Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro
Luciana da Paz dos Santos, Jamile Maiara da Silva Santos, Vanúzia Gonçalves Menezes, Ricássio de Sousa Barberino, Bruna Bortoloni Gouveia, Agnes Yasmin Pitombeira Cavalcante, Thae Lanne Barbosa Gama Lins, Rodrigo José de Sousa Gonçalves, Juliana Jales de Hollanda Celestino, Maria Helena Tavares de Matos
Abstract
The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the best results for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 h. The percentage of normal follicles decreased (P < 0.05) after 7 days of culture in all treatments compared to the fresh control. A significant reduction in the percentage of primordial follicles and an increase (P < 0.05) in the growing ones were observed in all treatments compared to fresh control. Furthermore, both the control medium and 1 ng/mL EGF promoted an increase (P < 0.05) in follicular activation compared to other EGF treatments. The PCNA-positive cells in the EGF treatment were higher (P < 0.05) than in fresh control and α-MEM+ . Pretreatment of ovarian tissue with PI3K inhibitor significantly inhibited (P < 0.05) αMEM+ -stimulated primordial follicle activation, but had no effect on EGF-stimulated activation (P > 0.05). In conclusion, PI3K pathway mediates the in vitro spontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.
Keywords
References
Adhikari D, Risal S, Liu K, Shen Y. 2013. Pharmacological inhibition of mTORC1 prevents overactivation of the primordial follicle pool in response to elevated PI3K signaling. PLoS One, 8(1):53810.
Andrade ER, Seneda M, Alfieri AA, Oliveira JA, Bracarense APFRL, Figueiredo JR, Toniolli R. 2005. Interactions of indole acetic acid with EGF and FSH in the culture of ovine preantral follicles. Theriogenology, 64:1104-1113.
Andrade PM, Chaves RN, Alves AMCV, Rocha RMP, Lima LF, Carvalho AA, Rodrigues APR, Campello CC, Gastal EL, Figueiredo JR. 2014. Effects of α-MEM and TCM-199 culture media and epidermal growth factor on survival and growth of goat and sheep preantral follicles cultured in vitro. Anim Reprod, 11:567-572.
Braw-Tal R, Yossefi S. 1997. Studies in vivo and in vitro on the initiation of follicle growth in the bovine ovary. J Reprod Fertil, 109:165-171.
Braw-Tal R. 2002. The initiation of follicle growth: the oocyte or the somatic cells?. Mol Cell Endocrinol, 187:11-18.
Cavalcante AYP, Gouveia BB, Barberino RS, Lins TLBG, Santos LP, Gonçalves RJS, Celestino JJH, Matos M.H.T. 2016. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue. Zygote, 24:578-582.
Celestino JJH, Bruno JB, Lima-Verde IB, Matos MHT, Saraiva MV, Chaves RN, Martins FS, Lima LF, Name KPO, Campello CC, Silva JRV, Báo SN, Figueiredo JR. 2009. Recombinant epidermal growth factor maintains follicular ultrastructure and promotes the transition to primary follicles in caprine ovarian tissue cultured in vitro. Reprod Sci, 16:239-246.
Celestino JJH, Bruno JB, Saraiva MVA, Rocha RMP, Brito IR, Duarte ABG, AraújoVR, Silva CMG, Matos MHT, Campello CC, Silva JRV, Figueiredo JR. 2011. Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles. Cell Tissue Res, 344:539-50.
Chaves RN, Martins DFS, Saraiva MVA, Celestino JJH, Lopes CAP, Correia JC, Lima-Verde IB, Matos MHT, Báo SN, Name KPO, Campello CC, Silva JRV, Figueiredo JR. 2008. Chilling ovarian fragments during transportation improves viability and growth of goat preantral follicles cultured in vitro. Reprod Fertil, 20:640-647.
Cushman RA, Wahl CM, Fortune JE. 2002. Bovine ovarian cortical pieces grafted to chick embryonic membranes: a model for studies on the activation of primordial follicles. Hum Reprod, 17:48-54.
Derrar N, Price CA, Sirard M-A. 2000. Effects of growth factors and co-culture with ovarian medulla on the activation of primordial in explants of bovine ovarian cortex. Theriogenology, 54:587-598.
