Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody

Abstract

Background: This study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms.

Methods: Porcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium.

Results: Treatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment.

Conclusions: These results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

Figure 1

Estradiol concentration by cultured porcine granulosa cells in response to anti-inhibin α-subunit antibody treatment at varying concentrations (0, 50, 100, 200, 300  μg/mL). Each value (plus SEM) represents the average of data from 6 independent culture experiments, each with 3 replicate wells. Means marked with different letters are significantly different (a-b, b-c, c-d, c-e, d-e: P < 0.01).

Figure 2

Estradiol concentration by cultured porcine granulosa cells in response to treatments of varying concentrations of anti-inhibin α-subunit antibody (0, 25, 50, and 100  μg/mL) and FSH (0, 10, 20, and 50 ng/mL). Each value (plus SEM) represents the average of data of 6 independent culture experiments, each with 3 replicate wells. Means marked with different letters are significantly different (a-b, b-c: P < 0.05; a-c, a-d, b-d, c-d: P < 0.01).

Figure 3

Proliferation (represented by the MTT OD value) of porcine granulosa cells following culture for varying times and in response to different concentrations (0, 50, 100, and 200  μg/mL) of anti-inhibin α-subunit antibody treatment. Each value (plus SEM) represents data of 6 independent culture experiments, each with 3 duplicate wells. Means marked with different letters are significantly different (a-b, b-c: P < 0.05; a-c: P < 0.01).

Figure 4

Expression levels of phosphorylated FOXL2, PKA, and Smad3 proteins in cultured porcine granulosa cells in response to varying concentrations (0, 50, and 200  μg/mL) of anti-inhibin α-subunit antibody treatment, normalized to β-actin expression. Each value (plus SEM) represents the average data of 4 independent culture experiments, each with 2 duplicate wells. Means marked with different letters are significantly different (a-b: P < 0.01).

Figure 5

mRNA expression levels of cultured porcine granulosa cells in response to varying concentrations (0, 50, and 200  μg/mL) of anti-inhibin α-subunit antibody treatment, normalized to β-actin expression. Each value (plus SEM) represents the average of data from 6 independent culture experiments, each with 3 duplicate wells. Means marked with different letters are significantly different (a-b: P < 0.05; a-c, a-d, b-c, c-d: P < 0.01).