Journal: Cell

Article Title: Optimal-transport analysis of single-cell gene expression identifies developmental trajectories in reprogramming

doi: 10.1016/j.cell.2019.01.006

Figure Lengend Snippet: (A) Log-likelihood ratio of obtaining iPSC vs non-iPSC fate on each day (x-axis) in 2i. Obox6+ cells in red. (B) Bright field and fluorescence images of iPSC colonies generated in 2i by overexpression of OKSM with either Zfp42 or Obox6 (or negative control). (C) Percentage of Oct4-EGFP+ colonies in 2i on day 16, for one of five experiments (Figure S6D). Error bars show standard deviation of three biological replicates. (D-F) Effect of varying concentration of GDF9 (red) vs control (grey) on (D) Oct4-EGFP+ colonies (error bars show standard deviation); (E) the strength of iPSC signature score in bulk RNA-Seq; and (F) cellular composition assayed by scRNA-seq. (G) Schematic of the reprogramming landscape in serum. Color indicates cell-set membership. Color of TFs indicates which cell set they regulate. Color of cytokine indicates the cell class to which they signal. See also Figure S6.

Article Snippet: Determination of paracrine effects of GDF9 on reprogramming To determine the effect of GDF9 on reprogramming, we plated secondary MEFs at a concentration of 5,000 cells per well of a 24-well plate and added either recombinant mouse GDF9 (R&D Systems, 739-G9-010, lot SOZ0516121) daily from day 8 onward, or control (0.1% Bovine Serum Albumin in 4 mM HCl, R&D Systems, RB04).

Techniques: Fluorescence, Generated, Over Expression, Negative Control, Standard Deviation, Concentration Assay, Control, RNA Sequencing

Journal: Advanced Science

Article Title: AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5‐POLG Axis

doi: 10.1002/advs.202307480

Figure Lengend Snippet: Obesity impairs oocyte maturation and mtDNA activity. A) Number of pups per litter born from female mice fed control (CON) and obesogenic diet (OB) (n = 12). B) H&E staining of ovaries and follicles. Scale bar 200 µm, 50 µm (n = 8). The number of primary, secondary and antral follicles per section of ovary in CON and OB females (n = 8). C, D) The mature oocytes were collected in the fallopian tubes, and the number of MII oocytes and the proportion of abnormal MII oocytes were counted. Stars indicate the abnormal oocytes, and scale bar 50 µm (n = 15). E) Immunoblotting of oocyte maturation indicators in MII oocytes, including BMP15, GDF‐9, active caspase‐7, and active caspase‐3. β‐Tubulin was used as a loading control (n = 5). F) Immunoblotting of mitochondrial fission and biomass indicators in MII oocytes, including Drp1, Drp1‐S616, VDAC and Tom20. β‐Tubulin was used as a loading control (n = 5). G) mtDNA content in mature oocytes (n = 6). H) mRNA expression of mtDNA genes involved in respiration chain complex (n = 6). I) O 2 consumption was measured in mature oocytes (n = 5; 70 oocytes were pooled per replicate). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; unpaired two‐tail Student's t test was used in analyses.

Article Snippet: [ , ] Membrane were probed using primary antibodies, including BMP‐15 (#18 982; Proteintech), GDF‐9 (af739; Novus), Caspase‐7 (sc‐56063; Snta‐Cruz), Caspase‐3 (#9662; Cell Signaling), β‐tubulin (#179 513; Abcam), DRP1 (#8570; Cell Signaling), p‐DRP1 (#3455; Cell Signaling), VDAC (#4866; Cell Signaling), TOM20 (sc‐17764; Santa Cruz), ATF5 (ab184923; Abcam), LonP1 (#15 440; Proteintech), POLG (ab128899; Abcam), HSP70 (#4872; Cell Signaling), AMPKa (D63G4; Cell Signaling), p‐AMPKa (#2535; Cell Signaling), GAPDH (#97 166; Cell Signaling).