Fujihara M, Comizzoli P, Keefer CL, Wildt DE, Songsasen N. 2014. Epidermal Growth Factor (EGF) Sustains In Vitro Primordial Follicle Viability by Enhancing Stromal Cell Proliferation via MAPK and PI3K Pathways in the Prepubertal, but Not Adult, Cat Ovary. Biol Reprod, 90:1-10
Gall L, Chene N, Dahirel M, Ruffini S, Boulesteix C. 2004. Expression of epidermal growth factor receptor in the goat cumulus-oocyte complex. Mol Reprod Dev, 67: 439-445.
Granville CA, Memmott RM, Gills JJ, Dennis PA. 2006. Handicapping the Race to Develop Inhibitors of the Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Pathway. Clin Cancer Res, 12:679-689.
Gutierrez CG, Ralph JH, Telfer EE, Wilmut I, Webb R. 2000. Growth and antrum formation of bovine preantral follicles in long-term culture in vitro. Biol Reprod, 62:1322-1328.
Hemamalini NC, Rao BS, Tamilmani G, D. Amarnath, R. Vagdevi, K.S. Naidu, K.K. Reddy, V.H. Rao. 2003. Influence of transforming growth factor-a, insulin-like growth factor-II, epidermal growth factor or follicle stimulating hormone on in vitro development of preantral follicles in sheep. Small Rumin Res, 50:11-22.
Hoffman GE, Wei LE, Luciane VS. 2008. The Importance of Titrating Antibodies for Immunocytochemical. Curr Protoc Neurosci, 45:2-12.
Hsueh AJW, Kawamura K, Cheng Y, Fauser BCJM. 2015. Intraovarian control of early folliculogenesis. Endocr Rev, 36:1-24.
John GB, Gallardo TD, Shirley LJ, Castrillon DH. 2008. Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth. Dev Biol, 321:197-204.
Keating AF, Mark CJ, Sen N, Sipes G, Hoyer PB. 2009. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a] anthracene in neonatal rat ovaries. Toxicol Appl Pharmacol, 241:127-134.
Kiley SC, Thornhill BA, Belyea BC, Neale K, Forbes MS, Luetteke NC, Lee DC, Chevalier RL. 2005. Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats. Kidney Int, 68:504-514.
Lan ZJ, Krause MS, Redding SD, Li X, Wu GZ, Zhou HX, Bohler HC, Ko C, Cooney AJ, Zhou J, Lei ZM. 2017. Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice. Mol Cell Endocrinol, 444:26-37.
Li J, Kawamurab K, Cheng Y, Liu S, Klein C, Liu S, Duanc E-K, Hsueh AJW. 2010. Activation of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A, 10:10280-10284.
Li-Ping Z, Da-Lei Z, Jian H, Liang-Quan X, Ai-Xia X, Xiao-Yu D, Dan-Feng T, Yue-Hui Z. 2010. Protooncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways. Reprod Biol Endocrinol, 8:66.
Liu L, Xie Y, Lou L. 2006. PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 Activation. Eur J Cell Biol, 85:367-374.
Mani AM, Fenwick MA, Cheng Z, Sharma MK, Singh D, Wathes DC. 2010. IGF1 induces upregulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol dependent kinase/AKT in bovine granulosa cells. Reproduction, 139:139-151.
Mao J, Smith MF, Rucker EB, Wu GM, McCauley TC, Cantley TC, Prather RS, Didion BA, Day BN. 2004. Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro. J Anim Sci, 82:1967-1975.
Maruo T, Ladines-Llave CA, Samoto T, Matsuo H, Manalo AS, Ito H, Mochizuki M. 1993. Expression of epidermal growth factor and its receptor in the human ovary during follicular growth and regression. Endocrinology, 132:924-931.
Monget P, Bobe J, Gougeon A, Fabre S, Monniaux D, Dalbies-Tran. R. 2012. The ovarian reserve in mammals: A functional and evolutionary perspective. Mol Cell Endocrinol, 356:2-12.
Morbeck DE, Flowers WL, Britt JH. 1993. Response of porcine granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and EGF in vitro. J Reprod Fertil, 99:577-584.
