Identification of Polymorphisms in GDF9 and BMP15 Genes in Jamunapari and Crossbred Goats in Bangladesh

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are two crucial fecundity genes 15 associated with litter size traits in the goat. Our previous study on GDF9 and BMP15 genes detected single nucleotide polymorphisms (SNPs) associated with litter size in Bangladeshi Black Bengal goats. In this study, Jamunapari and crossbred goats of Bangladesh were screened to identify polymorphisms in the GDF9 and BMP15 genes and to assess the association between identified SNPs and litter size. The genomic DNA from 100 goats (50 Jamunapari and 50 crossbred) was used in Polymerase Chain Reaction (PCR) to amplify the exon 2 of the GDF9 and exon 2 of the BMP15 gene. PCR products were sequenced employing the BigDye Terminator cycle sequencing protocol, to identify SNPs. A generalized linear model was utilized to perform the association analysis for identified SNPs and litter size. Seven SNPs were identified, of which four: C818CT, G1073A, G1189A and G1330T were in the GDF9 gene, three: G616T, G735A and G811A were in the BMP15 gene. G735A was a synonymous SNP, whereas the remaining were non-synonymous SNPs. Identified SNP loci in GDF9 were low polymorphic (PIC<0.25) while loci in BMP15 were moderately polymorphic (PIC≥0.25). The genotypes at the G1330T locus had a significant (p<0.05) difference in litter size in Jamunapari goat, but no significant difference was observed for all genotypes at other loci. This study provides additional molecular markers that would be useful for future research on the litter size trait in goats of Bangladesh.

Development and identification of candidate aptamers for growth differentiation factor 9

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.</p>

Development and identification of candidate aptamers for growth differentiation factor 9

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted growth factors that are essential for fertility and ovarian development in many mammalian species. It is hypothesised that the ratios of these growth factors within ovarian follicles are unique to each species and determine litter size in mammals. The current detection method for these proteins is by Western blotting with monoclonal antibodies, however this methodology generates semi-quantitative results at best. Systematic evolution via exponential enrichment (SELEX) is a method involving iterative rounds of affinity maturation. It has been used for generating synthetic DNA- and RNA-based sequences (aptamers) that are capable of recognising a target molecule with high sensitivity. To understand more about the biological functions of BMP15 and GDF9, reliable detection methods need to be developed so these proteins can be measured in biological samples. The generation of aptamers that recognise BMP15 and GDF9 of multiple species could enable a novel detection system to be developed to further explore the role of BMP15 and GDF9 in determining litter size in mammals. In this study, recombinant BMP15 and GDF9 proteins from species that vary in litter size were produced and purified by IMAC and HPLC for aptamer validation. Membrane-SELEX was used to generate candidate aptamers against recombinant human GDF9. After ten rounds of SELEX, seven candidate aptamers were identified and sequenced. Alignment of the sequences revealed a conserved region of 29bp in four of the seven candidate aptamers. These sequences also showed a high (85-90%) guanine/thymine content consistent with G-quadruplex forming aptamers, a tertiary structure that increases specificity to the target molecule. These four sequences also showed homology with that of a previously published aptamer for BMP15. This is interesting as BMP15 and GDF9 are both members of the transforming growth factor beta superfamily and as such, share a high peptide homology suggesting convergent evolution occurred in two unrelated SELEX experiments against related proteins. Whilst time constraints did not allow for characterisation of the resultant GDF9 aptamer candidates, nor their validation using recombinant ovine and mouse GDF9 produced herein, this study showed that GDF9 aptamer candidates were generated by SELEX that exhibited sequence similarities to an aptamer generated against a structurally-related protein.</p>

An investigation of the effects of <i>BMPR1B</i>, <i>BMP15</i>, and <i>GDF9</i> genes on litter size in Ramlıç and Dağlıç sheep

