Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis

in Reproduction

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

Abstract

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

Introduction

It is well established that gonadotropins participate in complex feedback loops required for spermatogenesis and folliculogenesis. Follicle-stimulating hormone (FSH) is important for the development of spermatogonia and steroidogenesis in Leydig cells. FSH also regulates the number of Sertoli and Leydig cells (Baker & O'Shaughnessy 2001, Baker et al. 2003). During folliculogenesis, granulosa cells respond differently to FSH depending on follicle size. In small preantral follicles, FSH induces granulosa cell proliferation. In larger follicles, however, FSH induces these cells to express the luteinizing hormone receptor and enzymes of the steroid biosynthesis pathway, such as P450 aromatase (Cyp19a1), P450 side chain cleavage enzyme (Cyp11a1), and STAR protein (Star) (Cortvrindt et al. 1997, Robker & Richards 1998, Spears et al. 1998, Kreeger et al. 2005, Kwintkiewicz et al. 2007). The mechanism(s) by which small follicle granulosa cells are responsive to only the mitogenic activity of FSH remain poorly understood. In preantral follicles, all granulosa cells have active Notch signaling (Johnson et al. 2001, Hahn et al. 2005, Vanorny et al. 2014), and thus, this pathway may mediate the proliferation response of granulosa cells to FSH in growing follicles by suppressing their differentiation and the expression of genes involved in steroidogenesis.

The evolutionarily conserved Notch signaling pathway has been shown to be required for follicle development and fertility. We, and others, have demonstrated that Notch2, Notch3, and the ligands Jagged1 (Jag1) and Jag2 are expressed dynamically in the cells of growing follicles (Johnson et al. 2001, Vorontchikhina et al. 2005, Vanorny et al. 2014). The downstream effector genes Hes1, Hes5, Hey1, Hey2, and HeyL overlap with Notch and its ligands in the granulosa cells (Johnson et al. 2001, Hahn et al. 2005). Several lines of evidence indicate that Notch signaling in the ovary is necessary for granulosa cell proliferation and normal follicle development. In mice deficient for lunatic fringe (Lfng), a modifier of the extracellular domain of the Notch receptors, Notch signaling was inhibited in granulosa cells, and these ovaries contained aberrant multi-oocyte follicles (MOFs) that did not undergo meiotic maturation properly (Hahn et al. 2005). In ex vivo ovary culture, the addition of γ-secretase inhibitors that block Notch signaling resulted in a loss of granulosa cell proliferation (Zhang et al. 2011). Notch2 is necessary for primordial follicle formation (Trombly et al. 2008) and Notch2 deficient ovaries had MOFs that demonstrated a lack of granulosa cell proliferation (Xu & Gridley 2013, Vanorny et al. 2014). A similar ovarian phenotype was found in mice with an oocyte-specific mutation of Jag1 (Vanorny et al. 2014).

Notch signaling pathway genes are also expressed in the developing testis and juvenile and adult Leydig cells (Dirami et al. 2001, von Schönfeldt et al. 2004, Lupien et al. 2006, Tang et al. 2008, Defalco et al. 2013). During gonadogenesis, Notch signaling regulates the differentiation of Leydig cells from progenitors (Tang et al. 2008). Inhibiting Notch signaling increased the number of Leydig cells, and conversely, expression of constitutively active Notch resulted in a loss of these cells pre- and postnatally (Tang et al. 2008, Defalco et al. 2013). Interestingly, gain- or loss-of-function Notch mutations resulted in both aberrant testis cord formation and loss of germ cells (Tang et al. 2008).

There are four Notch receptors (Notch1–4) in mammals and five ligands: Deltalike1 (Dll1), Dll3, Dll4, Jag1, and Jag2 (Kopan & Ilagan 2009). On ligand activation, the Notch intracellular domain (NOTCHICD) translocates to the nucleus and binds DNA in a complex with an obligate cofactor, RBPJK (Jarriault et al. 1995, Lu & Lux 1996, Ong et al. 2006), activating the transcription of its downstream target effectors, the HES and HEY proteins. The HEY and HES proteins are two families of basic helix–loop–helix Orange (bHLH-O) transcriptional repressors (Nakagawa et al. 2000, Iso et al. 2003, Kokubo et al. 2005). In this way, Notch signaling can both activate and repress gene transcription.

The HES and HEY proteins are able to repress transcription through several distinct mechanisms. They can form homodimers that bind to E (CANNTG) or N (CACNAG) boxes in target promoters and then recruit transcriptional repressor complexes (Sasai et al. 1992, Kageyama & Nakanishi 1997, Takata & Ishikawa 2003, Kokubo et al. 2005, Holderfield & Hughes 2008, Niwa et al. 2011). Alternatively, they can repress transcription through the formation of heterodimers with ubiquitously expressed class A bHLH factors, such as E12 and E47. The sequestration of these factors in nonfunctional heterodimers prevents the formation of heterodimers with tissue-specific bHLH activators, such as MYOD or PARAXIS (Leimeister et al. 2000, Iso et al. 2003). The HEY proteins also form complexes with non-bHLH transcription factors and repress their activity. For example, HEY1 binds to the androgen receptor (Belandia et al. 2005), and HEY2 binds to the aryl hydrocarbon receptor nuclear translocator (Chin et al. 2000). The HEY proteins have also been demonstrated to form complexes with GATA factors and repress expression of genes such as atrial naturietic factor, myosin heavy chain, and Müllerian inhibiting substance through GATA sites (Elagib et al. 2004, Kathiriya et al. 2004, Fischer et al. 2005, Ishiko et al. 2005, Martin et al. 2005, Shirvani et al. 2006, Xiang et al. 2006).

Transcription of genes in the steroid biosynthesis pathway is positively regulated in the gonads by steroidogenic factor 1 (referred to as SF1, gene symbol Nr5a1), a nuclear receptor and CREB/CREM (Ito et al. 1997, Gurates et al. 2003, Ragazzon et al. 2006, Schimmer & White 2010). In contrast, dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome X (NrOb1 gene symbol, referred to as Dax1) is a nuclear receptor that acts as a pan transcriptional inhibitor of steroid biosynthetic genes (Tremblay & Viger 2001a, Wang et al. 2001, Lalli & Sassone-Corsi 2003, Jo & Stocco 2004, Manna et al. 2009). Notch signaling could regulate steroid biosynthesis by acting on these regulators of steroidogenesis, either by inhibiting the transcription of SF1 or by up-regulating expression of Dax1.

GATA4, a zinc finger domain transcription factor, has a double zinc finger-binding domain that recognizes the GATA motif in promoters (review Tevosian (2014)). GATA4 is expressed in both Leydig and granulosa cells (Heikinheimo et al. 1997, Viger et al. 1998, 2004, Laitinen et al. 2000, Tremblay & Viger 2001a,b, Martin et al. 2005, Padua et al. 2014, 2015) and is a key positive transcriptional regulator of genes involved in steroid biosynthesis, including SF1, Cyp19a1, Star, 3B hydroxysteroid dehydrogenase (Hsd3b1), and Cyp11A1 (Wooten-Kee & Clark 2000, Tremblay & Viger 2001a,b, Tremblay et al. 2002, Bouchard et al. 2005, Martin et al. 2005, Bergeron et al. 2015, Schrade et al. 2015). Notch signaling has been demonstrated to inhibit GATA factor-mediated transactivation of cardiac, hematopoietic, and skeletal muscle specific promoters (Kathiriya et al. 2004, Fischer et al. 2005, Ishiko et al. 2005, Shirvani et al. 2006), indicating that the steroidogenic promoters could also be targets of the Notch pathway.

In the present study, we sought to examine the role of Notch and its HEY downstream effector proteins in the regulation of the expression of genes involved in gonadal steroidogenesis. Our data indicates that Notch signaling inhibits FSH-induced steroidogenic gene expression in small follicles. Also, activated Notch blocked the synthesis of progesterone in cultured MA-10 Leydig cells. A number of genes encoding important enzymes involved in the gonadal steroidogenesis pathway are positively regulated by GATA4 through direct transcriptional activation of the corresponding promoter sequences. We now show that GATA4-dependent activation of these promoters is also a target for Notch-mediated repression, further supporting a role for Notch signaling in the control of steroidogenesis.

Materials and methods

Cells

MA-10 cells, a Leydig tumor cell line (a kind gift from Dr Mario Ascoli, University of Iowa), were cultured in RPMI-1640 (Life Technologies) with 15% horse serum (GE Healthcare Hyclone, Logan, UT, USA), 20 mM HEPES (Sigma–Aldrich), 4 mM glutamine (GE Healthcare Hyclone), and 50 μg/ml gentamycin (Mediatech, Herndon, VA, USA) on gelatinized plates as described in Mizutani et al. (2006). NIH3T3 cells were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM (Life Technologies) with 10% newborn calf serum (Atlanta Biologicals, Flowery Branch, GA, USA) and Primocin (Invivogen, San Diego, CA, USA).

Plasmids

The GATA4 expression plasmid and promoter–luciferase constructs, mouse Star (−902 to +17), Cyp19a1 (−218 to +44), and human HSD3B2 (−224 to +53), have been described elsewhere (Tremblay & Viger 2001a,b, Tremblay et al. 2002, Martin et al. 2005). The pCS2+ -Notch1ICD and -Notch3ICD constructs were kind gifts of Dr Raphael Kopan (Washington University, St Louis, MO, USA). The mouse Notch2ICD (a kind gift of Dr Kathy Loomes, Children's Hospital of Philadelphia, Philadelphia, PA, USA) was cloned into the pCS2+ backbone. The Hey2 expression plasmid and deletion constructs Hey2-62 and Hey2-121 were cloned into the pCS2+ vector and confirmed by sequencing.

Animals

CD1 mice (Mus musculus) were bred and housed in a vivarium at Arizona State University (ASU) on a 10 h light:14 h darkness schedule with access to food and water ad libitum. ASU is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC). All procedures were carried out in compliance with the ASU Institutional Animal Care and Use Committee and AALAC under an approved research protocol.

Follicle culture

Ovaries from 14-day-old CD1 mice were harvested and follicles were mechanically isolated using a fine needle. Small preantral follicles that were ∼200 μm in diameter and had attached theca cells were chosen for culture, as described previously (Murray et al. 1998, Lenie et al. 2004). The follicles were cultured in α-MEM (Mediatech) with 5% FBS (Atlanta Biologicals), insulin, transferrin and selenium (ITS) (Sigma–Aldrich), 50 μg/ml ascorbic acid (Sigma–Aldrich), 0.3% BSA (Sigma–Aldrich), 2 mM glutamine (GE Healthcare HyClone), and penicillin/streptomycin (Mediatech) in the presence of 200 mIU/ml FSH (Sigma–Aldrich) under one of the following conditions: DMSO vehicle only, 25 or 50 μM N-(N-(3,5-difluorophenacetyl-l-alanyl))-(S)-phenylglycine t-butyl ester (DAPT), a Notch inhibitor (Sigma–Aldrich). Each treatment well received 20 follicles and each treatment was done in duplicate. After 48 h, total follicle RNA was purified for analysis by quantitative RT-PCR (qRT-PCR). The data are the results of three experiments.

