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J Zhejiang Univ Sci B. 2010 December; 11(12): 952–957.
PMCID: PMC2997404

Follicle-stimulating hormone promotes proliferation of cultured chicken ovarian germ cells through protein kinases A and C activation*

Abstract

The study was conducted to investigate the effects of follicle-stimulating hormone (FSH) on embryonic chicken ovarian germ cell proliferation and its possible involvements of protein kinases A (PKA) and C (PKC) pathways. Ovarian cells were treated with FSH alone or in the presence of forskolin (FRSK), PKA inhibitor (H89), PKC activator (PMA) or inhibitor (H7). The germ cell number was counted from micropictures. The immunocytochemistry of proliferating cell nuclear antigen (PCNA) was applied to identify the proliferating cells. The germ cell labeling index (LI) was determined for cell proliferation. The FSH treatment increased the germ cell number, and this stimulating effect was enhanced by FRSK or PMA, but inhibited by H89 or H7 in a dose-dependent manner. Moreover, the PCNA-LI showed parallel changes with germ cell numbers. This study suggests that FSH may stimulate proliferation of cultured chicken ovarian germ cells by activation of both the PKA and PKC signaling pathways.

Keywords: Follicle-stimulating hormone, Protein kinase A, Protein kinase C, Germ cell, Proliferation

1. Introduction

Follicle-stimulating hormone (FSH) plays a critical role in gonad development. FSH stimulates cell proliferation, growth, and DNA synthesis, enhances the secretion of steroids in the embryonic ovary, provokes structural changes of the ovary, and increases the cell numbers of different ovarian cell subpopulations of hatched chicks (Velázquez et al., 1997; Méndez-Herrera et al., 1998; Ohtani et al., 2001). FSH-binding cells have been detected on Day 6.5 of incubation in the left embryonic ovarian cells. With embryo development, the FSH-positive cells in the left ovary increased, which further stimulated the sex hormone synthesis and accelerated the germ cell development (Woods et al., 1991). The effects of FSH on ovarian granulosa cell proliferation, differentiation, and hormone secretion have been extensively investigated in vitro (Dorrington et al., 1988; Pedernera et al., 1999; Yogo et al., 2002). In our previous study, it was shown that gonadotropins, especially FSH, stimulated the germ cell proliferation (Xie et al., 2004). These findings suggest that FSH plays a promoter role in regulating growth and proliferation of ovarian cells. However, the biological function of FSH and its signal transduction in ovarian germ cells are still unknown.

The FSH signal transduction has been considered to be mainly mediated through cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase A (PKA) pathway (Yu et al., 2003; Hunzicker-Dunn and Maizels, 2006), which has been proposed as a first reaction tier by which FSH controls cellular proliferation, differentiation, and apoptosis. However, some recent studies reported that the diacylglycerol (DG)-protein kinase C (PKC) also plays an equal role in FSH action (Lu et al., 2001; Ali and Sirard, 2005; Jin et al., 2006). PKC signaling pathway was included in the desensitization of FSH-induced adenylate cyclase (AC) activity in rat sertoli cells (Eskola et al., 1998) and chick embryo testis cells (Peralta et al., 2009). Lu et al. (2001) reported that PKC, rather than PKA, was involved in FSH-mediated resumption of mouse cumulus cell-enclosed oocytes. In addition, FSH increases PKC activity in human ovarian cancer cells (Ohtani et al., 2001). These results suggest that the PKC signaling pathway, at least in part, is involved in FSH-stimulated cell proliferation and growth. Determining whether the PKC-mediated system is involved in the FSH-stimulated cell proliferation in cultured chicken ovarian germ cells still requires further investigation.

This study was carried out to investigate the underlying signal transduction mechanism of FSH on germ cell proliferation through the PKA and PKC-mediated systems by activation of PKA and PKC or blockade of PKA and PKC.

2. Materials and methods

2.1. Animals

Fertilized chicken eggs were purchased from a commercial hatchery and incubated at 38.5 °C with 60% relative humidity until Day 18.

2.2. Culture of ovarian cells

The dispersion and culture procedures of ovarian cells were described as elsewhere (Liu et al., 2006). Briefly, left ovary fragments were minced and digested. The dispersed cells were seeded at 5×104 cells/well in McCoy’s 5A medium (GIBCO BRL, CA, USA) containing 1.75 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 mmol/L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Sangon, Shanghai, China), 3×10−8 mol/L selenite, 5 µg/ml transferrin and 10 µg/ml insulin (Sigma, St. Louis, MO, USA). Cells were incubated at 39 °C under a water-saturated atmosphere containing 95% air and 5% CO2.

2.3. Treatment of cultured cells with chemicals

At the beginning of culture, cells were treated with different doses of FSH (1–1000 ng/ml, OVAGEN™, New Zealand), and then were incubated with forskolin (FRSK, 10−7–10−5 mol/L, activator of the adenylate cyclase, Sigma), H89 (10−7–10−5 mol/L, PKA inhibitor, Sigma), PMA (10−8–10−6 mol/L, PKC activator, Sigma), or H7 (10−7–10−5 mol/L, PKC inhibitor, Sigma) alone or in combination with an optimal dose of FSH.

2.4. Analysis of morphological changes

Phase-contrast images were captured with a CCD camera (Pixera Pro 150ES, USA) and Simple PCI advanced imaging software (Compix Inc., USA). The images were used to analyze the cell morphological changes. The germ cell number was counted in each image.

2.5. Immunocytochemistry of proliferating cell nuclear antigen (PCNA)

The cells were fixed and incubated overnight at 4 °C with mouse anti-PCNA antibody (Boster Co., Wuhan, China), followed by biotin-goat anti-mouse IgG as secondary antibody. Cells with nuclei that were brown to black were considered as positive. The labeling index (LI) was shown as the percentage of the germ cell number of PCNA-labeled nuclei to the total germ cell number.

2.6. Statistical analysis

Data were analyzed by analysis of variance (ANOVA) and Duncan’s multiple range tests using the SAS 8.0 software system and were expressed as the mean±standard error of mean (SEM). A significant difference was considered at P<0.05.

3. Results

3.1. Effect of FSH on germ cell proliferation

FSH (10–1000 ng/ml) treatment increased the germ cell number after 48-h culture (P<0.05), but lower concentrations of FSH (1 ng/ml) failed to increase the germ cell number (Fig. (Fig.1).1). The germ cells in the FSH-treated groups were more definite and had higher cuboidal form. The PCNA-staining was deeper after FSH treatment (Figs. (Figs.2a2a and and2b)2b) and the germ cell LI was markedly higher, relative to the control (P<0.05, Fig. Fig.33).

Fig. 1

Effects of FSH on proliferation of ovarian germ cells from embryonic chickens after 48-h culture

Fig. 2Fig. 2Fig. 2Fig. 2Fig. 2Fig. 2

Immunocytochemical staining of PCNA in embryonic chicken ovarian cells treated with FSH alone or combined with PKA/PKC activators/inhibitors after 48-h culture

Fig. 3

Changes of PCNA-LI in chicken ovarian germ cells after treatment with FSH alone or combined with PKA/PKC activators/inhibitors after 48-h culture

3.2. Effects of FRSK and H89 on FSH-stimulated germ cell proliferation

FRSK (10−7–10−5 mol/L) augmented the germ cell number (P<0.05, Fig. Fig.4a).4a). Treatment with FRSK and FSH resulted in a visible increase in germ cell number (P<0.05, Fig. Fig.4a)4a) and PCNA expression in all groups (Figs. (Figs.2b2b and and2c).2c). The LI showed an obvious synergic effect of FSH and FRSK on germ cell proliferation (P<0.05, Fig. Fig.3).3). Morphological analysis demonstrated that FSH-stimulated proliferation of the germ cells was visibly repressed by the treatment of H89. However, no significant changes were found among all groups of H89 (10−7–10−5 mol/L) alone, relative to the control (Fig. (Fig.4b).4b). H89 significantly depressed PCNA expression in the united groups compared with FSH alone after 48-h culture (Figs. (Figs.2b2b and and2d),2d), and the germ cell LI was visibly lower in the united groups of H89 and FSH than in the FSH group (P<0.05, Fig. Fig.33).

