Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro

Abstract

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2-1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte-cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0-100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

Keywords: Bone morphogenetic protein 15 (BMP15); Cumulus expansion; Growth differentiation factor 9 (GDF9); Oocyte growth; Pig.

Fig. 1.

Expression of GDF9 and BMP15 mRNAs in porcine oocyte–cumulus cell complexes (OCCs; A), oocytes (Oos; B), cumulus cells (CCs; C), and mural granulosa cells (MGCs; D) from early antral follicles (1.2–1.5 mm) and antral follicles (4.0–6.0 mm) was examined by RT-PCR (E) and qPCR (F and G). The RT-PCR product bands specific to porcine GDF9, BMP15, and GAPDH (internal control) mRNAs are shown in the top, middle, and bottom panels, respectively (E). Relative expression levels of GDF9 (F) and BMP15 (G) mRNAs are shown as the mean ± S.E. from more than three replicates. Porcine GAPDH was used as an internal control.

Fig. 2.

Typical morphologies of porcine oocyte–cumulus cell complexes (OCCs) cultured with GDF9 (A) and BMP15 (B) during growth culture. The top, middle, and bottom panels in each set of pictures show results from Day 0, 3, and 5 of incubation, respectively. The different concentrations (ng/ml) of growth factors are shown in increasing order from left to right. The scale bar represents 200 µm.

Fig. 3.

Effects of GDF9 and BMP15 on the diameters of porcine oocyte–cumulus cell complexes (OCCs) during growth culture (A and B), and the diameters of oocytes after growth culture (C and D). OCCs were cultured in the growth medium supplemented with GDF9 (A and C) and BMP15 (B and D). The diameters of OCCs excluding disintegrated complexes were measured at Day 0, 3, and 5. The diameters of OCCs including fully grown oocytes collected from antral follicles (4.0–6.0 mm) were measured as in vivo controls (FO) in (A) and (B). The diameters of growing oocytes (GO) from early antral follicles (1.2–1.5 mm) and FO were measured as in vivo controls in (C) and (D). GDF9 and BMP15 concentrations are shown in ascending order under each set of boxes. The numbers of OCCs or oocytes (n) used for each experiment are shown at the bottom of each box. The mean diameter ± S.E. is shown at the top of each box. Boxes with different letters (a–g) in each figure are significantly different (two-way ANOVA for OCC diameters, and one-way ANOVA for oocyte diameters; P < 0.05). The experiments were replicated more than three times.

Fig. 4.

Typical morphologies of the cumulus expansion of oocyte–cumulus cell complexes (OCCs, A–C). After growth culture for 5 days with 0, 10, 50, or 100 ng/ml GDF9 (B) or BMP15 (C), OCCs were futher cultured for maturation without GDF9 and BMP15. OCCs collected from early antral follicles (1.2–1.5 mm; A, GO) and antral follicles (4.0–6.0 mm; A, FO) were cultured in the maturation medium as in vivo controls. The scale bar represents 500 µm. Relative expression levels of LHCGR (D) and FSHR (E) mRNAs in porcine cumulus cells after 5 days of growth culture with GDF9 (100 ng/ml) or BMP15 (100 ng/ml). Cumulus cells from early antral follicles (GO: 1.2–1.5 mm; open bar) and antral follicles (FO: 4.0–6.0 mm; grey bar) were used as in vivo controls. Porcine GAPDH was used as an internal control. Values with different letters (a–c) are significantly different (one-way ANOVA; P < 0.05). As LHCGR mRNA of GO was not detected in several analyses (shown as N.D.), it was excluded from the statistical analysis. Data are shown as the mean ± S.E. from three replicates.