Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits

Abstract

Background: The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine.

Results: Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126-128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p </= 0.03) and another possibly associated with age at puberty (p = 0.09).

Conclusion: AKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits. The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064-DQ474068, GenBank:DQ494488-DQ494490 and GenBank:DQ487182-DQ487184].

Figure 1

Alignment of pig AKR1C mRNAs. Conserved residues T24, L54, Y55, H117, F118, W227, N306 and Y310 involved in substrate binding are highlighted in yellow [52] and residues highlighted in blue are invariant residues found in all AKRs [53]. Residues Asp274Asn in AKR1C1, Ile16Phe, Tyr216Asn, Val234Phe in AKR1C2 and Val97Phe in AKR1C3 in red are non-synonymous amino acid polymorphisms identified in cDNA and genomic sequence of Meishan and White composite pigs. Dashes indicate residues that are absent, shading indicates identity and asterisks represent 10 bp increments.

Figure 2

Phylogenetic relationship of AKR1C family members of human, mouse, cow and pig. Multiple sequence alignment was performed by ClustalW and neighbor-joining tree of full-length amino acid sequences is shown. Species is designated by a lower case letter preceding the AKR1C gene family member (h = human; b = bovine; p = porcine and m = mouse). Numbers at branches represent 1000 bootstrapped datasets and scale bar represents substitutions per site. Human genes are from the AKR1C cluster on chromosome 10p15, mouse and bovine genes are from chromosomes 13 in both species and the five pig genes on SSC10q described in this study. In general, except for AKR1CL2, paralogues within species are more related than are homologues among species.

Figure 3

Radiation hybrid maps of AKR1C genes on IMpRH_7000rad and IMNpRH2_12000rad panels. The IMpRH map is aligned to human chromosome 10p15 (inverted).

Figure 4

BAC contig map of region containing pig AKR1C genes. Location and orientation of pig genes is shown above. BAC clone ends are represented as ● for SP6 primer sequence and ⊢ as the T7 primer sequence end. Clones in dashed lined were not subcloned and sequenced but aligned by BAC-end sequences (BES). The human position on HSA10 is shown in megabases and gene order is shown below. Not all porcine BACs in this region are shown.

Figure 5

RT-PCR amplification of pig AKR1C genes in different tissues from a purebred Meishan sow at day 25 of gestation and mature testis using gene-specific primers (Hyp, hypothalamus; Spl, spleen; Plc, placenta; Mam, mammary tissue; Pan, pancreas; Pit, pituitary; Lng, lung; Lvr, liver; Ovr, ovary; Adr, adrenal; Kid, kidney; LD, longissimus dorsi muscle; End, endometrium; Brn, brain; Int, small intestine; Tst, testis). Amplicon sizes are given in Table 1; marker is a 100 bp ladder.