Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions

Abstract

The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.

Fig. 1.

Purification of h/mGDF9:BMP15 and initial testing of their activities in mouse granulosa cell assay. (A) A plasmid containing MYC-tagged GDF9 and FLAG-tagged BMP15 was transfected into HEK-293T cells to yield GDF9 (green), BMP15 (blue), and GDF9:BMP15 (bicolor). Use of anti-FLAG agarose allowed immunoprecipitation of BMP15 and GDF9:BMP15. (B) After immunoprecipitation, h/mBMP15 was detected by anti-FLAG, and h/mGDF9 was detected by anti-MYC. (C–H) Mouse granulosa cells were treated with no ligand (white), 100 ng/mL h/mBMP15 (h/mB15; blue), 100 ng/mL h/mGDF9 (h/mG9; green), 3 ng/mL hGDF9:BMP15 or 16 ng/mL mGDF9:BMP15 (h/mG9:B15; orange), and a mix of 100 ng/mL homodimers (h/mB15+h/mG9; yellow) for 5 h. Total RNA was extracted, and downstream ECM genes Ptx3 (C and F), Has2 (D and G), and Ptgs2 (E and H) were quantified by qPCR and are shown relative to the control (no ligand) sample. Data in C–H represent the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls not treated with ligand. (C–E) Induction of Ptx3, Has2, or Ptgs2 with hBMP15 versus combination treatment was not statistically significant. (F–H) Induction of Ptx3, Has2, or Ptgs2 with mGDF9 versus combination treatment was not statistically significant.

Fig. 2.

h/mGDF9:BMP15 dose-dependent effects in downstream ECM gene regulation. Mouse granulosa cells were treated with serial dilutions of hGDF9:BMP15 (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 ng/mL) or mGDF9:BMP15 (0.1, 0.3, 1.0, and 3.0 ng/mL). hBMP15 (100 ng/mL) or mGDF9 (10 ng/mL) was used as a positive control. Gene expression of Ptx3 (A and D), Has2 (B and E), and Ptgs2 (C and F) was measured to quantify ligand activities. Data represent the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls not treated with ligand.

Fig. 3.

h/mGDF9:BMP15 dose-dependent effects in OOX cumulus cell expansion. (A–E) Representative photographs of OOX cumulus cells treated with no ligand (A), 30 ng/mL hBMP15 (B), 30 ng/mL mGDF9 (C), 0.3 ng/mL hGDF9:BMP15 (D), or 0.3 ng/mL mGDF9:BMP15 (E) in the presence of EGF (10 ng/mL). (F–G) OOX cumulus cells were treated with 30 ng/mL homodimers and a 1:10 serial dilution of hGDF9:BMP15 (0.0003, 0.003, 0.03, 0.3, and 3.0 ng/mL) or mGDF9:BMP15 (0.3 and 3.0 ng/mL) to test dose-dependent effects in OOX cumulus cell expansion. Data in F and G represent the mean ± SEM (n = 10). ***P < 0.001 compared with controls not treated with ligand.

Fig. 4.

Identification of the h/mGDF9:BMP15 SMAD-signaling pathway and type 1 receptors in mouse granulosa cells. (A) Wild-type granulosa cells were treated with ligands (100 ng/mL hBMP15, 100 ng/mL mGDF9, 3 ng/mL hGDF9:BMP15, and 16 ng/mL mGDF9:BMP15) for 1 h. Anti–P-SMAD1/5/8 and anti–P-SMAD2/3 were used to detect the two SMAD-signaling pathways. Actin was used as the internal control. (B) Alk6^−/− granulosa cells were treated with the same ligands to examine the phosphorylation of SMAD1/5/8 and SMAD2/3. Actin was used as the internal control. (C–H) The ALK2/3/6 inhibitor LDN-193189 (100 nM) or the ALK4/5/7 inhibitor SB-505124 (1 μM) was coincubated with the ligands to test if the induction of downstream ECM genes Ptx3 (C and F), Has2 (D and G), and Ptgs2 (E and H) was abolished compared with controls not treated with inhibitor. Data in C–H represent the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Identification of the h/mGDF9:BMP15 type 2 receptor in mouse granulosa cells. (A) Ligands (100 ng/mL mGDF9 and 3 ng/mL h/mGDF9:BMP15) were incubated with 1 μg/mL BMPR2* (T.B.T), BMPR2, ACVR2A, or ACVR2B ECD. Anti–P-SMAD2/3 was used to compare SMAD2/3 phosphorylation levels among different type 2 receptor ECD treatments. Actin was used as the internal control. (B–G) BMPR2* ECD (1 μg/mL) was incubated with the ligands (100 ng/mL hBMP15, 100 ng/mL mGDF9, 3 ng/mL hGDF9:BMP15, and 16 ng/mL mGDF9:BMP15) to test if the induction of downstream ECM genes Ptx3 (B and E), Has2 (C and F), and Ptgs2 (D and G) was altered compared with controls without the ECD receptor. Data in B–G represent the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Identification of the h/mGDF9:BMP15 SMAD-signaling pathway and type 1 receptors in COV434 cells. (A) COV434 cells were treated with 100 ng/mL hBMP15, 100 ng/mL mGDF9, and 3 ng/mL h/mGDF9:BMP15 for 1 h. Anti–P-SMAD1/5/8 and anti–P-SMAD2/3 were used to detect the two P-SMAD–signaling pathways. Actin was used as the internal control. (B) COV434 cells were treated with 100 ng/mL hBMP15, 100 ng/mL hGDF9, and 3 ng/mL hGDF9:BMP15 and a mix of their homodimers (100 ng/mL) for 1 h. Anti–P-SMAD2/3 was used to define ligand activities. Actin was used as the internal control. (C and D) (Left) COV434 cells were treated with serial dilutions of hBMP15 or mGDF9 (1.0, 10, or 100 ng/mL) or h/mGDF9:BMP15 (0.1, 0.3, 1.0, or 3.0 ng/mL) to test the dose-dependent effect on SMAD2/3 phosphorylation. Actin was used as the internal control. (Right) Western blots of three independent experiments were quantified, and the data are shown as the mean ± SEM (n = 10). ***P < 0.001 compared with controls not treated with ligand. (E and F) The ALK2/3/6 inhibitor LDN-193189 (100 nM) or the ALK4/5/7 inhibitor SB-505124 (1 μM) was coincubated with the ligands to test if the induction of SMAD phosphorylation was abolished compared with controls with no inhibitor treatment. Actin was used as the internal control.

Fig. 7.

Models for BMP15 and GDF9 homodimers and GDF9:BMP15 heterodimers in regulating cumulus granulosa cell functions. (A) In human (and sheep), the GDF9:GDF9 homodimer has extremely low activity. Active BMP15:BMP15 homodimer binds to BMPR2 and ALK6 to up-regulate ECM genes minimally via a SMAD1/5/8 pathway, whereas the potent GDF9:BMP15 heterodimer likely binds to a BMPR2-ALK4-ALK6 receptor complex to transmit a signal through phosphorylation of SMAD2/3. (B) In mouse (and rat), BMP15:BMP15 is inactive, whereas the GDF9:GDF9 homodimer cooperates with the GDF9:BMP15 heterodimer to regulate granulosa cell function via a SMAD2/3 pathway starting from secondary follicles. The GDF9:GDF9 homodimer is likely the dominant (active) ligand in primary follicles.