This study was carried out to determine the presence of polymorphisms in genes affecting litter size. The SNPs in bone morphogenetic protein receptor type 1B (BMPR1B), bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) genes were detected in 60 uniparous and 60 multiparous ewes from Ramlıç and Dağlıç breeds. The ewes are maintained in nine public herds at the breeding station of the Afyonkarahisar Sheep and Goats Breeders' Association and lambed in two consecutive breeding seasons. PCR and DNA sequencing analyses were conducted, and 36, 4, and 11 SNPs in Ramlıç and 40, 3, and 11 SNPs in Dağlıç were detected in BMPR1B, BMP15, and GDF9 genes, respectively. A total of 16 SNPs in Ramlıç and 10 SNPs in Dağlıç breeds for three genes were found to be significant (P<0.05). The resulting analyses showed that four SNPs (g.49496G>A, c.1658A>C, c.2037C>T, c.2053C>T) of the BMPR1B gene and one deletion mutation (c.28_30delCTT) in the BMP15 gene of the Ramlıç breed as well as five SNPs (c.1487C>A, c.2492C>T, c.2523G>A, c.2880A>G, and c.2763G>A) of the BMPR1B gene of the Dağlıç breed have significant positive regression coefficients in the desired direction of the rare allele. The observed mutations have potential to be used as genetic markers in the selection of prolific animals for both breeds.

Serum Concentrations of GDF9 and BMP15 Across the Menstrual Cycle

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are TGF-β proteins that regulate key processes throughout folliculogenesis and are determinants of mammalian fecundity (1). They are uniquely produced predominantly by the oocyte and have potential clinical application as markers of oocyte quality and quantity (2). However, no studies have been conducted to assess whether serum concentrations alter across the different phases of the menstrual cycle, and thus if assessment should be confined to specific cycle stages. The aim of this study was to measure serum concentrations of these proteins during the menstrual cycle in women at different stages of reproductive life. Serum was collected every 1-3 days throughout the menstrual cycle from 41 healthy ovulatory women from three cohorts: menses to late luteal phase (21-29 years of age; n=16; University of Otago) and across one interovulatory interval (18-35 years of age; n=10; and 45-50 years of age; n=15; University of Saskatchewan), with simultaneous ultrasound scans confirming ovulation. Serum concentrations of GDF9, BMP15, estradiol, FSH, LH, progesterone, inhibin A and B and AMH were measured. GDF9 and BMP15 were detectable in 54% and 73% of women and varied 236- and 52-fold between women, respectively. To detect changes, mean concentrations and variances across the cycle were statistically modelled using a generalized additive model of location, shape and scale (GAMLSS). Across the menstrual cycle, there were minimal changes in serum GDF9 or BMP15 within a woman for all cohorts, with no significant differences detected in modelled mean concentrations. However, modelled variances were highest in the luteal phases of all women for BMP15 immediately following ovulation, regardless of age, suggesting a possible underlying cyclic pattern. These results suggest that serum BMP15 and GDF9 are not overtly affected by menstrual cycle dynamics but may be more stable in the follicular phase. Larger studies with more frequent sampling should establish if BMP15 and presumably GDF9 demonstrate clinically relevant cyclic variation. References: (1) Gilchrist RB et al., HRU 2008; 14:159-77. (2) Riepsamen AH et al., Endocrinol 2019; 160:2298-313.

Activation-induced cytidine deaminase is a possible regulator of cross-talk between oocytes and granulosa cells through GDF-9 and SCF feedback system

Activation-induced cytidine deaminase (AID, Aicda) is a master gene regulating class switching of immunoglobulin genes. In this study, we investigated the significance of AID expression in the ovary. Immunohistological study and RT-PCR showed that AID was expressed in murine granulosa cells and oocytes. However, using the Aicda-Cre/Rosa-tdRFP reporter mouse, its transcriptional history in oocytes was not detected, suggesting that AID mRNA in oocytes has an exogenous origin. Microarray and qPCR validation revealed that mRNA expressions of growth differentiation factor-9 (GDF-9) in oocytes and stem cell factor (SCF) in granulosa cells were significantly decreased in AID-knockout mice compared with wild-type mice. A 6-h incubation of primary granuloma cells markedly reduced AID expression, whereas it was maintained by recombinant GDF-9. In contrast, SCF expression was induced by more than threefold, whereas GDF-9 completely inhibited its increase. In the presence of GDF-9, knockdown of AID by siRNA further decreased SCF expression. However, in AID-suppressed granulosa cells and ovarian tissues of AID-knockout mice, there were no differences in the methylation of SCF and GDF-9. These findings suggest that AID is a novel candidate that regulates cross-talk between oocytes and granulosa cells through a GDF-9 and SCF feedback system, probably in a methylation-independent manner.

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle

The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.