GATA4 is a negative regulator of contractility in mouse testicular peritubular myoid cells

in Reproduction
Restricted access

Reduced contractility of the testicular peritubular myoid (PTM) cells may contribute to human male subfertility or infertility. Transcription factor GATA4 in Sertoli and Leydig cells is essential for murine spermatogenesis, but limited attention has been paid to the potential role of GATA4 in PTM cells. In primary cultures of mouse PTM cells, siRNA knockdown of GATA4 increased the contractile activity, while GATA4 overexpression significantly attenuated the contractility of PTM cells using a collagen gel contraction assay. Using RNA sequencing and qRT-PCR, we identified a set of genes that exhibited opposite expressional alternation between Gata4 siRNA vs nontargeting siRNA-treated PTM cells and Gata4 adenovirus vs control adenovirus-treated PTM cells. Notably, ion channels, smooth muscle function, cytokines and chemokines, cytoskeleton, adhesion and extracellular matrix were the top four enriched pathways, as revealed by cluster analysis. Natriuretic peptide type B (NPPB) content was significantly upregulated by GATA4 overexpression in both PTM cells and their culture supernatant. More importantly, the addition of 100 μM NPPB could abolish the promoting effect of Gata4 silencing on PTM cell contraction. Taken together, we suggest that the inhibitory action of GATA4 on PTM cell contraction is mediated at least partly by regulating genes belonging to smooth muscle contraction pathway (e.g. Nppb).

 

An official journal of

Society for Reproduction and Fertility

Sections

Figures

  • View in gallery

    GATA4 localization in PTM cells within mouse adult testes. (A) Immunofluorescence staining for a PTM cell marker MYH11 (green signals) on the tissue sections of 8-week-old testes. Nuclei were stained with DAPI. (B) GATA4 detection by immunohistochemical staining (brown signals) in 8-week-old testis sections. Scale bar, 50 μm. Arrowheads indicate PTM cells at the periphery of seminiferous tubules.

  • View in gallery

    PTM cell isolation and alternation of GATA4 expression. (A) Representative image of isolated cells staining with a PTM cell marker α-SMA (red signals). Nuclei were stained with DAPI. Scale bar, 10 μm. (B) Histogram illustrating the purity of isolated PTM cells, indicated by the percentage of α-SMA-positive cells. (C) Immunocytochemistry staining of GATA4 (brown signals) in Gata4 siRNA- and nontargeting siRNA-transfected PTM cells. Scale bar, 50 μm. (D) Relative mRNA level of Gata4 in Gata4 siRNA- and nontargeting siRNA-transfected PTM cells. (E) Cell staining of GATA4 in Gata4 adenovirus- and control adenovirus-infected PTM cells. Scale bar, 50 μm. (F) Altered mRNA level of Gata4 in PTM cells after Gata4 adenovirus infection. In B, D, F, data are presented as the mean ± s.e.m. ** indicates P < 0.01 by Student’s t test.

  • View in gallery

    Negative regulation of PTM cell contraction by GATA4. (A) Collagen contractility assay in PTM cells transfected with Gata4 siRNA or Gata4 adenovirus or their nontargeting controls for 2 days. (B) Quantitative data showing the contraction area (cm2) in Gata4 siRNA-, Gata4 adenovirus- and their control-treated PTM cells. Data are presented as the mean ± s.e.m. *indicates P < 0.05, ** indicates P < 0.01 by Student’s t test. (C) Immunofluorescence images showing the distribution of α-SMA in treated PTM cells. α-SMA filaments organized in strongly labeled stress fibers after Gata4 silencing. Scale bar, 10 μm.

  • View in gallery

    Gene expression profile of Gata4-silenced and -overexpressed PTM cells. (A) RNA sequencing discovering 91 GATA4-positively regulated genes and 32 genes that are negatively regulated by GATA4 in PTM cells. Table of biologic processes enriched in differentially expressed genes (KEGG analysis). (B, C, D and E) Expression levels (FPKM values) of selected gene sets, which are displayed with activities of ion channels (B), smooth muscle function (C), cytokines and chemokines (D), and cytoskeleton, adhesion and ECM (E) in Gata4 siRNA-, Gata4 adenovirus- and control-treated PTM cells. Red frames indicate genes that were further confirmed by qRT-PCR.