Enzyme immunoassay progesterone assay

MA-10 cells were seeded at 1×105 cells/well and cultured as above. These cells were transfected using Fugene6 (Invitrogen) at a 6:2 ratio with a maximum of 2 μg of DNA. Empty vector was used to equalize total DNA concentration across all transfections. At 48 h post-transfection the cells were transferred into serum-free medium and treated with 1 mM dibutyryl cAMP (dbcAMP; Sigma–Aldrich) or vehicle for 6 h. The culture medium was collected for enzyme immunoassay (EIA). The concentration of progesterone in the collected medium was determined using an EIA assay per the manufacturer's protocol (Cayman Chemicals, Ann Arbor, MI, USA). Cells were then lysed and RNA was isolated for qRT-PCR.

Quantitative RT-PCR

MA-10 cells were cultured, transfected, and treated with dbcAMP, as described above. MA-10 cells or intact follicles were lysed in TRIzol (Invitrogen) for RNA isolation, per the manufacturer's protocols. For these studies, three biological replicate experiments were performed. RNA was treated with DNase I and quantified by NanoDrop prior to cDNA synthesis using SuperScriptIII reverse transcriptase (Invitrogen). For each sample, 1 μg of RNA was used for cDNA synthesis. The cDNA was quantified using transcript-specific, intron-spanning primers and real-time PCR with Syber Green (Eurogentec, Fremont, CA, USA) on an ABI 7900 HT thermocycler using a 384-well format in 10 μl reactions. Products from each primer set were sequenced and analyzed by BLAST (National Center for Biotechnology Information (NCBI)) to verify their identity. Primer efficiency was determined using a standard curve. For each transcript, three biological replicates were assayed in triplicate. All samples were normalized to the Gapdh transcript and relative gene expression was calculated using ΔΔCq analysis (Haimes & Kelley 2010). For follicle RNA, the control sample was follicles cultured with FSH and vehicle. For MA-10 cells, the control sample was cells cultured without any treatment. Primer sequences for mouse genes are as follows: Gapdh, 5′-GGGAAGCCCATCACCATCTT-3′ and 5′-GCCCTTCTCCATGGTGGTGAA-3′; Dax1, 5′-GCCCTTTTCCTGCTGAGATTC-3′ and 5′-TCACAGCTTTGCACAGAGCAT-3′; SF1, 5′-CGCAACAACCTTCTCATTGAGA-3′ and 5′-TGGATCCCTAATGCAAGGAGTCT-3′; Star, 5′-AAGCTGTGTGCTGGAAGCTCCTAT-3′ and 5′-TGCTTCCAGTTGAGAACCAAGCAG-3′; Hsd3b1 (mouse homolog of human HSD3B2), 5′-ACACAAGGAAGGAATTCTCCAAGCTG-3′ and 5′-CCTCCAATAGGTTCTGGGTACCTT-3′; Cyp19a1, 5′-CAGCAAGTCCTCAAGCATGTTCCA-3′ and 5′-TTCCACCATTCGAACAAGACCAGG-3′; Hes1, 5′-CAACACGACACCGGACAAACCAAA-3′ and 5′-TGGAATGCCGGGAGCTATCTTTCT-3′; and Notch2, 5′-TGCTGTGGCTCTGGCTGT-3′ and 5′-TGTGGTAGGTAACACAGGTCCCT-3′.

Luciferase assays

NIH3T3 cells were plated at a density of 8×104 cells/ml in 24-well plates and cultured as described above. Cells were transfected the next day with Lipofectamine and Plus reagent following the manufacturer's protocol (Invitrogen). Each transfected well received 0.05 μg of GATA4 expression vector, 0.2 μg of steroidogenic gene promoter–luciferase reporter construct, and 0.025 μg of plasmids expressing NotchICDs or Hey proteins. All transfections included 0.05 μg of pCMV–eGFP (Invitrogen). Total plasmid DNA was kept at 0.4 μg/well in all transfections with the addition of empty vector. All assays were done in triplicate on the same plate. The control sample for these assays was transfection of the reporter only. At 48 h post-transfection, cells were lysed in Luciferase Cell Culture Lysis Buffer (Promega). Luciferase activity was measured for each well by reacting 20 μl of cell lysate with 100 μl of Luciferase Assay Buffer (Promega) in 96-well plates, using an FLx800 microplate reader (Biotek Instruments, Winooski, VT, USA). All samples were normalized to GFP expression. All data are the mean±s.d. of three experiments done in triplicate, and statistical significance was determined using a one-way ANOVA.

Bioinformatics techniques

Steroid enzyme promoter–luciferase constructs were sequenced and analyzed for the presence of transcription factor binding sites using the TFsearch database (Heinemeyer et al. 1998), which can be found at http://www.cbrc.jp/cbrc-databases.

Statistical analyses

All data are the mean±s.d. of a minimum of triplicate replicates from three biological experiments. Statistical significance was determined using one-way ANOVA except for qRT-PCR that was analyzed using two-way ANOVA, P<0.05 for statistical significance at the 95% confidence limit.

Results

FSH induces both proliferation and expression of steroidogenic genes in granulosa cells depending on follicle size (Robker & Richards 1998, Kwintkiewicz et al. 2007). Notch2 and Notch3 are expressed in all granulosa cells of small growing follicles (Johnson et al. 2001), and Notch signaling induces proliferation of many cell types, including granulosa cells (Zhang et al. 2011, Vanorny et al. 2014), thus, it is possible that Notch acts downstream of FSH. To examine this, we cultured preantral follicles from 14-day-old CD1 mice in the presence or absence of a Notch inhibitor, DAPT, that blocks ligand-mediated activation of Notch and relieves Notch-mediated repression of gene expression (Fig. 1). Follicles were cultured in the presence of FSH under one of the following conditions: DMSO vehicle only or 25 or 50 μM DAPT. After 48 h, total follicle RNA was purified and gene expression was analyzed using qRT-PCR. Hes1, whose transcription is up-regulated by active Notch signaling, was significantly inhibited by DAPT indicating that Notch signaling was blocked (Fig. 1). Conversely, Notch2 expression was increased through a reciprocal signaling pathway, as expected (Vanorny et al. 2014). Genes encoding enzymes important for the synthesis of gonadal steroids – Star, Cyp19a1, and Hsd3b1 (Payne & Hales 2004) – were significantly up-regulated in follicles cultured in 25 or 50 μM DAPT (Fig. 1). These data indicate that Notch signaling inhibits FSH-induced expression of steroidogenic genes in granulosa cells of small preantral follicles.

Figure 1
Figure 1

Notch signaling inhibits expression of steroidogenic genes in granulosa cells from preantral follicles. (A) Cartoon describing the assay. Preantral follicles were isolated from 14-day-old CD1 mice and 20 follicles were cultured in the presence of FSH and 0, 25, or 50 μM DAPT to block Notch signaling for 48 h. Gene-specific qRT-PCR was done on total follicle RNA. Hes1 transcription is repressed and demonstrates that DAPT inhibited Notch signaling. Notch2 transcription was increased, as expected. Hsd3b1, Star, and Cyp19a1 demonstrated increased transcription when cultured with FSH and DAPT. Significance was determined by ANOVA, P<0.05, *indicates that the sample is statistically different from the mock sample and **indicates 25 and 50 μm DAPT samples are statistically different from each other. Data are the results of three experiments, each done in triplicate.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

Because Notch pathway genes are also expressed in Leydig cells, the effect of Notch signaling on steroidogenesis was examined in MA-10 cells, a well-characterized Leydig tumor cell line that synthesizes progesterone when stimulated by hormones or dbcAMP (Ascoli 1981). MA-10 cells were transfected with the constitutively active intracellular domains of Notch1, Notch2, or Notch3. At 48 h post-transfection the cells were either stimulated with dbcAMP or vehicle and the medium was assayed for progesterone by EIA. Treatment with dbcAMP induced a >30-fold increase in progesterone secretion in untransfected cells (Fig. 2). A combination of dbcAMP treatment and transfection of Notch1ICD or Notch2ICD resulted in a significant reduction of progesterone synthesis (*P<0.05; Fig. 2). Notch3ICD followed this trend but was not statistically significant. Therefore, Notch signaling can inhibit synthesis of progesterone in MA-10 Leydig cells.

Figure 2
Figure 2

Activated Notch inhibits progesterone secretion in MA-10 cells. MA-10 cells were transfected with a Notch1–3ICD or empty vector. At 48 h post-transfection, cells were stimulated with dbcAMP to induce steroidogenesis and progesterone was detected in cell supernatants by EIA. Cells transfected with empty vector and treated with dbcAMP secreted a significant amount of progesterone compared to empty vector transfected cells (**P<0.001). When transfected with Notch1ICD or Notch2ICD, progesterone secretion by dbcAM-treated cells was significantly inhibited, as compared to dbcAMP-treated cells that received empty vector (*P<0.05). Notch3ICD decreased progesterone secretion in the presence of dbcAMP, but not significantly. Transfection with Notch1–3ICD without dbcAMP treatment had no effect on progesterone synthesis. Data are the means±s.e.m. of three experiments done in triplicate.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

Since Dax1 and SF1 are central regulators of steroidogenesis, we next determined if their expression was activated or inhibited, respectively, by Notch signaling. MA-10 cells were transfected with plasmids expressing NOTCH1–3ICD and induced with dbcAMP for 6 h, and RNA was isolated for qRT-PCR. As shown in Fig. 3, Dax1 mRNA levels decrease in response to dbcAMP, consistent with the previously reported decrease in protein (Jo & Stocco 2004, Manna et al. 2009). Transfection with any activated Notch receptor did not block or attenuate this effect. Similarly, neither dbcAMP nor activated Notch receptors had any effect on the expression of SF1 mRNA (Fig. 3). These data suggested that Notch may regulate the promoters of specific genes necessary for steroid biosynthesis because progesterone synthesis was inhibited (Fig. 2).

Figure 3
Figure 3

Activated Notch does not inhibit Dax1 or SF1 transcription. MA-10 cells were transfected with plasmids expressing Notch1–3ICD or empty vector and cultured in the presence or absence of dbcAMP, and total RNA was harvested for qRT-PCR using gene specific primers. Dax1 transcripts are decreased by dbcAMP but not by activated Notch. SF1 transcription was not affected by either dbcAMP or activated Notch. Data presented are the relative expression of three experiments done in triplicate ±s.d. *P<0.05 as compared to untreated MA-10 cells.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

Whether Notch could mediate repression of specific steroid biosynthesis genes was tested using the well-characterized promoters of Star, HSD3B2, and Cyp19a1 PII, which are active in granulosa cells and Leydig cells. These promoters are synergistically activated by GATA4 and SF1 (Tremblay & Viger 2001a,b, Bouchard et al. 2005, Martin et al. 2005, Kwintkiewicz et al. 2007, Schrade et al. 2015, Bergeron et al. 2015). Interestingly, GATA4 activity is negatively regulated by HEY2 in the heart and developing cardiovascular system, making it a possible target for Notch signaling in gonadal cells also (Kathiriya et al. 2004, Kokubo et al. 2005). NIH3T3 cells were used in these studies to reduce background signals due to endogenous GATA4, SF1, and DAX1 activity. Cells that were transfected with a plasmid expressing Gata4 and a Star promoter–luciferase reporter gene demonstrated greater than sixfold up-regulation of luciferase activity over the reporter alone. This activity was significantly inhibited by co-transfection with NOTCH1ICD, NOTCH2ICD, or NOTCH3ICD (Fig. 4A). Similarly, both the −222 to +55 HSD3B2- and the −284 to −23 Cyp19a1 PII-luciferase reporters were up-regulated 2.2- and fourfold, respectively, by GATA4. NOTCH1ICD, NOTCH2ICD, and NOTCH3ICD significantly inhibited the ability of GATA4 to activate these promoters (Fig. 4B and C). These observations are consistent with the MA-10 cell data and indicate that activated Notch can repress the promoters of specific genes in this pathway.

