Abstract
The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCKI, and RACI in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCKI, VAV, and RACI expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.
Similar content being viewed by others
References
McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21(2):200–214.
Hsueh AJ, Kawamura K, Cheng Y, Fauser BC. Intraovarian control of early folliculogenesis. Endocr Rev. 2015;36(1):1–24.
Richards JS, Pangas SA. The ovary: basic biology and clinical implications. J Clin Invest. 2010;120(4):963–972.
de Melo AS, Dias SV, Cavalli RC, et al. Pathogenesis of polycystic ovary syndrome: multifactorial assessment from the foetal stage to menopause. Reproduction. 2015;150(1):R11–R24.
Qiao J, Feng HL. Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence. Hum Reprod Update. 2011;17(1):17–33.
Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 2006;16(10):522–529.
Villalonga P, Ridley AJ. Rho GTPases and cell cycle control. Growth Factors. 2006;24(3):159–164.
Tomar A, Schlaepfer DD. Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol. 2009;21(5):676–683.
Goicoechea SM, Awadia S, Garcia-Mata R. I’m coming to GEF you: regulation of RhoGEFs during cell migration. Cell Adh Migr. 2014;8(6):535–549.
Maurya VK, Sangappa C, Kumar V, et al. Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouse. J Ovarian Res. 2014;7:32. doi:10.1186/1757-2215-7-32.
Jun JH, Joo CK. MicroRNA-124 controls transforming growth factor beta1-induced epithelial-mesenchymal transition in the retinal pigment epithelium by targeting RHOG. Invest Ophthalmol Vis Sci. 2016;57(1):12–22.
Jackson BC, Ivanova IA, Dagnino L. An ELMO2-RhoG-ILK network modulates microtubule dynamics. Mol Biol Cell. 2015;26(14):2712–2725.
Lee J, Park B, Kim G, et al. Arhgef16, a novel Elmo1 binding partner, promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation. Biochim Biophys Acta. 2014;1843(11):2438–2447.
Tzircotis G, Braga VM, Caron E. RhoG is required for both FcgammaR- and CR3-mediated phagocytosis. J Cell Sci. 2011;124(pt 17):2897–2902.
Morin A, Cordelieres FP, Cherfils J, Olofsson B. RhoGDI3 and RhoG: vesicular trafficking and interactions with the Sec3 Exocyst subunit. Small GTPases. 2010;1(3):142–156.
Hiramoto K, Negishi M, Katoh H. Dock4 is regulated by RhoG and promotes Rac-dependent cell migration. Exp Cell Res. 2006;312(20):4205–4216.
Murga C, Zohar M, Teramoto H, Gutkind JS. Rac1 and RhoG promote cell survival by the activation of PI3 K and Akt, independently of their ability to stimulate JNK and NF-kappaB. Oncogene. 2002;21(2):207–216.
Zhao L, Du X, Huang K, et al. Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice. Sci Rep. 2016;6:23972. doi:10.1038/srep23972.
Laurin M, Cote JF. Insights into the biological functions of Dock family guanine nucleotide exchange factors. Genes Dev. 2014;28(6):533–547.
Mettus RV, Rane SG. Characterization of the abnormal pancreatic development, reduced growth and infertility in Cdk4 mutant mice. Oncogene. 2003;22(52):8413–8421.
Honnma H, Endo T, Henmi H, et al. Altered expression of Fas/Fas ligand/caspase 8 and membrane type 1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis. 2006;11(9):1525–1533.
Anderson E, Lee MT, Lee GY. Cystogenesis of the ovarian antral follicle of the rat: ultrastructural changes and hormonal profile following the administration of dehydroepiandrosterone. Anat Rec. 1992;234(3):359–382.
Kirsch TM, Friedman AC, Vogel RL, Flickinger GL. Macrophages in corpora lutea of mice: characterization and effects on steroid secretion. Biol Reprod. 1981;25(3):629–638.
Campbell KL. Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular integrity and function. Biol Reprod. 1979;21(4):773–786.
Chen MJ, Chou CH, Chen SU, Yang WS, Yang YS, Ho HN. The effect of androgens on ovarian follicle maturation: dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARgamma-dependent PTEN expression. Sci Rep. 2015;5:18319. doi:10.1038/srep18319.:18319.
Voronina E, Lovasco LA, Gyuris A, Baumgartner RA, Parlow AF, Freiman RN. Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b. Dev Biol. 2007;303(2):715–726.
Yan Z, Lee GY, Anderson E. Influence of dehydroepiandrosterone on the expression of insulin-like growth factor-1 during cystogenesis in polycystic rat ovaries and in cultured rat granulosa cells. Biol Reprod. 1997;57(6):1509–1516.
Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR. Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells. Biol Reprod. 1997;56(4):891–897.
Li SK, Hearn MT. Isolation of thecal cells: an assessment of purity and steroidogenic potential. J Biochem Biophys Methods. 2000;45(2):169–181.
Magoffin DA, Erickson GF. Purification of ovarian theca-interstitial cells by density gradient centrifugation. Endocrinology. 1988;122(5):2345–2347.
Ortega I, Villanueva JA, Wong DH, et al. Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis. J Ovarian Res. 2014;7:21. doi:10.1186/1757-2215-7-21.
Vazquez-Cuevas FG, Zarate-Diaz EP, Garay E, Arellano RO. Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells. Reprod Biol Endocrinol. 2010;8:88. doi:10.1186/1477-7827-8-88.
Chadchan SB, Kumar V, Maurya VK, Soni UK, Jha RK. Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation. Mol Cell Endocrinol. 2016;425:69–83.
Kumar V, Maurya VK, Joshi A, Meeran SM, Jha RK. Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice. Biol Reprod. 2015;92(4):109.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–685.
Joshi A, Mahfooz S, Maurya VK, et al. PARP-1 during embryo implantation and its up-regulation by oestradiol in mice. Reproduction. 2014;147(6):765–780.
Spina-Purrello V, Giliberto S, Barresi V, Nicoletti VG, Giuffrida Stella AM, Rizzarelli E. Modulation of PARP-1 and PARP-2 expression by L-carnosine and trehalose after LPS and INFgamma-induced oxidative stress. Neurochem Res. 2010;35(12):2144–2153.
Murphy AM, Montell DJ. Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J Cell Biol. 1996;133(3):617–630.
Franz MB, Daube S, Keck C, Sator M, Pietrowski D. Small GTPases are involved in sprout formation in human granulosa lutein cells. Arch Gynecol Obstet. 2013;287(4):819–824.
Katoh H, Negishi M. RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo. Nature. 2003;424(6947):461–464.
Sidibe A, Polena H, Razanajatovo J, et al. Dynamic phosphorylation of VE-cadherin Y685 throughout mouse estrous cycle in ovary and uterus. Am J Physiol Heart Circ Physiol. 2014;307(3):H448–H454.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ubba, V., Soni, U.K., Chadchan, S. et al. RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model. Reprod. Sci. 24, 738–752 (2017). https://doi.org/10.1177/1933719116669057
Published:
Issue Date:
DOI: https://doi.org/10.1177/1933719116669057