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. 2010 Mar 25;24(6):1203–1217. doi: 10.1210/me.2009-0325

Periovulatory Expression of Hyaluronan and Proteoglycan Link Protein 1 (Hapln1) in the Rat Ovary: Hormonal Regulation and Potential Function

Jing Liu 1, Eun-Sil Park 1, Thomas E Curry Jr 1, Misung Jo 1
PMCID: PMC2875804  PMID: 20339004

Abstract

Periovulatory follicular matrix plays an important role in cumulus-oocyte complex (COC) expansion, ovulation, and luteal formation. Hyaluronan and proteoglycan link protein 1 (HAPLN1), a component of follicular matrix, was shown to enhance COC expansion in vitro. However, the regulatory mechanisms of periovulatory expression of Hapln1 and its role in periovulatory granulosa cells have not been elucidated. We first determined the periovulatory expression pattern of Hapln1 using pregnant mare serum gonadotropin/human chorionic gonadotropin (PMSG/hCG)-primed immature rat ovaries. Hapln1 expression was transiently induced both in intact ovaries and granulosa cells at 8 h and 12 h after hCG injection. This in vivo expression of Hapln1 was recapitulated by culturing preovulatory granulosa cells with hCG. The stimulatory effect of hCG was blocked by inhibition of protein kinase A, phosphatidylinositol-dependent kinase, p38 MAPK, epidermal growth factor signaling, and prostaglandin synthesis, revealing key mediators involved in LH-induced Hapln1 expression. In addition, knockdown of Runx1 and Runx2 expression by small interfering RNA or inhibition of RUNX activities by dominant-negative RUNX decreased hCG or agonist-induced Hapln1 expression. Chromatin immunoprecipitation assays verified the in vivo binding of RUNX1 and RUNX2 to the Hapln1 promoter in periovulatory granulosa cells. Luciferase reporter assays revealed that mutation of the RUNX binding sites completely obliterated the agonist-induced activity of the Hapln1 promoter. These data conclusively identified RUNX proteins as the crucial transcription regulators for LH-induced Hapln1 expression. Functionally, treatment with HAPLN1 increased the viability of cultured granulosa cells and decreased the number of the cells undergoing apoptosis, whereas knockdown of Hapln1 expression decreased granulosa cells viability. This novel finding indicates that HAPLN1 may promote periovulatory granulosa cell survival, which would facilitate their differentiation into luteal cells.

LH-dependent induction of Hapln1 expression is mediated through the activation of key ovulatory mediators and transcriptional regulators RUNX. HAPLN1 may promote periovulatory granulosa cell survival.

In response to the preovulatory gonadotropin surge, the cumulus-oocyte complex (COC) of periovulatory follicles expands by forming a hyaluronic acid (HA)-rich matrix surrounding the cumulus cells, which is crucial for successful ovulation and fertilization. To form the COC matrix, periovulatory granulosa and cumulus cells need to express and secrete these matrix proteins. These proteins then assemble into the matrix: HA deposits on the cumulus cell membranes, forms the structural backbone of the extracellular matrix (ECM), and interacts with several HA-binding proteins such as versican (1), inter-α-trypsin inhibitor (IαI) (2,3), TNFα-stimulated gene 6 (4), and pentraxin 3 (Ptx 3) (5,6). Mice containing a mutation in genes for several matrix components exhibited defective COC formation or organization, leading to compromised ovulation and/or fertilization (6,7,8,9).

HAPLN1, also named cartilage link protein 1 or link protein, was initially identified in the proteoglycan fraction extracted from bovine articular cartilage (10). It interacts with HA and the globular domains of proteoglycans, such as aggrecan (11), versican (12), and IαI (13), to form stable ternary complexes in a variety of ECM. HAPLN1 is essential for cartilage proteoglycan aggregate formation (14) and has a broad spectrum of biological functions, including chondrocyte differentiation (15) and cardiac development (16). The lack of Hapln1 in homozygous mice resulted in perinatal lethality, accompanied by severe chondrodysplasia (15) and cardiac malformation (16).

Hapln1 expression was detected in rat, mouse, and human ovaries (17,18,19). In rodent models, the immunoreactivity of HAPLN1 was increased in the mural granulosa and cumulus cell compartments of periovulatory follicles in vivo after hCG stimulation (17,18). In human granulosa-lutein cells, HAPLN1 protein levels were up-regulated in vitro by LH stimulation (19). The functional importance of HAPLN1 was assessed in the mouse COC matrix (20). Sun et al. (20) found that treatment with HAPLN1 peptide enhanced in vitro COC expansion via cross-linking HA-IαI complex on the surrounding cumulus cell matrix, suggesting its role as a stabilizer in the COC matrix.

