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The correlation of growth hormone (GH) mRNA abundance and expression of specific transcription factors was studied in pituitaries of panhypopituitary (Ames df/df and Snell dwJ/dwJ dwarf), isolated GH-deficient (lit/lit), and GH-overproducing (growth hormone-releasing hormone [GHRH] transgenic) mice compared with normal littermates. A fluorescence-based reverse transcriptase polymerase chain reaction assay was developed for seven target mRNAs: GH, prolactin (PRL), pro-opiomelanocortin (POMC), alpha-subunit of the glycoprotein hormones (alphaSU), Pit-1, Prop-1, and Zn-16. Amplification parameters for each of these primer pairs were determined in order to calculate initial mRNA transcript number. The reproducibility of the assay was found to be +/-10% for either Pit-1 or Zn-16 mRNAs measured in characterized murine GHFT1-5 somatotroph precursor cells. The cell extracts also showed an increased abundance of both Zn-16 and Pit-1 mRNAs when compared with whole pituitary extracts. Measurement of copy number in normal pituitaries showed that for every 10(6) GH or PRL mRNAs, there were 3 x 10(5) POMC, 4 x 10(4) alphaSU, 2 x 10(3) Pit-1, and only 70 Zn-16 or Prop-1 transcripts. Transcript abundance in GH-altered mice as a percentage of copy number per normal gland showed that POMC was significantly reduced in dwJ/dwJ (p0.01) and df/df (p0.05) mice. AlphaSU mRNA was reduced in df/df (p0.05), dwJ/dwJ (p0.05), and lit/lit (p0.05) mice, but not in GHRH-excess mice. PRL mRNA was not detected in dwarf mice, reduced to 52% of normal in lit/lit (p0.05), and unchanged in GHRH-excess animals. GH mRNA was not detected in dwarf mice, reduced to 1.3% in lit/lit (p0.005), and increased to 242% in GHRH-excess mice (p0.05). Pit-1 mRNA was not detected in dwarf mice, was 2.9% of normal in lit/lit (p0.005) mice, and increased to 200% in GHRH-excess mice (p0.05). Prop-1 was not present in dwarf mice, was decreased to 1.4% in lit/lit (p0.01), and increased to 223% in GHRH-excess mice (p0.05). Zn-16 abundance in df/df mice was significantly reduced (p0.05) to 4.8% of normals, to 6.3% of normals in dwJ/dwJ (p0.005), to 6.1% of normals in lit/lit (p0.005) mice, and significantly elevated in GHRH-excess mice to 197% (p0.05). Altered pituitary mRNA abundance was found for several products not previously measured, or thought not to be affected by these mutations. Correlation of GH mRNA abundance with transcription factor copy number showed a significant correlation for Pit-1, Prop-1, and Zn-16. These quantitative analyses provide the first in vivo evidence that Zn-16 mRNA abundance correlates with GH expression.

Rat Zn-15 is a transcription factor activating GH gene expression by synergistic interactions with Pit-1, named for 15 DNA-binding zinc fingers, including fingers IX, X, and XI that are responsible for GH promoter binding. In this study, a mouse cDNA for Zn-15 was characterized. The predicted 2192-amino acid mouse protein is 89% identical to rat (r) Zn-15 overall, and is 97% similar in the C-terminal domain necessary for binding the GH promoter. However, the mouse cDNA encodes 16 zinc fingers, and sequences of rZn-15 pituitary cDNAs were the same as the mouse (m) Zn-16; the rat sequence in GenBank has a one nucleotide offset of a 17-bp segment in the finger V region. The mouse and corrected rat sequences contain four tandemly repeated fingers in the N-terminus, each separated by seven amino acids, typical of zinc finger proteins of the transcription factor IIIA-type. Analysis of mZn-16 expression by RT-PCR showed that the mRNA is produced at similar levels in normal and GH-deficient Ames dwarf (Prop-1 ) mouse pituitaries at postnatal day 1. Mouse Zn-16 mRNA also was detected by ribonuclease protection assay in the pre-somatotrophic mouse cell line GHFT1-5. The Zn-16 protein is bipartite in that the N-terminal half displays tandem spacing typical of most zinc finger proteins, while the C-terminal portion contains long linkers between fingers that cooperatively bind to a DNA response element. Expression in early postnatal pituitary and in pre-somatotrophic cells suggests that Zn-16 could play a role in pituitary development prior to somatotroph differentiation.

Publisher Summary This chapter provides an overview of the study to investigate the use of RT-PCR to detect mRNAs relevant to elucidating the patterns of gene expression in the developing Ames dwarf pituitary. The use of RT-PCR for GH mRNA provides a rapid and reliable means of determining dwarfism at ages before body-size differences become apparent. The chapter presents a method that provides classification of dwarfs vs normals that is 100% correct for animals that could be identified by size. GH mRNA is not present in the Ames dwarf pituitary within three days of the initiation of transcription in the normal mouse pituitary. The Ames mutation appears to be involved in the blockage of GH transcription, implicating the involvement of Pit-1 in the effects of the mutation. The assays of Pit-1 confirmed the absence of detectable mRNA in dwarfs at 30 days of age or older. A study discussed in the chapter shows that Pit-1 is not detectable in dwarfs even at seven days of age.