The genes in question, BMP15 and GDF9, are among the most celebrated regulators of female fertility in all of mammals. Both encode growth factors secreted by the oocyte, the egg cell itself, and both belong to the transforming growth factor-beta superfamily, a large group of signaling proteins that orchestrate development throughout the body. During folliculogenesis, the process by which ovarian follicles mature, BMP15 and GDF9 are produced by oocytes within growing follicles and act on the surrounding granulosa and cumulus cells in a paracrine fashion. BMP15 drives granulosa cell proliferation, cumulus expansion, and follicular development, while GDF9, located on ovine chromosome 5, is essential for early follicle progression, normal ovulation, fertilization, and subsequent reproductive performance. The stakes are high: in GDF9-null mice, and in ewes carrying naturally occurring inactivating GDF9 mutations, follicular development arrests entirely beyond the primary stage.

What makes these genes especially tantalizing for sheep breeders is their dosage-sensitive behavior. Landmark studies in New Zealand and Irish flocks showed that mutations in BMP15 can increase ovulation rate in heterozygous carriers while rendering homozygous carriers completely infertile—an unusual genetic pattern that made BMP15 a poster child for prolificacy research. Similar associations emerged in Cambridge and Belclare sheep, and Chinese breeds such as the prolific Small-tailed Han sheep have been screened extensively for BMP15 and GDF9 variants. Given this pedigree, the Qinghai team reasoned that if Tibetan sheep carried functional variants in these genes, those variants might explain—or eventually help breeders improve—their characteristically small litters.

The study combined gene cloning, bioinformatic prediction, quantitative real-time PCR, and a genotyping technology called improved multiplex ligase detection reaction, or iMLDR. From Tibetan ewes raised on a single commercial farm in Gonghe County, the researchers collected jugular vein blood from 200 healthy adults aged three to four years, each with complete litter-size records spanning two to three parities. All ewes were naturally mated during the same autumn breeding season, with no exogenous hormones administered, and lambing records were verified by farm technicians. A separate group of six six-month-old ewes from the same flock provided twelve tissues—hypothalamus, heart, lung, kidney, liver, spleen, muscle, uterus, ovary, oviduct, rumen, and duodenum—snap-frozen in liquid nitrogen for exploratory expression profiling.

Cloning revealed predicted open reading frames of 468 base pairs within the BMP15 fragment and 405 base pairs within the GDF9 fragment, encoding 155 and 134 amino acids respectively. Because these lengths fall far short of the known full-length ovine precursor proteins, the sequences must be regarded as partial coding fragments rather than complete coding sequences—a limitation the authors emphasize throughout. Phylogenetic analysis confirmed the fragments belong squarely with domestic sheep: the predicted amino acid sequences showed the highest similarity to Ovis aries reference sequences, followed by yak, with the lowest similarity to domestic dog for BMP15 and to cichlid fish for GDF9. Bioinformatic prediction painted a contrasting portrait of the two protein fragments. The BMP15 fragment showed slight overall hydrophobicity, with a grand average of hydropathicity of 0.026, and carried one transmembrane-like segment near its N-terminus spanning roughly residues 10 through 27. The GDF9 fragment was hydrophilic overall, with a hydropathicity average of minus 0.323, and carried a predicted Sec-type signal peptide with a potential cleavage site between amino acids 25 and 26—a hallmark of proteins targeted to the classical secretory pathway. The BMP15 fragment was predicted to be stable, with an instability index of 35.21, while the GDF9 fragment, at 53.62, was predicted unstable. Both fragments were dominated by random coils and alpha-helices, and each carried 13 predicted phosphorylation sites.

The expression results delivered one genuine surprise. As expected, GDF9 transcript abundance was highest in the ovary, consistent with its established role as an oocyte-derived factor, followed by kidney and uterus. But BMP15, rather than peaking in the ovary, showed its highest expression in the oviduct—the tube where gamete transport, fertilization, and early embryo–maternal communication occur—with a relatively high signal also detected in the liver. The authors stress that these extra-ovarian BMP15 signals are preliminary observations from an exploratory analysis: the ewes’ estrous-cycle stages could not be determined, reference-gene stability was not systematically evaluated across tissues, and no protein-level or spatial-localization validation was performed. Still, the oviduct finding hints at a potential extra-ovarian expression pattern that previous work in goats, where BMP15 generally showed more tissue-restricted expression, had not suggested.

