Mol Biol Rep (2011) 38:17–21 DOI 10.1007/s11033-010-0072-8

An MspI polymorphism in the inhibin alpha gene and its associations with superovulation traits in Chinese Holstein cows Ke-Qiong Tang • Shu-Jing Li • Wu-Cai Yang • Jun-Na Yu • Li Han • Xiang Li • Li-Guo Yang

Received: 9 December 2009 / Accepted: 5 March 2010 / Published online: 18 March 2010 Ó Springer Science+Business Media B.V. 2010

Abstract To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, the PCR–RFLP method was applied to detect an A[G transition determining an MspI polymorphism at position 192 in the exon I of the bovine inhibin alpha (INHA) gene and evaluate its associations with superovulatory response in 118 Chinese Holstein cows treated for superovulation. Association analysis showed that cows with the GG genotype resulted in a significant increase in the number of ova (TNO) than AG and AA genotypes in the first (P = 0.023), second (P = 0.004) and third (P = 0.002) superovulation treatments and produced more transferable embryos (NTE) than that of AG and AA genotypes in the third (P = 0.045) superovulation treatment. Moreover, individuals with GG genotype produced more transferable embryos than AA (P \ 0.05) genotype in the second superovulation treatment and all

Electronic supplementary material The online version of this article (doi:10.1007/s11033-010-0072-8) contains supplementary material, which is available to authorized users. K.-Q. Tang  W.-C. Yang  J.-N. Yu  L. Han  X. Li  L.-G. Yang Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China S.-J. Li Beijing Amber Embryo Technology Co. Ltd, Beijing 100107, China L.-G. Yang (&) College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected]

cows without superovulation response were mutations with genotypes of AA and AG. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices. Keywords MspI

Inhibin alpha  Superovulation  RFLP 

Introduction Superovulation is a major component for successful embryo transfer technique which has been used worldwide to produce valuable bovine embryos for breeding [1]. However, the number of transferable embryos has not changed markedly in the last 20 yr, and the use of Multiple Ovulation and Embryo Transfer (MOET) technology in the animal industries is approaching a plateau. The major limitation to its development is its reliance on FSH-induced superovulation and the large variability in response to treatments [2]. The number of follicles that develop to ovulatory size depends on both the amount of FSH and the time of exposure to FSH [3, 4]. The release of FSH by the pituitary is in tune controlled by synergistic action of two of the major products of ovulatory follicles, inhibin and estradiol [5]. A reduction in circulating inhibin or in inhibin activity may result in an increased level of FSH and an increase in the number of follicles recruited [6–8]. Furthermore, inhibin had significant effect on expression of FSHR througu FSH action and inhibin A has been considered as an autocrine negative regulator of FSHR expression in the ovary [9]. The INHA gene encodes the a-subunit of inhibins has been widely research in human and animals [10]. Passive

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and active immunizations against inhibin a-subunit can affect FSHR expression to improve ovarian response to superovulation, and then lead to increase ovulation rate and higher yield of transferable embryos in heifers or cows [11–14]. Schneyer et al. [15] had demonstrated that proteins derived from the alpha-inhibin precursor modulate FSH binding to its receptor as well as its biological activity. Due to its role in negative feedback control of FSH, the INHA has been proposed as risk factors for premature ovarian failure (POF) [6]. A missense (A257T) of human INHA locus was first reported and detailed significant associations with POF, and function analysis found that the A257T mutation was sufficient to impair the binding affinity of inhibin to its receptors, and leaded to the subsequent inability to regulate the FSH level by negative feedback [10]. Moreover, two INHA SNPs of 129C[T and 675C[T were found in human and the association analysis indicated that the 129C[T SNP associated with susceptibility to POF [16]. In pigs, INHA gene linkage mapping to SSC15 and INHA SNPs have been considered as candidate gene markers for ovulation rates in pigs [17, 18]. Furthermore, one INHA SNP of G284A was identified and the association analysis represented that allele G was positively correlated with litter size, and therefore INHA gene was a major gene controlling the productivity of goat [19]. To date, no significant association of INHA polymorphisms with productivity traits of bovine has been revealed. Herein, this study reports for the first time the distribution of the MspI polymorphism of bovine gene in 118 Chinese Holstein cows and evaluate its association with superovulation traits, in order to investigate the prognostic significance of the INHA gene in superovulation responsiveness.

