Journal: Scientific Reports

Article Title: Changes in ovarian hardness and elasticity affect the development and function of secondary follicles

doi: 10.1038/s41598-026-39396-5

Figure Lengend Snippet: Effects of oocyte-secreted factors on secondary follicles cultured in 0.1 % alginate bead with viscosity 4-12 cP. A; The effect of oocyte-secreted factors on the development of secondary follicles with multilayered granulosa cells. Ten secondary follicles with multilayered granulosa cells were placed in each alginate bead and then were cultured for 7 days in culture medium containing 10 ng/ml ovine FSH, GDF9 (0, 10 and 100 ng/ml) and ITS Supplement in the presence of 1 % (v/v) FBS. The diameter of each secondary follicle was measured 7 days after culture. In the box plot: bar, × = median, box = 25 th to 75 th percentiles, whiskers = 10 th and 90 th percentile. N=3 animals for each treatment. The experiment was repeated three times. The different symbols represent significant differences (p < 0.05). *; Significant differences observed between days 0 and 7 in the 10 and 100 ng/mL GDF9 groups (p < 0.05). B; The effect of oocyte-secreted factors on the expression of marker genes about the follicular development, luteinization and inflammation/anti-inflammatory in granulosa cells of cultured secondary follicles. Ten secondary follicles with multilayered granulosa cells were placed in each alginate bead and then were cultured for 7 days in culture medium containing 10 ng/ml ovine FSH, GDF9 (0, 10 and 100 ng/ml) and ITS Supplement in the presence of 1 % (v/v) FBS. The diameter of each secondary follicle was measured 7 days after culture. Levels of mRNA were normalized to that of L19 . The values are the mean ± SEM. N=3 animals for each treatment. The experiment was repeated three times. The value of 0 ng/ml GDF9 was set as 1, and the data are presented as fold induction. *Significant differences observed between the 0 ng/mL and 10 or 100 ng/mL GDF9 groups (p < 0.05).

Article Snippet: Ten of secondary follicles were placed in one alginate bead and were cultured for 7 days in the medium (DMEM/F12 containing penicillin and streptomycin) containing 10 ng/ml ovine FSH (NIDDK, Torrance, CA, USA) and ITS Supplement (Sigma) in the presence of 1 % (v/v) FBS with or without mouse recombinant GDF9 (0, 10 and 100 ng/ml) (R&D Systems Inc., Minneapolis, MN) or Cytochalasin (0, 1 and 10 μM) (Sigma) at 37 °C and 5% CO 2 condition.

Techniques: Cell Culture, Viscosity, Expressing, Marker

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: NPJ Microgravity

Article Title: Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells

doi: 10.1038/s41526-023-00248-5

Figure Lengend Snippet: a Illustration of the strategy to label the Oo-Mvi by Gdf9-Cre;mTmG mouse model. The membrane-localized red fluorescent protein (mT) switches to green-fluorescent protein (mG) in oocytes of Gdf9-Cre;mTmG mouse to label oocyte membrane morphology. b Images of Gdf9-Cre;mTmG oocytes, showing the mushroom-like Oo-Mvi with vesicle tips distributed in the zona pellucida of oocytes in both the SMG and the NG groups. Scale bars, 30 μm. c 3D high-resolution images showing a decreased density of Oo-Mvi on the oocytes’ surface under SMG. Scale bars, 10 μm. d Numbers of Oo-Mvi reduced in SMG oocytes ( n = 8) compared to that in NG ( n = 8), showing a significantly reduced number of Oo-Mvi on oocytes in follicles after SMG treatment. p value = 0.00052. e High magnification showing that the length of Oo-Mvi in the SMG group was shorter than that in the NG group. Scale bars, 5 μm. f Quantification of the length of Oo-Mvi confirmed a dramatic decrease in the SMG group ( n = 30) compared to that in the NG group ( n = 30). p value = 0.00000000000000000052. The colors were inverted to black/white (b/w) to highlight Oo-Mvi in ( e ). Representative images are shown. Data is presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t -test and *** P < 0.001.

Article Snippet: In the GDF9-supplying experiment, we added GDF9 protein (500 ng/mL, 739-G9-010/CF, R&D) into follicle cultured medium for 2 days.

Techniques: Membrane, Two Tailed Test

Journal: NPJ Microgravity

Article Title: Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells

doi: 10.1038/s41526-023-00248-5

Figure Lengend Snippet: a Relative mRNA levels of Gdf9, Bmp15 , and Fgf8 under the SMG or the NG group, showing a decreased expression of OSFs in the SMG group ( n = 4). Gdf9: p value = 0.0081, Bmp15: p value = 0.0032, Fgf8: p value = 0.021. b Relative mRNA levels of Fscn1 and Myo10 under the SMG or the NG group, showing that the expression of GC-TZP forming related genes was downregulated after SMG treatment ( n = 4). Fscn1: p value = 0.025, Myo10: p value = 0.021. c Supplying GDF9 increased the PB1 ratio of oocytes (red arrowheads) from SMG treated follicles. Scale bars, 100 μm. d The ratio of PB1 in different groups, showing that the GDF9 supplement significantly increased the maturation of oocytes ( n = 38 in NG, n = 35 in SMG and n = 61 in SMG + GDF9). NG v.s. SMG: p value = 0.00021, SMG v.s. SMG + GDF9: p value = 0.021. Representative images of oocytes are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t -test in ( a , b ) and two-way ANOVA in ( d ). n.s. P ≥ 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: In the GDF9-supplying experiment, we added GDF9 protein (500 ng/mL, 739-G9-010/CF, R&D) into follicle cultured medium for 2 days.

Techniques: Expressing, Two Tailed Test

Journal: Scientific reports

Article Title: Cooperative effects of oocytes and estrogen on the forkhead box L2 expression in mural granulosa cells in mice.

doi: 10.1038/s41598-022-24680-x

Figure Lengend Snippet: Figure 4. Effects of BMP15 and GDF9 signals on FOXL2 expression in MGCs. (A) MGCs were cultured with or without recombinant BMP15/GDF9 (ODPFs) and E2 for 24 h (n = 4). (B) MGCs cultured with both oocytes and E2 were treated with or without an inhibitor of ALK5, SB431542 for 24 h (n = 4). (C) Comparison of the expression levels of FOXL2 protein between MGCs of Bmp15−/−/Gdf9+/− mice (DM) and those of littermate control Bmp15+/−/Gdf9+/− mice (Ctrl) (n = 3 each). The expression of FOXL2 protein was examined by western blotting (upper panel) and band intensities were quantified (lower panel). Asterisks or different letters (a and b) denote significant differences (p < 0.05).

Article Snippet: In some experiments, the basic culture medium was supplemented with 10–7 M of 17β-estradiol (Sigma-Aldrich), recombinant human BMP15 (50 ng/ml; R&D Systems, Minneapolis, MN, USA), recombinant mouse GDF9 (50 ng/ml; R&D Systems), and/or an inhibitor of ALK5, SB431542 (10 μM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Techniques: Expressing, Cell Culture, Recombinant, Comparison, Control, Western Blot