Protein kinase B is required for follicle-stimulating hormone mediated beta-catenin accumulation and estradiol production in granulosa cells of cattle
Introduction
The ovary is a dynamic reproductive organ that undergoes numerous changes during follicular maturation. Ovarian follicles develop in response to endocrine regulation by the hypothalamic–pituitary–gonadal axis, and several intra-ovarian factors. Pituitary derived FSH signals via a G-protein coupled receptor to initiate cAMP/protein kinase A (PKA) activity as well as induce additional intracellular signaling pathways to regulate granulosa cell (GC) target genes involved in proliferation, maturation, and estradiol (E2) synthesis (Hunzicker-Dunn and Maizels, 2006). In particular, phosphoinoside 3-kinase (PI3K) is an essential component of FSH signaling which leads to phosphorylation and activation of protein kinase B (PKB/AKT) (Richards et al., 2002, Gloaguen et al., 2011). Furthermore, FSH-treated rat GC transfected with a dominant negative AKT vector fail to induce aromatase mRNA and E2 production (Zeleznik et al., 2003). Additionally, binding of locally secreted insulin-like growth factor 1 (IGF-I) to tyrosine kinase receptors results in receptor auto-phosphorylation which ultimately leads to PI3K activation. Through a series of signaling cascades resulting from PI3K, AKT is phosphorylated on Thr308 and Ser473 which is important for its kinase activity (Alessi et al., 1996, Nicholson and Anderson, 2002).
Recent data indicate that FSH induction of CTNNB1 may be by a direct effect of increased AKT activity. Castañon et al. (2012) noted FSH-treated GC of cattle had increased protein accumulation of CTNNB1 and a tendency for an increase in AKT compared with non-treated controls. Therefore, the objective of the present study was to determine if FSH increased CTNNB1 accumulation in GC of cattle via activation of AKT. To accomplish this, AKT signaling was modulated using specific pathway activators and inhibitors. We report that AKT activity is required for FSH and IGF-I induction of CTNNB1 accumulation necessary in the regulation of E2 production from GC.
Section snippets
Tissue collection
Cattle ovaries were collected at a local federally inspected abattoir (Creekstone Farms, Arkansas City, KS) from non-pregnant cows and heifers not exposed to growth-promoting implants, ionophores, or antibiotics. For each biological replicate, paired ovaries were collected from 18 females and placed in individually marked whirl packs. A minimum of three biological replicates were performed for each experiment.
Ovaries at random stages of the estrous cycle were rinsed with 0.9% saline, followed
Results and discussion
A requirement of CTNNB1 as a key transcriptional co-factor in gonadotropin regulation of steroidogenesis has brought new perspectives to GC signal transduction. Previous data suggest CTNNB1 is hormonally regulated in developing cattle follicles, as relative amounts of CTNNB1 protein increase in large follicles producing greater amounts of E2 likely in response to endogenous FSH (Castañon et al., 2012). Likewise, CTNNB1 increased and total AKT protein abundance tended to increase in primary
Conclusion
In summary, findings in the present study extend the knowledge regarding how FSH regulates CTNNB1 protein in GC of cattle. Previous data suggested canonical wingless-type mouse mammary tumor virus integration site 2 (WNT2) or AKT signaling as two possible mechanisms for CTNNB1 accumulation, although WNT2 was not directly tested and therefore, cannot be completely discounted; the present studies reveal the important role of AKT-mediated CTNNB1 regulation. A model is proposed whereby signaling
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgements
This research was supported by the Oklahoma Agric. Exp. Sta., Stillwater (OKL02789); and Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR10-030S to JAHG. The authors would like to thank Creekstone Farms (Arkansas City, KS) for generous donation of cattle ovaries. We express the deepest gratitude to Dr. Carla Goad for assisting with statistical evaluation of data. Appreciation is also expressed to the NIDDK's National Hormone and Peptide Program and A.F. Parlow for
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