The yaks were obtained from the Songpan Bovine Breeding Farm in Sichuan, China. These animals were killed, and their tissues, including heart, liver, spleen, lung, kidney, mammary gland, uterus, oviduct, ovary, and testis (male = 3, female = 3), were collected and immediately frozen in liquid nitrogen until use.

Total RNA was extracted from tissues (11 difference tissues) using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The primers for amplification of yak ERα (ERα01 and ERα02) and ERβ (ERβ01 and ERβ02) gene coding sequences were based on the full-length sequences of bovine ERs, designed by Bacon designer (Table 1). cDNA synthesis was performed using PrimeScript reagent kit (TakaRa, Dalian, China) in a total volume of 10 μL of reaction mixture, containing 2 μL of 5× PrimeScript Buffer, 0.5 μL of PrimeScript RT enzyme Mix I, 0.5 μL of Oligo dT primer, 0.5 μL of random 6 mers, and 6.5 μL of total RNA. This cDNA was used as a template in reverse transcription-polymerse chain reaction (RT-PCR). The PCR reaction was performed in 25 μL volumes of the reaction mixture, containing 12.5 μL of PCR Mastermix, 1 μL of cDNA, 9.5 μL of ddH₂O, and 1 μL of each primer. The PCR procedure consisted of first denaturation step at 94°C for 5 min, followed by 35 amplification cycles (denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min 30 s), with a final elongation step at 72°C for 5 min. The expected fragment ERα 01 (820 bp), fragment ERα 02 (1,280 bp), fragment ERβ 01 (783 bp), and fragment ERβ 02 (875 bp) were extracted from the EZNA gel extraction kit (OMEGA, USA). The fragments were cloned into pMD19-T Vector (TakaRa, Dalian, China).

The nucleotide and deduced amino acid sequence identity was performed using LaserGene software package (DNASTAR, London, UK). The sequences of yak ERα and ERβ were aligned using the Multiple Sequence Alignment option in Clustal W. The neighbor-joining phylogenetic tree of ERα and ERβ was constructed using molecular evolutionary genetics analysis 5.

The tissue distributions of ERα and ERβ mRNA were examined in the heart, liver, spleen, lung, kidney, testis, mammary, oviduct, uterus, and ovary by quantitative RT-PCR using the SYBR Premix Ex Taq (TakaRa, China) with Bio-Rad Connect (iCycler iQ5 Real-time Detection System) in a 15 μL of reaction volume. GlycERαldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. The primers ERαQ, ERβQ, and GAPDH were designed using Bacon designer (Table 1). The reaction mixture for quantitative RT-PCR contained 7.5 μL of Green premix (TakaRa), 1.0 μL of cDNA, and 0.5 μL of each gene-specific primer, and ddH2O was used to adjust the total volume to 15 μL. Melting curve was performed to detect the specificity. PCR parameters were based on a three-step method: 94°C for 30 s; 40 cycle of amplification step (denaturation at 94°C for 5 s, annealing at 58°C for 25 s, and extension at 72°C for 25 s); and dissociation curve analysis at 95°C/10 s, 65°C to 95°C in 0.5°C intervals. Each sample was tested in triplicate. Serial dilutions of pooled cDNA samples of each tissue were used to generate the standard curves. The amplification efficiency between the target gene and reference gene is 97% to 99%. The expression of the target gene was compiled relative to the expression of GADPH by the relative quantification method 2^−ΔΔCt (Schmittgen and Livak, 2008).

The statistical significance of the variation was analyzed by one-way analysis of variance or Student’s t-test, followed by Tukey’s multiple comparison test. All the quantitative RT-PCR data were expressed as mean±SE, and significance was set at p<0.05.

The ERα PCR products were 820 and 1,280 bp, which were composed of 1,791 bp of open reading frame (ORF) coding 596 amino acids. The predicted molecular mass of yak ERα was 66.5 kDa. Multiple alignments were carried out based on amino acid sequences of ERα from Bos taurus, Gallus gallus, Homo sapiens, Mus musculus, Rattus norvegicus, alligator, Xenopus laevis, and zebrafish. ERα showed 99.5% and 91.1% amino acid identities with B. taurus and H. sapiens, whereas only 45.3% amino acid identity with zebrafish (Table 2). The ERβ PCR products were 783 and 875 bp, which contained 1,584 bp of ORF with a coding potential for 527 amino acid residues. The molecular mass of yak ERβ was 59.0 kDa. The protein sequence showed 99.0%, 70.5%, 78.8%, 80.9%, 96.5%, 87.8%, 79.6%, and 91.9% similarities to B. taurus, G. gallus, H. sapiens, M. musculus, R. norvegicus, alligator, X. laevis, and zebrafish, respectively (Table 2). Similar to most nuclear receptors, both ERα and ERβ contained six important domains that are labeled A though F (Figure 1). C and E domains of yak were highly conserved from fish to mammals between ERα and ERβ (C domain, 94% to 99% amino acid identity; E domain, 58% to 94%) in the six bindings (Figures 2 and 3). Both ERα and ERβ contained eight cysteine residues (Figure 4).

Based on the amino acid sequences of ERα and ERβ of other species, a phylogenetic tree was constructed using the neighbor-joining method (Figure 5). The phylogenetic analysis showed that all mammalian ERs formed an independent branch, whereas birds, reptile, amphibian, and fish formed another branch.

The two transcripts were expressed in a variety of tissues, but their expression levels varied (Figure 6). The expression of ERα was highest in the oviduct, followed by the uterus, mammary gland, spleen, stomach, and ovary, and lowest in the liver, lung, testis, spleen, and heart. The expression of ERβ was highest in the oviduct; intermediary in the uterus, testis, mammary gland, and ovary; and lowest in the spleen, mammary gland, heart, spleen, and liver. Moreover, in the same reproductive organs, the yERα mRNA level was higher than yERβ, except in the ovary.