Evaluation of associations of the polymorphism in the placenta- specific promoter 1.1 of the CYP19 gene in Black-and-White and Jersey cattle with milk production traits

The relationship between the SNP of the cytochrome P450 gene (CYP19-PvuII) and milk production traits of Black-and-White and Jersey cattle were analysed. A total of 437 cows were included in the study. A PCR-RFLP was used to genotype. The frequencies of genotypes and alleles for the Black-and-White cows were as follows: 0.8985 AA, 0.0977 AB, 0.0038 BB, and 0.9474 CYP19, 0.0526 CYP19. In the Jersey, all cows were genotyped as CYP19 (no polymorphism). There weren’t any associations between CYP19-PvuII polymorphism and milk production traits of the investigated cows.


Introduction
It is known that conversion of androgens into oestrogens is catalyzed by aromatase cytochrome P450 enzyme (VANSELOW et al., 1999) encoded by the CYP19 gene (FÜRBASS et al., 1997;KALBE et al., 2000), which was mapped to band q2.6 on chromosome 10 (VANSELOW et al., 2000).Characteristic for CYP19 is that it utilises different promoters in tissue-specific expression.Products of that gene are present in placenta, ovary, testes, adipose, bone (CONLEY and HINSHELWOOD, 2001).Different promoter regions correspond to different 5'-UTR transcripts but the coding region is identical for all tissues (KALBE et al., 2000).For example in placenta expression of aromatase is mainly driven by P1.1, a distal promoter (FÜRBASS et al., 2001) and P1.2, a promoter which is also active in ovary and brain (FÜRBASS et al., 1997;VANSELOW et al., 2000).Candidacy of CYP19 for milk production traits arise from the fact that oestrogen is involved (not directly) in lactogenesis but it influences mammary cells by increasing numbers of prolactin and growth hormone receptors.The level of oestrogen is associated with the activity of aromatase and CYP19 gene expression.
The aim of this study was to identify the CYP19-PvuII gene polymorphism in placental specific promoter P1.1 in Black-and-White (B&W) and Jersey cattle and its associations with milk production traits.

Materials and Methods
A total of 266 B&W cows with diverse proportion of Holstein Fresian (HF) genes (166 cows with 100% of HF and 100 cows with less than 100% of HF; in this group HF proportion ranging from 50 to 99%) and 171 pure-breed Jersey cows were genotyped using PCR-RFLP.Holstein Fresian breed have been introduced to the herd.The genomic DNA was isolated using Master Pure TM Kit (Epicentre Technologies).The analysed fragment of CYP19 gene was amplified using forward 5'-CTCTCGATGAGACAGGCTCC-3' and reverse 5'-ACAATGCTGGGTTCTGGACT-3' primers (VANSELOW et al., 1999).The PCR reactions contained approx.100 ng of genomic DNA, 0.5µM of each primer, 1xPCR buffer, 1.5 mM MgCl 2 , 200µM dNTP, 0.5 units of Taq polymerase (MBI Fermentas) and deionized water up to 20 µl.PCR product was digested with PvuII enzyme (Fermentas) at 37 o C/3 hours.The digestion products were separated by horizontal electrophoresis (120V, 50 minutes) through 2% agarose gels (PRONA) in 1xTBE and 1,0 µM ethidium bromide.The data of milk production traits in the first lactation were obtained from the farm documentation.Type III ANOVA was used (STATISTICA, 2005).Differences of means were tested with the multiple Duncan test.The following model was used: Y i j k l m n -305-day milk production record at 1 st lactation of cow o, µ-the overall mean, G i -the fixed effect of CYP19 genotype (i = 1,2), S j -the fixed effect of sire (j = 1, …74), HF k -the fixed effect of percentage of HF genes (k = 1,2)), YS l -the fixed effect of year-season of calving class (l = 1, …6), A m -age of calving, E i j k l m n -the random error

Results
In the group of Black-and-White cows the following DNA restriction fragments were obtained for the CYP19-PvuII polymorphism: 405 bp for the AA genotype (no digestion), 405, 327 and 78 bp for AB and 327 and 78 bp for BB genotype (Fig. ).All Jersey cows were genotyped as AA (Table 1).
Table 2 shows influence of genotypes (CYP19-PvuII polymorphism) on milk production traits in B&W cattle.The BB individual was excluded from the statistical analysis.Discussion Nowadays breeding and selection is complemented by the application of DNA marker information (COPPIETERS et al.,1999).In cattle a number of candidate genes and QTL regions for milk yield and composition have been detected (DYBUS, 2002;DYBUS et al., 2004;FREYER et al., 2003;GRUPE and SCHWERIN, 1998).Oestrogen together with other hormones stimulates mammary gland proliferation and causes the ducts lengthening and branching during first half of gestation.With progesterone oestrogen establish the conditions needed for geometric cell multiplication and are absolutely essential for mammary epithelial development and differentiation (TOPPER and FREEMAN, 1980).Placenta plays an important role in mammary gland development during the second half of pregnancy.This organ is one of the origins of oestrogen and activity of the product of CYP19, aromatase, is absolutely essential for conversion androgens to oestrogens.Expression of that gene in placenta is dependent on promoter 1.1, which is specific for this organ and it is hypotesed present that variation in the promoter region (P1.1) of CYP19 may affect milk yield.Frequencies of CYP19-PvuII alleles obtained in the present study were 0.9474 (CYP19 A ) and 0.0525 (CYP19 B ) for Black-and-White cattle and were similar to the frequencies obtained by KOMISAREK and DORYNEK (2002) for HF cattle.Higher frequencies of the CYP19 B (0.12) was noticed by VANSELOW et al. (1999).In this study no statistically differences between individuals of different CYP19 genotypes were found.In the present study, no associations between CYP19-PvuII gene and milk production traits were found for Black-and-White and Jersey cattle.However the distribution of genotypes was unfavourable to detect association with one allele showing very low frequency.Moreover, the gene effects may be small allowing to obtain significant association in large numbers of animals only.The influence of CYP19 AB and CYP19 BB variants on the milk production and reproduction traits should be evaluated further using other population and higher animal numbers.

Table 2 Least
square mean and standard deviation of milk production traits in B&W cows with different CYP19 genotypes (Mittelwerte und Standardabweichungen der Milchleistungsmerkmale bei B&W Kühen mit verschiedenen Genotypen CYP19-PvuII)