Estrogen receptor gene ( ESR ) and semen characteristics of boars

Estrogen receptor gene (ESR) which is localized in the swine chromosome 1, has been recognized as a “candidate” gene of reproductive traits. The aim of this study was to determine the mutations in the ESR/AvaI (ESR1) and ESR/PvuII (ESR2) gene in boars kept at the AI Station and its effect on selected quantitative and qualitative characters of the semen. The study included 217 boars maintained at the AI Station. The ESR genotypes were determined with PCR-RFLP. Two alleles ESR1 were identified: A (109 and 76 bp), B (76, 62 and 47 bp) and also two alleles ESR2: C (120 bp), D (65 and 55 bp), with the frequencies: 0.79 (A), 0.21 (B), 0.83 (C) and 0.17 (D) respectively. In the studied population of boars, the genotype AA was detected with the frequency 0.69, AB-0.21, BB-0.10 of ESR1 and CC-0.72, CD-0.23, DD-0.05 of ESR2. The analysis showed statistically significant differences (P≤0,01) between boars carrying different ESR1 and ESR2 genotypes and all semen traits.


Introduction
In younger time studies on porcine genome contributed to identification of polymorphic loci of the genes that may influence the level reproduction-relates traits in sows (KMIEĆ, et al., 2001;LINVILLE, et al., 2001;DRÖGEMÜLLER et al., 1999) and boars (MAĆKOWSKI, et al., 2004;KMIEĆ et al., 2003;SCHLINGMANN et al., 2002;KMIEĆ et al., 2001).Progress in genetics and molecular techniques has enabled application of DNA polymorphism by selection of animals known as marker-assisted selection (MAS).Estrogen receptor (ESR) gene which is localized on the first swine chromosome (p25-p24) is the "candidate gene" for reproductive traits.It belongs to intacellular group of receptors.In pigs and human the length of ESR protein is the same (595 aminoacids) and consists the same domains A, B, C, D, E and F (BÖKENKAMP et al., 1994).ESR plays an important role in many processes associated with reproduction.In females it influences on the maturation of milk gland during embryogenesis as well as Graafian follicle during menstruation cycle (TKACZYK and KALITA, 2001).In males ESR has impact on beginning and maintaining of spermatogenesis on different levels of hormone regulation: 1.hypothalmus-pituitary-testis axis, 2. Leydig, Sertoli and gametogenesis cells, 3. discharging tubule and epidydidymys (PAZIEWSKA and BILIŃSKA, 2003).The mutation within ESR gene results in changes leading to disturbance of reproduction (KMIEĆ et al., 2002).ROTHSCHILD et al. (1996) found associations between polymorphism in ESR/PvuII locus and litter size in pigs by using PCR-RFLP method.An AvaI and MspA1I polymorphisms were discovered in swine ESR gene by DRÖGEMÜLLER et al. (1997).Since that time many reports revealed status ESR gene as a "candidate" gene for performance traits in sows.Because estrogens have also significant impact on spermatogenesis, may presumed that mutations in its receptors cause changes in some semen parameters .The aim of the present study was to estimate the frequencies of ESR/AvaI and ESR/PvuII gene mutations and to find possible associations between different genotypes of these genes and characters of boars semen.

Materials and Methods
The experimental population included 217 boars kept at the same stations.Rearing and feeding conditions were equalized for all animals.Genomic DNA was isolated from blood leucocytes using Master Pure kit of Epicentre Technologies.ESR genotype was determined using the PCR procedure of SHORT et al. (1997) for ESR1 andDRÖGEMÜLLER et al. (1997) for ESR2.Each sample prepared for the polymerase chain reaction included: 1.5 µL 10x PCR buffer, 1.3 µL 1.5 mM MgCl 2 , 1.2 µL 10 mM dNTPs, 0.5 µL 10 µM forward primer, 0.5 µL 10 µM reverse primer, 1.5 µL genomic DNA, 1 µL Taq DNA polymerase (5 U), and 7.5 µL sterile deionised water.All reactions were performed on Biometra Cycler using the following temperature program: initial denaturation at 94ºC for 4 min, followed by denaturation at 94ºC for 40, primer annealing at 60ºC (for ESR1) or 55ºC (for ESR2), and primer extension at 72ºC, each for 30 sec, repeated for 30 cycles.A final extension step of 5 min at 72ºC completed the PCR amplification.Each sample was then digested with 5 I.U. of appropriate restriction endonuclease AvaI (ESR1) and PvuII (ESR2) (MBI Fermentas) at 37ºC overnight.The restriction fragment of DNA were separated by electrophoresis in 3% agarose gel, stained with ethidium bromide at 0.5 µg/ml, and visualized by u.v.transillumination and recorded with the use of the Vilber Lourmat system.The analysis of relationship between the ESR1 and ESR2 genotypes and ejaculate volume, sperm concentration, sperm alive percentage, number of live sperm in ejaculate were carried out by means of the GLM (General Linear Models) variation analysis procedure of the SAS ® calculation package.
The values of the studies traits were expressed as the means and their standard deviations.