Fig. 4Fig. 4Fig. 4Fig. 4

Effects of FRSK (a), H89 (b), PMA (c), and H7 (d) on FSH-stimulated increase in germ cell number of embryonic chicken ovary after 48-h culture

3.3. Effects of PMA and H7 on FSH-stimulated germ cell proliferation

No significant change was shown after the treatment with PMA (10−8 and 10−7 mol/L) alone, but PMA at 10−6 mol/L augmented the germ cell number (P<0.05, Fig. Fig.4c).4c). Combination of PMA and FSH induced a visible increase in germ cell number (P<0.05, Fig. Fig.4c)4c) and PCNA expression in all groups (Figs. (Figs.2b2b and and2e).2e). The germ cell LI also displayed a marked synergistic effect of FSH and PMA on germ cell proliferation (P<0.05, Fig. Fig.3).3). FSH-stimulated germ cell proliferation was blocked by treatments with H7, but there were no evident changes among all groups of H7 (10−7–10−5 mol/L) alone, relative to the control (Fig. (Fig.4d).4d). The H7 treatment reduced PCNA expression of germ cells in the united groups relative to FSH alone after 48-h culture (Figs. (Figs.2b2b and and2f),2f), and the germ cell LI was distinctly lower in the united groups of H7 and FSH than in the FSH group (P<0.05, Fig. Fig.33).

4. Discussion

The FSH exerts its action by binding to its receptor FSHR in target cells. Akazome et al. (2002) have detected the expression of FSHR mRNA in undifferentiated gonads of 4-d chick embryos. Our previous study showed that the FSHR mRNA level in embryonic chicken gonads began to increase at 18 d of incubation, and then reached a plateau around hatching (Mao et al., 2000). The expression of FSHR mRNA was mostly located in somatic cells, but partly in the germ cells of the ovary (Méndez et al., 2003). The AC-cAMP pathway has long been accepted as the sole second messenger when FSH regulates follicular development in the ovary (Hunzicker-Dunn and Maizels, 2006). To investigate this point, we examined the proliferating effect of FSH on cultured ovarian germ cells and further investigated the underlying signal transduction mechanism. We confirmed that the germ cell number was increased by FSH treatment and this stimulating effect was confirmed by increased germ cell LI. The results agreed with the findings that FSH induced the proliferation of 14-d chicken embryonic ovarian cells (Méndez et al., 2003). The FSH-stimulated proliferation of the germ cells was markedly increased by united treatment with a specific activator of the adenylate cyclase, FRSK, and there were noticeable changes in the germ cell number after the treatment with FRSK. To further support that the PKA-mediated system was involved in the FSH-stimulated cell proliferation in cultured ovarian germ cells, a specific inhibitor of PKA pathway, H89, was applied. The results showed that, H89 visibly blocked the FSH-stimulated proliferation of the germ cells. The conclusions agreed with the reports that H89 inhibited the promoting effects of FSH in sertoli cells (Crépieux et al., 2002). In summary, our results indicated that PKA plays an important role on the germ cell proliferation and was stimulated by FSH, since the treatment with H89 suppressed the FSH-stimulated cell proliferation while FRSK increased the FSH-stimulated cell proliferation.

As mentioned above, the PKC signaling pathway, at least in part, was involved in FSH-stimulated cell proliferation. The underlying signal transduction mechanism was studied by regulating the PKC-mediated system with PKC inhibitor (H7) or activator (PMA). H7 markedly suppressed the FSH-stimulated cell proliferation in cultured ovarian germ cells. The results are consistent with the findings that H7 inhibited the promoting effects of FSH in human epithelial ovarian cancer cells through PKC-mediated system (Ohtani et al., 2001). We also found that there was no evident change after the treatment with lower PMA alone, but an augmentation in the germ cell number with higher PMA. Combined administration of PMA and FSH resulted in a visible increase in the germ cell number and LI. These conclusions were consistent with the viewpoints that the treatment of ovarian germ cells and mouse spermatogonia with PMA and ginsenosides promoted the cell proliferation involving the PKC-mediated system (Liu and Zhang, 2006; Zhang et al., 2009). Hence, we propose that PKC-mediated system is involved in the FSH-stimulated cell proliferation in cultured chicken ovarian germ cells.

Though the PKA and PKC pathways are involved in the signal transduction of FSH, the mechanism may also be through other intracellular signaling cascades, including the phosphatidylinositol 3-kinase (PI3-K)/Akt and mitogen-activated protein kinase (MAPK) pathways (Pasapera et al., 2005; Alam et al., 2009). In granulosa cells, FSH mimicked some actions of insulin-like growth factor 1 (IGF-1) through phosphorylation and activation of protein kinase B (PKB)/Akt. Activation of these alternate pathways perceptibly leads to a more precise control of FSH-stimulated cell proliferation, steroidogenesis, and apoptosis in ovaries (Richards et al., 2002; Alam et al., 2009). Based on these hypotheses, FSH-induced germ cell proliferation might be mainly involving PKA and PKC-mediated pathways. However, in addition to PKA and PKC-mediated pathways, other signal transduction pathways that involve in the FSH-stimulated proliferation of germ cells require further studies.