Techniques: Activity Assay, Staining, Western Blot, Expressing

Journal: Advanced Science

Article Title: AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5‐POLG Axis

doi: 10.1002/advs.202307480

Figure Lengend Snippet: AMPK activation improves oocyte maturation and limits mtDNA heteroplasmy in obese females. A) Obese females were fed metformin in drinking water for 4‐weeks (OB+Metf). Immunoblotting of AMPKa, AMPKa phosphorylation at Thr‐172, BMP15, GDF‐9, ATF5, POLG and LonP1 proteins in mature oocytes of control (CON), obese (OB) and OB+Metf females. The β‐tubulin was used as a loading control (n = 5). B) H&E staining in ovary. The primary, secondary and mature oocytes were quantified in each ovary section (n = 5). C) Immunostaining of MitoSpy (green color) and TMRE (red color) in mature oocytes. Fluorescent intensity was qualified by ImageJ and normalized by MitoSpy/TMRE. Five mature oocytes were used in the analyses. Scar bar MitoSpy, 200 µm; bright field, 150 µm; TMRE, 80 µm. D) Number of pups per litter born from female mice. E) Co‐immunoprecipitation of ATF5 in measuring POLG in mature oocytes. ATF5 and POLG were immunoprecipitated followed with SDS‐PAGE separation and measured in immunoblotting. IgG was used as a negative control in immunoprecipitation. F) Percentage of mtDNA heteroplasmy and total mutation sites in mature oocytes from mtDNA‐sequencing (n = 5). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; one‐way ANOVA was used in analysis.

Article Snippet: [ , ] Membrane were probed using primary antibodies, including BMP‐15 (#18 982; Proteintech), GDF‐9 (af739; Novus), Caspase‐7 (sc‐56063; Snta‐Cruz), Caspase‐3 (#9662; Cell Signaling), β‐tubulin (#179 513; Abcam), DRP1 (#8570; Cell Signaling), p‐DRP1 (#3455; Cell Signaling), VDAC (#4866; Cell Signaling), TOM20 (sc‐17764; Santa Cruz), ATF5 (ab184923; Abcam), LonP1 (#15 440; Proteintech), POLG (ab128899; Abcam), HSP70 (#4872; Cell Signaling), AMPKa (D63G4; Cell Signaling), p‐AMPKa (#2535; Cell Signaling), GAPDH (#97 166; Cell Signaling).

Techniques: Activation Assay, Western Blot, Staining, Immunostaining, Immunoprecipitation, SDS Page, Negative Control, Mutagenesis, Sequencing

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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: 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-GAPDH (1:5000, HRP-60004, Proteintech, China), rabbit anti-GDF9 (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: Gene Expression, RNA Sequencing, Expressing, Immunostaining, Staining, In Vitro

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of the distinct presence of Cx43 and BMP-15 in the oophorons of each group (× 200) A-F: Cx43; G-L: BMP-15. A, G: control group; B, H: model group; C, I: positive group; D, J: low dose of BSJPP group; E, K: moderate dose of BSJPP group; F, L: high dose of BSJPP group. Positive group treated with premarin (0.03 mg/kg) once daily for 90 d. Con- trol and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Immunohistochemi- cal analysis clearly revealed the presence of Cx43 and BMP-15 in the oophorons of control mice and those treated with BSJPP (D, E) and premarin compared with the model group. BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Immunohistochemical staining, Control, In Vivo

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 4 Expression Cx43 and BMP-15 mRNA in the ovaries of mice in each group. 1: control group; 2: model group; 3: positive group; 4: low dose of BSJPP group; 5: moderate dose of BSJPP group; 6: high dose of BSJPP group. Positive group treated with pre- marin (0.03 mg/kg) once daily for 90 d. Control and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Compared with the model group, significantly upregulated expression of both Cx43 and BMP-15 was detected in the control group and the groups treated with BSJPP (M and L groups). BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Expressing, Control, In Vivo

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: