M. J. Argente

Identification of Single-Nucleotide Polymorphism in the Progesterone Receptor Gene and Its Association With Reproductive Traits in Rabbits

A total of 598 F2 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 5′-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.

Candidate gene analysis for reproductive traits in two lines of rabbits divergently selected for uterine capacity.

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.

Expression of progesterone receptor related to the polymorphism in the PGR gene in the rabbit reproductive tract

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.

Investigation of the oviductal glycoprotein 1 (OVGP1) gene associated with embryo survival and development in the rabbit.

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).

Analysis of the oviductal glycoprotein 1 polymorphisms and their effects on components of litter size in rabbits

The objective of this work was to study the effect of the oviductal glycoprotein 1 (OVGP1) genotype and mRNA expression on litter size and other fertility measures, as OVGP1 has positive effects on fertilization and early embryo development. We have analysed an F(2) cross of two lines of rabbits divergently selected for uterine capacity. The OVGP1 mRNA expression was analysed in both lines, but no differences were observed between them. The promoter region and mRNA were sequenced in the F(0) generation, and 17 polymorphic sites were found to co-segregate in three haplotypes (A, B and C). An association study was performed between several reproductive traits and a triallelic microsatellite identified in the promoter region as well as a non-synonymous SNP located in exon 11 [g.12944C>G (p.Arg468Gly)]. The alleles g.12944G and g.325(GT)(14)T(G)(5) of the B haplotype have a positive effect on the total number of kits born, number born alive, number of implanted embryos and foetal and prenatal embryo survival.

Identification of differentially expressed genes in the oviduct of two rabbit lines divergently selected for uterine capacity using suppression subtractive hybridization

Suppressive subtractive hybridization libraries from oviduct at 62 h post-mating of two lines of rabbits divergently selected for uterine capacity were generated to identify differentially expressed genes. A total of 438 singletons and 126 contigs were obtained by cluster assembly and sequence alignment of 704 expressed sequence tags (ESTs), of which 54% showed homology to known proteins of the non-redundant NCBI databases. Differential screening by dot blot validated 71 ESTs, of which 47 showed similarity to known genes. Transcripts of genes were functionally annotated in the molecular function and the biological process gene ontology categories using the BLAST2GO software and were assigned to reproductive developmental process, immune response, amino acid metabolism and degradation, response to stress and apoptosis terms. Finally, three interesting genes, PGR, HSD17B4 and ERO1L, were identified as overexpressed in the low line using RT-qPCR. Our study provides a list of candidate genes that can be useful to understanding the molecular mechanisms underlying the phenotypic differences observed in early embryo survival and development traits.