In conclusion, this study reveals that FSH promotes proliferation of cultured chicken ovarian germ cells, and the enhancement of PCNA expression further confirms its stimulatory effect. In addition, we demonstrate that PKA and PKC-mediated signal transduction system regulates the FSH-induced germ cell proliferation. Further work will be needed to identify other signaling pathways and the precise molecular mechanism of FSH action in germ cells.

Footnotes

*Project supported by the National Natural Science Foundation of China (No. 30871843) and the Key Project of the Science and Technology Department of Zhejiang Province, China (No. 2008C12050)

References

1. Akazome Y, Abe T, Mori T. Differentiation of chicken gonad as an endocrine organ: expression of LH receptor, FSH receptor, cytochrome P450c17 and aromatase genes. Reproduction. 2002;123(5):721–728. doi: 10.1530/rep.0.1230721. [PubMed] [Cross Ref]
2. Alam H, Weck J, Maizels E, Park Y, Lee EJ, Ashcroft M, Hunzicker-Dunn M. Role of the phosphatidylinositol-3-kinase and extracellular regulated kinase pathways in the induction of hypoxia-inducible factor (HIF)-1 activity and the HIF-1 target vascular endothelial growth factor in ovarian granulose cells in response to follicle-stimulating hormone. Endocrinology. 2009;150(2):915–928. doi: 10.1210/en.2008-0850. [PubMed] [Cross Ref]
3. Ali A, Sirard MA. Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone. Reproduction. 2005;130(3):303–310. doi: 10.1530/rep.1.00387. [PubMed] [Cross Ref]
4. Crépieux P, Martinat N, Marion S, Guillou F, Reiter E. Cellular adhesion of primary sertoli cells affects responsiveness of the extracellular signal-regulated kinases 1 and 2 to follicle-stimulating hormone but not to epidermal growth factor. Arch Biochem Biophys. 2002;399(2):245–250. doi: 10.1006/abbi.2002.2773. [PubMed] [Cross Ref]
5. Dorrington J, Chuma AV, Bendell JJ. Transforming growth factor β and follicle-stimulating hormone promote rat granulose cell proliferation. Endocrinology. 1988;123(1):353–359. doi: 10.1210/endo-123-1-353. [PubMed] [Cross Ref]
6. Eskola V, Ryhanen P, Savisalo M, Rannikko A, Kananen K, Sprengel R, Huhtaniemi I. Stable transfection of the rat follicle-stimulating hormone receptor complementary DNA into an immortalized murine Sertoli cell line. Mol Cell Endocrinol. 1998;139(1-2):143–152. doi: 10.1016/S0303-7207(98)00063-X. [PubMed] [Cross Ref]
7. Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in immature granulose cells that regulate target gene expression: branching out from protein kinase A. Cell Signal. 2006;18(9):1351–1359. doi: 10.1016/j.cellsig.2006.02.011. [PMC free article] [PubMed] [Cross Ref]
8. Jin S, Zhang M, Lei L, Wang C, Fu M, Ning G, Xia G. Meiosis activating sterol (MAS) regulate FSH-induced meiotic resumption of cumulus cell-enclosed porcine oocytes via PKC pathway. Mol Cell Endocrinol. 2006;249(1-2):64–70. doi: 10.1016/j.mce.2006.01.008. [PubMed] [Cross Ref]
9. Liu HY, Zhang CQ. Ginsenosides promote proliferation of cultured ovarian germ cells involving protein kinase C-mediated system in embryonic chickens. Asian-Aust J Anim Sci. 2006;19(7):958–963.
10. Liu HY, Zhang CQ, Zeng WD. Estrogenic and antioxidant effects of a phytoestrogen daidzein on ovarian germ cells in embryonic chickens. Domest Anim Endocrinol. 2006;31(3):258–268. doi: 10.1016/j.domaniend.2005.11.002. [PubMed] [Cross Ref]
11. Lu Z, Xia G, Zhang J. Protein kinase C, rather than protein kinase A is involved in follicle-stimulating hormone-mediated meiotic resumption of mouse cumulus cell-enclosed oocytes in hypoxanthine-supplemented medium. Mol Cell Endocrinol. 2001;182(2):225–232. doi: 10.1016/S0303-7207(01)00564-0. [PubMed] [Cross Ref]
