Associations between polymorphisms of growth hormone releasing hormone (GHRH) and pituitary transcription factor 1 (PIT1) genes and production traits of Limousine cattle

Associations between polymorphism of the bovine growth hormone releasing hormone (GHRH) and pituitary transcription factor 1 (PIT1) genes and production traits of Limousine cattle were analysed. A total of 130 calves were included in the study. PCR-RFLP method was used for genotyping. The frequencies of genotypes and alleles of PIT1 and GHRH were as follows: 0.0692 – AA, 0.4077 – AB, 0.5231 – BB, and 0.2731 for PIT1, 0.7269 for PIT1; 0.0154 – AA, 0.1692 – AB, 0.8154 – BB, and 0.1 for GHRH, 0.9 for GHRH. Associations between polymorphism and production traits of Limousine calves were found. Statistically significant differences (P ≤ 0.01) between individuals of different GHRH genotypes were found in relation to height at sacrum (cm) and height at withers (cm) at 210 day of age. The calves with AA genotype of GHRH were shorter (-8,14 and -8,33 cm) than AB and BB individuals (P ≤ 0.01). The small number of calves with the AA genotype did not enable important conclusions.


Introduction
Growth hormone (GH)-releasing factor (GRF, GHRH) is the hypothalamic peptide that specifically stimulates both synthesis and secretion of pituitary GH.After reaching the pituitary, GRF binds to its specific receptors on somatotrophs, and generates bursts of GH secretion episodically (FROHMAN et al., 1992).GUILLEMIN et al. (1982) have isolated a 44-amino acid peptide with GH-releasing activity from a human pancreatic tumor that had caused acromegaly.After the isolation of hGRF, the GRFs from other species were described.The sequence of bovine GRF (1-44-NH 2 ) differs from human GRF by only five residues (ESCH et al., 1983).MAYO et al. (1985) isolated and characterised the entire structure of the human gene encoding GHRH.The gene consists of five exons separated by interval introns and spanning 10 kb.In cattle, MOODY et al. (1995) identified a restriction fragment length polymorphism (RFLP) within PCR amplification product of the bovine GHRH gene.The bovine GHRH gene was sequenced and found to be 91 and 77% homologous to portions of exon 3 of the human and murine GHRH cDNA sequences, respectively.Linkage analysis determined that GHRH was linked to CSSM30 on bovine chromosome 13 (BARENDSE et al., 1994).Pit-1 (official nomenclature -POU1F1) is a member of the family (POU) transcription factors that regulate mammalian development.Pit-1, an approx.33-kilodalton pituitary-specific protein, contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for high-affinity DNA binding on the GH and PRL genes (HERR et al., 1988;ROSENFELD, 1991).Pit-1 activates GH and PRL gene expression, in part, through an N-terminal transactivation domain rich in hydroxylated amino acid residues (THEILL et al., 1989).During development, PIT1 gene expression precedes GH and PRL gene expression in the somatotroph and lactotroph, respectively, and is the major cell-specific activator of hormone expression from these cell types (NELSON et al., 1988;FOX et al., 1990).SCULLY et al. (2000) showed that whereas Pit-1 activates GH gene expression in one cell type, the somatotrope, it restricts its expression from another cell type, the lactotrope.OHTA et al. (1992) found that the human PIT1 gene spanned more than 14 kb and was divided into six exons ranging from 61 bp (exon 5) to 225 bp (exon 3); the five introns ranged in size from 0.7 kb (intron 4) to more than 7.5 kb (intron 2).The PIT1 gene is controlled by several factors that interacts with its 5' regulatory region, although autoregulation of the PIT1 gene itself also occurs as there are two Pit-1 binding sites in the 5' flanking region (CASTRILLO et al., 1991).RHODES et al. (1993) explored the molecular mechanism responsible for activation of the PIT1 gene in vivo.They demonstrated that an enhancer element, located more than 10 kb upstream of the transcriptional start site, was essential for pituitary-specific expression of the PIT1 gene in transgenic mice.RAJAS et al. (1998) characterized 12 kb of genomic DNA upstream of the PIT1 promoter.They identified a distal region that decreased the basal transcriptional activity of the PIT1 minimal promoter, indicating that this region behaves as a silencer.This distal regulatory region contains 3 Pit-1 autoregulatory elements.Bovine PIT1 cDNA has been sequenced by BODNER et al. (1988).PIT1 was sublocalized to the centromeric region of bovine chromosome 1, located midway between TGLA57 and RM95.In the bovine PIT1 gene the restriction fragment length polymorphism (for the HinfI restriction enzyme) was identified (MOODY et al., 1995).Molecular basis of this polymorphism was the point mutation (G→A) located within exon 6 of the PIT1 gene (DIERKES et al., 1998).Additionally, ZHAO et al. (2000) detected an SSCP polymorphism in intron 5 of this gene.RENAVILLE et al. (1997) showed that A allele (for the PIT1-HinfI polymorphism) was found to be superior for milk and protein yields and inferior for fat percentage in dairy cattle.In beef cattle, ZHAO et al. (2000) reported that PIT1-HinfI polymorphism appears to affect growth traits in Angus cattle and may be a candidate gene for use in MAS.Recently, ZWIERZCHOWSKI et al. (2001) showed no associations between PIT1-HinfI and growth performance and carcass traits of beef cattle.The aim of this study was to estimate the allelic frequencies at the bovine GHRH-HaeIII and PIT1-HinfI loci and to investigate the relationship of those polymorphisms and production traits of Limousine calves.

