Association between IGF1/TasI polymorphism and milk traits of Polish Holstein Friesian cows

The study was carried out on 658 Polish Holstein Friesian cows. A transversion of A→C in the P1 promoter region of bovine IGF1 gene at position 977 bp upstream from the start codon in exon 1 was identified using ACRS-PCR method. Reverse primer introduced an artificial TasI restriction site. Three genotypes (AA, AC, CC) were found in the analysed herd of dairy cows occurred with a frequency of 0.766, 0.213 and 0.021, respectively. In the presented study, the statistically significant differences between individuals of different IGF1/TasI genotypes were found in milk, fat and protein yield. In the 2nd and 3rd lactations, the cows carrying the CC genotype produced more milk than the AA individuals (P≤0.05). In the case of protein and fat yield, similar tendencies were observed.


Introduction
Most biological traits, such as milk, growth or reproductive traits, behavior and disease resistance, have a complex inheritance, which means that they are controlled by multiple genes and influenced by environmental factors.DNA polymorphisms are potentially useful as markers of quantitative trait loci (QTL).Studies have investigated polymorphisms within functional genes affecting milk production traits (milk yield, protein yield, fat yield, protein content and fat content) to determine whether the polymorphisms could be useful markers of QTL (Freyer et al. 2003).The somatotropic axis genes were often selected in these studies because of their biological significance on the quantitative traits of interest.
The growth hormone (GH) is synthesized in the pituitary gland and released to circulation (Dybus & Grzesiak 2006).GH has a mainly direct effect on the liver where is triggered a signal to synthesis and release of insulin like growth factor I (IGF-I) (Biereder et al. 1999, Kovacs et al. 2006).Although the increased expression of IGF-I in liver in response to GH is well characterized, the intracellular signaling pathways that mediate this effect have not been identified.One possibility is that GH-stimulated IGF-I expression is regulated by the JAK-STAT5b pathway (Davey et al. 2001).
The actions of IGF-I are modulated by a family of six high-affinity IGF binding proteins (IGFBP 1-6).In the target tissues non-bind IGF-I is known to interact with the insulinrelated receptors (IGF-IR, IGF-IIR or IR).The IGF-I type 1 receptor seems to be the proper »physiological« receptor (Baumrucker & Erondu 2000, Nedbal et al. 2000, Sirotkin et al. 2000).
In human, many clinical and experimental evidences suggest that the insulin-like growth factor receptor type I is involved in breast cancer etiology.In vitro, IGF-I/IGF-IR regulates breast cancer proliferation and survival (Sachdev & Yee 2001).In bovine mammary cell culture models, IGF-I is a potent mitogen involved in the regulation of epithelial cell proliferation and differentiation (Baumrucker & Stemberger 1989).IGF-I is believed to mediate galactopoietic effects from exogenous bovine GH (Sharma et al. 1994).However, in the mammary gland IGF-I can act in a paracrine or endocrine manner (Plath-Gabler et al. 2001).Thus the potential role of IGF-I in bovine mammary gland development and the stimulation milk synthesis has been studied extensively at the molecular level.
Insulin-like growth factor I is a 70 amino-acid, single chain polypeptide encoded by a single gene (EMBL accession no. X15726;Fotsist et al. 1989).The IGF1 gene is highly conserved across mammalian species.Mature IGF-I is encoded only by parts of exons 3 and 4. Transcripts derived from the exons 1 and 2 are alternately spliced onto exon 3 to generate two mRNA species (class 1 and class 2) and are regulated by distinct promoters (Adamo et al. 1991, Kim et al. 1991).There is extremely high conservation of both the nucleotide (93 %) and amino acid (96 %) sequences between bovine and human IGF-lA class.Mapped on chromosome 5 in cattle (Bishop et al. 1991), the IGF1 gene showed several polymorphisms as described previously (Zych et al. 2007).
The objectives of the present study were to estimate the allele and genotype frequencies of the IGF1/TasI polymorphism and to determine associations between these polymorphisms and milk production traits.No other papers were found concerning effects of IGF1/TasI polymorphism on milk production traits.