Results
Two ESR1 alleles were identified in boar herd under study: A and B. Three genotypes, namely AA, AB and BB were observed.The following lengths of restriction fragments were detected: 109 and 76 base pair (bp) for allele A, and 76, 62 and 47 bp for allele B. In the analyzed AI boars the alleles A and B occurred with a frequency of 0.79 and 0.21 respectively.The AA genotype occurred with a frequency of 0.69, AB with 0.21 and BB with 0.10 -Table 1.The polymorphism in the ESR2 gene was also detected.Two different alleles of the ESR2 gene were identified in the boar herd under study: alleles C and D that control the occurrence of three genotypes, namenly: CC , CD and DD.The lengths of restriction fragments detected during the experiment were as follows: 120 bp for allele C and 65 and 55 bp for allele D. The frequencies of the alleles and genotypes are presented in Table 2.  DRÖGEMÜLLER et al. (1997) in Duroc, German Landrace, German Yorkshire and Synthetic Line pigs.A also higher frequency (0.91) was observed by DVORAK et al. (1998).A similar frequency of allele C (0.83) was observed by SHORT et al. (1997) in Synthetic Line pigs.A higher frequency of allele C was revealed in Duroc (1.00), German Landrace (1.00) and Polish Landrace pigs (0.94) reported by DRÖGEMÜLLER et al. (2001), LINVILLE et al. (2001) and KMIEĆ et al. (2002) respectively.A lower frequency (< 0.60) was observed by LEGAULT et al. (1996), SHORT et al. (1997), andGIBSON et al. (2002) in Large White pigs.Table 3 presents means and their standard errors for the boars semen traits across ESR1 genotypes.Means in rows designated with the same letter differ significantly at P ≤ 0.01 Mean ejaculate volume for all the boars was 218.7 cm 3 falling within the range of 50-245 cm 3 reported by DUBIEL (1985).The highest mean ejaculate volume was found in AA (220.5 cm 3 ) , while the lowest in BB genotype -207.9 cm 3 (ESR1) boars and the recorded differences were statistically significant at P ≤ 0.01 (Table 3).
For ESR2 the greatest sperm concentration in ejaculates appeared in boars with DD genotype (610.1 x 10 6 /cm 3 ) and the lowest in boars with CC genotypes (595.2 x 10 6 /cm 3 ).The differences were confirmed statistically P ≤ 0.01 (Table 4).Average sperm alive percentage for all the analyzed boars was 72.4%.The highest sperm alive percentage (ESR1) was found for the boars with the AA genotype (72.8%), while the lowest for BB genotype (71.0%) and the recorded differences were statistically significant at P ≤ 0.01 (Table 3).
The last, but also important analyzed semen traits was number of live sperm in ejaculate.Mean number of live sperm in ejaculate was 91.4 x 10 9 .The greatest number appeared in boars with AA (91.6 x 10 9 ) and CC (92.3 x 10 9 ) genotype, while the smallest boars with BB (88.7 x 10 9 ) and CD (88.9 x 10 9 ) genotype for ESR1 and ESR2 respectively.The differences were confirmed statistically P ≤ 0.01 (Table 3, 4).

Conclusion
The analysis of relation between the genotype of estrogen receptor and the studied reproductive traits showed statistically significant differences (P ≤ 0.01) between all semen traits of boars carrying different ESR1 and ESR2 genotypes.The preliminary study showed that boars with AA (ESR1) and CC (ESR2) genotype produced ejaculates of large volume higher percentage and also higher number of sperm in ejaculate.The study suggest a possibility of using the existing polymorphisms in the estrogen receptor gene to improvement of some reproductive performance traits of boars.The results, however, should be verified by further research of ESR/AvaI and ESR/PvuII polymorphisms on a larger number of animals.

Table 1
The number and frequency of ESR1/AvaI genotype and alleles of boars under study (Frequenz der Genotypen und Allele von ESR1/AvaI in der untersuchten Eberpopulation)

Table 3
Values of studied semen characters in reference to ESR1/AvaI genotype (Beziehungen untersuchter Spermamerkmale zu den ESR1/AvaI Genotypen) Means in rows designated with the same letter differ significantly at P ≤ 0.01.