Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Granulosa cell tritiated thymidine incorporation following exposure to mature GDF9, mature BMP15 or pro-mature BMP15 at 100 ng/ml. Bars represent mean ± SEM and different lowercase letters indicate a statistically significant difference (P<0.05). Data were derived from 5 independent replicates.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Effect of supplementing COC during IVM with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 at 100 ng/ml dose, with and without FSH on oocyte developmental competence.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Control

Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Effect of supplementing COC during IVM with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 at 100 ng/ml dose, with and without FSH on blastocyst quality.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Control

Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Spent IVM medium were analysed for glucose and lactate levels post 23(mature BMP15 and pro-mature BMP15) or mature GDF9 at 100 ng/ml, in the absence or presence of FSH. A. Glucose uptake. B. Lactate production. Bars represent the mean ± SEM. Data were derived from 12 independent replicates for each treatment.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Effect of treatment of intact COCs with different forms of BMP15 (mature BMP15 and pro-mature BMP15) or mature GDF9 at 100 ng/ml, +/− FSH on intra-oocytemetabolic activity. A. Autofluorescence of NAD(P)H. B. Autofluorescence of FAD. C. REDOX ratio (FAD/NAD(P)H). D. GSH levels. Bars represent the mean ± SEM. Data were derived from 4 independent replicates for autofluorescence on intra-oocyte NAD(P)H, FAD, and REDOX ratio and 3 independent replicates for GSH levels. Columns with different superscripts are significantly different (P<0.05); a,b minus FSH, x–y plus FSH.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Activity Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

doi: 10.1371/journal.pone.0103563

Figure Lengend Snippet: Representative micrographs of intra-oocyte NAD(P)H (A, C) and FAD (B, D) autofluorescence and GSH fluorescence (E, F), after treatment with different forms of BMP15 (mature BMP15 and pro-mature BMP15) or mature GDF9 at 100 ng/ml, in the absence (A, B, E) or presence (C, D, F) of FSH.

Article Snippet: Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis, MN, USA).

Techniques: Fluorescence

Journal: Molecular and cellular endocrinology

Article Title: Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

doi: 10.1016/j.mce.2023.112049

Figure Lengend Snippet: Fig. 1. BMP15 and GDF9 ELISA development and validation. (A) Dose- response curves of cumulus cell and granulosa cell extracts in the BMP15 ELISA are non-parallel with human pro-BMP15. (B) Similar in the GDF9 ELISA, human GDF9 is non-parallel with cumulus cell and granulosa cell. (C,D) Extraction of proteins and DNA from cumulus cells was optimised using a range of sodium chloride concentrations. Data are presented as means of duplicate measures (black dots). The horizontal dotted line refers to the level of detection.

Article Snippet: The following reagents were obtained: recombinant human pro-BMP15 and pro-GDF9 (Monash University, Victoria, Australia), recombinant human and mouse mature GDF9 (8266-G9/CF and 739-G9, respectively) and human mature BMP5 (615-BM), BMP6 (507-BP) and BMP7 (354-BP) from R&D Systems (MN, USA), mature human BMP15 produced in E. coli (Catalogue Number CSB-EP002735HU, Cusabio, Houston,Tx, USA), streptavidin-labeled horseradish peroxidase enzyme conjugate (SNN2004) and tetramethylbenzidine (Life Technologies, MD, USA), phenylmethylsulfonyl fluoride (PMSF, Sigma, MO, USA), and Hoechst 33342 (Thermo Fisher Scientific, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Extraction

