Association of a missense mutation in the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers

Background Upon binding luteinizing hormone in the ovary, the luteinizing hormone/choriogonadotropin receptor (LHCGR) is necessary for follicular maturation and ovulation, as well as luteal function. We detected mutations in the LHCGR gene and evaluated their association with superovulation. Methods Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing, we examined polymorphisms in LHCGR and the genotypes associated with superovulation traits in 127 Chinese Holstein heifers. Results A G/T polymorphism (ss52050737) in exon 11 was significantly associated with the total number of ova and the number of transferable embryos. Conclusions LHCGR may be a new predictor for superovulation in Chinese Holstein heifers.

The technologies for achieving multiple ovulation and embryo transfer (MOET) are well established, and more than 500,000 embryos are produced annually from superovulated cows worldwide [16]. However, variability in the superovulation response among individuals continues to be one of the most frustrating problems with respect to embryo transfer in cattle [17]. Although great progress has been made toward understanding folliculogenesis and regulation of the hypothalamic-pituitarygonadal axis, and many approaches to superovulatory treatments have been explored, the high variability among individuals in ovarian responsiveness to the application of exogenous hormones remains a challenge. One reason for this variability is that superovulation traits are quantitative and are strongly affected by the environment. Adjustments to superovulation protocols related to luteinizing hormone/choriogonadotropin receptor (LHCGR) availability may increase the yield of bovine embryos [18]. The follicle stimulating hormone receptor (FSHR), inhibin alpha (INHA) and progesterone receptor (PGR) genes have been reported as predictors of superovulation in Chinese Holstein cows [19][20][21]. In addition, three single-nucleotide polymorphisms (SNPs) in exon 11 of the LHCGR gene are associated with bovine fertility traits [22], and four SNPs of G51656T, A51703G, A51726G and G51737A in intron 9 of the LHCGR gene had significant effects on the total number of ova (TNO) recovered from superovulated Holstein cows [23]. Therefore, LHCGR was examined further as a candidate gene for predicting response to superovulation in Chinese Holstein heifers. The objective of this study was to investigate the prognostic significance of LHCGR genotypes in superovulation responsiveness.

Experimental animals and sampling
A total of 127 Chinese Holstein heifers from 60.6 to 68.7 week of age old were obtained from four breeding farms of Beijing Dairy Cattle Center and used for superovulation treatment (see below). Blood samples were collected form each heifer aseptically from the caudal vein into a tube containing EDTA (ethylene diamine tetraacetic acid) as an anticoagulant. Genomic DNA was extracted using the RelaxGene Blood DNA System (Tiangen, Biotech, Co. Ltd.) and stored at −20°C.

Superovulation and embryo harvest
Superovulation was induced throughout the year except for the months of June, July and August using a 4-day regimen of decreasing doses (i.e. dose of 75, 75, 50, 50, 30, 30, 10 and 10 mg for each injection) of follicle stimulating hormone (Folltropin-V; Vetrepharm Canada Inc. , Belleville, ON, Canada) given at 12 h intervals beginning on day 4 after insertion of an intravaginal progesteronereleasing device (Day 0; CIDR, Inter Ag). A dose of 0.4 mg Prostaglandin F2α (Shanghai Institute of Planned Parenthood Research; SIPPR, Shanghai, China) was given to each heifer on the morning of day 6, and the CIDR device was removed that afternoon. Estrus was detected visually on the morning of day 8. At approximately 7:00 p.m. on day 8, each heifer was randomly artificially inseminated (AI) with the first of two straws from one of eight bulls containing sexed frozen-thawed sperm. AI with the second straw was performed 12 h later. On day 16, each uterine horn was flushed nonsurgically with 500 ml Dulbecco's phosphate-buffered saline and the recovered fluid was examined for oocytes or embryos under a stereomicroscope. Embryos were isolated and classified as transferable embryos or degenerating embryos according to the criteria of the International Embryo Transfer Society (IETS). Oocytes were defined as unfertilized ova. Heifers with no oocytes or embryos were defined as non-responders.