Materials and Methods Experimental animals and sampling All procedures involving animals were approved by the Animal Care and Use Committee of Huazhong Agricultural University. A total of 118 cows in Chinese Holstein breed (included 6 cows without superovulation response) were given two to four successive superovulation treatments in same protocol at monthly interval (Supplemental Table 1) at Beijing Amber Embryo Technology Co. Ltd. in China in 2007. Approximate 10 ml blood per cow was collected aseptically from the jugular vein and kept in a tube containing anticoagulant EDTA (Ethylene diamine tetraacetic acid) and held on ice until delivered to the laboratory. Genomic DNA were isolated from blood samples and stored at -20°C, following standard procedures [20].

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Superovulation procedure and Embryos harvest Gonadotrophin treatment began on day 4 after insertion of an intravaginal progesterone releasing device (PRID1; Ceva Sante Animale, France). Superovulation was induced by intramuscular injections of 15.6 ml FSH (Folltropin-V, Bioniche Animal Health Canada Inc.) administered in eight decreasing doses (2.7/2.7; 2.2/2.2; 1.7/1.7; 1.2/1.2) at intervals of 12 h. Each cow was twice given 0.6 mg of prostaglandin F2a (Pronaonlg F1, Pfizer, USA, 25 and 15 mg of dinoprost, respectively) along with the sixth and seventh FSH treatments. The PRID was removed following second prostaglandin treatment. Estrus detection by visual observation was carried out twice daily at approximately 12-h intervals for a minimum of 30 min per observation. Any cow standing to be mounted by a herd-mate was considered as being in heat. All cows were inseminated at 12 and 24 h after standing estrus using frozen-thawed semen from one bull of known good fertility. Uteri were non-surgically flushed on day 7 of post-AI (artificial insemination) by an experienced technician using standard techniques. Each uterine horn was flushed with 500 ml of phosphate buffer saline (PBS). In order to avoid forming permanent corpus lutea, PGF2a was administered to all cows immediately after the flushing procedure. Following flushing, the recovered lavage was filtered through an embryo filter (Miniflush Embryo Recovery System, mesh size 44 lm, Minitub, Germany). The fluid was examined for oocytes or embryos under a stereomicroscope and embryos were isolated and graded in A, B, C and D according to the criteria of the International Embryo Transfer Society (IETS). Grades A and B embryos were defined as transferable embryos, and grades C and D embryos as degenerate embryos. Oocytes were defined as unfertilized ova and counted on the basis of corpus lutea. Cows with no oocyte and embryo recovered in two successive superovulation treatments were defined as non responders or cows without superovulation response. The responsiveness of superovulation included the total number of ova (TNO), the number of transferable embryos (NTE), the number of unfertilized ova (NUO) and the number of degenerate embryos (NDE). PCR conditionns One reported SNP of A192G in the bovine INHA gene (GenBank number: rs41257116), which formed a MspI endonuclease restriction site (GGGAC), was selected as a marker to evaluate their effects on superovulation traits of Chinese Holstein cows. A pair of primers (F: 50 -GCCCT GTTTCTGGATGCC-30 and R: 50 -ATTCAACCCAACCT GCCTA-30 ) were synthesized to amplify a 249 bp product of the INHA exon 1. The PCR amplified fragment contained two common and one mutant MspI restriction sites

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at position 31, 154 and 75, respectively. The PCR was performed in a 20 ll reaction mixture containing 10 pmol primers, 200 lM of dNTP (deoxyribo nucleotide triphosphate), 2.5 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. The cycling protocol was 5 min at 95°C followed by 34 cycles (94°C for 45 s, 62°C annealing for 45 s, 72°C for 45 s), with a final extension at 72°C for 10 min.

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fitted in an animal model according to the founding that reproductive aging in cattle was associated with a reduced superstimulatory and superovulatory response [21]. The model showed as follow: yikj ¼ u þ Gi þ Nk þ Tj þ eikj where yikj was phenotypic value of traits; u was the population mean; Gi was fixed effect of genotype; Nk was fixed effect of age; Tj was fixed effect of contemporary group; eikj was random residual error.

