Patterns of expression of messenger RNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development and characterization of ovarian follicular populations in ewes carrying the Woodlands FecX2W mutation

Woodlands sheep have a putative genetic mutation (FecX2(W)) that increases ovulation rate. At present, the identity of FecX2(W) is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2(W) could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2(W) may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2(W) ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2(W) ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2(W) ewes. FecX2(W) ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2(W) mutation.     

Cloning and expression levels of genes relating to the ovulation rate of the Yunling black goat

This study investigated the nucleotide sequences and tissue expression levels of genes relating to the ovulation rate in Yunling black goats, a famous Yunnan province, China, local breed with low fecundity. Five genes, FSHB, FSHR, BMP15, BMPR1B, and ESR2, were investigated; the complete cDNA sequences of these genes were 390, 2088, 1185, 1509, and 1585 bp, respectively, and compared with Boer goats (a more fecund breed), the sequence identities were 99%, 99%, 99%, 100%, and 99%, respectively. There were two base differences in FSHB and BMP15, four in FSHR, and three in ESR2. There were fewer follicles and oocytes in Yunling black goats than in Boer goats. Expression levels of FSHB, FSHR, and BMP15 genes in Yunling black goats were lower, and expression levels of BMPR1B and ESR2 genes were higher. Serum FSH content was lower in Yunling black goats, but serum estrogen content was higher. Protein expression levels of FSHR, BMP15, BMPR1B, and ESR2 in ovary and pituitary correlated positively with gene mRNA expression levels. In Yunling black goats, the mRNA expression levels of FSHB, FSHR, and BMP15 positively correlated with litter size, but those of BMPR1B and ESR2 correlated negatively. Together, base changes and variations in mRNA and protein expression levels of genes relating to the ovulation rate result in low fecundity in the Yunling black goat. Reduced BMP15 and FSHR levels may be related to the observed fewer oocytes and, consequently, fewer follicles.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown that the transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) are crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

Screening for Booroola (FecB) and Galway (FecX) mutations in Indian sheep

This study reports the status of the Booroola (FecB) and Galway (FecX^G) mutations in Indian sheep breeds. The Kendrapada sheep (n = 46) was genotyped for the presence of FecB and FecX^G mutations, while the Garole (n = 34), Malpura (n = 30), and Decanni sheep (n = 15) for the FecX^G mutation. The FecB and FecX^G genotyping was carried out by forced restriction fragment length polymorphism PCR technique. In the present study, FecB mutation was discovered in the Kendrapada sheep of Orissa, which is now the second prolific sheep of India after the Garole. Out of 46 individuals of Kendrapada sheep, 26 were homozygous (BB), 15 heterozygous (B+) and 5 non-carriers (++) for the FecB mutation. The frequency of the FecB allele in this sample was about 0.73. Results indicated that the frequency of the FecB mutation is high, but the gene is not fixed in the population as reported in Garole sheep. None of sheep breeds carried the FecX^G mutation. The discovery of the FecB mutation in Kendrapada sheep will facilitate the use of FecB allele in improving the prolificacy of non-prolific sheep breeds of India.     

Screening for Booroola (FecB) and Galway (FecXG) mutations in Indian sheep

This study reports the status of the Booroola (FecB) and Galway (FecX^G) mutations in Indian sheep breeds. The Kendrapada sheep (n = 46) was genotyped for the presence of FecB and FecX^G mutations, while the Garole (n = 34), Malpura (n = 30), and Decanni sheep (n = 15) for the FecX^G mutation. The FecB and FecX^G genotyping was carried out by forced restriction fragment length polymorphism PCR technique. In the present study, FecB mutation was discovered in the Kendrapada sheep of Orissa, which is now the second prolific sheep of India after the Garole. Out of 46 individuals of Kendrapada sheep, 26 were homozygous (BB), 15 heterozygous (B+) and 5 non-carriers (++) for the FecB mutation. The frequency of the FecB allele in this sample was about 0.73. Results indicated that the frequency of the FecB mutation is high, but the gene is not fixed in the population as reported in Garole sheep. None of sheep breeds carried the FecX^G mutation. The discovery of the FecB mutation in Kendrapada sheep will facilitate the use of FecB allele in improving the prolificacy of non-prolific sheep breeds of India.     