Journal: Molecular and cellular endocrinology

Article Title: Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

doi: 10.1016/j.mce.2023.112049

Figure Lengend Snippet: Fig. 2. BMP15 and GDF9 structure and Western blot analysis of cumulus and granulosa cell extracts. Sequences of human pro-BMP15 (Panel A) and pro-GDF9 (Panel D). The pro- (roman text) and mature- (bold text) domains of human BMP15 and human GDF9 are shown. The residues are numbered according to the first residue of the signal peptide. Known and putative cleavage sites are depicted above the sequence. N- and O-linked glycosylation sites are also indicated (shaded in pink). The peptide sequences used to raise the BMP15 mAb#28A (A) and the two GDF9 mAbs#72B and #53/1 (D) are presented, as well as the mapped binding epitope of mAB#53/1 (D). Panel B: Western blots using biotinylated mAb#28A for detection of human pro- BMP15 and granulosa cell (GC) extracts. Each membrane was probed without or with immuno-neutralised mAb#28A using a spe cific neutralising peptide. Panels C,F: Quan tification of WB grey scale intensity and molecular weight calibrated to standard curves, without (solid lines) and with (dotted lines) antibody peptide neutralisation. Numbers on peaks correspond to the calcu lated molecular weights of bands. Panel E: Western blots using biotinylated mAb#72B for detection of human GDF9, pro-GDF9 and GC extracts. Each membrane was probed without or with immuno-neutralised mAb#72B using a specific neutralising pep tide. Red stars depict the bands which were fully neutralised in the GC extract samples.

Article Snippet: The following reagents were obtained: recombinant human pro-BMP15 and pro-GDF9 (Monash University, Victoria, Australia), recombinant human and mouse mature GDF9 (8266-G9/CF and 739-G9, respectively) and human mature BMP5 (615-BM), BMP6 (507-BP) and BMP7 (354-BP) from R&D Systems (MN, USA), mature human BMP15 produced in E. coli (Catalogue Number CSB-EP002735HU, Cusabio, Houston,Tx, USA), streptavidin-labeled horseradish peroxidase enzyme conjugate (SNN2004) and tetramethylbenzidine (Life Technologies, MD, USA), phenylmethylsulfonyl fluoride (PMSF, Sigma, MO, USA), and Hoechst 33342 (Thermo Fisher Scientific, MA, USA).

Techniques: Western Blot, Residue, Sequencing, Glycoproteomics, Binding Assay, Membrane, Molecular Weight

Journal: Molecular and cellular endocrinology

Article Title: Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

doi: 10.1016/j.mce.2023.112049

Figure Lengend Snippet: Fig. 3. Relationships between number of oocytes, levels of BMP15, GDF9 and DNA from cumulus cells. The relationships between total BMP15 (A), GDF9 (B), cumulus cell DNA (E) and oocyte number/patient, and between total BMP15 (C), GDF9 (D) and cumulus cell DNA are presented, as is the association between GDF9/CC DNA and BMP15/CC DNA (F). Each dot represents an in dividual patient, with closed circles (patient cohort 1) and open circles (patient cohort 2). Data represent linear regression analyses with regression lines plotted and corresponding p-values.

Article Snippet: The following reagents were obtained: recombinant human pro-BMP15 and pro-GDF9 (Monash University, Victoria, Australia), recombinant human and mouse mature GDF9 (8266-G9/CF and 739-G9, respectively) and human mature BMP5 (615-BM), BMP6 (507-BP) and BMP7 (354-BP) from R&D Systems (MN, USA), mature human BMP15 produced in E. coli (Catalogue Number CSB-EP002735HU, Cusabio, Houston,Tx, USA), streptavidin-labeled horseradish peroxidase enzyme conjugate (SNN2004) and tetramethylbenzidine (Life Technologies, MD, USA), phenylmethylsulfonyl fluoride (PMSF, Sigma, MO, USA), and Hoechst 33342 (Thermo Fisher Scientific, MA, USA).

