anti gdf9  (Bioss)

Journal: Animal Cells and Systems

Article Title: Generation of mouse and rat xenogeneic ovaries in vitro for production of mouse oocyte

doi: 10.1080/19768354.2024.2363601

Figure Lengend Snippet: Isolation of individual rat follicles for IVG culture and MII oocyte from IVM culture. (A) Representative images of isolation of individual follicles. (Scale bars 50 μm) (B) Representative follicle cultured on a Transwell-COL membrane on days 30–39 of culture. (Scale bars 200 μm) (C) Oocyte phase and expression of the Ddx4 and oocyte-specific marker GDF9 in vitro . (Scale bars 50 μm) (D) Image of the first polar body in MII oocyte. (Scale bar 50 μm) (E) Expression of α – tubulin in MII oocyte in vitro. (Scale bars 50 μm)

Article Snippet: The following reagents and chemicals were purchased from respective suppliers: Dulbecco’s modified Eagle’s medium (1×) (DMEM; Gibco, cat. no. 11965-092), phosphate-buffered saline (PBS; Gibco, cat. no. 10010-023), Minimum Essential Medium Eagle, alpha modification (1×) (α-MEM; Gibco, cat. no. 12571–063 and 32571-036), 2-mercaptoethanol (55 mM; Gibco, cat. no. 21985-023), fetal bovine serum (FBS; Gibco, cat. no. 16000-044), GMEM (1×) (Thermo, cat. no. 11710-035), KSR (15%) (Thermo, cat. no. 10828-028), retinoic acid (100 mM; Sigma, cat. no. R2625), GlutaMax (100×) (Thermo, cat. no. 32571-036), non-essential amino acids (100×) (Thermo, cat. no. 11140-050), sodium pyruvate (Gibco, cat. no. 11360-070), Y27632 (Peprotech, cat. no. 1293823), penicillin/streptomycin/L-glutamine (100×) (Gibco, cat. no. 10378-016), Insulin-Transferrin-Selenium (ITS; Thermo, cat. no. 41400-045), ascorbic acid (150 mM; TCI, cat. no. G0394), ICI182,780 (Tocris, cat. no. 1047), follicle-stimulating hormone (FSH; Sigma, cat. no. F4021), epidermal growth factor (EGF; R&D Systems, cat. no. 2028-EG-200), polyvinylpyrrolidone (PVP; Sigma, cat. no. PVP360), human chorionic gonadotropin (1,200 IU mL −1 ) (hCG; Sigma, cat. no. C8554), collagenase type IV (1 g mL −1 ; Gibco, cat. no. 17104-019), trypsin-EDTA, 0.25% (Gibco, cat. no. 25200-072), TCM 199(Gibco, cat. no. 11150-059), StemPro-34 SFM (1×), liquid (Gibco, cat. no. 10639-011), and hyaluronidase (500 µg ml −1 , TCI, cat. no. H0164), anti-Ddx4 (polyclonal, 1:500; Abcam), anti-GDF9 (polyclonal, 1:500; Invitrogen), anti-α-tubulin (monoclonal, 1:20, Thermo, cat no. 53-4502-82) fluorescently labeled (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA), DAPI (molecular probes, Thermo).

Techniques: Isolation, Cell Culture, Membrane, Expressing, Marker, In Vitro

Journal: Heliyon

Article Title: Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype

doi: 10.1016/j.heliyon.2024.e29879

Figure Lengend Snippet: Distribution of GDF9 in FF from the three PCOS phenotypes and control group. Data are presented as mean ± SD. d P < 0.01, compared with Control Group; a P < 0.01, compared with Control Group; b P < 0.05, compared with PCOS A; c P < 0.05, compared with PCOS B.

Article Snippet: Briefly, the CCs cell slices were fixed with 4 % paraformaldehyde in phosphate-buffered saline for 15 min, then immersed in 3 % hydrogen peroxide to block endogenous peroxidase activity, and subsequently blocked in goat serum for 1 h. The slices were then incubated overnight at 4 °C with primary antibodies rabbit anti-GDF9 (GDF9, Abcam, USA, 1:100), followed by incubation with biotin-labeled anti-rabbit secondary antibody for half an hour.

Techniques:

Journal: Heliyon

Article Title: Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype

doi: 10.1016/j.heliyon.2024.e29879

Figure Lengend Snippet: Expression of GDF9 in the CCs. (a) Immunohistochemical staining of GDF9 expressed in the control group, phenotype A, phenotype B and phenotype D. (Original magnification: × 400, bars, 50 μm). (b) Mean density of GDF9 in CCs of the three PCOS phenotypes and control group, a P < 0.01, compared with Control Group; b P < 0.01, compared with PCOS D; c P < 0.01, compared with Control Group; d P < 0.01, compared with PCOS D.