Material and methods
Six hundred and fifty-eight Polish Holstein-Friesian cows kept at the same farm located in the Lubuskie province were used in this study.Cows included in the analysis were required to have 305-day lactation (all animals completed 1st, 2nd and 3rd successive lactations).The blood was collected from the external jugular vein into K3EDTA-containing test tubes.Genomic DNA was isolated from the peripheral blood using the MasterPure Genomic DNA Purification Kit (Epicentre Technologies).
A 146-bp PCR product was amplified using the Biometra thermal cycler.The PCR reaction mixture and conditions were the same as those described previously by Zych et al. (2007).The Amplification Created Restriction Sites -Polymerase Chain Reaction (ACRS-PCR) primers, designed by Zych et al. (2007), were applied: IgfP1F 5' TCA TCC AGC TGA GAG ATT TGA AT 3' IgfP1R 5' TGT GTG TGT GTG TGT GTG TGA AT 3' The genotypes were identified by restriction analysis.The PCR-amplified DNA was digested with 5 units of the TasI restriction enzyme (10 U/µl, ↓AATT; MBI Fermentas/ABO, Gdansk, Poland) at 65 °C for 3 h.The products was separated on 2 % agarose gels (Prona Basic, le GQT) with ethidium bromide staining.The gels were visualized under UV light (312 nm) using the Vilber Lourmat transilluminator.
An association of the IGF1/TasI polymorphism with milk yield (in kg), milk fat and protein yield (in kg), as well as milk fat and protein content (percentage) was analysed.
The analysis of milk performance was based on the data obtained from the official milk recordings.Statistical calculations were performed using a General Linear Model (GLM).The following statistical model was used: where Y ijkl is the analysed trait, μ is the overall mean, G i is the fixed effect of IGF1 genotype (i=1, …3), CS j is the fixed effect of calving season (j=1, 2), s k is the random effect of sire (k=1, …254), β is the linear regression coefficient of calving age, x l is the calving age of a cow, A l is the mean calving age and e ijkl is the random error.
The significance of differences between particular traits for each lactation apart was tested using analysis of variance with Tukey's test (Statistica 9.0 PL software package).

Results
A transversion of A (allele A) to C (allele C) in the P1 promoter region of IGF1 gene was identified using ACRS-PCR method.Primer IgfP1R introduced an artificial TasI restriction site.Digestion of the 146 bp PCR product with the above-mentioned restriction nuclease resulted in two DNA bands (122 and 24 bp) for homozygote AA and three bands (146, 122 and 24 bp) for the AC heterozygote.The DNA amplified from homozygous CC animals remained undigested with TasI restrictase.Allele and genotype frequencies for analysed polymorphism are shown in Table 1.The AA genotype occurred with a frequency of 0.766, AC with 0.213 and CC with 0.021.In the present study, the statistically significant differences between individuals of different IGF1/TasI genotypes were found in milk, fat and protein yield.In the 2nd and 3rd 305-days lactations, the cows of the CC genotype produced more milk (+515 kg i +463 kg, respectively) than the AA individuals (P≤0.05).In the case of protein and fat yield, similar tendencies were observed.The cows of the homozygous IGF1 CC genotype yielded significantly more fat (+22 kg and +16 kg) and protein (both +14 kg) than cows carrying the AA genotype (significations were different depending on successive lactation).Moreover, in the 2nd lactation significant differences between AA and AC individuals in regard to protein concentration in milk were observed.No association was found between IGF1 RFLP-TasI and dairy production traits in the 1st lactation.

