RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model

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, DOCK1, and RAC1 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 DOCK1, VAV, and RAC1 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.

Keywords

RHOG DOCK1 ELMO1 RAC1 ovary PCOS DHEA theca and granulosa cells SKOV3

Get full access to this article

View all access options for this article.

References

McGee

Hsueh

. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21(2):200–214.

Hsueh

Kawamura

Cheng

Fauser

. Intraovarian control of early folliculogenesis. Endocr Rev. 2015;36(1):1–24.

Richards

Pangas

. The ovary: basic biology and clinical implications. J Clin Invest. 2010;120(4):963–972.

de Melo

Dias

Cavalli

. Pathogenesis of polycystic ovary syndrome: multifactorial assessment from the foetal stage to menopause. Reproduction. 2015;150(1):R11–R24.

Qiao

Feng

. 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

. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 2006;16(10):522–529.

Villalonga

Ridley

. Rho GTPases and cell cycle control. Growth Factors. 2006;24(3):159–164.

Tomar

Schlaepfer

. Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol. 2009;21(5):676–683.

Goicoechea

Awadia

Garcia-Mata

. I’m coming to GEF you: regulation of RhoGEFs during cell migration. Cell Adh Migr. 2014;8(6):535–549.

10.

Maurya

Sangappa

Kumar

. 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.

11.

Jun

Joo

. 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.

12.

Jackson

Ivanova

Dagnino

. An ELMO2-RhoG-ILK network modulates microtubule dynamics. Mol Biol Cell. 2015;26(14):2712–2725.

13.

Lee

Park

Kim

. Arhgef16, a novel Elmo1 binding partner, promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation. Biochim Biophys Acta. 2014;1843(11):2438–2447.

14.

Tzircotis

Braga

Caron

. RhoG is required for both FcgammaR- and CR3-mediated phagocytosis. J Cell Sci. 2011;124(pt 17):2897–2902.

15.

Morin

Cordelieres

Cherfils

Olofsson

. RhoGDI3 and RhoG: vesicular trafficking and interactions with the Sec3 Exocyst subunit. Small GTPases. 2010;1(3):142–156.

16.

Hiramoto

Negishi

Katoh

. Dock4 is regulated by RhoG and promotes Rac-dependent cell migration. Exp Cell Res. 2006;312(20):4205–4216.

17.

Murga

Zohar

Teramoto

Gutkind

. 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.

18.

Zhao

Huang

. 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.

19.

Laurin

Cote

. Insights into the biological functions of Dock family guanine nucleotide exchange factors. Genes Dev. 2014;28(6):533–547.

20.

Mettus

Rane

. Characterization of the abnormal pancreatic development, reduced growth and infertility in Cdk4 mutant mice. Oncogene. 2003;22(52):8413–8421.

21.

Honnma

Endo

Henmi

. 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.

22.

Anderson

Lee

. 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.

23.

Kirsch

Friedman

Vogel

Flickinger

. Macrophages in corpora lutea of mice: characterization and effects on steroid secretion. Biol Reprod. 1981;25(3):629–638.

24.

Campbell

. Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular integrity and function. Biol Reprod. 1979;21(4):773–786.

25.

Chen

Chou

Chen

Yang

. 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.

26.

Voronina

Lovasco

Gyuris

Baumgartner

Parlow

Freiman

. Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b. Dev Biol. 2007;303(2):715–726.

27.

Yan

Lee

Anderson

. 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.

28.

Duleba

Spaczynski

Olive

Behrman

. Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells. Biol Reprod. 1997;56(4):891–897.

29.

Hearn

. Isolation of thecal cells: an assessment of purity and steroidogenic potential. J Biochem Biophys Methods. 2000;45(2):169–181.

30.

Magoffin

Erickson

. Purification of ovarian theca-interstitial cells by density gradient centrifugation. Endocrinology. 1988;122(5):2345–2347.

31.

Ortega

Villanueva

Wong

. 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.

32.

Vazquez-Cuevas

Zarate-Diaz

Garay

Arellano

. 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.

33.

Chadchan

Kumar

Maurya

Soni

Jha

. Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation. Mol Cell Endocrinol. 2016;425:69–83.

34.

Kumar

Maurya

Joshi

Meeran

Jha

. Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice. Biol Reprod. 2015;92(4):109.

35.

Laemmli

. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–685.

36.

Joshi

Mahfooz

Maurya

. PARP-1 during embryo implantation and its up-regulation by oestradiol in mice. Reproduction. 2014;147(6):765–780.

37.

Spina-Purrello

Giliberto

Barresi

Nicoletti

Giuffrida Stella

Rizzarelli

. 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.

38.

Murphy

Montell

. Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J Cell Biol. 1996;133(3):617–630.

39.

Franz

Daube

Keck

Sator

Pietrowski

. Small GTPases are involved in sprout formation in human granulosa lutein cells. Arch Gynecol Obstet. 2013;287(4):819–824.

40.

Katoh

Negishi

. RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo. Nature. 2003;424(6947):461–464.

41.

Sidibe

Polena

Razanajatovo

. 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.