Figure 4
Figure 4

Activated Notch represses GATA4-mediated transcription of steroidogenic enzyme gene promoters. (A, B and C) NIH3T3 cells were transfected with a specific luciferase reporter gene construct, an expression plasmid for GATA4, and a specific activated Notch receptor, as indicated. After 48 h in culture, cells were lysed and luciferase activity was determined. GATA4 binds to the promoter regions of the Star, HSD3B2, and Cyp19a1 reporter constructs, inducing transcription. The GATA4-induced luciferase activity of each gene was inhibited significantly by co-transfection of Notch1–3ICD (*P<0.05). All data are the results of three experiments done in triplicate ±s.d. Schematics above the graphs depict the reporter constructs.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

Direct transcriptional repression via Notch signaling is mediated by the HES and HEY bHLH-O repressor proteins binding to N and E boxes in the promoters of genes (Iso et al. 2003, Grogan et al. 2008, Heisig et al. 2012, Leal et al. 2012). Using TFSearch, an algorithm that searches DNA sequences for enhancer binding sites (Heinemeyer et al. 1998), the promoter regions of Star, HSD3B2, and Cyp19a1 PII were analyzed for N boxes (CACNAG consensus) and E boxes (CANNTG consensus) that could potentially bind HEY family members. The Cyp19a1 PII promoter has two E boxes (Fig. 5). The HSD3B2 promoter has a single N box, CACAAG, but no E boxes. Finally, the Star promoter has an N box and three E boxes (Fig. 5). The GATA sites have been confirmed as GATA4-binding sites that activate transcription of these genes in other studies (Tremblay & Viger 2001a,b, Bouchard et al. 2005, Martin et al. 2005).

Figure 5
Figure 5

TFSearch analysis of steroidogenic gene promoters. The sequence of the promoters in each luciferase reporter gene is presented. Each promoter was examined using TFsearch for the presence of N and E boxes. All of the GATA sites (blue highlight) have been confirmed in other studies. Cyp19a1 has two E boxes (green highlight), HSD3B2 has an N box (red highlight), and Star has three E boxes and an N box.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

To determine if the HEY bHLH-O repressors could inhibit GATA4, transcriptional activation of the steroidogenesis gene promoters was tested using a similar approach to the above discussion. As can be seen in Fig. 6A, GATA4 highly induced Star-luciferase activity when transfected into 3T3 cells, and this activity was significantly inhibited by co-transfection with any of the three HEY proteins. Similarly, the HSD3B2 reporter demonstrated a >11-fold increase in luciferase activity when co-transfected with GATA4 but was significantly repressed by HEY1 and HEYL only. HEY2 had no effect on the HSD3B2 promoter. Co-transfection of any of the three HEY proteins with the Cyp19a1 PII-luciferase reporter gene and GATA4 showed decreased activity, but only HEY1 inhibited this promoter significantly (Fig. 6C).

Figure 6
Figure 6

Hey1, Hey2, and Heyl can inhibit promoters of steroid biosynthesis genes. (A, B and C) NIH3T3 cells were transfected with a luciferase reporter gene construct along with expression plasmids for GATA4 and a HEY protein. Cell lysates were made 48 h post-transfection and luciferase activity determined. GATA4 activates each luciferase reporter to high levels and this is significantly inhibited with specific Hey genes (*P<0.05, over bar). HEY1, HEY2, and HEYL differentially inhibit GATA4 mediated up-regulation of transcription of Star, Cyp19a1, and HSD3B1. All data are the results of three experiments done in triplicate ±s.d.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

HEY proteins also can form complexes with the GATA factors and repress transcription. To determine if HEY2 represses the Star promoter directly by binding to the E boxes or acts through a complex with GATA4, 3T3 cells were transfected with the Star-luciferase reporter, plasmids expressing GATA4, and either full length HEY2 or one of two mutants (Fig. 7A). The first mutant, HEY2-62, lacks the N terminus, including the basic domain, but has an intact HLH domain. This mutant can form a dimer and interact with GATA4 but cannot bind DNA on its own (Kathiriya et al. 2004). The second mutant, HEY2-121, lacks both the basic and HLH domains; it can neither dimerize nor bind DNA (Fig. 7A). When co-transfected with GATA4 and the Star-luciferase reporter gene, neither HEY2-62 nor HEY2-121 could inhibit GATA4-induced transcription (Fig. 7B). These data indicated that Hey proteins likely regulate activation of steroidogenesis genes through specific sites in this promoter.

Figure 7
Figure 7

Hey proteins repress gene promoter activity through DNA binding. (A) The truncation mutants HEY2-62 (b) is missing the basic domain only, and HEY2-121 is missing both the basic and helix–loop–helix domains (bHLH). (B) NIH3T3 cells were transfected with the Star-luciferase reporter along with plasmids that express GATA4 and HEY2, HEY2-62, or HEY2-121. HEY2 significantly inhibited GATA4-induced luciferase activity. The loss of either the basic or bHLH domains of Hey2 resulted in a loss of repression of the Star-luciferase reporter gene (*P<0.05) and indicated that DNA binding was necessary for HEY2-mediated inhibition of this promoter. All data are the results of three experiments done in triplicate ±s.d.

Citation: REPRODUCTION 150, 4; 10.1530/REP-15-0226

Discussion

Follicle maturation and growth includes an increase in the size of the oocyte and the proliferation and differentiation of granulosa cells, leading to antrum formation and increased steroidogenesis. Growth of the follicle is regulated by the interplay of signals between the oocyte and the granulosa cells. A combination of oocyte factors, FSH, and estradiol are necessary for granulosa cell proliferation in preantral growing follicles (Moor et al. 1980, Robker & Richards 1998, Britt et al. 2000, Kawashima et al. 2008, Murray et al. 2008, West-Farrell et al. 2009). Notch signaling has been shown to be important for granulosa cell proliferation. Disruption of Notch signaling in conditional mutants of Notch2 and Jag1 in the ovary resulted in a lack of granulosa cell proliferation (Zhang et al. 2011, Vanorny et al. 2014). DAPT, a biomimetic inhibitor that is bound by the presenilin component of γ-secretase, has been used to disrupt Notch signaling in vitro. This inhibitor preferentially targets amyloid precursor protein and Notch processing by γ-secretase (Berezovska et al. 2000, Hadland et al. 2001, Yang et al. 2008). Using DAPT to inhibit Notch signaling in ex vivo ovary culture inhibited granulosa cell proliferation and folliculogenesis was halted (Zhang et al. 2011, Manosalva et al. 2013). In our studies, DAPT treatment resulted in the up-regulation of steroid biosynthesis genes in small follicles (Fig. 1).

Based on our data, altered Notch signaling disrupts the normal process of steroid biosynthesis during folliculogenesis, and this is consistent with the defective folliculogenesis noted in Lfng−/− and conditional mutants of Notch2, Jag1, and Hes1 (Hahn et al. 2005, Manosalva et al. 2013, Xu & Gridley 2013, Vanorny et al. 2014). The data presented here demonstrates that Notch signaling has a role in the complex regulation of the expression of enzymes of the steroid biosynthesis pathway and likely regulates FSH-induced proliferation of granulosa cells (Fig. 1). This is consistent with the role of Notch signaling in other tissues, promotion of proliferation, and regulation of the timing of the fully differentiated state. We propose that the differential response to FSH, proliferation in small preantral follicles, and increased steroidogenesis in larger preantral and antral follicles is regulated, in part, by Notch signaling.

In gonadal cells DAX1, a repressor, and the activator, SF1, regulate expression of multiple genes involved in steroidogenesis, including Cyp19a1 PII, Star, Cyp11A1, and HSD3B2 (Tremblay & Viger 2001a, Wang et al. 2001, Lalli & Sassone-Corsi 2003, Jo & Stocco 2004, Manna et al. 2009). Our studies indicated that activated Notch receptors can inhibit steroid synthesis in cAMP-activated MA-10 Leydig cells and in granulosa cells of preantral follicles (Figs 1 and 2), thus it was possible that expression of either factor might be regulated by Notch. For example, Dax1 transcription could be up-regulated by the NotchICD/RBPJK complex, blocking activation of SF1 and steroidogenesis. Alternatively, SF1 transcription could be inhibited by the HES or HEY repressors, also blocking steroidogenesis. Treatment of MA-10 cells with dbcAMP results in a decrease in DAX1 protein levels, increasing the transcriptional activity of SF1 (Wang et al. 2002, Rao et al. 2003, Jo & Stocco 2004, Trbovich et al. 2004, Manna et al. 2009). Our observations indicated that Notch-mediated inhibition of steroid biosynthesis was not achieved by modulating the expression of these two major transcriptional regulators (Fig. 3) but rather by repressing expression of genes that encode important nodal points in gonadal steroid biosynthesis.

Steroidogenesis genes have well-studied promoters that are transactivated by GATA4 (Tremblay & Viger 2001a,b, Tremblay et al. 2002, Viger et al. 2004, 2008, Bergeron et al. 2015, Schrade et al. 2015). All three activated Notch receptors significantly inhibited HSD3B2, Star, and Cyp19a1 promoter activity (Fig. 4). This observation is consistent with work by others that showed that the transactivation ability of GATA4 can be inhibited by Notch through HEY2 (Kathiriya et al. 2004). We determined that the Cyp19a1 PII, HSD3B2, and Star promoters had E and N boxes that HEY repressors could bind (Fig. 5), and subsequent experiments demonstrated that there was a differential inhibition of the promoters. For example, the Star promoter was significantly inhibited by HEY1, HEY2, and HEYL, but the HSD3B2 promoter, which contains a single N box, was only inhibited by HEY1 and HEYL (Fig. 6). This is consistent with previous data that demonstrated that HEY2 does not bind to N boxes with the sequence CACAAG (Nakagawa et al. 2000). Consistently, the Cyp19a1 promoter was not inhibited by HEY2 or HEYL and the 5′ most E box had a sequence that was not bound by HEY2 in previous studies (Nakagawa et al. 2000). The second E box found in this promoter is very similar in sequence and thus is likely a poor target for the HEY proteins also. These data indicate that direct DNA binding to specific sites in the promoters of these genes by the HEY proteins is likely the mechanism for Notch-mediated regulation of steroidogenesis gene transcription. We further ruled out the likelihood of the HEY repressors acting through complex formation with GATA4 by using mutants of Hey2 (Fig. 7). Our data demonstrated that GATA4-induced luciferase activity was repressed only by full-length HEY2 and not by a mutant that could form a complex with GATA4 but not bind DNA. In previous studies, there were no N or E boxes in the promoters examined, so this might indicate that in the absence of direct binding, the HEY repressors will act through complex formation with GATA factors (Kathiriya et al. 2004, Shirvani et al. 2006).