  • View in gallery

    Dysregulation of genes implicated in ion channels and smooth muscle contraction in Gata4-overexpressed PTM cells. (A, B, C and D) Differential expression of genes belonging to ion channels, such as Kcnk12 (A), Kcnq5 (B), Atpla3 (C) and Nalcn (D) between Gata4 adenovirus- and control-treated PTM cells. (E, F, G and H) The mRNA levels of genes implicated in smooth muscle contraction, such as Nppb (E), Gpr4 (F), Ednrb (G) and Agt (H) in Gata4-overexpressed and control groups. (I) Analysis of the content of NPPB (pg/mL) in Gata4 adenovirus- or nontargeting control-transfected PTM cells and their culture media by ELISA assay. M, culture media; C, cells. (J) Quantitative data showing the contraction area (cm2) in control-, Gata4 siRNA alone-, Gata4 siRNA plus 100 μM NPPB-treated PTM cells. Data are presented as the mean ± s.e.m. * indicates P < 0.05, **indicates P < 0.01 by Student’s t test. (K) Hypothetical and simplified model of critical factors (e.g. NPPB) involved in the regulation of PTM contraction by GATA4.

References

AriesAWhitcombJShaoWKomatiHSalehMNemerM 2014 Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate. Cell Death and Disease 5 e1566. (https://doi.org/10.1038/cddis.2014.524)

BergeronFNadeauGVigerRS 2015 GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway. Reproduction 149 245257. (https://doi.org/10.1530/REP-14-0369)

BielinskaMSeehraAToppariJHeikinheimoMWilsonDB 2007 GATA-4 is required for sex steroidogenic cell development in the fetal mouse. Developmental Dynamics 236 203213. (https://doi.org/10.1002/dvdy.21004)

CarvajalJA 2014 The role of brain natriuretic peptide in maintaining myometrial quiescence during pregnancy. Experimental Physiology 99 489494. (https://doi.org/10.1113/expphysiol.2013.077446)

ChenSRLiuYX 2015 Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 149 R159R167. (https://doi.org/10.1530/REP-14-0481)

ChenSRLiuYX 2016 Myh11-Cre is not limited to peritubular myoid cells and interaction between Sertoli and peritubular myoid cells needs investigation. PNAS 113 E2352. (https://doi.org/10.1073/pnas.1602873113)

ChenSRChenMWangXNZhangJWenQJiSYZhengQSGaoFLiuYX 2013 The Wilms tumor gene, Wt1, maintains testicular cord integrity by regulating the expression of Col4a1 and Col4a2. Biology of Reproduction 88 56. (https://doi.org/10.1095/biolreprod.112.105379)

ChenLYBrownPRWillisWBEddyEM 2014 Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance. Endocrinology 155 49644974. (https://doi.org/10.1210/en.2014-1406)

ChenSRTangJXChengJMLiJJinCLiXYDengSLZhangYWangXXLiuYX 2015 Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling. Oncotarget 6 3701237027. (https://doi.org/10.18632/oncotarget.6115)

ChenLYWillisWDEddyEM 2016a Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development. PNAS 113 18291834. (https://doi.org/10.1073/pnas.1517994113)

ChenMLiJJiangFFuJXiaXDuJHuMHuangJShenB 2016b Orai1 forms a signal complex with BKCa channel in mesenteric artery smooth muscle cells. Physiological Reports 4 e12682. (https://doi.org/10.14814/phy2.12682)

ChenSRBatoolAWangYQHaoXXChangCSChengCYLiuYX 2016c The control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon’s head to tail. Cell Death and Disease 7 e2472. (https://doi.org/10.1038/cddis.2016.344)

DeFalcoTPotterSJWilliamsAVWallerBKanMJCapelB 2015 Macrophages contribute to the spermatogonial niche in the adult testis. Cell Reports 12 11071119. (https://doi.org/10.1016/j.celrep.2015.07.015)

FantoniGMorrisPLFortiGVannelliGBOrlandoCBarniTSestiniRDanzaGMaggiM 1993 Endothelin-1: a new autocrine/paracrine factor in rat testis. American Journal of Physiology 265 E267E274. (https://doi.org/10.1152/ajpendo.1993.265.2.E267)