Article Snippet: Briefly, the CCs cell slices were fixed with 4 % paraformaldehyde in phosphate-buffered saline for 15 min, then immersed in 3 % hydrogen peroxide to block endogenous peroxidase activity, and subsequently blocked in goat serum for 1 h. The slices were then incubated overnight at 4 °C with primary antibodies rabbit anti-GDF9 (GDF9, Abcam, USA, 1:100), followed by incubation with biotin-labeled anti-rabbit secondary antibody for half an hour.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Heliyon

Article Title: Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype

doi: 10.1016/j.heliyon.2024.e29879

Figure Lengend Snippet: Analysis of multiple factors affecting blastocyst formation and clinical pregnancy rate in the PCOS phenotypes.

Article Snippet: Briefly, the CCs cell slices were fixed with 4 % paraformaldehyde in phosphate-buffered saline for 15 min, then immersed in 3 % hydrogen peroxide to block endogenous peroxidase activity, and subsequently blocked in goat serum for 1 h. The slices were then incubated overnight at 4 °C with primary antibodies rabbit anti-GDF9 (GDF9, Abcam, USA, 1:100), followed by incubation with biotin-labeled anti-rabbit secondary antibody for half an hour.

Techniques:

Journal: Cell communication and signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Fig. 5 Effects of Gdf9 variants on antral follicle development after PMSG stimulation. A Appearance of Gdf9wt/wt, Gdf9S415T/S415T and Gdf9Q308X/

Article Snippet: Primary antibodies used were mouse antiHis-tag (1:1000, HA601079, Huabio, China) for detecting His10-GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques:

Journal: Cell communication and signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Fig. 5 Effects of Gdf9 variants on antral follicle development after PMSG stimulation. A Appearance of Gdf9wt/wt, Gdf9S415T/S415T and Gdf9Q308X/

Article Snippet: Primary antibodies used were mouse anti-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit antiGDF9 (1:1000, ab93892, abcam, UK) for detecting GDF9 precursor (51kDa) and mature GDF9 (15kDa) , rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor (calculated molecular weight: 45kDa, observed molecular weight according to the instruction manual: 50kDa), rabbit anti-BMP15 (1:2000, ab108413, abcam, UK) for detecting mature BMP15 (14kDa), rabbit anti-p-smad2 (1:2000, 18338T, CST, USA), rabbit anti- smad2 (1:2000, 5339T, CST, USA) and mouse anti-P4HA2 (1:1000, 66604-1-IG, Proteintech, China).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Clinical characteristics of individuals with GDF9 bi-allelic variants

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Mutagenesis, Biomarker Discovery

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: IVF/ICSI outcomes of individuals with GDF9 bi-allelic variants

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Injection

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Identification of GDF9 bi-allelic variants. A Pedigrees of two affected families. Squares and circles represent males and females respectively. Diamonds represent members whose gender is unknown. Solid symbols indicate the affected members, and open symbols represent unaffected members. The equal sign indicates infertility. Arrows indicate probands. Sanger sequencing chromatograms of GDF9 are shown below. Downward arrows indicate the corresponding variants. B Schematic map of the variant positions in GDF9 at the genomic and protein levels. C Conservation of the affected amino acids is indicated by the alignment of seven species. Red letter represents amino acids affected by the variants. Asterisk indicates that the amino acid is conserved between species

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Sequencing, Variant Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Effects of GDF9 variants on gene expression and GDF9-BMP15 interactions. A Western blots of mature GDF9 proteins expressed in follicular fluid collected from the first and second retrieval cycle of individual P1 and age-matched controls ( n =3). B ELISA of GDF9 in follicular fluid collected from the first retrieval cycle of individual P1 and age-matched controls ( n =6). C Western blots of GDF9 expression in HEK293T cells transfected with human GDF9 WT , GDF9 Q321X , GDF9 S428T , and GDF9 His209GlnfsTer6 respectively and their relative mature-GDF9 expression in culture medium (CM) (3 independent experiments). The amount of concentrated culture medium used for the detection was 35μl per sample per assay. lnfsTer6, His209GlnfsTer6. * P < 0.05. D Western blots of GDF9 precursors and mature GDF9 over time during in vitro cleavage assay (3 independent experiments). Total GDF9=GDF9 precursor+mature GDF9. E Western blots of BMP15 expression in HEK293T cells co-transfected with human BMP15 and human GDF9 WT , GDF9 Q321X , GDF9 S428T , and GDF9 His209GlnfsTer6 respectively and their relative BMP15 precursor expression in CM (3 independent experiments). lnfsTer6, His209GlnfsTer6. * P < 0.05. F Co-IP assay of the binding of GDF9 proteins to BMP15 protein. lnfsTer6, His209GlnfsTer6. * P < 0.05. G Western blots of p-smad2 and smad2 expression in human primary luteinized granulosa cells treated with purified recombinant pro-GDF9 WT -BMP15 and pro-GDF9 S428T -BMP15 protein respectively (3 independent experiments)

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, In Vitro, Cleavage Assay, Co-Immunoprecipitation Assay, Binding Assay, Purification, Recombinant

