Goats’ Litter Size Concerning Bone Morphogenetic Protein 15 Gene Variations

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure

This study examines the BMP15 (Bone Morphogenetic Protein 15) gene as a potential genetic marker for reproductive traits, specifically litter size, in Damani goats. Genomic DNA was extracted from 180 Damani goats, and a novel single-nucleotide polymorphism (SNP), g.557 G > A, was identified in the promoter region of BMP15. Statistical analysis revealed a significant association between the g.557 G > A SNP and litter size (P < 0.05), with individuals carrying the AA genotype exhibiting significantly larger litter sizes compared to GG and GA genotypes. Bioinformatics analysis predicted that the g.557 G > A SNP creates new transcription factor binding sites (TBS) for FOXM1, FOXP3, FOXA1, AHR, and GATA4, all of which are key regulators of ovarian function and folliculogenesis. These TBS likely enhance BMP15 expression, influencing follicular development and contributing to the observed increase in litter size. Phylogenetic analysis across nine species demonstrated a high degree of sequence conservation, indicating that BMP15 plays a critical and conserved role in reproductive function across species. The findings suggest that the g.557 G > A SNP could serve as a valuable molecular marker for improving reproductive traits in Damani goats and highlight its broader potential for marker-assisted selection in livestock breeding, particularly to enhance fertility and reproductive efficiency in various goats species.

The Bone Morphogenetic Protein 15 (BMP15) and Growth Differentiation Factor 9 (GDF9) genes play important roles in follicular development, ovulation rate, and litter size in goats. This study aimed to investigate polymorphisms (SNP) in the coding sequences of BMP15 and GDF9 genes and to find possible associations between identified polymorphisms and reproductive traits in Bangladesh’s Black Bengal goat (BBG). In total, 85 DNA samples of BBG were utilized for PCR amplification, gene fragments sequencing, and subsequent association studies using a generalized linear model implemented by R software. Sequence analysis revealed three SNPs (g.5875A&gt;G, g.6051G&gt;A, and g.6124C&gt;G) in the exon 2 of the BMP15 gene, having one nonsynonymous mutation (g.6124C&gt;G) that changed amino acid glutamic acid to glutamine (E270Q). Besides, the g.3764C&gt;T polymorphism identified in the GDF9 gene was nonsynonymous in nature, changing the amino acid alanine to valine (A273V). The association analysis revealed that litter size and kidding interval differed significantly (p&lt;0.05) for g.6051G&gt;A and g.6124C&gt;G SNP genotypes of the BMP15 gene in the BBG population. Besides, the combined genotypes derived from three BMP15 polymorphisms also significantly affected average litter size and at the 3rd parity. However, the g.3764C&gt;T SNP genotypes of the GDF9 gene showed significant association with only average service per conception. Taken together, the identified SNPs of BMP15 and GDF9 genes showed potentials that could be used as molecular markers for improving the reproductive traits of BBG.

Litter size is an important economic trait in the goat industry. Previousstudies on the bone morphogenetic protein 15 (BMP15) gene detected some single-nucleotide polymorphisms (SNPs) such as c.963AG thatwere associated with an increase in ovulation rate and litter size. The aimof this study was to conduct a meta-analysis on the effect of thispolymorphism on litter size. We gathered and pooled data from five eligiblepublished studies. To investigate the effect of c.963AG onlitter size, we utilized four different genetic models assuming dominant(GG GA vs. AA), recessive (GG vs. GA AA), additive (GG vs. AA) andco-dominant (GG AA vs. GA) model of inheritance. Data were analyzed underrandom-effects models based on the value. Furthermore, sensitivityanalysis was carried out to validate the stability of results. The resultsshowed that the c.963AG polymorphism is associated with littersize when applying a dominant model (standardized mean difference (SMD) is 0.815, 95 % CI [0.170,1.461], value 0.013) and also with an additive model (SMD 0.755, 95 % CI [0.111, 1.400], value 0.022). However, the effect of c.963AGpolymorphism was not significant under recessive (SMD 0.186, 95 % CI [0.195, 4.259], value 0.339) and co-dominant (SMD 0.119, 95 % CI [0.525, 0.288], value 0.568) models. Sensitivity analysis demonstratedthat dropping studies with wide confidence intervals affects overall resultsunder the assumption of an additive model. The meta-analysis results revealedthat the AA genotype could be positively connected with litter size in goats.