Determining that the genes necessary for steroid biosynthesis in developing follicles and Leydig cells are Notch targets is a novel observation with important implications. Notch signaling is important for the development of the male reproductive tract, and Leydig cells particularly (Dirami et al. 2001, Tang et al. 2008, Hahn et al. 2009, Defalco et al. 2013). Perturbation of Notch signaling in granulosa cells results in MOFs, defective meiotic maturation, and loss of oocytes (Hahn et al. 2005, Trombly et al. 2008, Manosalva et al. 2013, Xu & Gridley 2013, Vanorny et al. 2014). Because Notch2, Notch3, and Jag2 are not expressed in the oocyte, it is logical to conclude that these defects arise because altered Notch signaling disrupted the reciprocal communication between the oocyte and granulosa cells. Also, GATA factors have important roles in gonad development and the function and regulation of steroid biosynthetic genes (Viger et al. 2008, Padua et al. 2014, 2015, Tevosian 2014, Bergeron et al. 2015, Schrade et al. 2015). Conditional alleles of Gata4 resulted in impaired fertility in both adult female and male mice, Gata4−/− ovaries ovulated fewer oocytes and developed ovarian cysts, and there was decreased Cyp19a1 expression (Kyrönlahti et al. 2011a). When Gata4 and Gata6 are simultaneously deleted in granulosa cells, there is a complete lack of proliferation and no follicular development in the adult (Padua et al. 2014). Knockdown or deletion of Gata4 in Leydig cells resulted in decreased expression of genes involved in cholesterol synthesis and transport and steroid biosynthesis (Bergeron et al. 2015, Schrade et al. 2015). The disruption of GATA4 function or conditional loss of Gata4 in Sertoli cells in adult males also resulted in a loss of fertility and repression of steroid biosynthesis genes (Kyrönlahti et al. 2011b, Schrade et al. 2014, Bergeron et al. 2015, Padua et al. 2015). HEY2 has been demonstrated to be an important factor in the regulation of GATA-mediated transactivation of cardiac specific genes (Kathiriya et al. 2004, Fischer et al. 2005), and our data implicates the HEY proteins in the regulation of steroidogenesis postnatally. Because Notch genes are expressed in adrenal glands (de Mendonca et al. 2014) and the developing pituitary (Goldberg et al. 2011), the possibility that alterations to the Notch pathway can affect steroidogenesis and potentially the hypothalamic–pituitary–gonadal axis will be an important future consideration.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was funded in part by NICHD grant HD39850-01 to J Wilson-Rawls.

Acknowledgements

The authors would like to thank Drs Raphael Kopan, Kathy Loomes, and Mario Ascoli for reagents.

References

  • AscoliM1981Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. Journal of Biological Chemistry256179183.

    • Search Google Scholar
    • Export Citation
  • BakerPJO'ShaughnessyPJ2001Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice. Reproduction122227234. (doi:10.1530/rep.0.1220227)

    • Search Google Scholar
    • Export Citation
  • BakerPJPakarinenPHuhtaniemeiITAbelMHCharltonHMKumarTRO'ShaughnessyPJ2003Failure of normal Leydig cell development in follicle-stimulating hormone (FSH) receptor-deficient mice, but not FSHβ-deficient mice: role for constitutive FSH receptor activity. Endocrinology144138145. (doi:10.1210/en.2002-220637)

    • Search Google Scholar
    • Export Citation
  • BelandiaBPowellSMGarcía-PedreroJMWalkerMMBevanCLParkerMG2005Hey1, a mediator of notch signaling, is an androgen receptor corepressor. Molecular and Cellular Biology2514251436. (doi:10.1128/MCB.25.4.1425-1436.2005)

    • Search Google Scholar
    • Export Citation
  • BerezovskaOJackCMcLeanPAsterJCHicksCXiaWWolfeMSWeinmasterGSelkoeDJHymanBT2000Rapid Notch1 nuclear translocation after ligand binding depends on presenilin-associated γ-secretase activity. Annals of the New York Academy of Sciences920223226. (doi:10.1111/j.1749-6632.2000.tb06926.x)

    • Search Google Scholar
    • Export Citation
  • BergeronFNadeauGVigerRS2015GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway. Reproduction149245257. (doi:10.1530/REP-14-0369)

    • Search Google Scholar
    • Export Citation
  • BouchardMFTaniguchiHVigerRS2005Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells. Endocrinology14649054916. (doi:10.1210/en.2005-0187)

    • Search Google Scholar
    • Export Citation
  • BrittKLDrummondAECoxVADysonMWrefordNGJonesMEESimpsonERFindlayJ2000An age-related ovarian phenotype in mice with targeted disruption of Cyp 19 (Aromatase) gene. Endocrinology14126142623. (doi:10.1210/endo.141.7.7578)

    • Search Google Scholar
    • Export Citation
  • ChinMTMaemuraKFukumotoSJainMKLayneMDWatanabeMHsiehCMLeeME2000Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system. Journal of Biological Chemistry27563816387. (doi:10.1074/jbc.275.9.6381)

    • Search Google Scholar
    • Export Citation
  • CortvrindtRSmitzJVan SteirteghemAC1997Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro. Human Reproduction12759768. (doi:10.1093/humrep/12.4.759)

    • Search Google Scholar
    • Export Citation
  • DefalcoTSaraswathulaABriotAIruela-ArispeMLCapelB2013Testosterone levels influence mouse fetal Leydig cell progenitors through Notch signaling. Biology of Reproduction8891. (doi:10.1095/biolreprod.112.106138)

    • Search Google Scholar
    • Export Citation
  • DiramiGRavindranathNAchiMVDymM2001Expression of Notch pathway components in spermatogonia and Sertoli cells of neonatal mice. Journal of Andrology22944952. (doi:10.1002/j1939-4640.2001.tb03434.x)

    • Search Google Scholar
    • Export Citation
  • ElagibKEXiaoMHussainiIMDelehantyLLPalmerLARackeFKBirrerMJShanmugasundaramGMcDevittMAGoldfarbAN2004Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2. Molecular and Cellular Biology2477797794. (doi:10.1128/MCB.24.17.7779-7794.2004)

    • Search Google Scholar
    • Export Citation
  • FischerAKlattigJKneitzBDiezHMaierMHoltmannBEnglertCGesslerM2005Hey basic helix–loop–helix transcription factors are repressors of GATA4 and GATA6 and restrict expression of the GATA target gene ANF in fetal hearts. Molecular and Cellular Biology2589608970. (doi:10.1128/MCB.25.20.8960-8970.2005)

    • Search Google Scholar
    • Export Citation
  • GoldbergLBAujlaPKRaetzmanLT2011Persistent expression of activated notch inhibits corticotrope and melanotrope differentiation and results in dysfunction of the HPA axis. Developmental Biology3582332. (doi:10.1016/j.ydbio.2011.07.004)

    • Search Google Scholar
    • Export Citation
  • GroganSPOleeTHiraokaKLotzMK2008Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site. Arthritis and Rheumatism5827542763. (doi:10.1002/art.23730)

    • Search Google Scholar
    • Export Citation
  • GuratesBAmsterdamATamuraMYangSZhouJFangZAminSSebastianSBulunSE2003WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells. Molecular and Cellular Endocrinology2086175. (doi:10.1016/S0303-7207(03)00198-9)

    • Search Google Scholar
    • Export Citation
  • HadlandBKManleyNRSuDLongmoreGDMooreCLWolfeMSSchroeterEHKopanR2001Gamma-secretase inhibitors repress thymocyte development. PNAS9874877491. (doi:10.1073/pnas.131202798)

    • Search Google Scholar
    • Export Citation
  • HahnKLJohnsonJBeresBJHowardSWilson-RawlsJ2005Lunatic fringe null female mice are infertile due to defects in meiotic maturation. Development132817828. (doi:10.1242/dev.01601)

    • Search Google Scholar
    • Export Citation
  • HahnKLBeresBRowtonMJSkinnerMKChangYRawlsAWilson-RawlsJ2009A deficiency of lunatic fringe is associated with cystic dilation of the rete testis. Reproduction1377993. (doi:10.1530/REP-08-0207)

    • Search Google Scholar
    • Export Citation
  • Haimes J & Kelley M 2010 Demonstration of a ΔΔCq calculation method to compute relative gene expression from qPCR data. ThermoScientific Tech Note 1–4

  • HeikinheimoMErmolaevaMBielinskaMRahmanNANaritaNHuhtaniemiITTapanainenJSWilsonDB1997Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary. Endocrinology13835053514. (doi:10.1210/endo.138.8.5350)

    • Search Google Scholar
    • Export Citation
  • HeinemeyerTWingenderEReuterIHermjakobHKelAEKelOIgnatievaEVAnankoEAPodkolodnayaOAKolpakovF1998Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Research26362367. (doi:10.1093/nar/26.1.362)

    • Search Google Scholar
    • Export Citation
  • HeisigJWeberDEnglbergerEWinklerAKneitzSSungW-KWolfEEilersMWeiC-LGesslerM2012Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors. PLoS Genetics8e1002728. (doi:10.1371/journal.pgen.1002728)

    • Search Google Scholar
    • Export Citation
  • HolderfieldMTHughesCCW2008Crosstalk between vascular endothelial growth factor, Notch, and transforming growth factor-in vascular morphogenesis. Circulation Research102637652. (doi:10.1161/CIRCRESAHA.107.167171)

    • Search Google Scholar
    • Export Citation
  • IshikoEMatsumuraIEzoeSGaleKIshikoJSatohYTanakaHShibayamaHMizukiMEraT2005Notch signals inhibit the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through the induction of HES1. Journal of Biological Chemistry28049294939. (doi:10.1074/jbc.M406788200)

    • Search Google Scholar
    • Export Citation
  • IsoTKedesLHamamoriY2003HES and HERP families: multiple effectors of the Notch signaling pathway. Journal of Cellular Physiology194237255. (doi:10.1002/jcp.10208)

    • Search Google Scholar
    • Export Citation
  • ItoMYuRJamesonJL1997DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Molecular and Cellular Biology1714761483.