Genotyping of MspI INHA allele by PCR–RFLP Aliquots of 5 ll PCR products of INHA gene were digested with 5 U MspI (TaKaRa, Tokyo, Japan) for 8 h at 37°C following the supplier’s instructions. The digested products were detected by 8% PAGE (polyacrylamide gel electrophoresis) and the subsequent silver staining. To confirm the results obtained with the PCR–RFLP technique, six PCR products, three representative for GG and three for AG genotypes, were sequenced in both directions in an ABI PRIZM 377 DNA sequencer (Perkin-Elmer).

Table 1 Association analysis of bovine INHA genotypes with the first and second superovulation traits of the number of ova (TNO), transferable embryos (NTE), unfertilized ova (NUO) and degenerate embryos (NDE) in Chinese Holstein cows First

AA(25)

AG(80)

GG(13)

P value

NUO 0.840 ± 0.256b 1.487 ± 0.256b

2.923 ± 1.077a 0.048

NDE 1.600 ± 0.409 NTE 3.040 ± 0.515

2.462 ± 0.739 5.077 ± 1.179

1.875 ± 0.257 3.900 ± 0.461

0.479 0.240

TNO 5.480 ± 0.719a 7.262 ± 0.651ab 10.462 ± 1.475b 0.023

Statistical analysis

Second AA(25) NUO

Gene frequencies were determined by direct counting. The associations of the animal genotypes of INHA with superovulation traits including TNO, NDE, NTE and NUO were calculated by General Linear Model of SAS 8.1 (SAS Inst. Inc., Cary, NC). The significance of the differences among four superovulation treatments was determined by Duncan’s test at the significant level of 0.05 and 0.01 followed by one-way ANOVA program of SAS procedures. The ages of superovulated cows that were divided into three groups, namely 2.0–3.0 years, 3.1–4.0 years and 4.1–5.0 years was

NDE NTE TNO

AG(80) ab

1.346 ± 0.457

A

1.308 ± 0.383

a

2.962 ± 0.482

A

5.615 ± 0.879

P value

2.615 ± 1.124 0.065

A

3.615 ± 0.561B 0.007

ab

6.154 ± 1.400b 0.252

A

12.385 ± 1.492B 0.004

1.038 ± 0.230 1.873 ± 0.271 4.165 ± 0.530 7.076 ± 0.671

b

Note: Figure in brackets is the number of samples. Least square mean values (±SE) with different letters are significantly different with lower case superscripts at P \ 0.05 and upper case superscripts at P \ 0.01

Table 2 Association analysis of bovine INHA genotypes with the third and fourth superovulation traits of the number of ova (TNO), transferable embryos (NTE), unfertilized ova (NUO) and degenerate embryos (NDE) in Chinese Holstein cows Third AA(21)

AG(60)

NUO 0.857 ± 0.232

GG(11)

1.000 ± 0.261

P value

1.818 ± 0.600

0.224

NDE 1.333 ± 0.438a 1.667 ± 0.304a

3.273 ± 0.506b 0.013

NTE 4.190 ± 0.827a 3.733 ± 0.523a

7.364 ± 1.416b 0.045

A

TNO 6.381 ± 1.099

6.400 ± 0.620

12.456 ± 1.522B 0.002

Fourth AA(19)

AG(43)

GG(9)

NUO

Fig. 1 Representative genotyping INHA gene by a polyacrylamide gel electrophoresis. Strands with 123, 95 and 31 bp for AA genotype, 95, 79, 44 and 31 bp for GG genotype, 123, 95, 79, 44 and 31 bp for AG genotype appeared at this locus. M represented a marker with pBR322 DNA/MspI (TaKaRa, Tokyo, Japan)

GG(13) a

A

0.895 ± 0.366a 1.814 ± 0.353a

P value

4.00 ± 1.423b 0.012

NDE

2.211 ± 0.691 2.116 ± 0.353

2.889 ± 0.754

0.753

NTE

3.263 ± 0.577 4.163 ± .655

3.667 ± 1.280

0.772

TNO

6.368 ± 1.212a 8.093 ± 0.806ab 10.556 ± 1.464b 0.189

Note: Figure in brackets is the number of samples. Least square mean values (±SE) with different letters are significantly different with lower case superscripts at P \ 0.05 and upper case superscripts at P \ 0.01

