Mol Cell Endocrinol. 2017 Jan 15;440:25-33. doi: 10.1016/j.mce.2016.11.002.

Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle.

Totty ML1, Morrell BC1, Spicer LJ2.
1Department of Animal Science, Oklahoma State University, Stillwater, OK, 74078, USA.
2Department of Animal Science, Oklahoma State University, Stillwater, OK, 74078, USA. Electronic address:



To determine the mechanism by which fibroblast growth factor 9 (FGF9) alters granulosa (GC) and theca (TC) cell proliferation, cell cycle proteins that regulate progression through G1 phase of the cell cycle, cyclin D1 (CCND1) and cyclin-dependent kinase-4 (CDK4; CCND1’s catalytic partner), were evaluated. Ovaries were obtained from a local abattoir, GC were harvested from small (1-5 mm) and large (8-22 mm) follicles, and TC were harvested from large follicles. GC and TC were plated in medium containing 10% fetal calf serum followed by various treatments in serum-free medium. Treatment with 30 ng/mL of either FGF9 or IGF1 significantly increased GC numbers and when combined, synergized to further increase GC numbers by threefold. Abundance of CCND1 and CDK4 mRNA in TC and GC were quantified via real-time PCR. Alone and in combination with IGF1, FGF9 significantly increased CCND1 mRNA expression in both GC and TC. Western blotting revealed that CCND1 protein levels were increased by FGF9 in TC after 6 h and 12 h of treatment, but CDK4 protein was not affected. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway inhibitor, U0126, significantly reduced FGF9-induced CCND1 mRNA expression to basal levels. For the first time we show that CCND1 mRNA expression is increased by FGF9 in bovine TC and GC, and that FGF9 likely uses the MAPK pathway to induce CCND1 mRNA production in bovine TC.
KEYWORDS:Cattle; Cyclin D1 (CCND1); Cyclin-dependent kinase 4 (CDK4); Fibroblast growth factor 9 (FGF9); Granulosa cells; Theca cells


Replication of cells or mitosis is regulated by a complex series of events that are highly regulated with much precision. Many hormones and growth factors have been implicated in the regulation of mitosis including fibroblast growth factors (FGF) such as FGF9. We have been studying the role of FGF9 in the regulation of granulosa and theca cell mitosis [1, 2] as well as evaluating its role in ovarian follicular growth [3] and steroidogenesis [1, 2]. Normal cell proliferation is dependent on these processes to be carried out with exactness but when it does not, cancer can develop. An increase in cell proliferation is mediated by increased speed of the cell cycle which is divided into four phases: gap 1 (G1), synthesis (S), gap 2 (G2), and mitosis (M) (see Figure 1). Each of these phases are regulated by different proteins at specific checkpoints, insuring that the cell cycle progresses in the correct order, and only when the cell is healthy. Checkpoints include the restriction (R) point within G1, G1/S, S/G2, G2/M and M. The most important protein families for cell cycle regulation consist of cyclins (CCNs), cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDKIs). Cyclins are the regulatory subunit of CDKs and once they are bound together, the dimmer can activate its respective checkpoint to allow progression of the cell cycle. Each checkpoint is activated by specific proteins as follows: R point, CCND and CDK4 or -6; G1/S phase, CCNE and CDK2; S/G2 phase, CCNA and CDK2; G2/M phase, CCNA and CDK1; and M phase, CCNE and CDK1 as shown in Figure 1. Our results indicate that FGF9 acts on CCND1 at the beginning of the cell cycle [4]. These findings highlight the role FGF9 may play in regulating cell proliferation, and although further research is needed, these findings will help lead the way to development of intracellular targets of FGF9.



Figure 1. Schematic of the main components of the cell cycle in ovarian theca cells and the location of FGF9 action.



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[3] Schütz LF, Schreiber NB, Gilliam JN, Cortinovis C, Totty ML, Caloni F, Evans JR, Spicer LJ. Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle. J Dairy Sci. 2016; 99:9143-9151.

[4] Totty ML, Morrell BC, Spicer LJ. Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle. Mol Cell Endocrinol. 2017; 440:25-33.