed to study mechanisms affecting growth of in vivo-derived (IVD) and in vitro-produced (IVP) fetuses of cattle. Day-7 IVD or IVP cattle blastocysts were transferred to recipients, with pregnant females being slaughtered on Days 90 or 180 of gestation or allowed to undergo parturition. Uteri and contents were dissected and physically measured, and maternal and fetal plasma and amniotic and allantoic fluids were collected for IGF-1 and IGF-2 determinations, and IGFBP profile characterization. Transcripts for IGF-1 and IGF-2 mRNA in placental and fetal tissues, and IGF-1r and IGF-2r in placentomes were determined. There was a greater fetal weight in the IVP group, which was associated with greater IGF-1 and IGF-2 concentrations in maternal circulation, and changes in IGFBP profiles within fetal fluids. Day-90 IVP-derived fetuses were longer, had greater organ weights, larger placentomes, less placentome IGF-2r mRNA transcript, and greater maternal IGF-1 and IGF-2 concentrations than controls. On Day 180 and at parturition tissues from IVP-derived fetuses/calves were from larger uteri, with larger placentomes/fetal membranes, fetuses/calves weighed more, had greater fetal hepatic IGF-2 mRNA transcript, had less fetal plasma IGF-1 and greater allantoic IGF-2 concentrations, greater and lesser IGFBP activities in the allantoic and amniotic fluids, respectively, and greater glucose and fructose accumulation in fetal fluids. Components of the IGF system were differentially regulated not only according to the gestation period (Days 90 or 180) and fluid type (maternal or fetal plasma, amniotic or allantoic fluids), but also based on conceptus origin (IVP or IVD) in cattle.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.