Materials and Methods
A total of 130 limousine calves were genotyped.The calves were born between 1998-2001, and were offspring of 4 bulls and 80 cows.Crude DNA was isolated from blood samples using MasterPure TM kit (Epicentre Technologies).The PCR-RFLP method was used for the polymorphism located in the GHRH gene.The following primer sequences were designed on the basis of the nucleotide sequence of the GHRH gene (GenBank U29611) and Primer3 software (http://www-genome.wi.mit.edu/cgibin/primer/primer3.cgi/)namely: GHRHF -5'-TTCCCAAGCCTCTCAGGTAA-3' GHRHR -5'-GCGTACCGTGGAATCCTAGT-3' A 297-base pair (bp) fragment of the GHRH gene was amplified.The PCR reaction contained 100 ng of genomic DNA, 15 pmol of each primer, 2 µl 10 x PCR buffer (MBI Fermentas), 2.0 mM MgCl 2 , 200 µM dNTP and 0.5 units Taq-polymerase in a total volume of 20 µl.The following cycles were applied: denaturation -94 °C/5 min, followed by 30 cycles -94 °C/40 sec, primer anneling -60 °C/40 sec, PCR products synthesis -72 °C/40 sec, and final synthesis -72 °C/4 min using a DNA thermal cycler (Perkin Elmer Cetus Corp.).Amplified DNA was digested by mixing 15 µl of PCR product with 5 units of HaeIII (GG↓CC) enzyme (MBI Fermentas).The digestion products were separated by horizontal electrophoresis (90 volts, 50 minutes) through 3% agarose gels (Gibco BRL) in 1 x TBE and 1.0 µM ethidium bromide.A 451-base pair (bp) fragment of the PIT1 gene was amplified using forward 5'-AAACCATCATCTCCCTTCTT-3' and reverse 5'-AATGTACAATGTGCCTTCT GAG-3' primers (WOOLLARD et al., 1994).The PCR reaction contained 100 ng of genomic DNA, 15 pmol of each primer, 2 µl 10 x PCR buffer (MBI Fermentas), 1.5 mM MgCl 2 , 200 µM dNTP and 0.5 units Taq-polymerase in a total volume of 20 µl.The following cycles were applied: denaturation at 94.5°C/5 min, followed by 30 cycles at 94°C/40 sec, primer anneling at 56 °C/40 sec, PCR products synthesis at 72 °C/40 sec, and final synthesis at 72 °C/4 min using a DNA thermal cycler (Perkin Elmer Cetus Corp.).Amplified DNA was digested with HinfI (G↓ANTC) enzyme (MBI Fermentas).The digestion products were separated by horizontal electrophoresis (90 volts, 50 minutes) through 2% agarose gels (Gibco BRL) in 1 x TBE and 1.0 µM ethidium bromide.Data for production traits of calves, including body weight in 3, 210 and 365 day of life, height at sacrum, height at withers, chest girth in 3, 210 and 365 day of life and average daily gain for 3-210 and 3-365 day of life were obtained from the farm documentation.Statistical calculations were performed using procedures of SAS ® .Distribution frequencies of the two alleles were compared by Chi-square test.The effect of studied genotypes on the production traits of calves were analysed using GLM procedure.The used model was as follows: where: Y ijklm -analysed trait; µ -the overall mean, G i -the fixed effect of GHRH and PIT1 genotypes (i = 1,...3), S j -the fixed effect of sire, YS k -the fixed effect of year-season, P leffect of the sex, E ijklm -the random error.

