OVINE PLACENTAL LACTOGEN: ITS REGULATION AND FUNCTION

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Long-Term Objectives: Fetal growth rate not only determine the size of the offspring at birth, but also influences the ease of delivery and postnatal growth rate of the offspring. Unfortunately, our understanding of what factors influence prenatal development and the mechanism of action of these factors is limited at best. Existing evidence suggests that ovine placental lactogen (oPL) plays a significant role in modulating fetal metabolism. The long-term objectives are aimed at (1) understanding the biological functions, mechanisms of action and biochemical properties of proteins secreted by the placenta and (2) understanding the regulatory mechanisms under which placental products are made. This information could lead to methods to (1) reduce pregnancy failure and (2) modulate fetal growth rate. Specific Aims and Methods: (1) Purify to homogeneity and obtain amino acid sequence information on the OPL receptor. The OPL receptor will be purified from fetal liver collected at 120 days of gestation by a combination of anion-exchange, affinity, size-exclusion and reverse- phase chromatography. The purified receptor will be subjected to limited NH2-terminal vapor-phase amino acid sequencing. (2) Generate, isolate and sequence full-length CDNA'S to OPL receptor MRNA. Consensus sequences between the ovine growth hormone (OGH) receptor and bovine prolactin (bPRL) receptor CDNA'S will be used as primers for reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of fetal liver MRNA to generate OPL receptor specific CDNA probes to screen a fetal liver CDNA library. Alternatively, the GH and PRL receptor CDNA'S or oligonucleotide probes generated from the amino acid sequencing of the OPL receptor will be used to screen the CDNA library. Once a CDNA encoding the OPL receptor is isolated, it will be subjected to nucleotide sequencing by the dideoxy-chain terminating procedure. The primary amino acid sequence of the OPL receptor will be inferred, and then compared for sequence similarity with other protein hormone receptors. (3) Isolate and structurally characterize the gene(s) for OPL. Genomic clones for OPL will be mapped and the exons with adjacent flanking regions will be sequenced to define the MRNA splice sites. The transcriptional start site and gene copy number will be determined, and the 5'-upstream regulatory region (1 to 2 kb) will be sequenced to examine potential cis-acting regulatory elements. Future emphasis will be placed on examining the tissue-specific expression of the OPL gene, as well as examining the mechanism of action of OPL.