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.

BackgroundA shortened reproductive period and earlier menopause have been associated with type 2 diabetes. Growth differentiation factor 9(GDF9) and bone morphogenetic protein 15 (BMP15) gene mutations have been associated with earlier menopause. Therefore, this study aimed to evaluate the association between BMP15 and GDF9 mutations with impairing female fecundity in diabetic patients. The study subjects comprised 90 female diabetic patients and 60 female healthy controls. The physio-biochemical analysis was measured using enzymatic determination. A single-strand conformation polymorphism (SSCP) protocol was utilized to assess the pattern of genetic variations.ResultsGenotyping analysis of the BMP15 gene showed a heterogeneous pattern with the presence of two genotypes: AA and AC genotypes. Five novel missense single nucleotide polymorphisms (SNPs) were identified in the BMP15 gene: four SNPs detected in both genotypes, and Met4Leu, a specific SNP, was detected only in the AC genotype. Cumulative in silico tools indicated a highly deleterious effect for the Met4Leu on the mutant protein structure, function, and stability. Diabetes patients showed a significantly higher frequency of genotype AC. The physio-biochemical analysis of fasting plasma glucose (FBG), glycosylated hemoglobin (HbA1c), and luteinizing hormone (LH) were significantly higher (P < 0.05) in AC genotype than AA genotype.ConclusionsThe current research provides the first indication regarding the tight association of BMP15 polymorphism with the impairing female fecundity in the diabetic. A pivotal role is played by the novel (Met4Leu) SNP that can be used as a predictor for the impairing female fecundity of diabetes, while no polymorphism was found in exon 4 of the GDF9 gene.

The families of TGF-β (transforming growth factor-β) proteins are the most important growth factors in the ovary, and three related oocyte-derived members, namely GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15), and BMPR1B (bone morphogenetic protein receptor 1B), have been shown to be essential for follicular growth and ovulation. Although the essential role of these genes in determining litter size in sheep and mouse and in controlling folliculogenesis in human has been demonstrated, there is limited information on their action in other species, especially in bovine. Bovine is a monotocous specie, as humans, with one or sometimes two newborns per birth. The twinning is a complex trait determined by both genetic and environmental factors. This study aimed at investigating the nucleotide sequences of different fragments of GDF9, BMP15, and BMPR1B genes in Maremmana cows reared in Castelporziano Presidential Estate (Rome). In this herd, in the period between 1996 and 2008, a twinning rate of 12 % (on average) was observed. We identified nine single-nucleotide polymorphisms (SNPs), five in the coding region, and four in the noncoding region: Two polymorphisms caused non-synonymous mutations, g.6045 G>A (V202I) in the BMP15 gene, and g.231 T>C (L66S) in GDF9 gene. The mutation L66S was found only in cows with double birth. In the literature, there are different evidences that mutations in proregion of GDF9 protein could affect its correct function. A relationship between mutations in this region of protein and granulosa cells proliferation and oocyte development was hypothesized.

Effects of novel variants in BMP15 gene on litter size in Mongolia and Ujimqin sheep breeds

Prolificacy of most local goat breeds in China is low. Jining Grey goat is one of the most prolific goat breeds in China, it is an important goat breed for the rural economy. ASMT (acetylserotonin O‐methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are essential for animal reproduction. Single nucleotide polymorphisms (SNPs) of ASMT and ADAMTS1 genes in the highly prolific breed (Jining Grey goats), medium prolific breed (Boer goats and Guizhou White goats) and low prolific breeds (Angora goats, Liaoning Cashmere goats and Inner Mongolia Cashmere goats) were detected by polymerase chain reaction‐restriction fragment length polymorphism and sequencing. Two SNPs (g.158122T>C, g.158700G>A) of ASMT gene and two SNPs (g.7979798A>G, g.7979477C>T) of ADAMTS1 gene were identified. For g.158122T>C of ASMT gene, further analysis revealed that genotype TC or CC had 0.66 (p < 0.05) or 0.75 (p < 0.05) kids more than those with genotype TT in Jining Grey goats. No significant difference (p > 0.05) was found in litter size between TC and CC genotypes. The SNP (g.158122T>C) caused a p.Tyr298His change and this SNP mutation resulted in changes in protein binding sites and macromolecule‐binding sites. The improvement in reproductive performance may be due to changes in the structure of ASMT protein. For g.7979477C>T of ADAMTS1 gene, Jining Grey does with genotype CT or TT had 0.82 (p < 0.05) or 0.86 (p < 0.05) more kids than those with genotype CC. No significant difference (p > 0.05) was found in litter size between CT or TT genotypes. These results preliminarily indicated that C allele (g.158122T>C) of ASMT gene and T allele (g.7979477C>T) of ADAMTS1 gene are potential molecular markers which could improve litter size of Jining Grey goats and be used in goat breeding.

