Date of Award

2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

First Advisor

David H. Townson

Second Advisor

Douglas Johnson

Abstract

Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation versus apoptosis of granulosa cells (GCs), 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 has been largely overshadowed by current interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMPs) and anti-Mullerian hormone (AMH). In the current study, effects of TGFα on bovine GC proliferation, intracellular signaling and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5mm) from bovine ovaries were aspirated and the cells were initially plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 degrees Celsius in 5% CO2 for 3-4 days. Once confluent, the cells were sub-cultured to 96-, 12- or 6-well plates in serum-free conditions (insulin 100 ng/mL; transferrin 55 ng/mL; sodium selenite 6.7 pg/mL). 24-hour treatment of bovine GCs with TGFα (10 and 100ng/mL) stimulated cell proliferation compared to control (p<0.05; n = 7 ovary pairs). Cell proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 hours of treatment, as measured 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, pre-exposure of the bovine GCs to TGFα (100ng/mL) failed to prevent Fas Ligand (100ng/mL)-induced apoptosis, as determined by caspase 3/7 activity (P<0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bovine GCs from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting TGFα may be incapable of promoting the persistence of follicles during the process of follicular selection and deviation.

Language

en

Number of Pages

69 p.

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