T.-P. Yu

Neonatal Meishan pigs show POU1F1 genotype effects on plasma GH and PRL concentration

Chinese Meishan pigs develop rapidly with onset of puberty at less than 100 days of age, and have a smaller placental size and larger litter size as compared with British/Continental breeds. POU1F1 is a member of the POU-domain family gene and is a positive regulator for growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone beta (TSHbeta) in several mammalian species. To investigate the role of POU1F1 in controlling pig growth and reproduction traits, Meishan (MS) pigs segregating a MspI POU1F1 polymorphism were used to determine differences of GH and PRL at both mRNA and circulating hormone concentrations. Animals from nine litters were used to collect pituitary (n=60) and/or blood samples (n=80) at day 0, 15, and 30 after birth, and all animals were genotyped (CC, CD, DD) for the MspI POU1F1 polymorphism. Reverse transcriptase-polymerase chain reaction (RT-PCR) with standard curve quantification was used to quantify mRNA levels for GH, PRL, and two alternative POU1F1 transcripts, POU1F1-alpha, and POU1F1-beta. Radioimmunoassays were done to determine the circulating concentration of GH and PRL in blood plasma. Our results indicated a significant effect of POU1F1 genotype on circulating levels of both GH and PRL at birth, but not thereafter. The DD neonates had lower levels of GH, but higher levels of PRL, than other genotypes. POU1F1-alpha mRNA decreased (P<0.05) from days 0 to 30, which paralleled decreases (P<0.05) in GH mRNA as well as PRL and GH plasma levels over the same period. POU1F1-beta mRNA levels did not significantly change over this period. Correlations were significant between POU1F1-alpha mRNA and both GH mRNA and GH plasma concentration levels, as well as between the two POU1F1 mRNA isoforms. Results from this study add to our understanding of the role of POU1F1 in controlling pig development and reproduction.

CLONING OF THE FULL LENGTH PIG PIT1 (POU1F1) CDNA AND A NOVEL ALTERNATIVE PIT1 TRANSCRIPT, AND FUNCTIONAL STUDIES OF THEIR ENCODED PROTEINS

PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin β subunit (TSHβ). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3′ end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the Δ3PIT1 (missing entire exon 3), Δ4PIT1 (missing entire exon 4) and PIT1β (additional 26 amino acids inserted in front of exon 2) transcripts. The Δ4PIT1 and PIT1β transcripts have been found to encode functionally different proteins in rodents. The Δ3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig Δ3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and Δ3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig Δ3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between Δ3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that Δ3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel Δ3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.