Figure 1.
Purification and quantification of pro-mature BMP15.
A. Silver stained SDS-polyacrylamide gel of BMP15 samples. The pro-region appeared as two ∼40 KDa bands and the mature region as a ∼16 KDa band and as a ∼17 KDa band that has been reported to be O-linked glycosylated [68]. Lane 1: flow through. Lanes 2–3: wash 1–2. Lanes 4–7: eluted fractions 1–4. B. The processed mature region of pro-mature BMP15 was quantified by Western blotting [mab28 monoclonal antibody [42]] using the mature region of hBMP15 (R&D Systems) as a standard. Lanes 1–4: mature region of BMP15 (R&D Systems); 200, 100, 50 and 10 ng, respectively. Lanes 5–7: decreasing doses of the purified BMP15 pro-mature complex.
Figure 2.
Bioactivity of different BMP15 and GDF9 forms.
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.
Table 1.
Effect of graded doses of pro-mature BMP15 during IVM on oocyte developmental competence.
Table 2.
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.
Table 3.
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.
Figure 3.
COC glucose consumption and lactate production.
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.
Figure 4.
Quantification of intra-oocyte NAD(P)H, FAD, REDOX ratio and GSH.
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,bminus FSH, x–yplus FSH.
Figure 5.
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.