FehlmannTReinheimerSGengCSuXDrmanacSAlexeevAZhangCBackesCLudwigNHartM 2016 cPAS-based sequencing on the BGISEQ-500 to explore small non-coding RNAs. Clinical Epigenetics 8 123. (https://doi.org/10.1186/s13148-016-0287-1)

FilippiniATripicianoAPalombiFTetiAPanicciaRStefaniniMZiparoE 1993 Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway. Endocrinology 133 17891796. (https://doi.org/10.1210/endo.133.4.8404621)

FlenkenthalerFWindschüttlSFröhlichTSchwarzerJUMayerhoferAArnoldGJ 2014 Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the speramtogonial stem cell niche in man. Journal of Proteome Research 13 12591269. (https://doi.org/10.1021/pr400769z)

HakonarsonHGrunsteinMM 1998 Regulation of second messengers associated with airway smooth muscle contraction and relaxation. American Journal of Respiratory and Critical Care Medicine 158 S115S122. (https://doi.org/10.1164/ajrccm.158.supplement_2.13tac700)

IedaMFuJDDelgado-OlguinPVedanthamVHayashiYBruneauBGSrivastavaD 2010 Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142 375386. (https://doi.org/10.1016/j.cell.2010.07.002)

JeppsTAGreenwoodIAMoffattJDSandersKMOhyaS 2009 Molecular and functional characterization of Kv7 K+ channel in murine gastrointestinal smooth muscles. American Journal of Physiology: Gastrointestinal and Liver Physiology 297 G107G115. (https://doi.org/10.1152/ajpgi.00057.2009)

LiXXChenSRShenBYangJLJiSYWenQZhengQSLiLZhangJHuZY 2013 The heat-induced reversible change in the blood-testis barrier (BTB) is regulated by the androgen receptor (AR) via the partitioning-defective protein (Par) polarity complex in the mouse. Biology of Reproduction 89 12. (https://doi.org/10.1095/biolreprod.113.109405)

LoirandGCario-ToumaniantzCChardinPPacaudP 1999 The Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle. Journal of Physiology 516 825834. (https://doi.org/10.1111/j.1469-7793.1999.0825u.x)

ManiBKRobakowskiCBrueggemannLICribbsLLTripathiAMajetschakMByronKL 2016 Kv7.5 Potassium channel subunits are the primary targets for PKA-dependent enhancement of vascular smooth muscle Kv7 currents. Molecular Pharmacology 89 323334. (https://doi.org/10.1124/mol.115.101758)

MayerCAdamMGlashauserLDietrichKSchwarzerJUKöhnFMStraussLWelterHPoutanenMMayerhoferA 2016 Sterile inflammation as a factor in human male infertility: involvement of Toll like receptor 2, biglycan and peritubular cells. Scientific Reports 6 37128. (https://doi.org/10.1038/srep37128)

MayerhoferA 2013 Human testicular peritubular cells: more than meets the eye. Reproduction 145 R107R116. (https://doi.org/10.1530/REP-12-0497)

Mazaud GuittotSTetuALegaultEPilonNSilversidesDWVigerRS 2007 The proximal Gata4 promoter directs reporter gene expression to sertoli cells during mouse gonadal development. Biology of Reproduction 76 8595. (https://doi.org/10.1095/biolreprod.106.055137)

OatleyJMOatleyMJAvarbockMRTobiasJWBrinsterRL 2009 Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development 136 11911199. (https://doi.org/10.1242/dev.032243)

OrlandiACalzettaLDoldoETarquiniCMateraMGPasseriD 2015 Brain natriuretic peptide modulates calcium homeostasis and epidermal growth factor receptor gene signalling in asthmatic airways smooth muscle cells. Pulmonary Pharmacology and Therapeutics 31 5154. (https://doi.org/10.1016/j.pupt.2015.02.005)

PennyGMCochranRBPihlajokiMKyronlahtiASchradeAHakkinenMToppariJHeikinheimoMWilsonDB 2017 Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors. Reproduction 154 455467. (https://doi.org/10.1530/REP-17-0311)