    • Search Google Scholar
    • Export Citation
  • JarriaultSBrouCLogeatFSchroeterEHKopanRIsraëlA1995Signalling downstream of activated mammalian Notch. Nature377355358. (doi:10.1038/377355a0)

    • Search Google Scholar
    • Export Citation
  • JoYStoccoDM2004Regulation of steroidogenesis and steroidogenic acute regulatory protein in R2C cells by DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1). Endocrinology14556295637. (doi:10.1210/en.2004-0941)

    • Search Google Scholar
    • Export Citation
  • JohnsonJEspinozaTMcGaugheyRWRawlsAWilson-RawlsJ2001Notch pathway genes are expressed in mammalian ovarian follicles. Mechanisms of Development109355361. (doi:10.1016/S0925-4773(01)00523-8)

    • Search Google Scholar
    • Export Citation
  • KageyamaRNakanishiS1997Helix–loop–helix factors in growth and differentiation of the vertebrate nervous system. Current Opinion in Genetics & Development7659665. (doi:10.1016/S0959-437X(97)80014-7)

    • Search Google Scholar
    • Export Citation
  • KathiriyaISKingINMurakamiMNakagawaMAstleJMGardnerKAGerardRDOlsonENSrivastavaDNakagawaO2004Hairy-related transcription factors inhibit GATA-dependent cardiac gene expression through a signal-responsive mechanism. Journal of Biological Chemistry2795493754943. (doi:10.1074/jbc.M409879200)

    • Search Google Scholar
    • Export Citation
  • KawashimaIOkazakiTNomaNNishiboriMYamashitaYShimadaM2008Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro. Reproduction136921. (doi:10.1530/REP-08-0074)

    • Search Google Scholar
    • Export Citation
  • KokuboHMiyagawa-TomitaSJohnsonRL2005Hesr, a mediator of the Notch signaling, functions in heart and vessel development. Trends in Cardiovascular Medicine15190194. (doi:10.1016/j.tcm.2005.05.005)

    • Search Google Scholar
    • Export Citation
  • KopanRIlaganMXG2009The canonical Notch signaling pathway: unfolding the activation mechanism. Cell137216233. (doi:10.1016/j.cell.2009.03.045)

    • Search Google Scholar
    • Export Citation
  • KreegerPKFernandesNNWoodruffTKSheaLD2005Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose. Biology of Reproduction73942950. (doi:10.1095/biolreprod.105.042390)

    • Search Google Scholar
    • Export Citation
  • KwintkiewiczJCaiZStoccoC2007Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells. Molecular Endocrinology21933947. (doi:10.1210/me.2006-0446)

    • Search Google Scholar
    • Export Citation
  • KyrönlahtiAVetterMEulerRBielinskaMJayPYAnttonenMHeikinheimoMWilsonDB2011aGATA4 deficiency impairs ovarian function in adult mice. Biology of Reproduction8410331044. (doi:10.1095/biolreprod.110.086850)

    • Search Google Scholar
    • Export Citation
  • KyrönlahtiAEulerRBielinskaMSchoellerELMoleyKHToppariJHeikinheimoMWilsonDB2011bGATA4 regulates Sertoli cell function and fertility in adult male mice. Molecular and Cellular Endocrinology3338595. (doi:10.1016/j.mce.2010.12.019)

    • Search Google Scholar
    • Export Citation
  • LaitinenMAnttonenMKetolaI2000Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors. Journal of Clinical Endocrinology and Metabolism8534763483. (doi:10.1210/jcem.85.9.6828)

    • Search Google Scholar
    • Export Citation
  • LalliESassone-CorsiP2003DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation. Molecular Endocrinology1714451453. (doi:10.1210/me.2003-0159)

    • Search Google Scholar
    • Export Citation
  • LealMCSuraceEIHolgadoMPFerrariCCTarelliRPitossiFWisniewskiTCastañoEMMorelliL2012Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: implications for cellular Aβ metabolism. Biochimica et Biophysica Acta1823227235. (doi:10.1016/j.bbamcr.2011.09.014)

    • Search Google Scholar
    • Export Citation
  • LeimeisterCDaleKFischerAKlamtBHrabé de AngelisMRadtkeFMcGrewMJPourquiéOGesslerM2000Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Developmental Biology22791103. (doi:10.1006/dbio.2000.9884)

    • Search Google Scholar
    • Export Citation
  • LenieSCortvrindtRAdriaenssensTSmitzJ2004A reproducible two-step culture system for isolated primary mouse ovarian follicles as single functional units. Biology of Reproduction7117301738. (doi:10.1095/biolreprod.104.028415)

    • Search Google Scholar
    • Export Citation
  • LuFMLuxSE1996Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. PNAS9356635667. (doi:10.1073/pnas.93.11.5663)

    • Search Google Scholar
    • Export Citation
  • LupienMDiévartAMoralesCRHermoLCalvoEKayDGHuCJolicoeurP2006Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility. Developmental Biology300497511. (doi:10.1016/j.ydbio.2006.09.010)

    • Search Google Scholar
    • Export Citation
  • MannaPRDysonMTJoYStoccoDM2009Role of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 in protein kinase A- and protein kinase C-mediated regulation of the steroidogenic acute regulatory protein expression in mouse Leydig tumor cells: mechanism of action. Endocrinology150187199. (doi:10.1210/en.2008-0368)

    • Search Google Scholar
    • Export Citation
  • ManosalvaIGonzálezAKageyamaR2013Hes1 in the somatic cells of the murine ovary is necessary for oocyte survival and maturation. Developmental Biology375140151. (doi:10.1016/j.ydbio.2012.12.015)

    • Search Google Scholar
    • Export Citation
  • MartinLJTaniguchiHRobertNMSimardJTremblayJJVigerRS2005GATA factors and the nuclear receptors, steroidogenic factor 1/liver receptor homolog 1, are key mutual partners in the regulation of the human 3β-hydroxysteroid dehydrogenase type 2 promoter. Molecular Endocrinology1923582370. (doi:10.1210/me.2004-0257)

    • Search Google Scholar
    • Export Citation
  • de MendoncaPORCostaICLotfiCFP2014The involvement of Nek2 and Notch in the proliferation of rat adrenal cortex triggered by POMC-derived peptides. PLoS ONE9e108657. (doi:10.1371/journal.pone.0108657)

    • Search Google Scholar
    • Export Citation
  • MizutaniTShiraishiKWelshTAscoliM2006Activation of the lutropin/choriogonadotropin receptor in MA-10 cells leads to the tyrosine phosphorylation of focal adhesion kinase by a pathway that involves Src family kinases. Molecular Endocrinology20619630. (doi:10.1210/me.2005-0277)

    • Search Google Scholar
    • Export Citation
  • MoorRMPolgeCWilladsenSM1980Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. Journal of Embryology and Experimental Morphology56319335.

    • Search Google Scholar
    • Export Citation
  • MurrayAAGosdenRGAllisonVSpearsN1998Effect of androgens on the development of mouse follicles growing in vitro. Journal of Reproduction and Fertility1132733. (doi:10.1530/jrf.0.1130027)

    • Search Google Scholar
    • Export Citation
  • MurrayAASwalesAKESmithREMolinekMDHillierSGSpearsN2008Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids. Molecular Human Reproduction147583. (doi:10.1093/molehr/gam092)

    • Search Google Scholar
    • Export Citation
  • NakagawaOMcFaddenDGNakagawaMYanagisawaHHuTSrivastavaDOlsonEN2000Members of the HRT family of basic helix–loop–helix proteins act as transcriptional repressors downstream of Notch signaling. PNAS971365513660. (doi:10.1073/pnas.250485597)

    • Search Google Scholar
    • Export Citation
  • NiwaYShimojoHIsomuraAGonzálezAMiyachiHKageyamaR2011Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis. Genes and Development2511151120. (doi:10.1101/gad.2035311)

    • Search Google Scholar
    • Export Citation
  • OngC-TChengH-TChangL-WOhtsukaTKageyamaRStormoGDKopanR2006Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. Journal of Biological Chemistry28151065119. (doi:10.1074/jbc.M506108200)

    • Search Google Scholar
    • Export Citation
  • PaduaMBJiangTMorseDAFoxSCHatchHMTevosianSG2014Simultaneous gene deletion of Gata4 and Gata6 leads to early disrution of follicular development and germ cell loss in the murine ovary. Endocrinology91110. (doi:10.1210/en.2014-1907)

    • Search Google Scholar
    • Export Citation
  • PaduaMBJiangTMorseDAFoxSCHatchHMTevosianSG2015Combined loss of the GATA4 and GATA6 transcription factors in male mice disrupts testicular development and confers adrenal-like function in the testes. Endocrinology15618731886. (doi:10.1210/en.2014-1907)

    • Search Google Scholar
    • Export Citation
  • PayneAHHalesDB2004Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews25947970. (doi:10.1210/er.2003-0030)

    • Search Google Scholar
    • Export Citation
  • RagazzonBLefrançois-MartinezA-MValPSahut-BarnolaITournaireCChambonCGachancard-BouyaJ-LBegueR-JVeyssièreGMartinezA2006Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoid-producing adrenocortical cell lines derived from targeted tumorigenesis. Endocrinology14718051818. (doi:10.1210/en.2005-1279)

    • Search Google Scholar
    • Export Citation
  • RaoRMJoYLeers-SuchetaSBoseHSMillerWLAzharSStoccoDM2003Differential regulation of steroid hormone biosynthesis in R2C and MA-10 Leydig tumor cells: role of SR-B1-mediated selective cholesteryl ester transport. Biology of Reproduction68114121. (doi:10.1095/biolreprod.102.007518)

    • Search Google Scholar
    • Export Citation
  • RobkerRLRichardsJS1998Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biology of Reproduction59476482. (doi:10.1095/biolreprod59.3.476)

    • Search Google Scholar
    • Export Citation
  • SasaiYKageyamaRTagawaYShigemotoRNakanishiS1992Two mammalian helix–loop–helix factors structurally related to Drosophila hairy and enhancer of split. Genes and Development626202634. (doi:10.1101/gad.6.12b.2620)

    • Search Google Scholar
    • Export Citation
  • SchimmerBPWhitePC2010Minireview: Steroidogenic factor 1: its roles in differentiation, development, and disease. Molecular Endocrinology2413221337. (doi:10.1210/me.2009-0519)

    • Search Google Scholar
    • Export Citation
  • von SchönfeldtVWistubaJSchlattS2004Notch-1, c-kit and GFRα-1 are developmentally regulated markers for premeiotic germ cells. Cytogenetic and Genome Research105235239. (doi:10.1159/000078194)

    • Search Google Scholar
    • Export Citation
  • SchradeAKyrönlahtiAAkinrinadeOPihlajokiMHäkkinenMFischerSAlastaloT-PVelagapudiVTopparifJWilsonDB2015GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells. Endocrinology15618601872. (doi:10.1210/en.2014-1931)

    • Search Google Scholar
    • Export Citation
  • ShirvaniSXiangFKoibuchiNChinMT2006CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6. Biochemical and Biophysical Research Communications339151156. (doi:10.1016/j.bbrc.2005.10.190)

    • Search Google Scholar
    • Export Citation
  • SpearsNMurrayAAAllisonVBolandNIGosdenRG1998Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro. Journal of Reproduction and Fertility1131926. (doi:10.1530/jrf.0.1130019)

    • Search Google Scholar
    • Export Citation
  • TakataTIshikawaF2003Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression. Biochemical and Biophysical Research Communications301250257. (doi:10.1016/S0006-291X(02)03020-6)

    • Search Google Scholar
    • Export Citation
  • TangHBrennanJKarlJHamadaYRaetzmanLCapelB2008Notch signaling maintains Leydig progenitor cells in the mouse testis. Development13537453753. (doi:10.1242/dev.024786)

    • Search Google Scholar
    • Export Citation
  • TevosianSG2014Transgenic mouse models in the study of reproduction: insights into GATA protein function. Reproduction148R1R14. (doi:10.1530/REP-14-0086)

    • Search Google Scholar
    • Export Citation
  • TrbovichAMMartinelleNO'NeillFHPearsonEJDonahoePKSlussPMTeixeiraJ2004Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance. Journal of Steroid Biochemistry and Molecular Biology92199208. (doi:10.1016/j.jsbmb.2004.07.002)

    • Search Google Scholar
    • Export Citation
  • TremblayJJVigerRS2001aNuclear receptor Dax-1 represses the transcriptional cooperation between GATA-4 and SF-1 in Sertoli cells. Biology of Reproduction6411911199. (doi:10.1095/biolreprod64.4.1191)

    • Search Google Scholar
    • Export Citation
  • TremblayJJVigerRS2001bGATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements. Endocrinology142977986. (doi:10.1210/endo.142.3.7995)

    • Search Google Scholar
    • Export Citation
  • TremblayJJHamelFVigerRS2002Protein kinase A-dependent cooperation between GATA and CCAAT/enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity. Endocrinology14339353945. (doi:10.1210/en.2002-220413)

    • Search Google Scholar
    • Export Citation
  • TromblyDWoodruffTMayoK2008Suppression of Notch signaling in the neonatal mouse ovary decreases primordial follicle formation. Endocrinology15010141024. (doi:10.1210/en.2008-0213)

    • Search Google Scholar
    • Export Citation
  • VanornyDAPrasasyaRDChalpeAJKilenSMMayoKE2014Notch signaling regulates ovarian follicle formation and coordinates follicular growth. Molecular Endocrinology28499511. (doi:10.1210/me.2013-1288)

    • Search Google Scholar
    • Export Citation
  • VigerRSMertineitCTraslerJMNemerM1998Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter. Development12526652675.