Prolificacy genotypes at BMPR 1B,BMP15 and GDF9 genes in North African sheep breeds

The aim of this research was to investigate the genetic structure at BMPR 1B, BMP15 and GDF9 prolificacy genes in five sheep breeds reared in Tunisia: Barbarine, Queue Fine de L’Ouest, Noire de Thibar, Sicilo-Sarde and D’man. Genomic DNA of 204 sheep was investigated for the FecB^B (BMPR 1B), FecX^R, FecX^H, FecX^I, FecX^L, FecX^G, FecX^B (BMP15) and FecG^H (GDF9) mutations. The sequence variability of the different DNA fragments utilised for genotyping was further investigated by Single Stranded Conformation Polymorphism (SSCP) and sequencing. All the above-mentioned mutations were absent in the five sheep breeds examined. SSCP analysis and sequencing allowed the detection of two nucleotide variations. A non-functional mutation (T/C transition at nt 747 of BMP15 cDNA known as B3) was found at the BMP15 gene, in the Noire de Thibar breed; this mutation was first detected in the Belclare sheep. A new nucleotide change G/A at nt 1159 of BMP15 cDNA, causing the amino acid change A119T in the mature peptide, was detected in the Barbarine breed for the first time. The highly prolific D’man ewes were monomorphic for the absence of all the known prolificacy alleles.     

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.     

Screening of indigenous goats for prolificacy associated DNA markers of sheep

The present study was undertaken to explore the genetic basis of caprine prolificacy and to screen indigenous goats for prolificacy associated markers of sheep in BMPR1B, GDF9 and BMP15 genes. To detect the associated mutations and identify novel allelic variants in the candidate genes, representative samples were collected from the breeding tract of indigenous goat breeds varying in prolificacy and geographic distribution. DNA was extracted and PCR amplification was done using primers designed or available in literature for the coding DNA sequence of candidate genes. Direct sequencing was done to identify the genetic variations. Mutations in the candidate genes associated with fecundity in sheep were not detected in Indian goats. Three non-synonymous SNPs (C818T, A959C and G1189A) were identified in exon 2 of GDF9 gene out of which mutation A959C has been associated with prolificacy in exotic goats. Two novel SNPs (G735A and C808G) were observed in exon 2 of BMP15 gene.     

单碱基编辑系统介导欧拉藏绵羊GDF9、FecB多胎基因的定点突变

本研究旨在利用单碱基编辑系统(single base editing system)实现欧拉藏绵羊成纤维细胞FecB和GDF9基因靶位点A到G和C到T的碱基替换并检测其编辑效率。首先设计合成靶向欧拉藏绵羊FecB和GDF9基因的sgRNA序列,再分别连接至epi-ABEmax、epi-BE4max质粒,构建载体并电转至欧拉藏绵羊成纤维细胞,最后对阳性细胞FecB和GDF9基因进行Sanger测序鉴定靶位点突变结果,并通过T-A克隆估算单碱基编辑系统的编辑效率。结果显示获得了靶向欧拉藏绵羊FecB和GDF9基因的sgRNA,并构建使欧拉藏绵羊FecB和GDF9基因单碱基突变的载体,FecB基因靶位点编辑效率为39.13%,GDF9基因靶位点(G260、G721、G1184)编辑效率分别为10.52%、26.67%和8.00%。本研究运用单碱基编辑系统在欧拉藏绵羊成纤维细胞上实现了FecB和GDF9基因靶位点突变,为改良欧拉藏绵羊一胎多羔的繁殖性状奠定理论基础。     

Improving productivity of Malpura breed by crossbreeding with prolific Garole sheep in India

Garole, a prolific small sized sheep breed of India was introduced in the Mutton Project of the Institute during 1997 to explore the possibility of incorporating fecundity gene into Malpura (M) sheep of semi-arid region of Rajasthan. The data on reproduction and production traits of M and Garole × Malpura (GM) half-breds was used in the study to assess the improvement in evolved crossbred stock. The overall percent gain in prolificacy in GM was 52.38, which increased to 75.73 in third parity. The GM compared to M ewes also weaned more lambs per ewe lambed (1 versus 1.41). The lambing rate averaged 91.75 and 148.21% in M and GM ewes, respectively. The twin lambing percentage was only 4.71% in M flock while; it reached up to 51.10% in GM. Moreover, 5.72% of GM ewes produced triplets also. The genetic group non-significantly affect ewe efficiency (EE), but GM produced 3.3% more total lambs weight per ewe lambed at weaning and 6.15% more total lamb weight per ewe lambed at 6-month of age. The body weights at different ages of GM half-breds were lower (P < 0.01) compared to contemporary M lambs. The parity of ewes significantly affects body weight from birth weight to 12-month of age. The survivability of GM half-breds was almost at par with the local M sheep. Results revealed that the fecundity genes responsible for increasing prolificacy have been incorporated into the GM and it might prove a valuable germplasm towards evolving a new prolific strain of sheep. The crossing of Garole with M increased the ewe efficiency.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

Genome‐wide association study to identify genomic regions affecting prolificacy in Lori‐Bakhtiari sheep

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome‐wide association study was performed on estimated breeding values for litter size in Lori‐Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium‐density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism‐specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori‐Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.     

