Mol Biol Rep (2013) 40:579–584 DOI 10.1007/s11033-012-2095-9

Effects of PGR and ESRa genotypes on the pregnancy rates after embryo transfer in Luxi cattle Ke-Qiong Tang • Wu-Cai Yang • Bin Pai Shu-Jing Li • Long Chen • Li-Guo Yang

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Received: 9 May 2012 / Accepted: 3 October 2012 / Published online: 19 October 2012 Ó Springer Science+Business Media Dordrecht 2012

Abstract Progesterone receptor (PGR) and estrogen receptor alpha (ESRa), which mediate the biological effects of the steroid hormones progesterone and estrogen, play a central role in the establishment and maintenance of pregnancy. The objectives of this study were to detect bovine PGR and ESRa genes polymorphisms and analyze their relationships with the pregnancy rates after embryo transfer and the hormone concentrations at the day of embryo transfer. One reported SNP of PGR G59752C and a novel SNP of ESRa G75935C were analyzed in 132 recipients of Luxi cattle. For the PGR gene, recipients with g.59752 GG and g.59752 GC genotypes had obviously higher pregnancy rates than g.59752 CC genotype. For the ESRa gene, recipients with g.75935 GC and g.75935 CC genotypes had obviously higher pregnancy rates than g.75935 GG genotype. Furthermore, the same tendency was observed for these two genes that the same genotype groups with high pregnancy rates had high progesterone concentration and low estrogen concentration at the day of embryo transfer. These results showed for the first time that PGR G59752C and ESRa G75935C polymorphisms had obvious K.-Q. Tang  W.-C. Yang  B. Pai  L.-G. Yang (&) Key Laboratory under Education Ministry of China for Agricultural Animal Genetics, Breeding, and Reproduction, Wuhan, China e-mail: [email protected] K.-Q. Tang e-mail: [email protected] Present Address: K.-Q. Tang  W.-C. Yang  B. Pai  L.-G. Yang College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China S.-J. Li  L. Chen Beijing Amber Embryo Technology Co. Ltd., Beijing 100107, China

effects on the pregnancy rates after embryo transfer, and indicated that PGR G59752C and ESRa G75935C polymorphisms could be potential markers for recipient selection of embryo transfer. Keywords Luxi cattle  Embryo transfer  PGR  ESRa  Pregnancy rates

Introduction Embryo transfer (ET) has become the most powerful tool for animal breeders and animal scientists to improve genetic construction of their animal herds. In the bovine, more than 800,000 embryos were transferred annually in worldwide [1], and 80 % Holstein bulls admitted to the young sire testing program in Canada were produced by ET [2]. However, embryonic survival rates after ET varies widely from 40 to 70 % [3, 4]. In the past decade, numerous new technologies have been adopted within the ET industry [2, 5], and the effects of embryo grade, season, nutrition, synchronization, age, conditions of embryo storage and embryo transfer technician on the pregnancy rates of ET have been elucidated [6–9]. But, the conception rates following embryo transfer has changed very little over the years, ET is still an expensive, inefficient technology [10]. Candidate gene approach has been widely used in genetic association and biomarker from animals to humans [11], and many candidate genes of animal reproduction traits have been detected, such as ESR [12], GDF10 [13], GnRHR [14] and so on. So, it looks meaningful for recipient selection to use candidate gene approach to identify potential markers that are correlated with the pregnancy rates after ET. Progesterone and estrogen play a central role in the regulation of the reproductive events associated with establishment

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and maintenance of pregnancy [15–17]. Progesterone has been widely used to treat women with threatened miscarriage and presumed progesterone deficiency to improve expectations for ongoing pregnancy [18, 19]. Low progesterone (P4) concentrations after ovulation or a delay in the rise of P4 had significant negative effects on the embryo development and survival [20, 21], and finally resulted in a decrease in pregnancy rates [22, 23]. Furthermore, previous studies found that P4 levels at day of ET were significantly correlated with the pregnancy rate [24–27], and Nishigai et al. [28] reported that the pregnancy rate tended to rise when the blood estrogen (E2) concentration was low at the day of ET, and indicated that high blood P4 concentration and low blood E2 concentration at the day of ET were very important for recipient selection. Owing to the central role of progesterone and estrogen actions in establishment and maintenance of pregnancy, the progesterone receptor (PGR) and estrogen receptor (ESR) genes appeared to be good candidate for mutations associated with the pregnancy rates after ET. Estrogen receptor alpha (ESRa), which is the one forms of ESR, has a higher affinity for estrogen and is the predominant form in normal endometrium [29]. Through gene targeting demonstrated that PGR and ESRa were essential for the female reproductive activities associated with progesterone and estrogen, respectively [15, 30–32], and expression analysis revealed an important role of PGR in the regulation of embryo development [33]. In addition, PGR and ESRa polymorphisms significantly associated with spontaneous abortion [34, 35]. Therefore, PGR and ESRa were chosen as candidate genes to investigate their effects on the pregnancy rates after ET. PCR–RFLP and sequencing methods were used to detect variants of the bovine PGR and ESRa genes, and evaluate its relationships with the pregnancy rates after ET in order to identify potential markers for recipient selection of ET.

