Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P < 0.001), averaging 2.1 ± 0.1 when compared to 1.2 ± 0.1 in wild-type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P < 0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P < 0.001), averaging 1.61 ± 0.07 in heterozygotes and 1.29 ± 0.03 in wild-type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise.

Mechanisms of action of the principal prolific genes and their application to sheep production.

The prolificacy variation in sheep makes it an excellent animal model to understand the mechanisms regulating ovulation rate. Identification of mutations responsible for the increased prolificacy of the Inverdale, Booroola, Javanese, Cambridge and Belclare sheep open new avenues of investigation for the paracrine control of folliculogenesis. To date, all known mutations are in genes from ligands or receptors of the transforming growth factor beta superfamily, and point to the bone morphogenetic protein family of peptides as local regulators of ovarian follicle growth. The mechanism of action of the mutated genes is not fully understood, but results in the ovulation of a higher number of follicles with smaller diameter and fewer granulosa cells than that of the wildtype, thus speeding the differentiation of ovulatory follicles. Comparisons of the performance of Booroola-crossed flocks in different countries showed that carriers of the prolificacy mutation have higher ewe productivity but also higher perinatal mortality and lighter weight lambs. Their economic impact on the sheep industry depends on farm environment and management. Nevertheless, the diagnostic tests now available to identify the genetic mutations resulting in increased ovulation rate, will simplify the introduction of these mutations and their monitoring in flocks for research and commercial purposes.