None of polymorphism of ovine fecundity major genes FecB and FecX was tested in goat

The polymorphism of mutation Q249R in BMPR-IB gene (FecB) and loci FecX^I, FecX^H, FecX^G, FecX^B in BMP15 gene was analyzed by forced PCR-RFLP method in 550 individuals from 6 flocks or breeds of goats with litter size varied from 1.4 to 2.7 including Boer (209), Haimen (128), second generation of Boer goat crossed with Huanghuai goat (82), Huanghuai (71), Nubi (37) and Matou (23) goat. None of mutations was detected in these goat breeds and their crossbreed. These results suggest that fecundity of goat is not linked to the same loci in BMPR-IB and BMP15 as sheep. Therefore, it is necessary to seek for other genes or loci in order to develop marker assistance selection techniques and study the prolific mechanism of the goat.     

The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecL^L. Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecL^L carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecL^L. B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecL^L/FecL^L ewes at mRNA and protein levels. In FecL^L carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.     

Intra-cervical application of Misoprostol at estrus alters the content of cervical hyaluronan and the mRNA expression of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and cyclooxygenase-2 in the ewe

The complex anatomy the of ovine cervix limits the success of transcervical artificial insemination in sheep, but Misoprostol (a PGE₁ analogue) relaxes the cervix and facilitates transcervical artificial insemination. However, the mechanism by which Misoprostol causes cervical relaxation is not known. This study examined if intra-cervical Misoprostol altered the hyaluronan content and the mRNA expression of COX-2, LHR, or FSHR in the cervix of the estrus ewe. Estrus was synchronized in cyclic ewes with progestagen pessaries and 48 h after sponge removal ewes were treated intra-cervically with 0 (controls), 200, or 400 μg Misoprostol. Hyaluronan content was determined by ELISA and mRNA expression of LHR, FSHR, and COX-2 was analyzed by in situ hybridization using digoxigenin-11-uridine-5′-triphosphate labeled riboprobes. The hyaluronan content of the cervix was significantly higher in sheep that received 200 (P < 0.05) or 400 (P < 0.05) μg Misoprostol compared to controls. Moreover, it was significantly (P < 0.05) higher in the vaginal region compared to mid and uterine regions. Misoprostol increased (P < 0.05) the mRNA expression of LHR and COX-2 but not FSHR. The expression for all three genes was highest in the vaginal region and lowest in uterine region. The luminal epithelium and circular smooth muscle layers had higher (P < 0.05) expression for LHR, FSHR, and COX-2 mRNAs, and the sub-epithelial stroma had the lowest (P < 0.05). We propose that the intra-cervical application of Misoprostol induces the mRNA expression of LHR, FSHR, and COX-2 through a positive feedback loop. The data suggest that softening of the cervix by Misoprostol is caused by an increase in the hyaluronan content of the cervix.     

In vitro regulation of sheep ovarian surface epithelium (OSE) proliferation by local ovarian factors

The ovarian surface epithelium (OSE) forms a lining around the entire ovary and actively participates in the ovulatory cycle. To investigate how specific growth factors and hormones affect OSE proliferation, the present study used sheep as a model to examine the effects of follicular and luteal products on the proliferation of sheep OSE cells in culture, and to analyse the influences of large antral follicles and corpora lutea (CL) on the expression of gonadotrophin receptors (FSHR and LHR) in the OSE. In the present study, follicular fluids from medium and large follicles, and extracts of corpora lutea stimulated the growth of OSE cells. The results of the present study showed that factors in the follicular fluid can induce OSE proliferative activity, and this stimulation effect could not be attributed to steroids in the follicular fluid since oestrogen and progesterone treatments failed to stimulate OSE cells. The expression of LH and FSH receptors over large follicles (5 mm or larger) was two and four times higher than those over stroma and CL, respectively. In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated by, at least in part, the up-regulation of gonadotrophin receptors, and facilitated by the action of mitogenic glycopeptides and growth factors, but not steroids.     

Regulation of ovulation rate in mammals: contribution of sheep genetic models

Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate.     

DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation

Recent discoveries that high prolificacy in sheep carrying the Booroola gene (FecB) is the result of a mutation in the BMPIB receptor and high prolificacy in Inverdale sheep (FecX(I)) is the result of a mutation in the BMP15 oocyte-derived growth factor gene have allowed direct marker tests to be developed for FecB and FecX(I). These tests were carried out in seven strains of sheep (Javanese, Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge) in which inheritance patterns have suggested the presence of major genes affecting prolificacy and in the prolific Garole sheep of India, which have been proposed as the ancestor of Australian Booroola Merinos. The FecB mutation was found in the Garole and Javanese sheep but not in Thoka, Woodlands, Olkuska, Lacaune, Belclare, and Cambridge sheep. None of the sheep tested had the FecX(I) mutation. These findings present strong evidence to support historical records that the Booroola gene was introduced into Australian flocks from Garole (Bengal) sheep in the late 18th century. It is unknown whether Javanese Thin-tailed sheep acquired the Booroola gene directly from Garole sheep from India or via Merinos from Australia. The DNA mutation test for FecB will enable breeding plans to be developed that allow the most effective use of this gene in Garole and Javanese Thin-tailed sheep and their crosses.