Stimulatory effects of TGFα in granulosa cells of bovine small antral follicles

Abstract

Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation and apoptosis of granulosa cells (GC) and can influence whether small antral follicles continue their growth or undergo atresia. Transforming growth factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function; yet its investigation has been largely overshadowed by emerging interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMP) and anti-Mullerian hormone (AMH). Here, effects of TGFα on bovine GC proliferation, intracellular signaling, and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5 mm) from slaughterhouse specimens of bovine ovary pairs were aspirated and the cells were plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 °C in 5% CO2 for 3 to 4 d. Once confluent, the cells were sub-cultured for experiments (in 96-, 12-, or 6-well plates) in serum-free conditions (DMEM/F12 medium with ITS). Exposure of the bGC to TGFα (10 or 100 ng/mL) for 24 h stimulated cell proliferation compared to control (P < 0.05; n = 7 ovary pairs). Proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 h of treatment, as evidenced by phosphorylated ERK1/2 expression (P < 0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, P < 0.05). Additionally, prior exposure of the bGC to TGFα (100 ng/mL) failed to prevent Fas Ligand (100 ng/mL)-induced apoptosis, as measured by caspase 3/7 activity (P < 0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bGC from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting that TGFα may be incapable of promoting their persistence in follicles during the process of follicular selection/dominance.

Keywords: apoptosis; bovine; follicle; granulosa cell; mitogen-activated protein kinase (MAPK) signaling; transforming growth factor-alpha.

Figure 1.

Effects of TGFα on proliferation of bovine GC and MAPK/ERK signaling. (A) Relative increase in cell number after 24H of culture was measured by MTS assay (absorbance at 490 nm). Different letters denote differences among groups (P < 0.05; n = 7 ovary pairs). (B) Immunoblot and quantitation of phosphorylated ERK1/2 (pERK1/2) expression relative to total ERK. (Left) Immunoblot of pERK1/2 and total ERK expression by bovine GC after exposure to TGFα and/or U0126 for 2H. (Right) Expression of pERK1/2 relative to total ERK as measured by densitometry. Asterisk denotes difference relative to Control (CTRL; P < 0.05; n = 3 ovary pairs).

Figure 2.

Effects of MAPK signaling on proliferation of bovine GC and Fas ligand-induced apoptosis. (A) Relative increase in cell number after 24 h of culture was measured by MTS assay (absorbance at 490 nm). Different letters denote differences among groups (P < 0.05; n = 4 ovary pairs). (B) (Left) Time-series images (100×) of cultures of bovine GC after pretreatment with TGFα for 24 h, followed by exposure to FasL for up to 6 h. Note abundance of phase-bright cells (i.e., dying cells) as early as 3 h after exposure to FasL. (Right) Bar graph depicting incidence of apoptosis of bovine GC (Caspase 3/7 activity) after pretreatment with TGFα (100 ng/mL) for 24 h, followed by exposure to FasL for up to 6 h. Different letters denote differences among groups (P < 0.05; n = 7 ovary pairs).