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Table 3 Effect of repeated FSH superovulation treatments on superovulation traits of the number of ova (TNO), transferable embryos (NTE), unfertilized ova (NUO) and degenerate embryos (NDE) in Chinese Holstein cows Trait

First

Second

Third

Fourth

P value

TNO

7.237 ± 0.506

7.339 ± 0.541

7.120 ± 0.544

8.057 ± 0.623

0.643

NTE

3.848 ± 0.357

4.118 ± 0.406

4.272 ± 0.437

3.914 ± 0.456

0.892

NUO

1.509 ± 0.221

1.280 ± 0.223

1.065 ± 0.193

1.871 ± 0.295

0.076

NDE

1.881 ± 0.210

1.941 ± 0.216

1.783 ± 0.236

2.271 ± 0.298

0.473

Note: Data represent least square means and standard errors

Results and discussion In order to better describe different genotypes of A[G mutation of bovine INHA gene, we name the alleles as INHA-A and INHA-G. The mutation A[G (ACCGAAG to ACCGGAG) locates at position 192 in exon I forms an MspI endonuclease restriction site (CCGG). Therefore, the MspI digestion of amplified products shows three fragments (123, 95 and 31 bp) for INHA-A allele, four fragments (95, 79, 44 and 31) for INHA-G allele (Fig. 1). Frequencies of A and G alleles are 0.551 and 0.449 in analyzed population and all non responders belonged to genotypes of AA and AG (Supplemental Table 2). The results of association analysis between the INHA gene and superovulation traits in Chinese Holstein cows were summarized in Tables 1 and 2. Cows with the GG genotype had a significantly higher TNO than AG and AA genotypes in the first (P = 0.023), second (P = 0.004) and third (P = 0.002) superovulation treatments (Tables 1 and 2). At the same time, NTE in GG individuals was significantly higher than that of AG and AA in the third (P = 0.045) superovulation treatment (Table 2). Cows with GG genotype produced more transferable embryos than AA genotype (P \ 0.045) in the third superovulation treatment. Moreover, individuals with GG genotype had better performance in NTE in the first, second and fourth superovulation treatments (Tables 1 and 2). The G allele had a favorable, positive effect on superovulation traits of TNO and NTE. Conversely, GG genotype was also associated with NUO in first (P = 0.048), second (P = 0.065) and fourth (P = 0.012) superovulation treatments and produced significant increase in NDE than AA and AG genotypes in the second (P = 0.007) and third (P = 0.013) superovulation treatments (Tables 1 and 2). We consider that these associations can be explained by the following two possible reasons. (1) Although this mutation doesn’t change amino acid sequence, it possibly decreases inhibin concentrations by affecting the expression of the INHA gene and the stability of the INHA transcription, and then resulted in an increase of the concentrations of FSH by removing the negative feedback to the pituitary. It has been observed that the synonymous SNP can affect in vivo

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protein folding and consequently function, as well as gene expression and phenotype [22–27]. (2) The negative effects on NUO and NDE, positive effects on TNO and NTE might be explained by the positive genetic correlation existed between these traits. In this MspI polymorphisms, all non responders belonged to genotypes of AA and AG, so it was implied that individuals with A allele were more likely to be non-responders. It is an interesting phenomenon worth for further investigation. Moreover, the association analysis revealed that contemporary group only had a significant effect on NDE in the third superovulation, and there were no significant correlation existed between age and superovulation traits (Supplemental Table 3 and Table 4). At the same time, no significant difference existed among four superovulation treatments (Table 3) in our experiment. The result indicates that the repeated superovulations with the same gonadotrophin for four consequent superovulation treatments at interval of 1 month did not lead to a decreased response, which implies that the effect of repeated superovulation on ovarian response depends strongly on the genetic background of cows. However, further studies with multiple superovulations encompassing longer periods are needed. In this study, we first reported the MspI polymorphism in bovine INHA gene, and its correlation with the superovulation traits in bovine. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices. Acknowledgments This work was supported by both of the National High Technology Research and Development Program of China (863 program) (No.2007AA10Z152) and National Key Technology R&D Program (No. 2006BAD04A02-11).

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