PIT1-HinfI gene polymorphism and its associations with milk production traits in polish Black-and-White cattle

Associations between polymorphism localised in the six exon of PIT1 gene (PIT1-HinfI) and milk production traits of Black-and-White cattle were analysed. A total of 900 cows were included in the study. PCR-RFLP method was used. The frequencies of the genotypes and alleles were as follows: 0.054 for AA, 0.377 for AB and 0.569 for BB, and 0.243 for PIT1 and 0.757 – PIT1. There were no associations between PIT1-HinfI polymorphism and milk production traits of the cows.


Introduction
Pit-1 (official nomenclature -POU1F1) is a member of the POU-family of transcription factors that regulate mammalian development.Pit-1, an approx.33kilodalton pituitary-specific protein, contains two domains, termed POU-specific and POU-homeo, which are both necessary for high-affinity DNA binding to promoters of 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 somatotrophic and lactotrophic cells, respectively, and is the major cell-specific activator of hormone expression from these cell types (SUPOWIT et al., 1992).The inhibition of Pit-1 synthesis markedly decreased both GH and PRL expression and proliferation of somatotropic and lactotropic cell lines (CASTRILLO et al., 1991).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.The PIT1 gene is controlled by several factors that interact 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 silent mutation (G→A) located within exon 6 of the PIT1 gene (DIERKES et al., 1998).RENAVILLE et al. (1997) showed that the 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.ZWIERZCHOWSKI et al. (2002) showed that the allele A of the Pit1 locus positively affected milk production traits.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 marker assisted selection (MAS).ZWIERZCHOWSKI et al. (2001) andDYBUS et al. (2003) found 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 PIT1-HinfI locus of the bovine PIT1 gene and to investigate the relationship of this polymorphism and milk production traits Black-and-White cows.
The data for 305-day milk production in the first, second and third lactation, including production of milk, milk fat and milk protein and proportions of milk fat, milk protein and sum of milk fat and protein, were obtained from the farm documentation.Statistical calculations were performed using procedures of SAS ® .Differences in the frequencies of PIT1 genotypes (AA, AB and BB) in analysed herds of cows were tested with the chi-square test of independence.The effect of PIT1 genotypes on the milk production traits of the cows were analysed using General Linear Model (GLM) procedure.The model used was as follows:  The BB genotype was the most frequent in all the studied herds (0.531-0.603), followed by the heterozygotic AB (0.328-0.421), whereas the AA was the least frequent (0.043-0.069).The frequency of the PIT1 A ranged from 0.233 to 0.261 (Table 2).The genetic equilibrium in the studied population was not disturbed.The size of certain genotypes of the PIT1 was not statistically different from the theoretical one.
No differences in genotype and allele frequencies were found between herds (AAχ 2 4;0,05 = 1,07, ABχ 2 4;0,05 = 2,11, BBχ 2 4;0,05 = 0,89).Table 4 shows the influence of the PIT1-HinfI polymorphism on milk production traits in the B&W cows.Discussion Frequencies of PIT1-HinfI alleles obtained in this study were similar to the frequencies obtained earlier for Black-and-White cattle.Higher frequency of the PIT1 A (0.32) was observed in study carried out by DIERKES et al. (1998).Somewhat higher frequency of the PIT1 A (0.26) was observed in the studies of MOODY et al. (1995) andKLAUZIŃSKA et al. (1999); 0.25 in the studies carried out by ZWIERZCHOWSKI et al. (2002) andOPRZĄDEK et al. (2003).Slightly lower frequency of the PIT1 A (0.18, 0.15 and 0.15) was observed by RENAVILLE et al. (1997), WOOLLARD et al. (1994) and HORI-OSHIMA and BARRERAS-SERRANO (2002), respectively.Pit-1 transcription factor is a component of the GH cascade, also called "somatotropic axis".It has been described as the critical cell-specific transcription factor responsible for activating expression of the prolactin (PRL) and growth hormone (GH) genes in the anterior pituitary gland.Because the PRL and the GH are essential for mammary gland development and milk yield, the Pit-1 gene has a potential to explain genetic variations in dairy traits (ZWIERZCHOWSKI et al., 2002).RENAVILLE et al. (1997) showed that the allele A of the PIT1 gene was found to be superior for milk and protein yields and inferior for fat percentage.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.These authors also showed that RFLP in PIT1 gene is a promising new possibility to select for increased protein yield and, to a lesser extent, milk yield through selection for the allele A. PARMENTIER et al. (1999) demonstrated significant superiority of the allele A for milk and protein yield, but an inferiority for fat yield.HORI-OSHIMA and BARRERAS-SERRANO (2002) found that the animals with the AA genotype for Pit-1/HinfI polymorphism had higher milk yield.Similar results were published by ZWIERZCHOWSKI et al. (2003) who showed that both genotypes with allele A at the PIT1 locus positively affected all milk production traits studied.The AB genotype was superior for the milk yield and for daily yield of all milk components, while genotype AA was shown to positively affect their concentrations.Recently, DE MATTOS et al. (2004) found that the heterozygous HinfI (AB) sires were superior in relation to the HinfI BB sires for milk fat production (P<0.05).In our study, no associations between RFLP in PIT1 gene and milk production traits were found for B&W cattle.Bearing in mind the above mentioned results, it is difficult to indicate which allele of PIT1-HinfI polymorphism should be favoured in the improvement of production traits of Black-and-White cattle.

Table 1
Characteristics of the investigated population of dairy cows (Charakteristik der untersuchten Milchkuhpopulationen)

Table 4
Mean and standard deviation of milk production traits in cows carrying different PIT1-HinfI genotypes.(Mittelwerte und Standardabweichungen der Milchleistungsmerkmale bei Kühen mit verschiedenen Genotypen