Results
The following DNA restriction fragments were obtained for the GHRH-HaeIII polymorphism: 242 and 55 bp for the AA genotype, 242, 194, 55, 48 bp for the AB genotype, and 194, 55, 48 bp for the BB (Fig. 1).Table 1 shows frequencies of genotypes and alleles of the GHRH-HaeIII and PIT1-HinfI obtained in this study.The analysis of relationships between the polymorphism at the GHRH and PIT1 genes and production traits of Limousine calves found that the level of the analysed traits was significantly influenced by the sire, year/season, sex and genotype (P < 0.01 and P < 0.05) -Table 2. All those factors were included in the statistical model which was used to analyse the relationships between the GHRH and PIT1 genes polymorphism and production traits.* -significance of differences at P ≤ 0.05; ** -significance of differences at P ≤ 0.01.

M P AA AA AB AB BB BB
Table 3 shows the influence of the GHRH and PIT1 genes polymorphisms on production traits in Limousine cattle.Values in lines with the same index differ significantly; capitals -P ≤ 0.01, small letters -P ≤ 0.05.

Discussion
Frequencies of GHRH-HaeIII alleles obtained in this study were 0.1 (GHRH A ) and 0.9 (GHRH B ). Higher frequency of the GHRH A (0.70) in Angus breed, and lower (0.07) in Hereford was observed by MOODY et al. (1995).In case of PIT1-HinfI polymorphism frequency of the PIT1 A allele obtained in this study (0.27) were higher than observed in study carried out by ZWIERZCHOWSKI et al. (2001) for Limousine cattle -0.22.In the other cattle breeds, the following frequencies of PIT1 A allele were observed by ZWIERZCHOWSKI et al. (2001): 0.22 -Charolaise, 0.25 -Simmental, 0.27 -Hereford and 0.35 -Red Angus;MOODY et al., (1995): 0.45 -Angus, 0.26 -Holstein, 0.21 -Hereford, 0.18 -Gelbvieh, 0.10 -Brahman;KLAUZIŃSKA et al. (1999): 0.26 -Polish Black&White;RENAVILLE et al. (1997): 0.18 -Holstein. RENAVILLE et al. (1997) showed that the A allele was found to be superior for milk and protein yields, inferior for fat percentage, and superior for body depth, angularity, and rear leg set, which is difficult to explain.A canonical transformation revealed that Pit-1 had three actions, one linked to milk yield traits and angularity, a second linked to body depth and rear leg set, and a third linked to lower fat yields and to higher angularity.In beef cattle, ZHAO et al. (2000) reported that PIT1-HinfI polymorphism appears to affect growth traits in Angus cattle and may be a candidate gene for use in MAS.Recently, ZWIERZCHOWSKI et al. (2001) showed no associations between PIT1-HinfI and growth performance and carcass traits of beef cattle.In humans, different mutations of the PIT1 gene have been reported in patients with familial pituitary hypoplasia (PFÄFFLE et al., 1992) or with sporadic combined pituitary hormone deficiency (RADOVICK et al., 1992).Mutations in the Pit1 gene are responsible for the dwarf phenotypes in mice (LI et al., 1990).In our study, no associations between RFLP in PIT1 gene and production traits of Limousine calves were found.A small number of animals with the AA genotype (n=8) makes it impossible to draw important conclusions.ZIMMERMAN et al. (1993) described congenital gigantism due probably to central hypersecretion of GRF (GHRH).Normal at birth (4.4 kg, 53 cm), the male patient was 182 cm tall with a weight of 99.4 kg at the age of 7 years.In this study, statistically significant differences (P ≤ 0.01) between individuals of different GHRH genotypes were found only in reference to height at sacrum (cm) and height at withers (cm) at 210 th day of age.The calves with AA genotype of GHRH were shorter (-8,14 and -8,33 cm) than AB and BB individuals (P ≤ 0.01).The small number of calves (n=2) with the AA genotype did not allow drawing conclusions.Bearing in mind the results obtained in this study, it should be stressed that the usefulness of PIT1-HinfI and GHRH-HaeIII polymorphisms for the improvement of production traits of Limousine cattle seems questionable.The obtained results, however, should be verified by further investigations on a greater number of animals.

Table 1
Frequency of genotypes and alleles of the GHRH and PIT1 genes in Limousine cattle (Frequenz von Genotypen und Allelen von GHRH und PIT1 beim Limousine-Rind)