    • Search Google Scholar
    • Export Citation
  • VigerRSTaniguchiHRobertNMTremblayJJ2004Role of the GATA family of transcription factors in andrology. Journal of Andrology25441452. (doi:10.1002/j.1939-4640.2004.tb02813.x)

    • Search Google Scholar
    • Export Citation
  • VigerRSGuittotSMAnttonenMWilsonDBHeikinheimoM2008Role of the GATA family of transcription factors in endocrine development, function, and disease. Molecular Endocrinology22781798. (doi:10.1210/me.2007-0513)

    • Search Google Scholar
    • Export Citation
  • VorontchikhinaMAZimmermannRCShawberCJTangHKitajewskiJ2005Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns5701709. (doi:10.1016/j.modgep.2005.02.001)

    • Search Google Scholar
    • Export Citation
  • WangZJJeffsBItoMAchermannJCYuRNHalesDBJamesonJL2001Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1. PNAS9879887993. (doi:10.1073/pnas.141543298)

    • Search Google Scholar
    • Export Citation
  • WangXJDysonMTMondilloCPatrignaniZPignataroOStoccoDM2002Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells. Molecular and Cellular Endocrinology1885563. (doi:10.1016/S0303-7207(01)00748-1)

    • Search Google Scholar
    • Export Citation
  • West-FarrellERXuMGombergMAChowYHWoodruffTKSheaLD2009The mouse follicle microenvironment regulates antrum formation and steroid production: alterations in gene expression profiles. Biology of Reproduction80432439. (doi:10.1095/biolreprod.108.071142)

    • Search Google Scholar
    • Export Citation
  • Wooten-KeeCRClarkBJ2000Steroidogenic factor-1 influences protein deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophopshate-responsive regions of the murine steroidogenic acute regulatory protein gene. Endocrinology14113451355. (doi:10.1210/endo.141.4.7412)

    • Search Google Scholar
    • Export Citation
  • XiangFSakataYCuiLYoungbloodJMNakagamiHLiaoJKLiaoRChinMT2006Transcription factor CHF1/Hey2 suppresses cardiac hypertrophy through an inhibitory interaction with GATA4. American Journal of Physiology. Heart and Circulatory Physiology290H1997H2006. (doi:10.1152/ajpheart.01106.2005)

    • Search Google Scholar
    • Export Citation
  • XuJGridleyT2013Notch2 is required in somatic cells for breakdown of ovarian germ-cell nests and formation of primordial follicles. BMC Biology1113. (doi:10.1186/1741-7007-11-13)

    • Search Google Scholar
    • Export Citation
  • YangTArslanovaDGuYAugelli-SzafranCWeimingX2008Quantification of γ-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein. Molecular Brain11528. (doi:10.1186/1756-6606-1-15)

    • Search Google Scholar
    • Export Citation
  • ZhangC-PYangJ-LZhangJLiLHuangLJiS-YHuZ-YGaoFLiuY-X2011Notch signaling is involved in ovarian follicle development by regulating granulosa cell proliferation. Endocrinology15224372447. (doi:10.1210/en.2010-1182)

    • Search Google Scholar
    • Export Citation

R M George is now at GE Healthcare Life Sciences, 7th Floor Brigade Metropolis, Whitefield, Bangalore 560048, India

K L Hahn is now at STEMCELL, Ste. 400, 570 West Seventh Avenue, Vancouver, British Columbia, Canada V5Z 1B3

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    Notch signaling inhibits expression of steroidogenic genes in granulosa cells from preantral follicles. (A) Cartoon describing the assay. Preantral follicles were isolated from 14-day-old CD1 mice and 20 follicles were cultured in the presence of FSH and 0, 25, or 50 μM DAPT to block Notch signaling for 48 h. Gene-specific qRT-PCR was done on total follicle RNA. Hes1 transcription is repressed and demonstrates that DAPT inhibited Notch signaling. Notch2 transcription was increased, as expected. Hsd3b1, Star, and Cyp19a1 demonstrated increased transcription when cultured with FSH and DAPT. Significance was determined by ANOVA, P<0.05, *indicates that the sample is statistically different from the mock sample and **indicates 25 and 50 μm DAPT samples are statistically different from each other. Data are the results of three experiments, each done in triplicate.

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    Activated Notch inhibits progesterone secretion in MA-10 cells. MA-10 cells were transfected with a Notch1–3ICD or empty vector. At 48 h post-transfection, cells were stimulated with dbcAMP to induce steroidogenesis and progesterone was detected in cell supernatants by EIA. Cells transfected with empty vector and treated with dbcAMP secreted a significant amount of progesterone compared to empty vector transfected cells (**P<0.001). When transfected with Notch1ICD or Notch2ICD, progesterone secretion by dbcAM-treated cells was significantly inhibited, as compared to dbcAMP-treated cells that received empty vector (*P<0.05). Notch3ICD decreased progesterone secretion in the presence of dbcAMP, but not significantly. Transfection with Notch1–3ICD without dbcAMP treatment had no effect on progesterone synthesis. Data are the means±s.e.m. of three experiments done in triplicate.

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    Activated Notch does not inhibit Dax1 or SF1 transcription. MA-10 cells were transfected with plasmids expressing Notch1–3ICD or empty vector and cultured in the presence or absence of dbcAMP, and total RNA was harvested for qRT-PCR using gene specific primers. Dax1 transcripts are decreased by dbcAMP but not by activated Notch. SF1 transcription was not affected by either dbcAMP or activated Notch. Data presented are the relative expression of three experiments done in triplicate ±s.d. *P<0.05 as compared to untreated MA-10 cells.

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    Activated Notch represses GATA4-mediated transcription of steroidogenic enzyme gene promoters. (A, B and C) NIH3T3 cells were transfected with a specific luciferase reporter gene construct, an expression plasmid for GATA4, and a specific activated Notch receptor, as indicated. After 48 h in culture, cells were lysed and luciferase activity was determined. GATA4 binds to the promoter regions of the Star, HSD3B2, and Cyp19a1 reporter constructs, inducing transcription. The GATA4-induced luciferase activity of each gene was inhibited significantly by co-transfection of Notch1–3ICD (*P<0.05). All data are the results of three experiments done in triplicate ±s.d. Schematics above the graphs depict the reporter constructs.

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    TFSearch analysis of steroidogenic gene promoters. The sequence of the promoters in each luciferase reporter gene is presented. Each promoter was examined using TFsearch for the presence of N and E boxes. All of the GATA sites (blue highlight) have been confirmed in other studies. Cyp19a1 has two E boxes (green highlight), HSD3B2 has an N box (red highlight), and Star has three E boxes and an N box.

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    Hey1, Hey2, and Heyl can inhibit promoters of steroid biosynthesis genes. (A, B and C) NIH3T3 cells were transfected with a luciferase reporter gene construct along with expression plasmids for GATA4 and a HEY protein. Cell lysates were made 48 h post-transfection and luciferase activity determined. GATA4 activates each luciferase reporter to high levels and this is significantly inhibited with specific Hey genes (*P<0.05, over bar). HEY1, HEY2, and HEYL differentially inhibit GATA4 mediated up-regulation of transcription of Star, Cyp19a1, and HSD3B1. All data are the results of three experiments done in triplicate ±s.d.

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    Hey proteins repress gene promoter activity through DNA binding. (A) The truncation mutants HEY2-62 (b) is missing the basic domain only, and HEY2-121 is missing both the basic and helix–loop–helix domains (bHLH). (B) NIH3T3 cells were transfected with the Star-luciferase reporter along with plasmids that express GATA4 and HEY2, HEY2-62, or HEY2-121. HEY2 significantly inhibited GATA4-induced luciferase activity. The loss of either the basic or bHLH domains of Hey2 resulted in a loss of repression of the Star-luciferase reporter gene (*P<0.05) and indicated that DNA binding was necessary for HEY2-mediated inhibition of this promoter. All data are the results of three experiments done in triplicate ±s.d.

  • AscoliM1981Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. Journal of Biological Chemistry256179183.

    • Search Google Scholar
    • Export Citation
  • BakerPJO'ShaughnessyPJ2001Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice. Reproduction122227234. (doi:10.1530/rep.0.1220227)

    • Search Google Scholar
    • Export Citation
  • BakerPJPakarinenPHuhtaniemeiITAbelMHCharltonHMKumarTRO'ShaughnessyPJ2003Failure of normal Leydig cell development in follicle-stimulating hormone (FSH) receptor-deficient mice, but not FSHβ-deficient mice: role for constitutive FSH receptor activity. Endocrinology144138145. (doi:10.1210/en.2002-220637)

    • Search Google Scholar
    • Export Citation
  • BelandiaBPowellSMGarcía-PedreroJMWalkerMMBevanCLParkerMG2005Hey1, a mediator of notch signaling, is an androgen receptor corepressor. Molecular and Cellular Biology2514251436. (doi:10.1128/MCB.25.4.1425-1436.2005)

    • Search Google Scholar
    • Export Citation
  • BerezovskaOJackCMcLeanPAsterJCHicksCXiaWWolfeMSWeinmasterGSelkoeDJHymanBT2000Rapid Notch1 nuclear translocation after ligand binding depends on presenilin-associated γ-secretase activity. Annals of the New York Academy of Sciences920223226. (doi:10.1111/j.1749-6632.2000.tb06926.x)

    • Search Google Scholar
    • Export Citation
  • BergeronFNadeauGVigerRS2015GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway. Reproduction149245257. (doi:10.1530/REP-14-0369)

    • Search Google Scholar
    • Export Citation
  • BouchardMFTaniguchiHVigerRS2005Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells. Endocrinology14649054916. (doi:10.1210/en.2005-0187)