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Effects of Gdf9 variants on fertility and follicular development. A Average number of pups produced per cage (each cage contains 2 females and 1 male) by females Gdf9 wt/wt ( n =3), Gdf9 Q308X/Q308X ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) when paired with wild-type males. * P < 0.05. B Average litter sizes of Gdf9 wt/wt n =9) and Gdf9 S415T/S415T ( n =8) when paired with wild-type males. * P < 0.01. C Appearance of wild-type and variant ovaries as viewed through the stereoscope. D H&E staining of wild-type and mutant ovaries. E Follicle counts and follicle counts per area (mm 2 ) of Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) mice (PrF: Primordial follicles, PF: Primary follicle, SF: Secondary follicle, AF: Antral follicle, AtF: Atretic follicle). * P < 0.05

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Produced, Variant Assay, Staining, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Effects of Gdf9 variants on antral follicle development after PMSG stimulation. A Appearance of Gdf9 wt/wt , Gdf9 S415T/S415T and Gdf9 Q308X/S415T ovaries 48 hours after PMSG stimulation as viewed through the stereoscope. B Appearance of cumulus-oocyte complex collected 12 hours after HCG injection. C H&E staining of Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) ovaries 48 hours after of PMSG stimulation. The images shown were the sections where the largest follicle of each ovary was located. The follicle diameter, average thickness of the mural granulosa cell layer and cumulus cell layer were calculated for the largest follicle. * P < 0.05. D Ki-67 immunostaining in Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) ovaries. The ki67 positivity rates of cumulus cells and mural granulosa cells in large follicles, and the ki67 positivity rates of granulosa cells in small follicles were calculated separately. * P < 0.05. E TUNEL (red) and DAPI (blue) immunofluorescence in Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) ovaries. The dotted lines outline the boundaries of granulosa cells. Arrows indicate TUNEL-positive cells. TUNEL positivity rates of granulosa cells in each antral follicle were calculated

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Injection, Staining, Immunostaining, TUNEL Assay, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Abnormal estrogen secretion and atypical endometrial hyperplasia caused by Gdf9 variants. A Serum estradiol levels in Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) mice at 10-12 weeks old during proestrus. * P < 0.05. B Serum testosterone levels in Gdf9 wt/wt ( n =3), Gdf9 S415T/S415T ( n =3) and Gdf9 Q308X/S415T ( n =3) mice at 10-12 weeks old during proestrus. C The mRNA expression of Fshr , Lhcgr , Star , Cyp11a1 , Cyp17a1 , Hsd3b2 , and Cyp19a1 of ovaries obtained from Gdf9 wt/wt ( n =3) and Gdf9 Q308X/S415T ( n =3) mice at 10-12 weeks old. * P < 0.05. D The mRNA expression of Fshr , Lhcgr , Star , Cyp11a1 , Hsd3b2 , and Cyp19a1 of mouse granulosa cells collected 48 hours after PMSG stimulation. * P < 0.05. E STAR (yellow) and DAPI (blue) immunofluorescence in Gdf9 wt/wt and Gdf9 Q308X/S415T ovaries. Small (diameter ≤300μm) and large (diameter >300μm) follicles were shown respectively. FD, follicle diameter. F H&E staining of uteri obtained from young (10-12 week) and old (32-34 week) Gdf9 wt/wt and Gdf9 Q308X/S415T mice at the stage of proestrus

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Altered gene expression in granulosa cells of Gdf9 Q308X/S415T mice explored by RNA sequencing. A Differentially expressed genes (DEGs) in granulosa cells collected from Gdf9 wt/wt and Gdf9 Q308X/S415T mice (three samples in each group, and each samples contains granulosa cells from three mice) 48 hours after PMSG stimulation. DEGs were defined as genes with adjusted P value < 0.05 and |log2FC|>1. B,C Enrichment analysis of GO-BP (Gene Ontology- Biological Process) terms ( B ) and Reactome pathways ( C ) using GSEA method. Normalized enrichment score (NES)>0 represent terms/pathways up-regulated in Gdf9 Q308X/S415T mice. NES<0 represent terms/pathways down-regulated in Gdf9 Q308X/S415T mice. D FPKM (Fragments Per Kilo bases per Million reads) of Has2 , P4ha2 , Cldn15 , and Aqp5 in granulosa cells collected from Gdf9 wt/wt and Gdf9 Q308X/S415T mice as a result of RNA-sequencing. * P < 0.05. E Expression of P4ha2 in granulosa cells of Gdf9 wt/wt ( n =3) and Gdf9 Q308X/S415T ( n =3) mice after 48 hours of PMSG stimulation detected by qPCR. * P < 0.05. F P4HA2 immunostaining in ovaries of Gdf9 wt/wt and Gdf9 Q308X/S415T mice. The integrated density of P4HA2 staining of cumulus cells (CC) and mural granulosa cells (GC) were calculated separately. * P < 0.001. G mRNA levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours. SB-431542 is an inhibitor of ALK5, which is the receptor of GDF9. * P < 0.001. H Protein levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours and 48 hours. Meanwhile, protein levels of STAR and CYP19A1 after 48 hours treatment were shown. * P < 0.05

Article Snippet: Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His 10 -GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor.

Techniques: Gene Expression, RNA Sequencing, Expressing, Immunostaining, Staining, In Vitro