Reddy P, Adhikari D, Zheng W, Liang S, Hämäläinen T, Tohonen V, Ogawa W, Noda T, Volarevic S, Huhtaniemi I, Liu K. 2009. PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles. Human Mol Genetics, 18:2813-2824
Roy SK, Greenwald GS. 1990. Immunohistochemical localisation of epidermal growth factor-like activity in the hamster ovary with a polyclonal antibody. Endocrinology, 126:1309-1317.
Ryan EK, Glister C, Lonergan P, Martin F, Knight PG, Evans ACO. 2008. Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep. J Ovarian Res, 1:1-13
Santos LP, Barros VRP, Cavalcante AYP, Menezes VG, Macedo TJS, Santos JMS, Araújo VR, Queiroz MAA, Matos MHT. 2014. Protein localization of epidermal growth factor in sheep ovaries and improvement of follicle survival and antrum formation in vitro. Reprod Dom Anim, 49:783-789.
Shea LD, Woodruff TK, Shikanov A. 2014. Bioengineering the ovarian follicle microenvironment. Annu Rev Biomed Eng, 16:29-52.
Silva J.R.V., Van Den Hurk R., Matos M.H.T., Santos R.R., Pessoa C., Moraes M.O., Figueiredo J.R. 2004. Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue. Theriogenology, 61:1691-1704.
Silva JRV, van den Hurk R, van Tol HTA, Roelen BAJ, Figueiredo JR. 2006. The Kit ligand/c-Kit receptor system in goat ovaries: Gene expression and protein localization. Zygote, 14:317-328.
Silva CMG, Castro SV, Faustino LR, Rodrigues GQ, Brito IR, Rossetto R, Saraiva MVA, Campello CC, Lobo CH, Souza CEA, Moura AAA, Donato MAA, Peixoto CA, Figueiredo JR. 2013. The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles. Res Vet Sci, 94:453-461.
Silva JRV, Van den Hurk R, Figueiredo JR. 2016. Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals. Dom Anim Endocrinol, 55:123-135.
Singh B, Rutledge JM, Armstrong DT. 1995. Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles. Mol Reprod Dev, 40:391-399.
Sobinoff AP, Nixon B, Roman SD, McLaughlim EA. 2012. Staying Alive: PI3K Pathway Promotes Primordial Follicle Activation and Survival in Response to 3MC-Induced Ovotoxicity. Toxicol Sci 128, 258-271.
Telfer EE, Zelinski MB. 2013. Ovarian follicle culture: advances and challenges for human and nonhuman primates. Fertil Steril, 99:1523-1533.
Wandji SA, Eppig JJ, Fortune JE. 1996a. FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. Theriogenology, 45:817-832.
Wandji SA, Srsen V, Voss AK, Eppig JJ, Fortune JE. 1996b. Initiation in vitro of growth of bovine primordial follicles. Biol Reprod, 55:942-948.
Zhang H, Risal S, Gorre N, Busayavalasa K, Li X, Shen Y, Bosbach B, Branstrom M, Liu K. 2014. Somatic Cells Initiate Primordial Follicle Activation and Govern the Development of Dormant Oocytes in Mice. Current Biology, 24:2501-2508.
Zhao Q, Ma Y, Sun NX, Ye C, Zhang Q, Sun SH, Xu C, Wang F, Li W. 2014. Exposure to bisphenol A at physiological concentrations observed in Chinese children promotes primordial follicle growth through the PI3K/Akt pathway in an ovarian culture system. Toxicol in Vitro, 28:1424-1429.
Zhao X, Dai W, Zhu H, Zhang Y, Cao L, Ye Q, Lei P, Shen G. 2006. Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane. Cell Biol Int, 30:653- 658.
Zhao Y, Zhang Y, Li J, Zheng N, Xu X, Yang J, Xialand G, Zhang M. 2017. MAPK3/1 participates in the activation of primordial follicles through mTORC1- KITL signaling. J Cell Physiol, 233(1):226-237.
Facebook
Google+
Twitter
LinkedIn
Mendeley
StumbleUpon
CiteULike
Reddit
Email