QianYLiuSGuanYPanHGuanXQiuZLiLGaoNZhaoYLiX 2013 Lgr4-mediated Wnt/beta-catenin signaling in peritubular myoid cells is essential for spermatogenesis. Development 140 17511761. (https://doi.org/10.1242/dev.093641)

RebourcetDO’ShaughnessyPJPitettiJLMonteiroAO’HaraLMilneLTsaiYTCruickshanksLRiethmacherDGuillouF 2014 Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis. Development 141 21392149. (https://doi.org/10.1242/dev.107029)

ReinlELCabezaRGregoryIACahillAGEnglandSK 2015 Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women. Molecular Human Reproduction 21 816824. (https://doi.org/10.1093/molehr/gav038)

RomanoFChiarenzaCPalombiFFilippiniAPadulaFZiparoEDe CesarisP 2006 Platelet-derived growth factor-BB-induced hypertrophy of peritubular smooth muscle cells is mediated by activation of p38 MAP-kinase and of Rho-kinase. Journal of Cellular Physiology 207 123131. (https://doi.org/10.1002/jcp.20554)

RomanoFGambaraGDe CesarisPZiparoEPalombiFFilippiniA 2007 Endothelin induces functional hypertrophy of peritubular smooth muscle cells. Journal of Cellular Physiology 212 264273. (https://doi.org/10.1002/jcp.21028)

SanadaHYonedaMYatabeJWilliamsSMBartlettJWhiteMJCordonLNFelderRARisnerGMArmandoI 2016 Common variants of the G protein-coupled receptor type 4 are associated with human essential hypertension and predict the blood pressure response to angiotensin receptor blockade. Pharmacogenomics Journal 16 39. (https://doi.org/10.1038/tpj.2015.6)

SchellCAlbrechtMSpillnerSMayerCKunzLKohnFMSchwarzerUMayerhoferA 2010 15-Deoxy-delta 12-14-prostaglandin-J2 induces hypertrophy and loss of contractility in human testicular peritubular cells: implications for human male fertility. Endocrinology 151 12571268. (https://doi.org/10.1210/en.2009-1325)

SchradeAKyronlahtiAAkinrinadeOPihlajokiMHakkinenMFischerSAlastaloTPVelagapudiVToppariJWilsonDB 2015 GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells. Endocrinology 156 18601872. (https://doi.org/10.1210/en.2014-1931)

SchradeAKyronlahtiAAkinrinadeOPihlajokiMFischerSRodriguezVMOtteKVelagapudiVToppariJWilsonDB 2016 GATA4 regulates blood-testis barrier function and lactate metabolism in mouse sertoli cells. Endocrinology 157 24162431. (https://doi.org/10.1210/en.2015-1927)

SunXTommasiEMolinaDSahRBronihanKBDizDPetrovicS 2016 Deletion of proton-sensing receptor GPR4 associates with lower blood pressure and lower binding of angiotensin II receptor in SFO. American Journal of Physiology: Renal Physiology 311 F1260F1266. (https://doi.org/10.1152/ajprenal.00410.2016)

TripicianoAFilippiniABallariniFPalombiF 1998 Contractile response of peritubular myoid cells to prostaglandin F2alpha. Molecular and Cellular Endocrinology 138 143150. (https://doi.org/10.1016/S0303-7207(98)00010-0)

WelshMSaundersPTAtanassovaNSharpeRMSmithLB 2009 Androgen action via testicular peritubular myoid cells is essential for male fertility. FASEB Journal 23 42184230. (https://doi.org/10.1096/fj.09-138347)

WelterHKampferCLaufSFeilRSchwarzerJUKohnFMMayerhoferA 2013 Partial loss of contractile marker proteins in human testicular peritubular cells in infertility patients. Andrology 1 318324. (https://doi.org/10.1111/j.2047-2927.2012.00030.x)

WelterHHuberALaufSEinwangDMayerCSchwarzerJUKohnFMMayerhoferA 2014 Angiotensin II regulates testicular peritubular cell function via AT1 receptor: a specific situation in male infertility. Molecular and Cellular Endocrinology 393 171178. (https://doi.org/10.1016/j.mce.2014.06.011)

Index Card

PubMed

Google Scholar

Related Articles

Altmetrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 3 3 3
Full Text Views 236 236 85
PDF Downloads 68 68 19