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles in individuals <40 years old, and is a major cause of infertility in females. Genetic factors are considered to be responsible for the development of POF, however, the exact pathogenesis remains to be elucidated in the majority of cases. In the present study, the single nucleotide polymorphisms (SNPs) of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), inhibin βB (INHBB) and follicle stimulating hormone receptor (FSHR) genes were investigated, and their association with POF in a Chinese Hui population of the Ningxia Hui Autonomous Region in western China was evaluated. Peripheral blood samples were collected from 63 patients diagnosed with POF (POF group) and 58 normal control individuals (control group), from which the genomic DNA was isolated. The GDF9, BMP15, INHBB and FSHR genes were amplified using polymerase chain reaction assays, and their SNPs were determined by sequencing. In the four SNPs identified across the GDF9 loci, D57Y (169G>T), rs1049127 (546G>A), rs254286 (447C>T) and rs254285 (969C>G), the frequencies of the 546G>A genotype and allele A were significantly higher in the POF group, compared with the normal control group (34.92, vs. 6.90%; P<0.05 and 19.05, vs. 3.23%; P<0.05, repsectively), while no significant differences were observed in the occur rence of the c.447C>T and c.969C>G mutations between the two groups (60.32, vs. 50% and 50.79, vs. 55.17%, repsectively). The c.169G>T mutation within the GDF9 gene was only detected in two patients with POF, and the mutation did not occur in the normal control group. A total of three SNPs were detected within the BMP15 gene, including rs3810682 (−9C>G), rs79377927 (788_789insTCT) and rs17003221 (852C>T), and no significant differences were observed in the frequencies of the 9C>G and 852C>T genotypes between the POF and control groups (7.94, vs. 6.90% and 4.76, vs. 3.45%, respectively). The 788_789insTCT genotype was detected in only two patients with POF. A novel mutation, c.1095C>A, was identified in exon 2 of the INHBB gene, however, no significant difference was found in the occurrence of the mutation between the two groups (30.16, vs. 22.41%; P>0.05). The rs6165 (919G>A) and rs6166 (2039G>A) SNPs were detected in exon 10 of the FSHR gene; however, no significant difference was observed in the genotype frequencies between the two groups (92.06, vs. 91.38% and 96.83, vs. 93.10%, respecrively). These results demonstrated that GDF9 c.169G>T (D57Y), c.546G>A (rs1049127), and BMP15 rs79377927 (788_789insTCT) were associated with POF in the Chinese Hui population.

In horses, multiple ovulation resulting in implantation of multiple embryos is adverse. However, understanding the mechanisms underlying initiation of multiple ovulation (MO) is advantageous and is related to an increase in efficiency of embryo transfer techniques. It has been postulated that MO may have a genetic background. Two major genes: bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are considered to play a crucial role in folliculogenesis and controlling the ovulation rate. Thus, the aim of the presented study was to identify the variation within equine BMP15 and GDF9 genes to verify their potential role on spontaneous, repetitive multiple ovulations in mares. In addition, variation screening of investigated genes in population of Thoroughbred and Arabian breeds was performed together with establishment of transcript abundance of BMP15 and GDF9 genes in equine ovarian tissue. Sanger sequencing of Arabian and Thoroughbred mares divided according to ovulation rate, revealed occurrence of 3 SNPs in BMP15 and STS in GDF9 genes. The PCR-RLFP and statistical analysis indicated that none of the genotype frequencies were significant in any breeds and none of them were claimed as functional according to ovulation rate. Furthermore, evaluation of transcript abundance by RT -PCR of both genes in ovarian tissues showed that expression of both genes was similar but GDF9 was significantly expressed in growing follicles with 21-30 mm diameter and in ovarian parenchyma, which suggest their potential role in folliculogenesis.