Statistical analysis
Association of the genotype of the mutation with superovulation traits was calculated using the General Linear Model of PASW Statistics 18 (SPSS Inc. , Chicago, Illinois, USA). The factors considered were genotype, yard, age, season, and bull. The statistical model was where Y jkhi was the phenotypic value of traits, u was the population mean, G j was the fixed effect of genotypes, A k was the effect of age and acted as a covariate, S h was the fixed effect of season (spring, autumn and winter), D i was the effect of the yard, B l was the effect of the bull and acted as a random variable, and e jkhil was the random residual error. For the TNO, the factor of the bull was removed from the model.

Genotypic and allelic frequencies
After DNA sequencing, we found a single mutation (ss52050737) in the 1049 bp fragment amplified with primer set 1. Genotyping of the mutation was performed by SSCP with primer set 2, and the corresponding three genotypes were named GG, GT, and TT ( Figure 1). Products with three genotypes were sequenced respectively for sake of avoiding the false positive of SSCP (Figure 2). The frequencies of the G and T alleles were 0.398 and 0.602, respectively, and the frequencies of the GG, GT, and TT genotypes were 0.150, 0.496, and 0.354, respectively.

Association of genotypes with superovulation traits
The numbers of non-responders with the GG, GT, and TT genotypes were 2, 7, and 4 respectively. The results of association analysis between genotypes and superovulation traits are shown in Table 1. Heifers with the GG and GT genotypes had a significantly higher total number of ova (TNO) than those with the TT genotype (P < 0.05), and heifers with the GG genotype had a significantly higher number of transferable embryos (NTE) than those with GT and TT (P < 0.05). Heifers with the GG genotype had a smaller number of unfertilized ova (NUO) than those with the GT and TT genotypes, although only the difference between GG and GT was significant. There were no significant differences in the number of degenerate embryos (NDE) among the three genotypes.

Discussion
The use of peripubertal donors in embryo transfer (ET) programs has been studied, and no differences in TNO or NTE have been found among heifers that were older than 10 months of age [24]. Furthermore, reproductive and lactational traits of heifers subjected to embryo transfer do not differ from their untreated full siblings [24]. Thus, the heifers in this study can be used as donors in embryo transfer programs without compromising their subsequent reproductive or lactational performance [24]. Hayakawa et al. (2009) compared the outcomes of superovulation and embryo transfer with sexed and unsexed sperm and observed no difference between the two groups for superovulation traits or pregnancy rates [25]. These observations are consistent with results of our previous study [26]. For AI in our study, sexed frozen-thawed sperm from different bulls was used randomly to inseminate heifers.
Heifers with the GG genotype had significantly higher TNO and NTE than those with the TT genotype, indicating that the G allele had a favorable, positive effect on these two superovulation traits. Three SNPs (ss52050737,  ss52050738, and ss52050739) in exon 11 of LHCGR and their significant associations with variations in cattle fertility were described [22], and our results further demonstrated the SNP (ss52050737), in particular, was associated with a positive response to superovulation. As a response to superovulatory treatment in cattle, more ovarian follicles initiate growth, granulosa cells proliferate and acquire follicle stimulating hormone receptors, and LHRs are induced, permitting the granulosa cells to later respond to LH [1]. Subsequently, several events occur: resumption of oocyte meiosis, transformation of the steroid enzyme complex from production of estrogen to production of progesterone, follicular rupture, and finally corpus luteum formation when serum LH levels increase during the preovulatory LH surge [1]. In cattle, moreover, LH may promote cytoplasmic maturation or competence of the oocyte as well as embryonic development after fertilization, both in vivo and in vitro [4,5,27,28]. The presence of LHRs in bovine oocytes, embryos, and blastocysts has been reported [5]. Bovine oviducts also express LHRs, and their activation results in increased synthesis of oviductal glycoprotein [29], which binds to embryos to enhance their development [30][31][32][33][34]. Thus, LH may enhance embryonic development through both direct and indirect mechanisms after binding to its receptor. As a G protein-coupled receptor, binding of the LHCGR allows dissociation of membrane-bound cognate G proteins that regulate phospholipase C, adenylyl cyclase, and ion channels, which in turn control cellular inositol phosphates, cAMP, Ca 2+ , and other secondary messengers [8]. Exon 11, in which SNP ss52050737 is located, encodes the intracellular domain of the LHCGR protein. Therefore, a missense mutation may change the structure of the intracellular region of the LHCGR and reduce the effect of LH to explain the functional relevance of this mutation to variation in response to superovulation and phenotype. However, the biological mechanism by which the mutation influences the function of LHCGR requires further investigation.