Materials and methods Experimental cattle and sampling This study was conducted in Beijing Amber Embryo Technology Co. Ltd., Beijing, China. One hundred and thirty-two Luxi cattle with no relatedness in three generations were recipients of embryos. Genomic DNA was extracted from blood samples using phenol–chloroform standard protocols. Blood samples without anticoagulant were allowed to clot overnight at room temperature and the serum was frozen at –20 °C until it was assayed for P4 and E2 concentrations. Synchronisation of oestrus in recipients One hundred and thirty-two Luxi cattle received a CIDR device (New Zealand DEC Co.,Ltd.) on random days of the

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estrous cycle (day 0), an injection of 0.6 mg prostaglandin F2a (Qilu Animal Health Products Co., Ltd., China) was given on day 9, and devices were removed on day 12. Estrus detection by visual observation was carried out on day 13, and embryos were transferred 6.5–8.5 days after detection of estrus. Embryo transfer Recipients only were used if they had been observed in standing estrus and possessed a palpable corpus luteum (CL) at the time of transfer. A single frozen-thawed Wagyu cattle embryo was transferred transcervically with a standard l/4 mL embryo transfer instrument into the uterine horn ipsilateral to the CL. All embryos were assigned a developmental stage and quality grade according to standards of the International Embryo Transfer Society (Savoy, IL). Developmental stage codes were: 3 = early morula; 4 = morula; 5 = early blastocyst; and, 6 = blastocyst. Quality codes were: 1 = symmetrical and spherical embryo mass with individual blastomeres that were uniform in size, color, and density with at least 85 % of the cellular material intact (excellent or good); and, 2 = moderate irregularities in overall shape of embryonic mass or in size, color and density of individual cells with at least 50 % of the cellular material intact (fair). Embryos receiving stage codes of B3 or C6 or poor quality embryos (quality score of 3 or 4) were excluded from the study. Pregnancy diagnosis was performed 40 d after embryo transfer by ultrasonography. Primer synthesis and PCR conditions One reported SNP of PGR G59752C [36] located in intron 3 and a novel SNP of ESRa G75935C located in intron 4 were selected as potential markers for pregnancy rates after ET. Primers sequences (F: 50 -GTGAATTTGCTCCAAGATTC-30 and R: 50 -GCCCGACCTTCCCATAA-30 ) and reaction conditions for C59752G polymorphism were selected according to previous study [37]. Primers (F: 50 -GGTA GCAACAAGGGAAAGTCAAGT-30 and R: 50 -GAAACACAAAATGGCATGCTC AC-30 ) were designed by Primer 5.0 to find ESRa G75935C mutation. PCR was performed in a total volume of 20 ll containing 10 pmol primers, 200 lM of dNTP (deoxyribo nucleotide triphosphate), 2 ll of 109 reaction buffer which contained 1.5 mM of MgCl2, 0.5 unit of Taq-DNA polymerase (Promega, Madison, WI), and 50 ng of genomic DNA as template. After denaturation at 94 °C for 5 min, 34 amplification cycles were performed comprising denaturation at 94 °C for 45 s, annealing at 64 °C for 45 s, and extension at 72 °C for 45 s, followed by a further 10 min extension at 72 °C. PCR products were electrophoretically

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detected on 1.5 % agarose gel (5 V/cm) by staining with GelRed. Genotyping Novel polymorphism of ESRa G75935C was genotyped by sequencing. To detect the SNP PGR59752, the PGR PCR products were digested with AdeI [37] (TaKaRa, Tokyo, Japan). The digestion mixture contained 4 lL PCR products, 19 digestion buffer, 3.0 U of each enzyme and digested at 37 °C overnight. Fragments were separated on 2 % agarose gels and visualized with GelRed staining.