The bone morphogenetic protein-15 (BMP-15) gene is thought to be one of the important candidate genes involved in premature ovarian failure (POF). However, to date, there has been no consensus on the relationship between mutations in BMP-15 and ovarian dysfunction. To analyse mutations in the BMP-15 gene in Chinese women with POF. We sequenced the BMP-15 gene protein coding region of 92 patients and 76 healthy controls that cycle regularly. No mutations were found in the BMP-15 gene protein coding region. There was no difference in the incidence of single nucleotide polymorphism (SNP) rs 17003221 (CT) in exon 2 (p>0.05), or SNP rs (3810682CG: ss16336587) in the putative promoter region of exon 1, between the two groups. However, the allele gene frequency in SNP rs (3810682CG: ss16336587) was C (97.92%) and G (2.08%), respectively, in Chinese women, which is different from other races. Our findings indicate that mutations in BMP-15 exons, or changes in BMP-15 pro-peptide, are rare in Chinese women with POF. In addition, our data suggest that the 2 SNPs are not related to POF in Chinese women, while providing evidence for SNP variation between different races.

The Small Tailed Han is a prolific local sheep breed in China. The bone morphogenetic protein receptor IB (BMPR-IB) gene, which affects the fecundity of Booroola Merino sheep, and the bone morphogenetic protein 15 (BMP-15) gene, which affects the fecundity of Inverdale, Hanna, Belclare, Cambridge, and Lacaune sheep, were studied as candidate genes associated with the prolificacy of Small Tailed Han sheep. Single nucleotide polymorphisms of BMPR-IB and BMP-15 genes were detected in Small Tailed Han ewes (n = 188) by PCR-RFLP. The combined effect of the 2 genes on the prolificacy of Small Tailed Han sheep was studied. The results indicated that the same FecB mutation (Q249R) occurred in the BMPR-IB gene in Small Tailed Han ewes as found in Booroola Merino ewes. The Small Tailed Han ewes with genotypes FecB(B)/FecB(B) and FecB(B)/FecB(+) had 1.40 (P < 0.01) and 1.11 (P < 0.01) more lambs, respectively, than those with genotype FecB(+)/FecB(+). The same FecX(G) mutation (Q239Ter) of the BMP-15 gene was found in Small Tailed Han ewes as in Belclare and Cambridge ewes. The Small Tailed Han ewes with the heterozygous mutant FecX(G)/FecX(+) had 0.55 (P < 0.01) more lambs than those with the wild-type FecX(+)/FecX(+). The Small Tailed Han ewes carrying mutations in both BMPR-IB and BMP-15 genes had greater litter size than those with either mutation alone. In view of our results, marker-assisted selection using both BMPR-IB and BMP-15 genes is warranted to increase litter size in sheep and will be of considerable economic value to sheep producers.

The main purpose of this study was to assess the presence of the previously reported single nucleotide polymorphisms (SNPs) in the sheep growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes and their association with litter size at lambing in Iran-Black sheep breed. Blood samples were taken from 120 Iran-Black ewes. DNA extraction was conducted using a modified salting-out method. DNA fragments with sizes of 462bp and 141bp for the GDF9 and BMP15 genes were amplified using PCR with specific primers, respectively. The PCR-RFLP approach was adopted for detecting the genotypes. The results indicated that the SNP in the exon 2 of BMP15 is a monomorphic locus in Iran-Black sheep. However, the substitution of G to A nucleotide was determined in the GDF9 locus. Digestion of the 462bp PCR product from exon 1 of theGDF9 using the HhaI restriction enzyme produced fragments of 52, 156, and 254bp. However, DNA fragments containing the A nucleotide yielded only two fragments (52 and 410bp). The heterozygous animals for this mutation in GDF9 locus had fragments of all four sizes (52, 156, 254, and 410bp). The frequency (0.75) of the wild type allele (+) in GDF9 locus was higher than the frequency (0.25) of mutant allele (G). The observed frequencies for the GG, G+ and ++ genotypes were 0.05, 0.40 and 0.55, respectively. The association results indicated that the mutation of GDF9 gene has a substantial impact on lambing rate and the Iran-Black ewes with the GG and G+ genotypes had higher lambing rate than those with the ++ genotype. Thus, a gene assisted selection program to improve lambing rate in this breed can be designed based on the GDF9 gene mutation. Key words: BMP15, GDF9, Litter Size, Iran-Black Sheep

A single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation