Combined Loss of the GATA4 and GATA6 Transcription Factors in Male Mice Disrupts Testicular Development and Confers Adrenal-Like Function in the Testes

Combined Loss of the GATA4 and GATA6Transcription Factors in Male Mice Disrupts TesticularDevelopment and Confers Adrenal-Like Function inthe Testes

Maria B. Padua, Tianyu Jiang, Deborah A. Morse, Shawna C. Fox,Heather M. Hatch, and Sergei G. Tevosian

Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville,Florida 32610

The roles of the GATA4 and GATA6 transcription factors in testis development were examined bysimultaneously ablating Gata4 and Gata6 with Sf1Cre (Nr5a1Cre). The deletion of both genesresulted in a striking testicular phenotype. Embryonic Sf1Cre; Gata4flox/flox Gata6flox/flox (condi-tional double mutant) testes were smaller than control organs and contained irregular testis cordsand fewer gonocytes. Gene expression analysis revealed significant down-regulation of Dmrt1 andMvh. Surprisingly, Amh expression was strongly up-regulated and remained high beyond postnatalday 7, when it is normally extinguished. Neither DMRT1 nor GATA1 was detected in the Sertoli cellsof the mutant postnatal testes. Furthermore, the expression of the steroidogenic genes Star,Cyp11a1, Hsd3b1, and Hsd17b3 was low throughout embryogenesis. Immunohistochemical anal-ysis revealed a prominent reduction in cytochrome P450 side-chain cleavage enzyme (CYP11A1)-and 3�-hydroxysteroid dehydrogenase-positive (3�HSD) cells, with few 17�-hydroxylase/17,20lyase-positive (CYP17A1) cells present. In contrast, in postnatal Sf1Cre; Gata4flox/flox Gata6flox/flox

testes, the expression of the steroidogenic markers Star, Cyp11a1, and Hsd3b6 was increased, buta dramatic down-regulation of Hsd17b3, which is required for testosterone synthesis, was ob-served. The genes encoding adrenal enzymes Cyp21a1, Cyp11b1, Cyp11b2, and Mcr2 were stronglyup-regulated, and clusters containing numerous CYP21A2-positive cells were localized in the in-terstitium. These data suggest a lack of testis functionality, with a loss of normal steroidogenic testisfunction, concomitant with an expansion of the adrenal-like cell population in postnatal condi-tional double mutant testes. Sf1Cre; Gata4flox/flox Gata6flox/flox animals of both sexes lack adrenalglands; however, despite this deficiency, males are viable in contrast to the females of the samegenotype, which die shortly after birth. (Endocrinology 156: 1873–1886, 2015)

In most mammals, inheritance of the Y chromosome as-sures commitment to a male fate. Sex determination be-

comes realized at midgestation through the expression ofthe Y chromosome testis-determining gene Sry. SRY-de-

pendent activation of the transcription factor sex-deter-mining region Y-box 9 (SOX9) orchestrates a cascade ofevents leading to differentiation of the Sertoli cell popu-lation that guides the conversion of the bipotential em-

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived November 13, 2014. Accepted February 6, 2015.First Published Online February 10, 2015

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Abbreviations: AMH, anti-Müllerian hormone; BrdU, bromodeoxyuridine; CYP17A1, 17�-hydroxylase/17,20 lyase; CYP11A1, cytochrome P450 side-chain cleavage enzyme;CYP21A2, cytochrome P450, family 21, subfamily A, polypeptide 2; DHH, desert hedge-hog; DMRT1, doublesex and mab-3-related transcription factor 1; E, embryonic day;FOXL2, forkhead box L2; GATA4, GATA binding protein 4; GATA6, GATA binding protein6; GH2AX, gamma histone variant H2AX; H&E, hematoxylin and eosin; 3�HSD, 3�-hy-droxysteroid dehydrogenase; IF, immunofluorescence; Insl3, insulin-like factor 3; MT-hAMH, transgenic male mice overexpressing AMH; MVH, mouse vasa homolog; NR5A1/AD4BP, nuclear receptor subfamily 5, group A, member 1/adrenal 4-binding protein; PND,postnatal day; qPCR, quantitative RT-PCR; SF1, steroidogenic factor 1; SOX9, sex-deter-mining region Y-box 9; Sry, sex determining region of Chr Y; STAR, steroidogenic acuteregulatory protein; WT1, Wilms’ tumor 1.

O R I G I N A L R E S E A R C H

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bryonic gonad into testes rather than ovaries (1, 2). Aftersex determination, the testis forms two separate compart-ments, the testicular cords and the interstitial region. Theinterstitial region lies outside of the testis cords and con-tains several cell types, most notably the steroidogenic fe-tal Leydig cells (2). Normal development of fetal Leydigcell progenitors depends on paracrine signaling instruc-tions emanating from the Sertoli cells to initiate steroid-ogenesis (3). The master regulator steroidogenic factor 1(SF1) (SF1/NR5A1/Ad4BP, henceforth SF1) is at the helm ofthe steroidogenic expression program in several endocrineorgans, including the testis, where it is the first geneticmarker that gives steroid-synthesizing cells their distinc-tive identity and controls their metabolism, proliferation,and survival (4).

In vertebrates, 6 GATA transcription factors act as keyregulators of the development of multiple tissues. Two ofthese proteins, GATA4 and GATA6, are expressed in thesomatic cells of the embryonic testis (5). Early in gonadaldevelopment, GATA4 in association with its cofactorFOG2/ZFPM2 (friend of GATA/zinc finger protein mul-tiple 2) acts to promote sex determination and testis dif-ferentiation (6). The Cre-LoxP loss-of-function geneticapproach has been applied to clarify the role of GATA4 intestis differentiation using testis-specific Cre drivers to di-rect Gata4 gene deletion (7, 8). Sf1Cre; Gata4flox/flox

males develop partially descended small testes, exhibit ashort anogenital distance, and are infertile. The morphol-ogy of the Sf1Cre; Gata4flox/flox testis cords is irregular,with numerous immature Sertoli cells being observedwithin them. The expression of Dmrt1, one of the keytranscription factors in the male pathway (9, reviewed inRef. 10), is absent throughout embryogenesis (8). Sf1Cre(11) effectively deleted Gata4 as early as embryonic day(E)11.5–E12.5 in the precursors of Sertoli and Leydig cells(8). In contrast, in Amrh2Cre; Gata4flox/flox males, no ob-vious defects were observed during embryonic or earlypostnatal testis development, and the external genitaliaand testicular descent were normal. Adult Amrh2Cre;Gata4flox/flox males develop age-dependent infertility, ac-companied by testicular atrophy and vacuolization of theseminiferous tubules (7). Amhr2Cre is expressed in fetalSertoli cells and in Sertoli and Leydig cells postnatally (12);however, the extent of deletion in Sertoli vs Leydig cellsvaried depending upon the gene studied (7, 12). Therefore,it is possible that the absence of a prenatal testicular phe-notype is the result of a delayed or mosaic Amhr2Cre-mediated recombination in the fetal testes (reviewed inRef. 13).

Although the involvement of GATA4 in regulating Ser-toli cells is incontrovertible, the cell-autonomous role ofthis protein in the steroidogenic interstitial cells is less

clear. XY GATA4-null embryonic stem cells are unable todifferentiate into Leydig cells (14); however, interstitialcells expressing Leydig steroidogenic enzymes developnormally in mice deficient in the GATA4 protein (8). Thepresence of Gata6 in the developing mouse testis has beenlong documented (5, 15), but no specific regulatory func-tion has been assigned to GATA6 in any testicular lineage.Given that GATA6 is coexpressed with GATA4 in thetestis, it is unknown whether their functions completelyoverlap or whether GATA6 plays an independent role intestis development. To address these questions, we carriedout a deletion of both Gata4 and Gata6 in the mouseembryonic testis. Here, we report that these proteins ex-hibit several overlapping functions in the Sertoli and Ley-dig cells of the testis.

Materials and Methods

Generation of mouse strainsProcedures involving live animals were approved by the In-

stitutional Animal Care and Use Committees of University ofFlorida. The Gata4flox/flox and Gata6flox/flox “flox” mice wereobtained from The Jackson Laboratory repository. The trans-genic Sf1Cre mice (a gift from late Dr Parker) harbor Sf1 (Nr5a1)regulatory elements driving Cre expression within a bacterialartificial chromosome (BAC) (11). Strains carrying Sf1Cre-me-diated deletions were produced by crossing flox mice withSf1Cre-containing animals, followed by backcross to generatehomozygous deletions. Sf1Cre; Gata6flox/flox mice are fertile, butSf1Cre; Gata4flox/flox mice are sterile (16). Therefore, Sf1Cre;Gata4flox/� Gata6flox/flox males were backcrossed with “doubleflox” Gata4flox/flox Gata6flox/flox females to generate condi-tional double mutants (Sf1Cre; Gata4flox/flox Gata6flox/flox).Gata4flox/flox Gata6flox/flox animals were used as experimentalcontrols. The mice were maintained in a mixed 129/C57BL/6genetic background. The primers used for genotyping (Inte-grated DNA Technologies) are listed in Supplemental Table 1.

First-strand cDNA synthesis and quantitativeRT-PCR (qPCR)

Gonad-mesonephros complexes (for E13.5) and testes werecollected at different stages of development (E15.5 and E18.5and postnatal day [PND]4, PND9, and PND47) from controlsand Sf1Cre; Gata4flox/flox Gata6flox/flox animals for RNA ex-traction. The conditions are described in Supplemental Materialsand Methods. The primers used (Integrated DNA Technologies)are listed in Supplemental Table 2.

Immunofluorescence (IF)Testes were collected from control and Sf1Cre; Gata4flox/flox

Gata6flox/flox animals (n � 3 from each genotype) at differentstages of development (E13.5, E15.5, and E18.5 and PND4,PND7, and PND30). IF experiments were carried out as previ-ously described (16, 17). The primary antibodies and experi-mental conditions are listed in the supplemental antibody table.

1874 Padua et al Role of GATA4 and GATA6 in Testicular Development Endocrinology, May 2015, 156(5):1873–1886


Hematoxylin and eosin (H&E) stainingTestes from controls and Sf1Cre; Gata4flox/flox Gata6flox/flox

mice (n � 2 from each genotype) were harvested at PND7,PND17, and PND30 for histological analysis. Tissue sectionswere processed as previously described (17).

ImmunohistochemistryImmunohistochemical reactions were performed with the

ImmPRESS polymerized reporter enzyme staining system kit(Vector Laboratories, Inc), which uses peroxidase for detection.The procedure is described in detail in Supplemental Materialsand Methods.

Intratesticular testosterone concentrationThe intratesticular testosterone concentration was deter-

mined using the competitive Cayman’s testosterone enzyme im-munoassay kit (Cayman Chemical Co), following the manufac-turer’s guidelines. The procedure is described in theSupplemental section.

Bromodeoxyuridine (BrdU) incorporation andTerminal deoxynucleotidyl transferase dUTP NickEnd Labeling (TUNEL) assays

These procedures are described in Supplemental Materialsand Methods.

Whole-mount in situ hybridizationThe procedure is described in Supplemental Materials and

Methods.

Results

Absence of doublesex and mab-3-relatedtranscription factor 1 (DMRT1) expression in theE13.5 Sertoli cells of Sf1Cre; Gata4flox/flox

Gata6flox/flox testisIn the testis, GATA4 is already present in the somatic

cells at E10.5 (8, 18, 19). Extending earlier observations(5, 15), we show that GATA6 is detected in the Sertoli andinterstitial cells of control testis at E13.5 (Figure 1A).GATA4 and GATA6 are coexpressed in the Sertoli cellsand in some interstitial (presumably Leydig cells) and coe-lomic epithelial cells (Figure 1A). Sf1Cre-mediated recom-bination is highly effective in the embryonic testis (com-pare Figure 1, A and F), and expression of the GATA4 andGATA6 proteins was no longer detectable in the somaticcells ofSf1Cre;Gata4flox/flox Gata6flox/flox testes as earlyas

Figure 1. Gene expression analysis of E13.5 control and Sf1Cre; Gata4flox/flox Gata6flox/flox testes. Representative sections of control (A–E) andSf1Cre; Gata4flox/flox Gata6flox/flox (F–J) testes at E13.5. Testicular sections were stained with antibodies against GATA4 (green) and GATA6 (red) (Aand F); DMRT1 (green) and SF1 (red) (B and G); AMH (green) and SOX9 (red) (C and H); the pluripotent germ cell marker OCT3/4 (green) and WT1(red) (D and I); and the universal germ cell marker MVH (red) (E and K). Nuclei were stained with DAPI (blue). Scale bars represent 100 �m. TC,testicular cords. K, Quantitative analysis of gene expression in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at E13.5. The examined genes were Amh,Dhh, Dmrt1, Mvh, and Sox9. The results are shown as the mean � SEM of the fold change relative to controls for at least 4 biological replicates(n � 4), with significance considered at **, P � .01.

doi: 10.1210/en.2014-1907 endo.endojournals.org 1875


E13.5. As described previously (8, 20), residual coelomicepithelial cells in the double mutant testis remained pos-itive for GATA4 or GATA6, with some of these cells ex-pressing both proteins (Figure 1F). The efficiency ofSf1Cre in achieving the deletion of Gata genes remainedhigh on all subsequent embryonic and PNDs examined(compare Figure 2, A and D and G and J, for E15.5 andE18.5, respectively, and figures 4 and 5 PND4 andPND30, respectively, below).

In mice, DMRT1 is expressed in the genital ridge inboth sexes until approximately E14.5, when it becomestestis specific and is detected in both Sertoli and germ cells(9, 21). IF experiments revealed that DMRT1 is expressedin both the Sertoli cells (by colocalization with SF1) andgonocytes of testes (Figure 1B). In contrast, the only cells

expressing DMRT1 in E13.5 Sf1Cre; Gata4flox/flox

Gata6flox/flox testis were germ cells, whereas the Sertolicells were devoid of DMRT1 staining (Figure 1G). A sim-ilar pattern of expression for DMRT1 was observed insubsequent stages of embryonic development (compareFigure 2, B and E and H and K, for E15.5 and E18.5,respectively). Accordingly, gene expression analysis viaquantitative reverse transcription-polymerase chain reac-tion also revealed significant down-regulation of Dmrt1(P � .01) in all embryonic stages evaluated (Figures 1Kand 2, M and N).

In males, anti-Müllerian hormone (AMH) is responsi-ble for the regression of the Müllerian ducts and is secretedby fetal and early postnatal Sertoli cells (reviewed in in Ref.22). The expression of Amh in mice begins at E11.5 (22–

Figure 2. The analysis of somatic gene expression at E15.5 and 18.5 in Sf1Cre; Gata4flox/flox Gata6flox/flox testes. A–L, Representative images oftesticular sections from controls (A–C and G–I) and Sf1Cre; Gata4flox/flox Gata6flox/flox mice (D–F and J–L) at E15.5 (A–F) and E18.5 (G–L). Thesections were stained for GATA4 (green) and GATA6 (red) (A, D, G, and J); DMRT1 (green) and SF1 (red) (B, E, H, and K); and AMH (green) andSOX9 (red) (C, F, I, and L). Nuclei were stained with DAPI (blue). Scale bars represent 100 �m. M and N, Gene expression analysis via qPCR inSf1Cre; Gata4flox/flox Gata6flox/flox testes at E15.5 (M) and E18.5 (N). The examined transcripts were Amh, Dhh, Dmrt1, Mvh, Sf1, Sox9, Gata4, andGata6. The results are graphed as the mean � SEM of the fold change relative to controls, from n � 5 for E15.5 and n � 4 for E18.5 biologicalreplicates, with significance considered at *, P � .05; **, P � .01; and ***, P � .001.



24). During embryogenesis (E13.5 to E18.5), AMH wasexpressed by the Sertoli cells of both the controls and thedouble mutant testes (Figures 1, C and H, and 2, C and Fand I and L). Early in development (E13.5), the expressionof Amh in Sf1Cre; Gata4flox/flox Gata6flox/flox testes wasno different from that in controls. In contrast, Amh ex-pression was significantly up-regulated (P � .01) in thedouble mutant testes at E15.5 (Figure 2M); this trend con-tinued at E18.5, although it was not significant (Figure2N).

Similar to AMH, the SOX9 transcription factor is ex-pressed by the pre-Sertoli cells and is a major protein pro-moting their subsequent differentiation (25, 26). SOX9 isfirst detectable in the bipotential gonad, and at E11.5, itsexpression becomes notably up-regulated in the testes anddown-regulated in the ovaries (1). Previous work demon-strated that Amh expression is directly controlled bySOX9 through its binding site in the Amh promoter (27).SOX9 was immunolocalized to the Sertoli cells, with nodetectable changes in the pattern of expression in Sf1Cre;Gata4flox/flox Gata6flox/flox testes compared with the con-trols in all embryonic stages examined (Figures 1, C and H,and 2, C, F, I, and L). Similarly, quantitative assessment ofSox9 expression did not reveal any significant changes indouble mutant testes relative to the controls (Figures 1Kand 2, M and N). Another important signaling moleculeproduced by Sertoli cells is the desert hedgehog (DHH)protein. DHH is required for the differentiation of ste-roidogenic fetal Leydig cells (28, 29). In mice, Dhh ex-pression in the testis is detected at E11.5 and continuesthroughout embryogenesis (6, reviewed in Ref. 3). Theexpression of Dhh in Sf1Cre; Gata4flox/flox Gata6flox/flox

testes was normal (Figures 1K and 2, M and N).

Abnormal testis cord architecture and decreasednumbers of gonocytes in Sf1Cre; Gata4flox/flox

Gata6flox/flox embryonic testisAt E13.5, we observed no notable difference in the

overall number of primordial germ cells (by IF staining forthe mouse vasa homolog [MVH], the pluripotent germ cellmarker, POU domain, class 5, transcription factor 1(OCT3/4), and via qPCR) between control and doublemutant testis (compare Figure 1, D, E, I, and J, respec-tively). However, an irregular distribution of gonocytes inthe disorganized testis cords of the Sf1Cre; Gata4flox/flox

Gata6flox/flox testis was already prominent. A dramaticreduction in the overall number of gonocytes became ap-parent in subsequent stages of embryonic development(E15.5 and 18.5) (compare Figure 3, C and G and D andH). Accordingly, significant down-regulation of the Mvhtranscript was detected at both E15.5 and E18.5 (Figure 2,M and N).

The smaller size of Sf1Cre; Gata4flox/flox Gata6flox/flox

testes compared with the control organs was notable at theearliest stage we analyzed, E13.5 (Figures 1, A and F, and2, G and J). To determine whether cell proliferation iscompromised in the double mutant testes, we used BrdUDNA labeling. Numerous BrdU-labeled cells were ob-served in both and Sf1Cre; Gata4flox/flox Gata6flox/flox tes-tes at E15.5 (Supplemental Figure 1, A–C and F–H) andE17.5 (Supplemental Figure 1, D, E, I, and J). Colocaliza-tion of BrdU-positive cells with the Wilms’ tumor 1 (WT1)protein showed that somatic (mostly Sertoli) cells prolif-erate normally in both genotypes at E15.5 (SupplementalFigure 1, D and H). The ratio of BrdU-labeled cells to4�,6-diamidino-2-phenylindole (DAPI)-positive cells did

Figure 3. Decrease in gonocyte numbers and loss of the testis architecture in Sf1Cre; Gata4flox/flox Gata6flox/flox males. Representative sections ofcontrol (A, C, E, and G) and Sf1Cre; Gata4flox/flox Gata6flox/flox (B, D, F, and H) testes at E15.5 (A–D) and E18.5 (E–H). The sections were stained forAMH (green) and the universal germ cell marker MVH (red) (A, B, E, and F); and Laminin (green) and MVH (red) (C, D, G, and H). Nuclei werestained by DAPI (blue). Scale bars represent 100 �m. TC, testis cords.



not differ between the control and double mutant testes atE17.5 (Supplemental Figure 1K)

In contrast, analysis of cell death using TUNEL stainingrevealed more apoptotic nuclei in Sf1Cre; Gata4flox/flox

Gata6flox/flox testes (Supplemental Figure 2, B and C) thanin controls (Supplemental Figure 2A) at E15.5. Numerousapoptotic nuclei were localized proximal to the coelomicepithelium at both embryonic points examined (E15.5 andE17.5) (Supplemental Figure 2, B and E). Similarly, agreater number of gonocytes (identified based on colocal-ization with MVH) was undergoing cell death in the dou-ble mutant testes (Supplemental Figure 2C).

GATA1 is not expressed in the Sertoli cells ofSf1Cre; Gata4flox/flox Gata6flox/flox testes

The GATA1 protein figures prominently in hemato-poietic development and is required for normal erythroidand megakaryocytic development (reviewed in Ref. 30).GATA1 is absent from the developing gonad but becomesrobustly expressed in the testis shortly after birth. Curi-ously, the Sertoli cells of the postnatal testis are the onlyknown extrahematopoietic site of Gata1 expression (31,32, reviewed in Refs. 33, 34). It has previously been re-ported that GATA1 is dispensable for Sertoli cell functionand for the expression of a number of testis-specific genes(35, 36). We considered the possibility that upon the de-

letion of both Gata4 and Gata6, Gata1 could display acompensatory function for Sertoli gene expression. Unex-pectedly, we observed that after the deletion of Gata4 andGata6, GATA1 expression in Sertoli cells did not com-mence as normal in the postnatal double mutant testis(compare Figures 4, B and C and G and H, and 5, D andH). The absence of Gata1 expression was corroboratedthrough qPCR (P � .001) (Figures 4K and 5I). Thus, ourmodel allows the analysis of testis gene expression in theabsence of all 3 GATA factors. To verify that the absenceof GATA1 alone is insufficient to exert changes in thesomatic or germ cells, we examined gene expression inthe Gata1 transgenic model in which the transgene res-cues Gata1 expression exclusively in the hematopoieticcell compartment of the Gata1-null mice, but nowhereelse in the animal (ensuring the survival of the otherwiselethal Gata1 gene deletion) (35). We observed no dif-ferences in the expression patterns of the GATA4,GATA6, and H2AX proteins (Supplemental Figure 3);AMH expression was also not elevated in the absence ofGATA1.

Similarly, the DMRT1 protein was virtually absent inthe Sertoli cells of postnatal Sf1Cre; Gata4flox/flox

Gata6flox/flox testes (compare Figures 4, D and L, and 5, Nand S). The DMRT1-positive cells remaining in doublemutant testes were mostly spermatogonial cells; only rare

Figure 4. GATA1 expression is lost in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at PND4. Representative images of testicular sections from controls(A–E) and Sf1Cre; Gata4flox/flox Gata6flox/flox mice (I–M) at PND4. The sections were stained for GATA4 (green) and GATA6 (red) (A and F); GATA1(green) and WT1 (red) (B and G); DMRT1 (green) and SF1) (red) (D and I); and AMH (MIS; green) and SOX9 (red) (E and J). C and H, Highermagnifications of B and G, respectively. The arrow in I points to the few DMRT1 and SF1 double-positive cells in the double mutant testis. Scalebars represent 100 �m (B, F, G, I, and J), 50 �m (A, D, and E), or 20 �m (C and H). K, Examination of gene expression via qPCR in Sf1Cre;Gata4flox/flox Gata6flox/flox testes at PND4. The examined transcripts were Amh, Dmrt1, Gata1, Mvh, Sox9, Gata4, and Gata6. The results are shownas the mean � SEM of the fold change relative to controls from at least n � 3 biological replicates, with significance considered at **, P � .01and ***, P � .001.



DMRT1-positive cells were observed among the Sertolicells (double DMRT1; SF1-positive) (Figure 4I, arrow). Inagreement with these results, gene expression analysis re-vealed significant down-regulation of Dmrt1 expression(P � .001) at all postnatal stages evaluated (Figures 4Kand 5T). It has been reported that loss of Dmrt1 expres-sion in the Sertoli cells (but not germ cells) leads to sexreversal, defined by ectopic expression of the female-spe-cific transcription factor FOXL2 in the Sertoli cells ofPND28 mouse testes (37). Although the expression of theDmrt1 gene was dramatically down-regulated and proteinstaining was virtually absent in Sf1Cre; Gata4flox/flox

Gata6flox/flox Sertoli cells at all developmental stages ex-amined, IF experiments did not detect FOXL2-positivecells in the PND30 double mutant testis (data not shown).Although qPCR at PND47 detected a tendency for theincreased expression of Foxl2, it was not statistically sig-nificant (P � .07) (Figure 5T).