Fig. 1 Representative genotyping bovine PGR gene by a polyacrylamide gel electrophoresis. Lanes 1, 3 and 6 represent GG genotype, Lanes 2, 4, 5 and 7 represent GC genotype, and lane 8 represents CC genotype. Marker: 50 bp DNA Ladder

Hormone assays by radioimmunoassay Serum concentrations of P4 and E2 in recipients at day of embryo transfer were measured by radioimmunoassay (Diagnostic Products Corporation, Beijing North Institute of Biotechnology, Beijing, China). Sensitivity was B3 pg/mL for E2 and B0.05 ng/mL for P4, whereas the intra- and interassay coefficients of variation were \10 and \15 % for each assay.

Statistical analysis Gene frequencies were determined by direct counting and Hardy–Weinberg equilibrium was analyzed using v2-test by SAS 8.1 software. Pregnancy rates among three genotypes of PGR gene were compared using Chi-square analysis. The differences between genotypes in the concentrations of E2 and P4 were analyzed by the one-way ANOVA. All analyses were performed using the SAS software. Statistical significance was determined based on a P value of 0.05. Data were presented as mean ± SD.

Results Genotypic and allelic frequencies Polymorphism of PGR G59752C forms the restriction site recognized by endonuclease AdeI and resulted in three genotypes named g.59752 GG (869 bp), g.59752 CG (207, 662 and 869 bp), g.59752 CC (207 and 662 bp) (Fig. 1). The frequency of three genotypes showed a tendency of g.59752 GG [ g.59752 GC [ g.59752 CC (Table 1). Polymorphism of ESRa G75935C was genotyped by sequencing (Fig. 2). The frequency of three genotypes showed a tendency of g.75935 GC [ g.75935 CC [ g. 75935 GG. These two polymorphisms were in Hardy– Weinberg equilibrium (Table 1).

Table 1 The allelic and genotypic frequencies for PGR G59752C and ESRa G75935C mutations in Luxi cattle Locus

Genotype

Genotypic frequency

Allele frequency

v2-value

PGR G59752C

GG (65)

0.492

G 0.682

2.149 (P [ 0.05)

GC (50)

0.389

C 0.318

CC (17)

0.129

GG (11)

0.083

G 0.364

GC (74)

0.561

C 0.636

CC (47)

0.356

ESRa G75935C

5.77 (P [ 0.05)

Fig. 2 Sequence of genotypes GG, GC and CC of G75935C in bovine ESRa gene

Effect of genotypes on pregnancy rates and hormone concentrations Pregnancy rates and concentrations of E2 and P4 at day of ET of different genotypes are presented in Tables 2 and 3. For the PGR gene, the G59752C mutation had no significant effect on the pregnancy rates and concentrations of E2 and P4. But, recipients with g.59752 GG (50.77 %; 33 of 65) and g.59752 GC (50.0 %; 25 of 50) genotypes had obviously higher pregnancy rates after ET than g.59752 CC (35.30 %; 6 of 17) genotype (Table 2). For the ESRa gene, recipients with g.75935 GC (50.00 %; 37 of 74) and g.75935 CC (48.94 %; 23 of 47) genotypes had obviously higher pregnancy rates after ET than g.75935 GG (35.30 %; 4 of 11) genotype (Table 2). Furthermore, the same tendency was observed for these two genes that the same genotype groups with high pregnancy rates had high P4 concentration and low E2 concentration at the day of embryo transfer (Table 3).

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Table 2 The pregnant rates of freezed embryo of different PGR and ESRa genotypes after being transferred PGR

No. of transferred

Pregnant rate (%)

ESRa

No. of transferred

Pregnant rate (%)

g.59752 GG

65

50.77 (33/65)

g.75935 GG

11

36.36 (4/11)

g.59752 GC

50

50.00 (25/50)

g.75935GC

74

50.00 (37/74)

g.59752 CC

17

35.30 (6/17)

g.75935CC

47

48.94 (23/47)