    • Search Google Scholar
    • Export Citation
  • BrittKLDrummondAECoxVADysonMWrefordNGJonesMEESimpsonERFindlayJ2000An age-related ovarian phenotype in mice with targeted disruption of Cyp 19 (Aromatase) gene. Endocrinology14126142623. (doi:10.1210/endo.141.7.7578)

    • Search Google Scholar
    • Export Citation
  • ChinMTMaemuraKFukumotoSJainMKLayneMDWatanabeMHsiehCMLeeME2000Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system. Journal of Biological Chemistry27563816387. (doi:10.1074/jbc.275.9.6381)

    • Search Google Scholar
    • Export Citation
  • CortvrindtRSmitzJVan SteirteghemAC1997Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro. Human Reproduction12759768. (doi:10.1093/humrep/12.4.759)

    • Search Google Scholar
    • Export Citation
  • DefalcoTSaraswathulaABriotAIruela-ArispeMLCapelB2013Testosterone levels influence mouse fetal Leydig cell progenitors through Notch signaling. Biology of Reproduction8891. (doi:10.1095/biolreprod.112.106138)

    • Search Google Scholar
    • Export Citation
  • DiramiGRavindranathNAchiMVDymM2001Expression of Notch pathway components in spermatogonia and Sertoli cells of neonatal mice. Journal of Andrology22944952. (doi:10.1002/j1939-4640.2001.tb03434.x)

    • Search Google Scholar
    • Export Citation
  • ElagibKEXiaoMHussainiIMDelehantyLLPalmerLARackeFKBirrerMJShanmugasundaramGMcDevittMAGoldfarbAN2004Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2. Molecular and Cellular Biology2477797794. (doi:10.1128/MCB.24.17.7779-7794.2004)

    • Search Google Scholar
    • Export Citation
  • FischerAKlattigJKneitzBDiezHMaierMHoltmannBEnglertCGesslerM2005Hey basic helix–loop–helix transcription factors are repressors of GATA4 and GATA6 and restrict expression of the GATA target gene ANF in fetal hearts. Molecular and Cellular Biology2589608970. (doi:10.1128/MCB.25.20.8960-8970.2005)

    • Search Google Scholar
    • Export Citation
  • GoldbergLBAujlaPKRaetzmanLT2011Persistent expression of activated notch inhibits corticotrope and melanotrope differentiation and results in dysfunction of the HPA axis. Developmental Biology3582332. (doi:10.1016/j.ydbio.2011.07.004)

    • Search Google Scholar
    • Export Citation
  • GroganSPOleeTHiraokaKLotzMK2008Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site. Arthritis and Rheumatism5827542763. (doi:10.1002/art.23730)

    • Search Google Scholar
    • Export Citation
  • GuratesBAmsterdamATamuraMYangSZhouJFangZAminSSebastianSBulunSE2003WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells. Molecular and Cellular Endocrinology2086175. (doi:10.1016/S0303-7207(03)00198-9)

    • Search Google Scholar
    • Export Citation
  • HadlandBKManleyNRSuDLongmoreGDMooreCLWolfeMSSchroeterEHKopanR2001Gamma-secretase inhibitors repress thymocyte development. PNAS9874877491. (doi:10.1073/pnas.131202798)

    • Search Google Scholar
    • Export Citation
  • HahnKLJohnsonJBeresBJHowardSWilson-RawlsJ2005Lunatic fringe null female mice are infertile due to defects in meiotic maturation. Development132817828. (doi:10.1242/dev.01601)

    • Search Google Scholar
    • Export Citation
  • HahnKLBeresBRowtonMJSkinnerMKChangYRawlsAWilson-RawlsJ2009A deficiency of lunatic fringe is associated with cystic dilation of the rete testis. Reproduction1377993. (doi:10.1530/REP-08-0207)

    • Search Google Scholar
    • Export Citation
  • Haimes J & Kelley M 2010 Demonstration of a ΔΔCq calculation method to compute relative gene expression from qPCR data. ThermoScientific Tech Note 1–4

  • HeikinheimoMErmolaevaMBielinskaMRahmanNANaritaNHuhtaniemiITTapanainenJSWilsonDB1997Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary. Endocrinology13835053514. (doi:10.1210/endo.138.8.5350)

    • Search Google Scholar
    • Export Citation
  • HeinemeyerTWingenderEReuterIHermjakobHKelAEKelOIgnatievaEVAnankoEAPodkolodnayaOAKolpakovF1998Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Research26362367. (doi:10.1093/nar/26.1.362)

    • Search Google Scholar
    • Export Citation
  • HeisigJWeberDEnglbergerEWinklerAKneitzSSungW-KWolfEEilersMWeiC-LGesslerM2012Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors. PLoS Genetics8e1002728. (doi:10.1371/journal.pgen.1002728)

    • Search Google Scholar
    • Export Citation
  • HolderfieldMTHughesCCW2008Crosstalk between vascular endothelial growth factor, Notch, and transforming growth factor-in vascular morphogenesis. Circulation Research102637652. (doi:10.1161/CIRCRESAHA.107.167171)

    • Search Google Scholar
    • Export Citation
  • IshikoEMatsumuraIEzoeSGaleKIshikoJSatohYTanakaHShibayamaHMizukiMEraT2005Notch signals inhibit the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through the induction of HES1. Journal of Biological Chemistry28049294939. (doi:10.1074/jbc.M406788200)

    • Search Google Scholar
    • Export Citation
  • IsoTKedesLHamamoriY2003HES and HERP families: multiple effectors of the Notch signaling pathway. Journal of Cellular Physiology194237255. (doi:10.1002/jcp.10208)

    • Search Google Scholar
    • Export Citation
  • ItoMYuRJamesonJL1997DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Molecular and Cellular Biology1714761483.

    • Search Google Scholar
    • Export Citation
  • JarriaultSBrouCLogeatFSchroeterEHKopanRIsraëlA1995Signalling downstream of activated mammalian Notch. Nature377355358. (doi:10.1038/377355a0)

    • Search Google Scholar
    • Export Citation
  • JoYStoccoDM2004Regulation of steroidogenesis and steroidogenic acute regulatory protein in R2C cells by DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1). Endocrinology14556295637. (doi:10.1210/en.2004-0941)

    • Search Google Scholar
    • Export Citation
  • JohnsonJEspinozaTMcGaugheyRWRawlsAWilson-RawlsJ2001Notch pathway genes are expressed in mammalian ovarian follicles. Mechanisms of Development109355361. (doi:10.1016/S0925-4773(01)00523-8)

    • Search Google Scholar
    • Export Citation
  • KageyamaRNakanishiS1997Helix–loop–helix factors in growth and differentiation of the vertebrate nervous system. Current Opinion in Genetics & Development7659665. (doi:10.1016/S0959-437X(97)80014-7)

    • Search Google Scholar
    • Export Citation
  • KathiriyaISKingINMurakamiMNakagawaMAstleJMGardnerKAGerardRDOlsonENSrivastavaDNakagawaO2004Hairy-related transcription factors inhibit GATA-dependent cardiac gene expression through a signal-responsive mechanism. Journal of Biological Chemistry2795493754943. (doi:10.1074/jbc.M409879200)

    • Search Google Scholar
    • Export Citation
  • KawashimaIOkazakiTNomaNNishiboriMYamashitaYShimadaM2008Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro. Reproduction136921. (doi:10.1530/REP-08-0074)

    • Search Google Scholar
    • Export Citation
  • KokuboHMiyagawa-TomitaSJohnsonRL2005Hesr, a mediator of the Notch signaling, functions in heart and vessel development. Trends in Cardiovascular Medicine15190194. (doi:10.1016/j.tcm.2005.05.005)

    • Search Google Scholar
    • Export Citation
  • KopanRIlaganMXG2009The canonical Notch signaling pathway: unfolding the activation mechanism. Cell137216233. (doi:10.1016/j.cell.2009.03.045)

    • Search Google Scholar
    • Export Citation
  • KreegerPKFernandesNNWoodruffTKSheaLD2005Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose. Biology of Reproduction73942950. (doi:10.1095/biolreprod.105.042390)

    • Search Google Scholar
    • Export Citation
  • KwintkiewiczJCaiZStoccoC2007Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells. Molecular Endocrinology21933947. (doi:10.1210/me.2006-0446)

    • Search Google Scholar
    • Export Citation
  • KyrönlahtiAVetterMEulerRBielinskaMJayPYAnttonenMHeikinheimoMWilsonDB2011aGATA4 deficiency impairs ovarian function in adult mice. Biology of Reproduction8410331044. (doi:10.1095/biolreprod.110.086850)

    • Search Google Scholar
    • Export Citation
  • KyrönlahtiAEulerRBielinskaMSchoellerELMoleyKHToppariJHeikinheimoMWilsonDB2011bGATA4 regulates Sertoli cell function and fertility in adult male mice. Molecular and Cellular Endocrinology3338595. (doi:10.1016/j.mce.2010.12.019)

    • Search Google Scholar
    • Export Citation
  • LaitinenMAnttonenMKetolaI2000Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors. Journal of Clinical Endocrinology and Metabolism8534763483. (doi:10.1210/jcem.85.9.6828)

    • Search Google Scholar
    • Export Citation
  • LalliESassone-CorsiP2003DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation. Molecular Endocrinology1714451453. (doi:10.1210/me.2003-0159)

    • Search Google Scholar
    • Export Citation
  • LealMCSuraceEIHolgadoMPFerrariCCTarelliRPitossiFWisniewskiTCastañoEMMorelliL2012Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: implications for cellular Aβ metabolism. Biochimica et Biophysica Acta1823227235. (doi:10.1016/j.bbamcr.2011.09.014)

    • Search Google Scholar
    • Export Citation
  • LeimeisterCDaleKFischerAKlamtBHrabé de AngelisMRadtkeFMcGrewMJPourquiéOGesslerM2000Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Developmental Biology22791103. (doi:10.1006/dbio.2000.9884)

    • Search Google Scholar
    • Export Citation
  • LenieSCortvrindtRAdriaenssensTSmitzJ2004A reproducible two-step culture system for isolated primary mouse ovarian follicles as single functional units. Biology of Reproduction7117301738. (doi:10.1095/biolreprod.104.028415)

    • Search Google Scholar
    • Export Citation
  • LuFMLuxSE1996Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element. PNAS9356635667. (doi:10.1073/pnas.93.11.5663)

    • Search Google Scholar
    • Export Citation
  • LupienMDiévartAMoralesCRHermoLCalvoEKayDGHuCJolicoeurP2006Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility. Developmental Biology300497511. (doi:10.1016/j.ydbio.2006.09.010)

    • Search Google Scholar
    • Export Citation
  • MannaPRDysonMTJoYStoccoDM2009Role of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 in protein kinase A- and protein kinase C-mediated regulation of the steroidogenic acute regulatory protein expression in mouse Leydig tumor cells: mechanism of action. Endocrinology150187199. (doi:10.1210/en.2008-0368)

    • Search Google Scholar
    • Export Citation
  • ManosalvaIGonzálezAKageyamaR2013Hes1 in the somatic cells of the murine ovary is necessary for oocyte survival and maturation. Developmental Biology375140151. (doi:10.1016/j.ydbio.2012.12.015)

    • Search Google Scholar
    • Export Citation
  • MartinLJTaniguchiHRobertNMSimardJTremblayJJVigerRS2005GATA factors and the nuclear receptors, steroidogenic factor 1/liver receptor homolog 1, are key mutual partners in the regulation of the human 3β-hydroxysteroid dehydrogenase type 2 promoter. Molecular Endocrinology1923582370. (doi:10.1210/me.2004-0257)

    • Search Google Scholar
    • Export Citation
  • de MendoncaPORCostaICLotfiCFP2014The involvement of Nek2 and Notch in the proliferation of rat adrenal cortex triggered by POMC-derived peptides. PLoS ONE9e108657. (doi:10.1371/journal.pone.0108657)

    • Search Google Scholar
    • Export Citation
  • MizutaniTShiraishiKWelshTAscoliM2006Activation of the lutropin/choriogonadotropin receptor in MA-10 cells leads to the tyrosine phosphorylation of focal adhesion kinase by a pathway that involves Src family kinases. Molecular Endocrinology20619630. (doi:10.1210/me.2005-0277)

    • Search Google Scholar
    • Export Citation
  • MoorRMPolgeCWilladsenSM1980Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. Journal of Embryology and Experimental Morphology56319335.