Continuous expression of AMH and atypicaldistribution of spermatogonia in Sf1Cre;Gata4flox/flox Gata6flox/flox testes after PND7

AMH is expressed in the mouse testis throughout em-bryonic development until birth, when its expression be-gins to decline. We noted that at PND7 AMH protein issharply reduced in the Sertoli cells of control testes (Figure5, B and C) and becomes completely absent by PND30(Figure 5M). In contrast, the expression of AMH in theSertoli cells of Sf1Cre; Gata4flox/flox Gata6flox/flox testesremained high postnatally (Figure 5, F and R, for PND7and PND30, respectively). qPCR experiments verified theIF data and corroborated significant up-regulation ofAmh expression at PND9 (P � .05) (Figure 5I) and PND47(P � .01) (Figure 5T). No postnatal changes in the ex-pression of its major regulator SOX9 (27), determinedeither via IF at PND7 (Figure 5, D and H) or qPCR atPND9 (Figure 5I), were observed in double mutant testes.

Figure 5. Persistent AMH expression in Sf1Cre; Gata4flox/flox Gata6flox/flox testes after PND7. Representative images of testicular sections fromcontrols (A–D and J–N) and Sf1Cre; Gata4flox/flox Gata6flox/flox mice (E–H and O–S) at PND7 (A–H) or PND30 (J–S). PND7 sections were stained withH&E (A and E) and with antibodies against AMH (green) and phosphorylated histone family protein H2A (G-H2AX) (red) (B and F); or againstGATA1 (green) and SOX9 (red) (D and H). C and G, Higher magnifications of B and F, respectively. PND30 sections were stained with H&E (J andO) and with antibodies against GATA4 (green) and GATA6 (red) (L and Q); AMH (green) and MVH (red) (M and R); or against DMRT1 (green) andSF1 (red) (N and S). DAPI (blue) was used for nuclear staining. K and P, Higher magnifications of J and O, respectively. In panels R and S, arrowspoint to the remaining spermatogonia in Sf1Cre; Gata4flox/flox Gata6flox/flox testes. Scale bars represent 200 �m (A, E, J, and O), 100 �m (F, H, L–N,and Q–S), 50 �m (B, D, K, and P), or 20 �m (C and G). I and T, Analysis of gene expression via qPCR in Sf1Cre; Gata4flox/flox Gata6flox/flox testes atPND9 (I) and PND47 (T). The analyzed genes were Amh, Dmrt1, Foxl2, Gata1, Mvh, Sox9, Gata4, and Gata6. The bar graphs represent themean � SEM of the fold change relative to controls from at least n � 3 biological replicates, with significance considered at *, P � .05; **, P �.01; and ***, P � .001.



These results are similar to those obtained at the prenataltime points we have evaluated.

Postnatal Sf1Cre; Gata4flox/flox; Gata6flox/flox testes areremarkably underdeveloped (compare Figure 5, A and Jand E and O, respectively). In particular, at PND30, thediameter of the seminiferous tubules is markedly smallerin the double mutant testis than in controls (compare Fig-ure 5, K and P), with fewer spermatogonia and Sertoli(somatic) cells being observed within each testicular cord.Furthermore, in the postnatal testes, localization of thespermatogonia adjacent to the basement membrane of thetestis cords is evident (G-H2AX-positive cells in Figure 5,B and C; cells with large purple nuclei in Figure 5K; andDMRT1-positive cells in Figure 5N), whereas in PND7Sf1Cre; Gata4flox/flox Gata6flox/flox testes, only rare sper-matogonia migrated to the basement membrane (Figure 5,F and G). The numbers of germ cells continued to declinein conditional double mutant testes, and at later postnatalstages, the expression of Mvh became significantly lower(P � .01) (Figure 5T); very few spermatogonia were de-tected by IF (Figure 5, R and S, arrows).

The steroidogenic pathway is compromised inSf1Cre; Gata4flox/flox Gata6flox/flox fetal testes

Interstitial Leydig cells are responsible for the produc-tion of testosterone that ensures the persistence of theWölffian ducts and stimulates their subsequent differen-tiation into organs of the male reproductive tract (re-viewed in Ref. 38). Several enzymes have been implicatedin the synthesis of testosterone from its precursor choles-terol, including the steroidogenic acute regulatory protein(STAR), cytochrome P450 side-chain cleavage enzyme(CYP11A1), 17�-hydroxylase/17,20 lyase (CYP17A1),3�-hydroxysteroid dehydrogenase (3�HSD), and 17�HSD(38). Two populations of Leydig cells are recognized in ro-dents: the fetal population, which arises after sex determi-nation and declines shortly after birth, and the adult popu-lation, which emerges during the first 2 weeks after birth andremains throughout adulthood (3, 39). Unlike adult Leydigcells, the fetal Leydig population is presumed to synthesizetestosterone in a pituitary-independent manner (40).

Immunohistochemical assessment of steroidogenic en-zymes in control testes showed robust staining corre-sponding to the CYP11A1, 3�HSD, and CYP17A1 pro-teins in the interstitial fetal Leydig cells at E15.5 and E18.5(Figure 6, A–C and J–L, respectively). In contrast, in theSf1Cre; Gata4flox/flox Gata6flox/flox testes, we observed amajor reduction in the number of cells expressingCYP11A1 and 3�HSD at both embryonic developmentalstages examined (Figure 6, D and E and M and N). More-over, only occasional CYP17A1-positive cells were im-munolocalized in the double mutant testis at E15.5 (Figure

6, F and I), which became completely undetectable atE18.5 (Figure 6O). qPCR confirmed the significant down-regulation of all of the steroidogenic genes examined, atboth E13.5 and E15.5 (Figure 6, P and Q), in the Sf1Cre;Gata4flox/flox Gata6flox/flox testes. In agreement with theseresults, whole-mount in situ hybridization experimentsperformed at E15.5 demonstrated the same strong down-ward trend of Cyp11a1, Cyp17a1, and Hsd17b3 RNAexpression in the double mutant testes (Supplemental Fig-ure 4). Similarly, at E18.5, most of the steroidogenic geneswere down-regulated, including Cyp11a1 (P � .01),Hsd3b1 (P � .01), and Hsd17b3 (P � .001); only Hsd3b6was significantly up-regulated (P � .001) (Figure 6R).

Leydig cells also express insulin-like factor 3 (Insl3), apeptide hormone that is critical for testicular descent (41;reviewed in Refs. 22, 42). In mice, Insl3 has been detectedas early as E12.5 (43). Quantitative analysis of Insl3 ex-pression in Sf1Cre; Gata4flox/flox Gata6flox/flox testes re-vealed a significant reduction of the transcript throughoutembryogenesis (E13.5, P � .05; E15.5, P � .001; andE18.5, P � .05) (Figure 6, P–R), which may explain theundescended intraabdominal position of the double mu-tant testes proximal to the kidneys (Supplemental Figure5, A and B). This phenotype is similar, but more severethan that of the Sf1Cre; Gata4flox/flox males, in which thetestes partially descend (8); however, distinct from Sf1Cre;Gata6flox/flox males in which testicular development isnormal (Supplemental Figure 6).

Increased expression of steroidogenic genes inSf1Cre; Gata4flox/flox Gata6flox/flox postnatal testes

Before birth, a profound decrease in the steroidogeniccompetence of the Sf1Cre; Gata4flox/flox Gata6flox/flox

double mutant testis is observed (Figure 6). In contrast,CYP11A1- and 3�HSD-positive cells become abundant inthe interstitial region of the double mutant testis at PND4(Figure 7, D and E, respectively). Gene expression analysisvia qPCR confirmed the significant up-regulation of theexpression of the steroidogenic genes Star (P � .001),Cyp11a1 (P � .01), and Hsd3b6 (P � .001) in doublemutant testes (Figure 7M).

Abundant CYP11A1- and 3�HSD-positive cells werealso present in the testes of the Sf1Cre; Gata4flox/flox

Gata6flox/flox animals at PND30; however, the distribu-tion of steroidogenic cells was notably different from thatat PND4. Most the CYP11A1- and 3�HSD-positive cellswere clustered proximal to the coelomic epithelium (Fig-ure 7, J and K, respectively), with only scatteredCYP11A1- and 3�HSD-positive cells being localized inthe interstitial region. Additionally, qPCR experimentsconducted at PND47 (Figure 7N) showed that althoughthe expression of some markers of steroidogenic cells



(Cyp11a1 and Hsd3b6) did not differ from that in controltestes, others (Star and Hsd3b1) were significantly up-regulated in the double mutant testes (P � .01 and P � .05,respectively).

Intriguingly, Hsd3b6 expression has been associatedwith adult Leydig cells (44, 45; however, see Ref. 46).Because we observed premature (E18.5) up-regulation ofHsd3b6 as well as an increase in the steroidogenic cellpopulation in Sf1Cre; Gata4flox/flox Gata6flox/flox testes atPND4, we evaluated the possibility that adult Leydig cellsappear precociously in the double mutant testes. Normaladult Leydig cell function is dependent on pituitary LHand requires expression of the LH receptor, Lhr (40). Weexamined the expression of Lhr via qPCR and found it tobe down-regulated in the double mutant testes at bothPND4 and PND47 (P � .01 and P � .05, respectively).Additionally, similar to the embryonic points we evalu-ated, no CYP17A1-positive cells were observed in theSf1Cre; Gata4flox/flox Gata6flox/flox testes at PND4, withsuch cells only rarely being detected at PND30 (Figure 7,

F and L), and the expression of Hsd17b3 was significantlylower (P � .001) at PND4 and PND47 (Figure 7, M andN). Furthermore, testosterone synthesis was significantlyreduced in the double mutant testes, as assessed via ELISA(Figure 7O), and testosterone-responsive tissues, such asseminal vesicles and submaxillary glands, were severelyaffected in Sf1Cre; Gata4flox/flox Gata6flox/flox males (Sup-plemental Figure 5, C–E). In summary, these data suggestthat premature differentiation of adult Leydig cells inSf1Cre; Gata4flox/flox Gata6flox/flox animals is an unlikelyexplanation for the increased activity of the selected ste-roidogenic genes observed in the double mutant testes.

Overexpression of adrenal genes and clusters ofCYP21A2 cells in conditional double mutant testes

It has long been proposed that steroidogenic adreno-cortical and testis cells are derived from a common pro-genitor population of the adrenogonadal primordium (4,47, 48). However, it was only recently demonstrated thatthe fetal mouse testis harbors a limited number of cells that

Figure 6. Analysis of steroidogenic enzyme expression during embryogenesis in Sf1Cre; Gata4flox/flox Gata6flox/flox testes. Sections of control (A–Cand J–L) and Sf1Cre; Gata4flox/flox Gata6flox/flox (D–F and M–O) testes at E15.5 (A–F) and E18.5 (J–O) were stained for CYP11A1 (A, D, J, and M),3�HSD (B, E, K, and N), and CYP17A1 (C, F, L, and O). G–I, Higher magnifications of D–F, respectively. Note the reduced number of CYP11A1-positive (D and M), 3�HSD-positive (E and N), and CYP17A1-positive (F and O) cells in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at bothdevelopmental stages. Scale bars represent 100 �m (A–F and J–O) and 50 �m (G–I). P–R, qPCR analysis of changes in the expression of Star,Cyp11a1, Hsd3b1, Hsd3b6, Hsd17b3, and Insl3 in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at E13.5 (P), E15.5 (Q), and E18.5 (R). The results areplotted as the mean � SEM of the fold change relative to controls from at least n � 3 biological replicates for E13.5 and E15.5 and n � 4biological replicates for E18.5, with significance considered at *, P � .05; **, P � .01; and ***, P � .001.



express Cyp11b1 and Cyp21a1, which are genes that en-code enzymes required for corticosteroid synthesis (49,50). Interestingly, Sf1Cre; Gata4flox/flox Gata6flox/flox an-imals do not develop adrenal glands (S.G. Tevosian, E.Jiménez, H.M. Hatch, T. Jiang, D.A. Morse, S.C. Fox,M.B. Padua, manuscript submitted); however, males sur-vive and live normal lifespans, in contrast to their femalelittermates, which die shortly after birth (17).

We hypothesized that steroidogenic gene expression inthe testes of Sf1Cre; Gata4flox/flox Gata6flox/flox animalsstems from the expansion of their adrenal-like population.As early as PND4, we detected overexpression of the ad-renal genes Mc2r (P � .001), Cyp21a1 (P � .001),Cyp11b1 (P � .01), and Cyp11b2 (P � .01) in Sf1Cre;Gata4flox/flox Gata6flox/flox testes (Figure 8I), and the sametrend was observed at later stages (Figure 8J). Histologicalanalysis of the double mutant testis at PND17 revealed thepresence of clusters of hypertrophic cells localized in theinterstitial region, proximal to the coelomic epithelium(Figure 8F, arrowheads). CYP21A2, a key enzyme com-mon to the synthesis of the adrenocortical hormones cor-ticosterone and aldosterone, was similarly immunolocal-ized in the interstitial region at PND17 and PND30,within the cells clustered in the subepithelial zone (Figure8, G and H, arrows). We concluded that the steroidogenic

expression observed in the testes of the Sf1Cre; Gata4flox/flox

Gata6flox/flox animals is derived not from the fetal or adultLeydig cells but from the expanded adrenocortical-likepopulation.

Discussion

Previously, we and others demonstrated that the GATA4transcription factor is required for the normal develop-ment and function of the reproductive organs of bothsexes, ie, the testes (7, 8) and ovaries (16, 51). Now, weshow that the deletion of both GATA transcription factorsGATA4 and GATA6 within the somatic compartment ofthe testis reveals a synergistic function for these proteins intestis differentiation. Male of the Sf1Cre; Gata4flox/flox

Gata6flox/flox genotype develop small, nondescended tes-tes, with irregular testis cords, and only a low number ofgonocytes/spermatogonia are found at puberty. Not sur-prisingly, these conditional double mutant males aresterile.

Our data suggest that the reduction in the size of thedouble mutant testes is caused by an imbalance betweencell proliferation and cell death. Although the proportionof proliferating cells in embryonic Sf1Cre; Gata4flox/flox

Figure 7. Analysis of steroidogenic enzymes in the postnatal Sf1Cre; Gata4flox/flox Gata6flox/flox testis. Sections of control (A–C and J–L) andSf1Cre; Gata4flox/flox Gata6flox/flox (D–F and M–O) testes at PND4 (A–F) or PND30 (G–L) were stained for CYP11A1 (A, D, G, and J), 3�HSD (B, E, H,and K), and CYP17A1 (C, F, I, and L). Scale bars represent 200 �m (G–L) and 100 �m (A–F). M and N, qPCR analysis of changes in the expressionof Lhr, Star, Cyp11a1, Hsd3b1, Hsd3b6, and Hsd17b3 in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at PND4 (M) and PND47 (N). The bar graphsrepresent the mean � SEM of the fold change relative to controls from at least n � 3 biological replicates for both developmental stages, withsignificance considered at *, P � .05; **, P � .01; and ***, P � .001. O, Intratesticular testosterone concentrations (pg/mL) in controls (black bar)and Sf1Cre; Gata4flox/flox Gata6flox/flox (gray bar) animals at PND � 120. The bar graph shows the mean concentration adjusted per mg of testiculartissue � SEM from n � 3 biological replicates of each genotype. The data were analyzed using Student’s t test, with significance considered at***, P � .001.



Gata6flox/flox testes does not differ from that in controls, agreater number of apoptotic nuclei were detected in boththe somatic and germ cells of double mutant testes. Theprecocious death of gonocytes at E17.5 is likely to bethe main reason for the low number of spermatogonia inthe adult testes. It is possible that the survival of gonocytes/spermatogonia was negatively affected by the disorgani-zation of the testis cords in the mutants. Disorganizedtestis cords are known to disrupt the positioning and in-teraction of Sertoli cells and gonocytes/spermatogoniawithin them (52). In addition, there is experimental evi-dence suggesting the importance of Leydig cells in main-taining testis cord structure and ensuring germ cell sur-vival (29, 53–55). Sf1Cre; Gata4flox/flox Gata6flox/flox

testes are devoid of fetal and adult Leydig cells. It is pos-sible that in addition to the viability, the development ofthe germ cells in Sf1Cre; Gata4flox/flox Gata6flox/flox testesis also affected. However, we did not specifically assess thestatus of germ cell differentiation in the double mutanttestes beyond their ability to initiate G-H2AX expression.

The transcription factor SOX9 is a key regulator ofSertoli cell differentiation (reviewed in Refs. 1, 56). Thelevels of SOX9 were not affected in Sf1Cre; Gata4flox/flox

Gata6flox/flox testes. However, we observed abundant ap-optotic nuclei near the coelomic epithelium in the embry-onic double mutant testes. Sertoli cells differentiate fromprecursors derived from the coelomic epithelium (57) thatexpress the GATA4 protein (18). Sf1Cre-mediated loss ofGATA proteins may preferentially affect the viability ofthe transitional Sertoli cell progenitors.

Unlike SOX9, DMRT1 and GATA1 were stronglydown-regulated in the Sertoli cells of Sf1Cre; Gata4flox/flox

Gata6flox/flox testis. We previously showed that DMRT1 islost from the Sertoli cells of Sf1Cre; Gata4flox/flox testes butonly during embryogenesis (8). This pattern differs fromthat in the double mutant testis, where somatic DMRT1 isalso undetectable postnatally (eg, at PND47), suggestinga role for GATA6 and/or GATA1 in the postnatal expres-sion of Dmrt1. The GATA1 testis-specific promoter ele-ment contains a conserved GATA site (58), and it is likelythat GATA1 is a direct target of GATA4 and GATA6 inSertoli cells.

In contrast, AMH is highly up-regulated in the postna-tal Sf1Cre; Gata4flox/flox Gata6flox/flox testis. It has beenproposed that GATA proteins are required for the regu-lation of Amh expression (59). Here, we show that AMHis expressed in the Sertoli cells of the embryonic testis in theabsence of GATA4 and GATA6 and is ectopically ex-pressed in the adult testis in the absence of all 3 GATAproteins (GATA1, GATA4, and GATA6). We concludethat Amh gene expression does not require GATA func-tion in males.

Interestingly, previously described transgenic malemice overexpressing AMH (MT-hAMH) exhibit a lownumber of mature Leydig cells and significant reduction ofserum testosterone; hence, their virilization is incomplete(60, 61). These characteristics resemble the phenotype ofthe Sf1Cre; Gata4flox/flox Gata6flox/flox males, in which theexternal genitalia were underdeveloped (data not shown)and the concentration of intratesticular testosterone wasdramatically reduced. We also showed that the expressionof Hsd17b3, the enzyme responsible for testosterone syn-thesis, was significantly down-regulated. However, inMT-hAMH animals, Lhr expression is increased 5-fold,

Figure 8. Adrenocortical genes are overexpressed in Sf1Cre; Gata4flox/flox Gata6flox/flox testes as early as PND4. Representative sections of control(A–D) and Sf1Cre; Gata4flox/flox Gata6flox/flox (E–H) testes at PND17 (A–C and E–G) or PND30 (D and H) were stained with H&E (A and E) and forCYP21A2 (C, D, G, and H). B and F, Higher magnifications of A and E, respectively. The arrowheads in F indicate a cluster of hypertrophic cellslocalized in the interstitial region. Scale bars represent 200 �m (A, C–E, G, and H) or 100 �m (B and F). I–J, Quantitative changes in the expressionof the adrenal transcripts Mc2r, Cyp21a1, Cyp11b1, and Cyp11b2 in Sf1Cre; Gata4flox/flox Gata6flox/flox testes at PND4 (I) and PND47 (J). The resultsare graphed as the mean � SEM of the fold change relative to controls from at least n � 3 biological replicates, with significance considered at**, P � .01 and ***, P � .001.



some steroidogenic enzymes are down-regulated, and thediameter of the seminiferous tubules and spermatogenesisare normal (61). Thus, the MT-hAMH testicular pheno-type is distinctly different from that observed in theSf1Cre; Gata4flox/flox Gata6flox/flox testes. Hence, it ishighly unlikely that the up-regulation of AMH in theSf1Cre; Gata4flox/flox Gata6flox/flox testes is solely respon-sible for their phenotype.

In Sf1Cre; Gata4flox/flox Gata6flox/flox testes, 2 distinctpatterns of the expression of genes encoding steroidogenicenzymes could be distinguished: embryonic and postnatal,with both patterns differing from those in the controls. Inthe embryonic double mutant testes, a strong decline in theexpression of most steroidogenic enzymes is observed(with only Hsd3b6 being overexpressed). In contrast, thesame steroidogenic gene set is up-regulated in the Sf1Cre;Gata4flox/flox Gata6flox/flox testis right after birth. We ex-plored the possibility of precocious differentiation of adultLeydig cells in double mutant testes based on the earlyup-regulation of Hsd3b6, which is known to be expressedin adult, but not fetal Leydig cells (44, 45). This possibilitywas found to be inconsistent with the overall gene expres-sion pattern in the early postnatal testis of the double mu-tants. For example, LH is required for normal adult Leydigcell function, and the LH receptor is up-regulated in adultLeydig cells (40). However, Lhr was down-regulated inthe postnatal Sf1Cre; Gata4flox/flox Gata6flox/flox testes.Moreover, in addition to the expression of Hsd3b6 inadult Leydig cells, recent work revealed robust Hsd3b6expression in the adrenal glands (46). Yamamura et al alsoestablished that another Hsd3b isoform, Hsd3b1, is ex-pressed much more efficiently by adrenocortical cells (46)compared with the Leydig cells (45, 46). Hsd3b1 expres-sion was increased in the postnatal Sf1Cre; Gata4flox/flox

Gata6flox/flox testes. In addition, the Sf1Cre; Gata4flox/flox

Gata6flox/flox testes expressed common enzymes requiredfor the androgenic and glucocorticoid/mineralocorticoidpathways (Star, Cyp11a1 and Hsd3b6, and Hsd3b1),whereas the level of Hsd17b3, the gene encoding the keyenzyme for testosterone synthesis, was significantlyreduced.

In summary, these data suggest that the cellular clustersfound in Sf1Cre; Gata4flox/flox Gata6flox/flox testes ex-pressing steroidogenic enzymes are not Leydig cells, but anadrenocortical-like population. These clusters are likelyderived from the expansion of the rare adrenal-like cellspresent in the developing testis (49, 50). In the normaltestis, the significance of the presence of these cells is cur-rently unknown. It has been hypothesized that these cellsare merely misallocated to the testes during the separationof the adreno-gonadal primordia (49). However, a role for

these cells in normal testis development cannot beexcluded.

In contrast to the androgen synthesis pathway, which isnotably compromised in the Sf1Cre; Gata4flox/flox

Gata6flox/flox testis, the corticosteroid and mineralocorti-coid pathway is fully active in the testis of these animals,with overexpression of the adrenal enzymes Cyp21a1,Cyp11b1, Cyp11b2, and Mcr2 being observed. This is themost parsimonious explanation for the normal lifespan ofthe Sf1Cre; Gata4flox/flox Gata6flox/flox males, whereastheir female littermates all die within 2 weeks after birth(17). Intriguingly, human patients with congenital adrenalhyperplasia develop testicular adrenal rest tumors that ex-press the adrenal cortex-specific genes CYP11B1,CYP11B2, and MC2R (62). However, and distinct fromSf1Cre; Gata4flox/flox Gata6flox/flox testis, testicular adre-nal rest tumors also express RNA for HSD17B3 andINSL3 (62).

Recently, Pihlajoki et al (64) described Sf1Cre;Gata6flox/flox mice, in which the Gata6 gene was deletedusing an Sf1Cre line of mice generated previously (63),FVB-Tg(Nr5a1-cre)2Lowl/J) that differs from the Sf1Cremouse line used in this work (11). These animals had noobvious testicular phenotype but developed small adrenalglands with compromised steroidogenic adrenal function(64). Interestingly, the adrenal glands of the Sf1Cre;Gata6flox/flox mice expressed gonadal-like transcripts,such as Amhr2, Inha, Inhba, and Inhbb (64). Gonadec-tomy of Sf1Cre; Gata6flox/flox males led to an increase inthe adrenal expression of Amhr2, Lhcgr, and Cyp17 (64).Taken together, these data suggest a role for GATA4 andGATA6 in establishing and maintaining the characteristicsteroidogenic cell identities of gonads and adrenals.

Acknowledgments

Address all correspondence and requests for reprints to: Dr Ser-gei G. Tevosian, Department of Physiological Sciences, Collegeof Veterinary Medicine, University of Florida, Gainesville, FL,32610. E-mail: [email protected].

Present address for D.A.M.: Department of Applied Physiol-ogy and Kinesiology, College of Health and Human Perfor-mance, University of Florida, Gainesville, FL, 32611.

This work was supported by the National Institutes of HealthGrant HD042751.

Disclosure Summary: The authors have nothing to disclose.

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Combined Loss of the GATA4 and GATA6 Transcription Factors in Male Mice Disrupts Testicular Development and Confers Adrenal-Like Function in the Testes