The heart of the study, however, lay in its association analysis. From the team’s prior whole-genome resequencing data for Tibetan sheep, four single-nucleotide polymorphisms were selected: two in BMP15, located on the X chromosome, and two in GDF9, on chromosome 5. Standardized annotation using the Ensembl Variant Effect Predictor against the ovine ARS-UI_Ramb_v3.0 reference genome classified the variants as two synonymous and two missense changes. The missense variants were BMP15 p.Leu251Pro, a leucine-to-proline substitution at amino acid position 251, and GDF9 p.Arg87His, an arginine-to-histidine substitution at position 87. The synonymous variants altered codons from GTG to GTA and CTA to CTG without changing the encoded valine and leucine residues. Probing the potential impact of the missense substitutions with SIFT, the team found both predicted to be tolerated, with scores of 0.16 and 0.12 respectively—preliminary evidence that these substitutions may not disrupt protein function.

Genotyping all 200 ewes by iMLDR on an ABI 3730XL analyzer revealed a population with limited variation. Three loci showed very low heterozygosity, with the synonymous BMP15 variant and the missense GDF9 variant each carried by only seven heterozygous animals out of 200. The GDF9 synonymous locus was the most polymorphic, with heterozygosity of 0.43 and a polymorphic information content of 0.34. No locus deviated significantly from Hardy–Weinberg equilibrium. When the researchers modeled the relationship between genotype and litter size using a General Linear Model that accounted for parity effects, the verdict was unambiguous: none of the four SNPs showed a significant association with litter size, with all P values exceeding 0.05 and partial eta-squared effect sizes ranging from less than 0.001 to 0.008—indicating very small genotype effects. Haplotype reconstruction identified three haplotypes for each gene, but linkage disequilibrium analysis showed weak allelic correlation between the two SNPs within each gene: although the D-prime statistic reached 1.0 for both pairs, the r-squared values were a mere 0.001 for BMP15 and 0.039 for GDF9.

Why did the famous prolificacy genes come up empty? The authors point to several plausible explanations. First, Tibetan sheep produce so few twins that phenotypic variation in litter size is extremely limited, reducing statistical power to detect genotype–phenotype associations even when they exist. Second, while 200 ewes is a respectable sample, it may still be insufficient for SNPs with small genetic effects, particularly when some genotypes occur at very low frequencies—the GG genotype at one BMP15 locus appeared in only three animals, and the CT genotype at another in only seven. Third, and perhaps most fundamentally, both missense variants were predicted by SIFT to be tolerated, suggesting these particular loci may simply not be causal variants affecting litter size, nor in strong linkage disequilibrium with functional variants that do. The findings echo a broader lesson from prolificacy genetics: dosage-sensitive mutations that dramatically alter ovulation rate in some breeds, such as the BMP15 mutations of Cambridge and Belclare sheep or the FecB mutation of Booroola Merinos, are not necessarily present—or relevant—in every population.

The study’s real contribution is baseline data. By characterizing partial sequences, exploratory expression profiles, population-genetic parameters, haplotypes, and linkage disequilibrium for BMP15 and GDF9 in Tibetan sheep, the team has laid groundwork for future identification and validation of reproduction-related functional variants in this high-altitude breed. The authors call for full-length coding sequence cloning, protein-level validation with Western blotting and immunohistochemistry, larger sample sizes, and in vivo and in vitro functional assays to clarify the complete molecular structures and biological functions of these oocyte-secreted growth factors. Until then, the genetic roots of Tibetan sheep’s modest fertility remain an open question—one shaped, the evidence suggests, not by single famous mutations but by the complex interplay of hypoxia adaptation, energy metabolism, endocrine regulation, and ovarian function forged across thousands of generations on the Roof of the World.

Subject of Research: Genetics

Article Title: Genetic variants of BMP15 and GDF9 genes and their association with litter size in Tibetan sheep

Article References: Zhang, L., Zhang, Y., Deng, Y., He, N., Sun, R., & Zhang, J. (2026). Genetic variants of BMP15 and GDF9 genes and their association with litter size in Tibetan sheep. Frontiers in Animal Science, 7, Article 1904014. https://doi.org/10.3389/fanim.2026.1904014

Image Credits: AI Generated

DOI: 10.3389/fanim.2026.1904014

Keywords: Genetic, variants, BMP15, GDF9, genes, association, litter, size, Tibetan, sheep, scientific research