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Researchers Link Four Variants to Conception Rate in Dairy Cows

NEW YORK (GenomeWeb News) – Four variants — two of which are within gap junction-related genes — are linked to conception rate in cows, a team of Japanese researchers reported in the Proceedings of the National Academy of Sciences today.

Those two genes, PKP2 and CTTNBP2NL, the team added, may affect whether embryos may implant. The other two variants the group uncovered, SETD6 and CACNB2, are located within neuroendocrine-related genes, which are involved in stimulating the release of follicle-stimulating hormone from the anterior pituitary gland. FSH, in turn, regulates reproductive processes.

"Our GWAS uncovered unexpected roles for these genes and provides a potential solution for the problem of declining conception rates in the livestock industry," the researchers led by Yoshikazu Sugimoto at the Shirakawa Institute of Animal Genetics in Fukushima said.

Dairy cattle, Sugimoto and his colleagues noted, have been bred for their milk yield, rather than for breeding capability, and the dairy industry has recently reported declining fertility in cows. Additionally, they said that candidate-gene approaches have generated long lists of genes that may be involved in conception in cows.

To search for a more specific group of genes linked to conception and fertility, Sugimoto and his colleagues conducted a genome-wide association study that compared 192 cows with high conception rates to 192 cows with low conception rates. They genotyped these 384 cows at 54,001 SNPs, and found six loci on five different chromosomes that correlated with conception rate. Two of those SNPs were not located near any known genes and were excluded from further analysis.

For each of the four remaining loci, the researchers examined and sequenced the exons as well as 5' and 3' UTR regions surrounding the loci to home in on potentially causal variants.

On chromosome 5, a deletion in PKP2 — a demosomal plaque protein whose expression affects Cx43 levels — popped out as a possible causal variant. Sugimoto and his colleagues genotyped an additional 1,034 cows and 2,528 sires and found that cattle with the deletion allele had higher conception rates. In addition, in a bovine endometrial cell line, the researchers noted increased PKP2 activity and higher mRNA levels were associated with the deletion allele, as compared to the wild-type allele. Further, they noted that the lower the PKP2 expression level, the lower the expression of Cx43, a gap junction protein, and the less calcium was diffused.

In addition, a variant on chromosome 3 in INRA-641, which is located in the 3'UTR of CTTNBP2NL, also appeared to affect conception in cows. Again, expression levels of CTTNBP2NL varied for one allele as compared to the other, suggesting to the researchers that those differences in expression could affect conception.

CTTNBP2NL, the researchers noted, has an unknown function, though, they determined that it interacts with PP2Ac, a protein phosphatase2A catalytic subunit, in bovine cells. Indeed, knockdown of CTTNBP2NL led to decreased phosphatase activity while its overexpression led to increased phosphatase activity.

One of the molecules PP2Ac targets is Cx43, the researchers said, and cells that over expressed CTTNBP2NL had lower levels of Cx43 phosphorylation.

Sugimoto and his colleagues examined the effects of these two genes in mice. PKP2, they found, improves cell communication through Cx43 while CTTNBP2NL inhibits it, also through Cx43. Cx43, they added, is expressed in mouse uterine stomal cells on day 5 of pregnancy, within 12 hours of the start of implantation.

By adding siPKP2 or CTTNBP2NL to mouse uteri, the researchers observed decreased Cx43 or decreased phosphorylation of Cx43, respectively. Through immunohistochemical assays, they determined that siPKP2 and CTTNBP2NL inhibits embryo implantation.

"PKP2 and CTTNBP2NL influence implantation through gap junction communication," the researchers wrote, noting that "[g]ap junction communication in the cumulus-oocyte complex of the ovary is also important for oocyte maturation and pregnancy outcome." They added that future studies should examine what molecules travel through gap junctions in the uterus and whether PKP2 and CTTNBP2NL play roles in oocyte maturation.

Meanwhile, the researchers picked up a signal on chromosome 18 that they whittled down to a variant in SETD6, whose protein product catalyzes the methylation of lysine in the RelA subunit of NF-κB. Among its many duties, NF-κB is involved in the transcriptional regulation of gonadotropin-releasing hormone, which, in turn, stimulates the release of follicle stimulating hormone and luteinizing hormone. They suggested that SETD6 might control FSH secretion via the transcriptional regulation of gonadotropin-releasing hormone.

A fourth region on chromosome 13 was traced to a deletion in CACNB2, and the researchers noted that cows with the deletion had higher conception rates than cows without it. Knockdown experiments of CACNB2 led to a decrease in FSH expression while over-expression of CACNB2 led to an increase of FSH expression.

Further, they reported that cows with both the deletion in CACNB2 and the SETD6 variant had even higher levels of FSH, indicating that CACNB2 may influence conception through the SETD6 pathway.

"Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry," Sugimoto and his colleagues wrote.