    • Search Google Scholar
    • Export Citation
  • MurrayAAGosdenRGAllisonVSpearsN1998Effect of androgens on the development of mouse follicles growing in vitro. Journal of Reproduction and Fertility1132733. (doi:10.1530/jrf.0.1130027)

    • Search Google Scholar
    • Export Citation
  • MurrayAASwalesAKESmithREMolinekMDHillierSGSpearsN2008Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids. Molecular Human Reproduction147583. (doi:10.1093/molehr/gam092)

    • Search Google Scholar
    • Export Citation
  • NakagawaOMcFaddenDGNakagawaMYanagisawaHHuTSrivastavaDOlsonEN2000Members of the HRT family of basic helix–loop–helix proteins act as transcriptional repressors downstream of Notch signaling. PNAS971365513660. (doi:10.1073/pnas.250485597)

    • Search Google Scholar
    • Export Citation
  • NiwaYShimojoHIsomuraAGonzálezAMiyachiHKageyamaR2011Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis. Genes and Development2511151120. (doi:10.1101/gad.2035311)

    • Search Google Scholar
    • Export Citation
  • OngC-TChengH-TChangL-WOhtsukaTKageyamaRStormoGDKopanR2006Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. Journal of Biological Chemistry28151065119. (doi:10.1074/jbc.M506108200)

    • Search Google Scholar
    • Export Citation
  • PaduaMBJiangTMorseDAFoxSCHatchHMTevosianSG2014Simultaneous gene deletion of Gata4 and Gata6 leads to early disrution of follicular development and germ cell loss in the murine ovary. Endocrinology91110. (doi:10.1210/en.2014-1907)

    • Search Google Scholar
    • Export Citation
  • PaduaMBJiangTMorseDAFoxSCHatchHMTevosianSG2015Combined loss of the GATA4 and GATA6 transcription factors in male mice disrupts testicular development and confers adrenal-like function in the testes. Endocrinology15618731886. (doi:10.1210/en.2014-1907)

    • Search Google Scholar
    • Export Citation
  • PayneAHHalesDB2004Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews25947970. (doi:10.1210/er.2003-0030)

    • Search Google Scholar
    • Export Citation
  • RagazzonBLefrançois-MartinezA-MValPSahut-BarnolaITournaireCChambonCGachancard-BouyaJ-LBegueR-JVeyssièreGMartinezA2006Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoid-producing adrenocortical cell lines derived from targeted tumorigenesis. Endocrinology14718051818. (doi:10.1210/en.2005-1279)

    • Search Google Scholar
    • Export Citation
  • RaoRMJoYLeers-SuchetaSBoseHSMillerWLAzharSStoccoDM2003Differential regulation of steroid hormone biosynthesis in R2C and MA-10 Leydig tumor cells: role of SR-B1-mediated selective cholesteryl ester transport. Biology of Reproduction68114121. (doi:10.1095/biolreprod.102.007518)

    • Search Google Scholar
    • Export Citation
  • RobkerRLRichardsJS1998Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biology of Reproduction59476482. (doi:10.1095/biolreprod59.3.476)

    • Search Google Scholar
    • Export Citation
  • SasaiYKageyamaRTagawaYShigemotoRNakanishiS1992Two mammalian helix–loop–helix factors structurally related to Drosophila hairy and enhancer of split. Genes and Development626202634. (doi:10.1101/gad.6.12b.2620)

    • Search Google Scholar
    • Export Citation
  • SchimmerBPWhitePC2010Minireview: Steroidogenic factor 1: its roles in differentiation, development, and disease. Molecular Endocrinology2413221337. (doi:10.1210/me.2009-0519)

    • Search Google Scholar
    • Export Citation
  • von SchönfeldtVWistubaJSchlattS2004Notch-1, c-kit and GFRα-1 are developmentally regulated markers for premeiotic germ cells. Cytogenetic and Genome Research105235239. (doi:10.1159/000078194)

    • Search Google Scholar
    • Export Citation
  • SchradeAKyrönlahtiAAkinrinadeOPihlajokiMHäkkinenMFischerSAlastaloT-PVelagapudiVTopparifJWilsonDB2015GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells. Endocrinology15618601872. (doi:10.1210/en.2014-1931)

    • Search Google Scholar
    • Export Citation
  • ShirvaniSXiangFKoibuchiNChinMT2006CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6. Biochemical and Biophysical Research Communications339151156. (doi:10.1016/j.bbrc.2005.10.190)

    • Search Google Scholar
    • Export Citation
  • SpearsNMurrayAAAllisonVBolandNIGosdenRG1998Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro. Journal of Reproduction and Fertility1131926. (doi:10.1530/jrf.0.1130019)

    • Search Google Scholar
    • Export Citation
  • TakataTIshikawaF2003Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression. Biochemical and Biophysical Research Communications301250257. (doi:10.1016/S0006-291X(02)03020-6)

    • Search Google Scholar
    • Export Citation
  • TangHBrennanJKarlJHamadaYRaetzmanLCapelB2008Notch signaling maintains Leydig progenitor cells in the mouse testis. Development13537453753. (doi:10.1242/dev.024786)

    • Search Google Scholar
    • Export Citation
  • TevosianSG2014Transgenic mouse models in the study of reproduction: insights into GATA protein function. Reproduction148R1R14. (doi:10.1530/REP-14-0086)

    • Search Google Scholar
    • Export Citation
  • TrbovichAMMartinelleNO'NeillFHPearsonEJDonahoePKSlussPMTeixeiraJ2004Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance. Journal of Steroid Biochemistry and Molecular Biology92199208. (doi:10.1016/j.jsbmb.2004.07.002)

    • Search Google Scholar
    • Export Citation
  • TremblayJJVigerRS2001aNuclear receptor Dax-1 represses the transcriptional cooperation between GATA-4 and SF-1 in Sertoli cells. Biology of Reproduction6411911199. (doi:10.1095/biolreprod64.4.1191)

    • Search Google Scholar
    • Export Citation
  • TremblayJJVigerRS2001bGATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements. Endocrinology142977986. (doi:10.1210/endo.142.3.7995)

    • Search Google Scholar
    • Export Citation
  • TremblayJJHamelFVigerRS2002Protein kinase A-dependent cooperation between GATA and CCAAT/enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity. Endocrinology14339353945. (doi:10.1210/en.2002-220413)

    • Search Google Scholar
    • Export Citation
  • TromblyDWoodruffTMayoK2008Suppression of Notch signaling in the neonatal mouse ovary decreases primordial follicle formation. Endocrinology15010141024. (doi:10.1210/en.2008-0213)

    • Search Google Scholar
    • Export Citation
  • VanornyDAPrasasyaRDChalpeAJKilenSMMayoKE2014Notch signaling regulates ovarian follicle formation and coordinates follicular growth. Molecular Endocrinology28499511. (doi:10.1210/me.2013-1288)

    • Search Google Scholar
    • Export Citation
  • VigerRSMertineitCTraslerJMNemerM1998Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter. Development12526652675.

    • Search Google Scholar
    • Export Citation
  • VigerRSTaniguchiHRobertNMTremblayJJ2004Role of the GATA family of transcription factors in andrology. Journal of Andrology25441452. (doi:10.1002/j.1939-4640.2004.tb02813.x)

    • Search Google Scholar
    • Export Citation
  • VigerRSGuittotSMAnttonenMWilsonDBHeikinheimoM2008Role of the GATA family of transcription factors in endocrine development, function, and disease. Molecular Endocrinology22781798. (doi:10.1210/me.2007-0513)

    • Search Google Scholar
    • Export Citation
  • VorontchikhinaMAZimmermannRCShawberCJTangHKitajewskiJ2005Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns5701709. (doi:10.1016/j.modgep.2005.02.001)

    • Search Google Scholar
    • Export Citation
  • WangZJJeffsBItoMAchermannJCYuRNHalesDBJamesonJL2001Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1. PNAS9879887993. (doi:10.1073/pnas.141543298)

    • Search Google Scholar
    • Export Citation
  • WangXJDysonMTMondilloCPatrignaniZPignataroOStoccoDM2002Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells. Molecular and Cellular Endocrinology1885563. (doi:10.1016/S0303-7207(01)00748-1)

    • Search Google Scholar
    • Export Citation
  • West-FarrellERXuMGombergMAChowYHWoodruffTKSheaLD2009The mouse follicle microenvironment regulates antrum formation and steroid production: alterations in gene expression profiles. Biology of Reproduction80432439. (doi:10.1095/biolreprod.108.071142)

    • Search Google Scholar
    • Export Citation
  • Wooten-KeeCRClarkBJ2000Steroidogenic factor-1 influences protein deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophopshate-responsive regions of the murine steroidogenic acute regulatory protein gene. Endocrinology14113451355. (doi:10.1210/endo.141.4.7412)

    • Search Google Scholar
    • Export Citation
  • XiangFSakataYCuiLYoungbloodJMNakagamiHLiaoJKLiaoRChinMT2006Transcription factor CHF1/Hey2 suppresses cardiac hypertrophy through an inhibitory interaction with GATA4. American Journal of Physiology. Heart and Circulatory Physiology290H1997H2006. (doi:10.1152/ajpheart.01106.2005)

    • Search Google Scholar
    • Export Citation
  • XuJGridleyT2013Notch2 is required in somatic cells for breakdown of ovarian germ-cell nests and formation of primordial follicles. BMC Biology1113. (doi:10.1186/1741-7007-11-13)

    • Search Google Scholar
    • Export Citation
  • YangTArslanovaDGuYAugelli-SzafranCWeimingX2008Quantification of γ-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein. Molecular Brain11528. (doi:10.1186/1756-6606-1-15)

    • Search Google Scholar
    • Export Citation
  • ZhangC-PYangJ-LZhangJLiLHuangLJiS-YHuZ-YGaoFLiuY-X2011Notch signaling is involved in ovarian follicle development by regulating granulosa cell proliferation. Endocrinology15224372447. (doi:10.1210/en.2010-1182)

    • Search Google Scholar
    • Export Citation