Techniques:

Journal: Molecular and cellular endocrinology

Article Title: Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

doi: 10.1016/j.mce.2023.112049

Figure Lengend Snippet: Fig. 4. Associations between oocyte number and maternal age with oocyte BMP15 and GDF9 production. Associations between BMP15/CC DNA (A, D), GDF9/CC DNA (B,E) and their ratio (C,F) with oocyte number (A-C) and maternal age (D-F) are presented. Each dot represents an individual patient, with closed circles (patient cohort 1) and open circles (patient cohort 2). Data represent linear regression analyses with regression lines plotted and corresponding p-values.

Article Snippet: The following reagents were obtained: recombinant human pro-BMP15 and pro-GDF9 (Monash University, Victoria, Australia), recombinant human and mouse mature GDF9 (8266-G9/CF and 739-G9, respectively) and human mature BMP5 (615-BM), BMP6 (507-BP) and BMP7 (354-BP) from R&D Systems (MN, USA), mature human BMP15 produced in E. coli (Catalogue Number CSB-EP002735HU, Cusabio, Houston,Tx, USA), streptavidin-labeled horseradish peroxidase enzyme conjugate (SNN2004) and tetramethylbenzidine (Life Technologies, MD, USA), phenylmethylsulfonyl fluoride (PMSF, Sigma, MO, USA), and Hoechst 33342 (Thermo Fisher Scientific, MA, USA).

Techniques:

Journal: Maturitas

Article Title: New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing.

doi: 10.1016/j.maturitas.2020.06.004

Figure Lengend Snippet: Fig. 1. Likely pathogenic variants in GDF9. 1a: Familial pedigree of Patient 2. The proband is indicated by an arrow. Small solid black circles represent voluntary termination of pregnancy (II5, II6, and II7) or miscarriage (III3). Information on height is given for individuals I1, I2, II1, II2, II3 and II4. Years of birth are indicated for the sibling II1, II2, II3, and II4. GH: growth hormone. 1b: Sequencing results of Patient 2. Top: IGV visualization of the GDF9 variants in Patient 2, showing variants physically close to each other. Middle: Sanger sequencing of GDF9 in Patient 2 confirming the presence of heterozygous variants. Bottom: Variant phasing, showing phased variants, and confirming compound heterozygosity (presence of one variant per clone, each clone corresponding to one line). 1c: Gene diagram showing variants position with respect to previously reported variants involved in POI (premature ovarian insufficiency), DOR (diminished ovarian reserve), PCOS (polycystic ovarian syndrome), and DZT (dizygotic twin). Reported variants are heterozygous except one homozygous (hom.) NH2: Amino terminal domain, COOH: Carboxy terminal domain. One star: functional studies showing weak reduction of expression, two stars: functional studies showing strong reduction of expression, three stars: functional studies showing loss of expression, underline: non-sense variants, bold: variants observed in our patient. 1d: Western blot of GDF9 in vitro expression. Left: Variants impair protein production/secretion in conditioned media. Detection of WT GDF9 (rhGDF9: recombinant human and GDF9-His8: HIS-tagged GDF9) and poor/absent detection of GDF9-P374L and GDF9-L265* (black box), indicating the variants impair the production of GDF9 and/or its secretion into conditioned media. Middle: Analysis of precursor and mature GDF9 intracellularly. Successful processing of WT HIS-tagged GDF9 (3). Detection of precursor GDF9-P374L without detection of mature protein suggesting impaired processing (4). Absence of detection of precursor and mature GDF9-L265* confirming lack of expression resulting from premature stop codon (5). Right: Variants impair BMP15-GDF9 interaction intracellularly. Detection of HIS-tagged BMP15 and WT GDF9 indicating they complement each other. Weak detection of HIS-tagged BMP15 and either GDF9-P374L or GDF9-L265* (black box), indicating the complementary interaction is not achieved with mutant GDF9.

Article Snippet: Recombinant human GDF9 mature domain (R&D Systems, Minneapolis, MN, USA - 8266-G9-010) was used as reference.

Techniques: Sequencing, Variant Assay, Functional Assay, Expressing, Western Blot, In Vitro, Recombinant, Mutagenesis