Discussion
Polymorphisms in the promoter region, apart from missense mutations in the coding regions, are more likely to have a direct association with performance traits than intronic polymorphisms or silent mutations in the coding region.Candidate genes approach utilize genes that have well known biological functions related to the development or physiology of an important trait.In the study of Lien et al. (2000) the most convincing QTL peak was observed for a region in the middle part of chromosome 5 close to the insulin-like growth factor 1 (IGF1) gene.It is possible that IGF-I has different effects at different stages of growth, because expression of the IGF1 gene is developmentally and physiologically regulated (Werner et al. 1994, Sirotkin et al. 2000).Additionally, nutritional stress significantly decreases the expression of the IGF1 gene and action of IGF-I at multiple steps (Underwood et al. 1994).
In the study of Plath-Gabler et al. (2001) the involvement of the IGFs system in mammary development and lactation of the cow, the temporal expressions of IGF-I and -II, its receptor type 1 (IGFR-1), IGF-binding proteins (IGFBPs) -1 to -6 and GH receptor (GHR) mRNA were examined.This was carried out for different stages of mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland.The most of the IGF1 mRNA was expressed by adipocytes (about 80 % of them showed a distinct IGF-I immunohistochemical staining).These results lead to conclude that paracrine/autocrine IGF-I is an important local factor for remodelling of the bovine mammary gland during involution and inactive during mammogenesis, lactogenesis and galactopoiesis.That may suggest an importance of endocrine IGF-I (produced by the liver) as primary mediator of galactopoietic effect of exogenous GH (Schams et al. 1991).
In most tissues IGF1 mRNA is only derived from exon 1 (P1 promoter), whereas both promoters (P1 and P2) are active in the liver (Shemer et al. 1992).Exon 2 has a classic signal peptide that should confer efficient secretion of mature peptide in contrast to the exon 1 derived long signal peptide, which may impede efficient IGF-I secretion.The function of class 2 IGF1 mRNA is to provide an efficient mechanism for rapid hepatic synthesis of IGF-I in response to GH (O'Sullivan et al. 2002).
The aim of this study was to search for possible associations between a SNP (single nucleotide polymorphism), the A/C transversion at position 977 bp upstream from the ATG codon in exon 1 (according to the GenBank sequence AF210383) in the 5'-noncoding region of the IGF1 gene (P1 promoter) and performance in milk production of Polish Holstein-Friesian cattle.The results indicated slightly effect of IGF1/TasI polymorphism on milk protein and fat yields, with preference of C allele.However, the effects of these polymorphisms on beef or milk production traits were not yet investigated by the other scientists.
The dinucleotide (CA) n repeat polymorphism located 989 bp to 954 bp upstream from the ATG codon in the 5' region of bovine IGF1 gene (Kirkpatrick 1992) is closely linked to regulatory elements of the IGF1 gene (Ge et al. 2001).In some microsatellites, the repeat units are interrupted by nonrepeat sequences, producing kind of a imperfect microsatellites.Occurrence of an AATA interrupt in the IGF1 microsatellite (P1 promoter) was investigated in a number of Artiodactyl species, namely pigs, camels, deer, cattle, goats, and sheep (Shariflou & Moran 2000).IGF1/TasI polymorphism is localized within »AATA« interrupt of (CA) n microsatellite, form two variants, (CA) 6 AATA(CA) n or (CA) 7 TA(CA) n for A and C alleles, respectively.Conservation of this interrupt in an other number of Artiodactyl species remains further investigation.In fact, close proximity of the microsatellite to the transcription sites and the ability of the CA repeats to form a Z-DNA structure indicate an important functional role for this microsatellite as a potential enhancer element (Nordheim & Rich 1983, Hamada et al. 1984).Ge et al. (1997) detected and then reported (Ge et al. 2001) a significant effect of the IGF1/ SnaBI polymorphism, located 512 bp upstream from the ATG codon in the regulatory region of the IGF1 gene on growth traits in Angus cattle.Little is also known about the effect of the IGF1/SnaBI polymorphism on milk traits in cattle.However, in the several study, cows with the A allele were always significantly associated with a higher milk, fat and protein yield (Siadkowska et al. 2006, Grzelak et al. 2007, Zych 2007, doctoral thesis).In contrast, no association was found between IGF1 RFLP-SnaBI and dairy production traits in Holstein cattle (Hines et al. 1998).
In conclusion, like the other polymorphisms, the utility of IGF1/TasI polymorphism is strongly limited by low frequency of the rare C allele.The effect of the mentioned locus is probably masked by loci with stronger positive or negative effects.Rather, the A/C transversion may be only considered as a genetic marker, linked to other polymorphisms located closely in the same regulatory region of IGF1 gene.More tests are needed among random populations of calves to verify the associated effects of IGF1/TasI polymorphism as the potential genetic marker.

Table 1
The number and frequency of IGF1/TasI genotypes and alleles of cows under study

Table 2
Means and standard deviations of milk performance traits in cows with different IGF1/TasI genotypes Means within rows bearing the same letters differ significantly at: small letters P≤0.05, capitals P≤0.01.
Zych S (2007) Expression and polymorphism of selected genes encoding somatotropic axis in connection with milk production traits of Black-and White cows with different share of HF genes.Doctoral thesis, The West Pomeranian University of Technology, Szczecin, Poland Received 7 January 2010, accepted 29 November 2010.