12. Mao X, Zhang C, Saito N, Shimada K. Expression of messenger ribonucleic acids of luteinizing hormone and follicle-stimulating hormone receptors in the embryonic and posthatch gonads of chicken. Jpn Poult Sci. 2000;37(4):212–220.
13. Méndez MC, Ramírez M, Varela AR, Chávez B, Pedernera E. Follicle stimulating hormone increases cell proliferation in the ovary and testis of the chick embryo. Gen Comp Endocrinol. 2003;133(2):181–188. doi: 10.1016/S0016-6480(03)00169-2. [PubMed] [Cross Ref]
14. Méndez-Herrera MC, Támez L, Candido A, Reyes-Esparza JA. Follicle stimulating hormone increases somatic and germ cell number in the ovary during chick embryo development. Gen Comp Endocrinol. 1998;111(2):207–215. doi: 10.1006/gcen.1998.7108. [PubMed] [Cross Ref]
15. Ohtani K, Sakamoto H, Kikuchi A, Nakayama Y, Idei T, Igarashi N, Matukawa T, Satoh K. Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system. Cancer Lett. 2001;166(2):207–213. doi: 10.1016/S0304-3835(00)00713-8. [PubMed] [Cross Ref]
16. Pasapera AM, Jiménez-Aguilera MP, Chauchereau A, Milgrom E, Olivares A, Uribe A, Gutiérrez-Sagal R, Ulloa-Aguirre A. Effects of FSH and 17β-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells. J Steroid Biochem Mol Biol. 2005;94(4):289–302. doi: 10.1016/j.jsbmb.2004.11.011. [PubMed] [Cross Ref]
17. Pedernera E, Solis L, Peralta I, Velázquez PN. Proliferative and steroidogenic effects of follicle-stimulating hormone during chick embryo gonadal development. Gen Comp Endocrinol. 1999;116(2):213–220. doi: 10.1006/gcen.1999.7352. [PubMed] [Cross Ref]
18. Peralta I, Romano MC, Velázquez PN. Signaling pathways involved in the effect of follicle-stimulating hormone on chick embryo testis cell. Poult Sci. 2009;88(2):380–386. doi: 10.3382/ps.2007-00402. [PubMed] [Cross Ref]
19. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falende AE, Lo YK, Sharma SC. Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res. 2002;57(1):195–220. doi: 10.1210/rp.57.1.195. [PubMed] [Cross Ref]
20. Velázquez PN, Peralta I, Pedernera E. Proliferative effect in vitro of follicle-stimulating hormone on the left ovary of the chick embryo. Gen Comp Endocrinol. 1997;105(1):40–49. doi: 10.1006/gcen.1996.6796. [PubMed] [Cross Ref]
21. Woods JE, Damianides-Keenan M, Thommes RC. FSH- and TSH-binding cells in the ovary of the developing chick embryo. Gen Comp Endocrinol. 1991;82(3):487–494. doi: 10.1016/0016-6480(91)90324-Y. [PubMed] [Cross Ref]
22. Xie MN, Zhang CQ, Zeng WD, Mi YL. Effects of follicle-stimulating hormone and 17β-estradiol on germ cell development of chicken embryonic ovary in culture. Comp Biochem Phys A. 2004;139(4):521–526. doi: 10.1016/j.cbpb.2004.10.017. [PubMed] [Cross Ref]
23. Yogo K, Ogawa T, Akiyama M, Ishida N, Takeya T. Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells. FEBS Lett. 2002;531(2):132–136. doi: 10.1016/S0014-5793(02)03441-5. [PubMed] [Cross Ref]
24. Yu Y, Li W, Han Z, Luo M, Chang Z, Tan J. The effect of follicle-stimulating hormone on follicular development, granulose cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary. Theriogenology. 2003;60(9):1691–1704. doi: 10.1016/j.theriogenology.2003.08.001. [PubMed] [Cross Ref]
25. Zhang DL, Wang KM, Zhang CQ. Ginsenosides stimulated the proliferation of mouse spermatogonia involving activation of protein kinase C. J Zhejiang Univ-Sci B. 2009;10(2):87–92. doi: 10.1631/jzus.B0820133. [PMC free article] [PubMed] [Cross Ref]

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