Elsevier

Animal Reproduction Science

Volume 163, December 2015, Pages 97-104
Animal Reproduction Science

Protein kinase B is required for follicle-stimulating hormone mediated beta-catenin accumulation and estradiol production in granulosa cells of cattle

https://doi.org/10.1016/j.anireprosci.2015.10.003Get rights and content

Abstract

Follicle-stimulating hormone regulation of ovarian estradiol (E2) production requires involvement of beta-catenin (CTNNB1), a transcriptional co-factor. In cultured granulosa cells (GC) of cattle, FSH treatment increased protein abundance of CTNNB1 as well as protein kinase B (AKT), a molecule known to regulate components of the CTNNB1 degradation complex. However, whether FSH induction of CTNNB1 is through direct modulation of AKT remains to be determined. To investigate specific contributions of AKT to CTNNB1 accumulation, GC were treated with insulin-like growth factor-I (IGF-I), a well-established AKT activator, in the presence or absence of FSH. Granulosa cells treated with FSH, IGF-I, and IGF-I plus FSH had increased CTNNB1 accumulation compared with controls (P  0.02; n = 6). E2 medium concentrations were greater (P = 0.09; n = 4) in FSH treated cells compared to controls (166 and 100 ± 28 pg/mL, respectively). Treatment with IGF-I and IGF-I plus FSH increased (P < 0.01) E2 to comparable concentrations. Subsequently, GC treated with lithium chloride (LiCl), a pharmacological activator of AKT, provided a response consistent with IGF-I treated cells, as LiCl, FSH, and FSH plus LiCl increased CTNNB1 accumulation compared with non-treated controls (P  0.03; n = 3). In contrast, inhibition of AKT signaling with LY294002 suppressed the ability of FSH and IGF-I to regulate CTNNB1. Additionally, LY294002 treatment reduced FSH and IGF-I mediated E2 medium concentrations (P  0.004). These results demonstrate that activation of AKT is required for gonadotropin regulation of CTNNB1 accumulation and subsequent ovarian E2 production.

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

References (35)

  • D.R. Alessi et al.

    Mechanism of activation of protein kinase B by insulin and IGF-1

    EMBO J.

    (1996)
  • J. Baker et al.

    Effects of an Igf1 gene null mutation on mouse reproduction

    Mol. Endocrinol.

    (1996)
  • B. Bao et al.

    Changes in messenger ribonucleic acid encoding luteinizing hormone receptor, cytochrome P450-side chain cleavage, and aromatase are associated with recruitment and selection of bovine ovarian follicles

    Biol. Reprod.

    (1997)
  • J. Behrens et al.

    Functional interaction of beta-catenin with the transcription factor LEF-1

    Nature

    (1996)
  • K.M. Brown et al.

    Lithium: the pharmacodynamic actions of the amazing ion

    Ther. Adv. Psychopharmacol.

    (2013)
  • B.I. Castañon et al.

    Follicle-stimulating hormone regulation of estradiol production: possible involvement of WNT2 and beta-catenin in bovine granulosa cells

    J. Anim. Sci.

    (2012)
  • E. Chalecka-Franaszek et al.

    Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
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