A total of 598 F 2 does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1-8 were amplified and sequenced. One SNP was found in the promoter region, 2464G.A, three SNPs in the 59-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes.

The association of the 2464G > A SNP found in the promoter region of the rabbit progesterone receptor gene with progesterone receptor (PR) expression was evaluated by Western blot analysis. This SNP was associated with 2 lines divergently selected for uterine capacity, the high line selected to increase uterine capacity and the low line selected to decrease uterine capacity. Two progesterone isoforms were obtained using a commercial monoclonal antibody: the PR-B isoform described previously in rabbits, and the PR-A isoform, not described previously in rabbits. The GG genotype, the genotype more frequent in the high line, showed less PR-B and PR-A expression than the AA genotype in the oviduct (GG/AA(PR-B) = 0.81 and GG/AA(PR-A) = 0.73) and uterus (around 0.70 in both isoforms). The GA genotype showed similar PR-A expression in both tissues and also similar PR-B expression in the oviduct to the GG genotype. Conversely, the GG genotype showed less PR-B expression than the GA genotype in the uterus (GG/GA(PR-B) = 0.79). Similar expression of both PR isoforms was found in the uterus at d 2 and 3 of gestation; meanwhile, an increase of both isoforms was observed in the oviduct. Similar PR-A expression was observed in the ampulla and isthmus; meanwhile, the PR-B expression in the isthmus was double that in the ampulla.

The objective of this work was to analyze 3 functional candidate genes for reproduction in 2 lines of rabbits divergently selected by uterine capacity. Both lines were selected for 10 generations. The selection was then relaxed until the 17th generation, when it was compounded by 61 and 63 does of the High and Low lines, respectively. We sequenced the SCGB1A1 gene, which encodes the main protein secreted by the rabbit in the uterus and seems to play an important role in implantation. We found 6 SNP in the promoter region cosegregating in 2 haplotypes in both lines with similar frequency. We also analyzed IGF1 mRNA because of its effects on embryo development, but we did not find any polymorphism between individuals of the 2 lines. The third gene analyzed was the TIMP1, which encodes a protein involved in many biological processes related to reproduction. We determined the sequence of its promoter region and found 1 SNP (g.1423A>G) segregating with different frequencies in both lines (0.60 for allele A in the High line and 0.82 for allele G in the Low line). The association study performed in an F(2) population (n = 598) generated by the cross of the 2 lines of rabbits revealed that the AA genotype had 0.88 embryos more than the GG genotype at 72 h of gestation. The difference increased to 2.23 embryos at implantation, but no difference was found between genotypes at birth. These results suggest that TIMP1 could be a candidate gene for embryo implantation and embryo survival.

The OVGP1 is an oviductal glycoprotein that has positive effects on fertilization and early embryo development. We have amplified and sequenced the rabbit OVGP1 gene, which spans 13 kb and it is formed by 11 exons and 10 introns. To find polymorphisms, a region encompassing the promoter to intron 1 has been sequenced in 22 rabbits of the H, V, R, and A Spanish lines. We have identified five SNPs and one triallelic microsatellite in the promoter region, and three SNPs and one dinucleotide INDEL in intron 1. The 10 polymorphic sites cosegregate forming two haplotypes. The allelic frequencies of the microsatellite have been analyzed in 98 rabbits belonging to the four lines and the three alleles were found in all the lines. The relative quantification of the OVGP1 mRNA in liver, kidneys, lungs, skeletal muscle, ovary, uterus, and oviduct reveals that the OVGP1 expression in the oviduct is 5,500 higher than in the uterus or ovary, whereas a low level of basal expression is detected in non-reproductive tissues. We have also compared the mRNA expression between samples of oviducts taken from non-mated rabbit and samples of oviducts at different stages of the early pregnancy, but no significant differences were found.

An association study was performed in rabbits between early embryo survival and development, and the nonconservative SNP 12944C>G located in exon 11 and the triallellic microsatellite [(GT)(15)T(G)(5), (GT)(14)T(G)(5), and (GT)(11)T(G)(7))] located in the promoter region of the oviductal glycoprotein 1 (OVGP1) gene. We analyzed an F(2) cross of 2 lines of rabbits divergently selected for uterine capacity. A total of 172 and 159 females were slaughtered at 48 and 72 h of gestation, respectively, to determine whether OVGP1 influences ovulation rate, fertilization rate, early embryo survival, and embryonic stage of development. The results of the SNP indicated that all genotypes showed similar early embryo survival and a similar embryonic stage of development at 48 h of gestation. However, at 72 h of gestation, the GG genotype showed greater early embryo survival than the CC genotype (0.56 embryos) and their embryos presented less embryonic development. Analysis of the microsatellite was performed to ascertain the presence or absence of the allele (GT)(14)T(G)(5). At both stages of gestation, the (GT)(14)T(G)(5)/(GT)(14)T(G)(5) genotype showed greater early embryo survival (0.94 and 1.54 embryos at 48 and 72 h of gestation, respectively) and less embryonic development than the homozygous genotypes without the allele (GT)(14)T(G)(5).