P [ 0.05

P value

P value

P [ 0.05

Discussion Progesterone and estrogen play a central role in the regulation of the reproductive events associated with establishment and maintenance of pregnancy [15–17], so mutations in the PGR or ESR gene could affect reproductive ability. De Vivo et al. [38] detected a polymorphism of G331A in promoter region of human PGR gene, and found this mutation associated with the breast cancer. PGR PROGINS variants act as risk-modulating factors in several progesterone-dependent neoplasms, such as ovarian cancer and endometrial cancer [39, 40]; functional studies demonstrated that PROGINS variants reduced the response of PGR to progesterone by affecting gene expression and mRNA stability [41, 42]. Furthermore, the PROGINS variant of V660L was also significantly associated with an increased likelihood of repeated miscarriage [34, 43–45]. In cattle, PGR polymorphisms had significant effects on fertilization and embryonic survival rates in vitro studies [36], and significantly associated with superovulation traits [37]. In this study, statistical analysis revealed that recipients with PGR g.59752 GG and g.59752 GC genotypes had obviously higher pregnancy rates after ET than g.59752 CC genotype. Although this SNP investigated in this study is intronic, that means they do not change the structure of the encoded protein. But, previous studies shown that SNPs in introns can affect in gene expression, phenotype and

consequently function [46–48]. McKenna et al. [49] reported that the PROGINS Alu insertion in the G intron of the PGR gene could increase mRNA stability and transcribe to a more stable and transcriptionally active protein. So the PGR G59752C variation might decrease the PGR mRNA stability and then influenced the actions of progesterone, and finally resulted in a lower pregnancy rates after ET, but further verification is needed. Furthermore, our results found that recipients with g.59752 GG and g.59752 GC genotypes had high level of P4 concentration and low level of E2 concentration at day of embryo transfer. This may be due to the PGR G59752C variation influenced the actions of progesterone and then decreased the inhibitory effect of progesterone on hypothalamus and anterior pituitary, and finally increased the secretion of estrogen [50–52]. Low P4 concentrations after ovulation or a delay in the rise of P4 had significant negative effects on the embryo development and survival [20, 21], and P4 levels at day of embryo transfer has been found in correlation with the pregnancy rate [25–27]. This could be another reason why recipients with g.59752 GG and g.59752 GC genotypes had obviously higher pregnancy rates after ET than g.59752 CC genotype. ESRa mutations have been identified in spontaneous abortion and in human breast cancer [53]. The PvuII polymorphism located in intron 1 of ESRa in particular significantly associated with an increased risk of spontaneous abortion [54], and functional studies found that the T-allele of the PvuII RFLP eliminates a functional binding site for the transcription factor B-myb, and then resulted in lower ESRa transcription [55]. Furthermore, Schuit et al. [56] reported that the ESRa PvuII T-allele significantly associated with decreased plasma E2 levels in an allele dose-dependent manner in postmenopausal women. In this study, the results showed that recipients with ESRa g.75935 GC and g.75935 CC genotypes had low E2 concentration and high P4 concentration at the day of embryo transfer. Although this SNP investigated in this study is intronic, this mutation may destroy a functional binding

Table 3 Effects of different bovine PGR and ESRa genotypes on serum concentrations of estrogen and progesterone Hormone

Genotype at PGR G59752C

P value

g.59752 GG

g.59752 GC

g.59752 CC

Estrogen (pg/mL)

3.300 ± 0.436

3.429 ± 0.521

4.643 ± 0.897

[0.05

Progesterone (ng/mL)

0.184 ± 0.037

0.191 ± 0.043

0.121 ± 0.034

[0.05

Hormone

Genotype at ESRa G75935C

P value

g.75935 GG

g.75935 GC

g.75935 CC

Estrogen (pg/mL)

4.937 ± 1.518

3.848 ± 0.439

2.653 ± 0.381

[0.05

Progesterone (ng/mL)

0.115 ± 0.045

0.172 ± 0.035

0.204 ± 0.043

[0.05

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site and then decreased the expression of ESRa, and finally resulted in low levels of E2 [55, 56]. Furthermore, low levels of E2 decreased the inhibitory effect of estrogen on the activity of 3 beta-hydroxysteroid dehydrogenase, and then increased the secretion of progesterone [57]. Nishigai et al. [28] reported that the pregnancy rate tended to rise when the blood estrogen concentration was low at the day of ET, and indicated that it was very important to select bovine recipients with the high blood P4 concentration and with the low blood E2 concentration at the day of ET for improvement of the pregnancy rate. So, this may be the reason why recipients with ESRa g.75935 GC and g.75935 CC genotypes had obviously higher pregnancy rates after ET than g.75935 CC genotype. In conclusion, our results showed for the first time that PGR G59752C and ESRa G75935C polymorphisms had obvious effects on the pregnancy rates after ET, and indicated that PGR G59752C and ESRa G75935C polymorphisms could be potential markers for recipient selection of ET. However, larger sample sizes are required to corroborate this finding. Acknowledgments This work was supported by Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (No.2009SC005) and the earmarked fund for Modern Agro-industry Technology Research System (nycytx-10).

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