In addition to its prominent role as an ECM stabilizer, Hapln1 is also found to be differentially expressed in cancer cells and associated with the growth and progression of malignant tumors (21,22,23). In particular, recent studies suggested the possible involvement of HAPLN1 in cell growth and survival. The mRNA levels of Hapln1 were down-regulated in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions that resulted in chondrocyte apoptosis (24). In human chondrosarcoma cells, Hapln1 expression was significantly reduced by IL-1α, which decreased cell proliferation (25). In the ovary, periovulatory granulosa cells become resistant to apoptosis, which is crucial for luteinization (26,27,28). Therefore, it is important to investigate whether HAPLN1 plays a role in granulosa cell survival in response to the LH surge.

Previously, Sun et al. (29) showed that FSH and IGF-I can increase HAPLN1 protein levels by activating phosphatidylinositol-dependent kinase (PI3K)/Akt signaling pathways in rat granulosa cell cultures, suggesting a potential role of HAPLN1 during follicle development. More recently, we found that knockdown of LH-induced RUNX1 by small interfering RNA (siRNA) resulted in reduced levels of Hapln1 mRNA in preovulatory rat granulosa cell cultures (30). RUNX1 belongs to RUNX family of transcription factors. In mammals, there are three Runx genes: Runx1, Runx2, and Runx3. The three RUNX proteins encoded from these genes share conserved DNA-binding domain referred to as Runt-homology domain, which recognizes the specific consensus DNA sequences (31). RUNX transcription factors play central roles in developmental control of cell proliferation and differentiation in various tissues (32). In the ovary, Runx1 and Runx2 expression was highly up-regulated in periovulatory granulosa cells after the LH surge (30,33). Moreover, the LH-induced RUNX1 and RUNX2 were found to be involved in the expression of several periovulatory genes (30,33). Therefore, it is conceivable that RUNX1 and/or RUNX2 are involved in transcriptional up-regulation of Hapln1 in periovulatory follicular cells.

Based on previous findings from our laboratory and others, we hypothesize that 1) the LH surge induces Hapln1 expression in periovulatory granulosa and cumulus cells; 2) the periovulatory Hapln1 expression is mediated by LH-induced downstream signaling molecules and transcription factors, including RUNX1 and/or RUNX2 in periovulatory follicular cells; and 3) HAPLN1 promotes the survival of periovulatory granulosa cells. To test these hypotheses, we first determined the expression profile of Hapln1 in periovulatory ovaries collected at various time points before and after hCG injection using pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature rats. After characterization of the Hapln1 expression pattern, we investigated the cellular/molecular mechanism(s) by which Hapln1 expression is regulated in periovulatory granulosa cells and COCs using both in vivo and in vitro experimental models. Last, we examined a potential role of HAPLN1 in periovulatory granulosa cell survival using an in vitro model.

Results

hCG-induced Hapln1 expression in whole ovaries and periovulatory granulosa cells

To determine the periovulatory expression pattern of Hapln1, levels of mRNA and protein were assessed using whole ovaries collected at different times after hCG administration by real-time PCR and Western blot analyses, respectively. Hapln1 mRNA levels were increased more than 100-fold at 8 h and 12 h after hCG and decreased by 24 h (Fig. 1A). HAPLN1 protein levels were increased at 8 h and remained elevated throughout the periovulatory period (Fig. 1B).

Figure 1.

Figure 1

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Up-regulation of Hapln1 expression by hCG in rat ovaries and granulosa cells in vivo. Periovulatory ovaries and granulosa cells were collected at 0, 4, 8, 12, or 24 h after hCG injection from PMSG-primed immature rats. The expression of Hapln1 mRNA (A and C) and HAPLN1 protein (B and D) were measured by real-time PCR and Western blot, respectively, as described in Materials and Methods. Levels of mRNA for Hapln1 were normalized to the L32 in each sample (mean ± sem; n = 3 independent samples). β-Actin was used as a loading control for Western blot. Bars with no common superscripts are significantly different (P < 0.05).

To determine the relative expression of Hapln1 in the granulosa cell compartment, granulosa cells were isolated from ovaries obtained at various times after hCG administration. The levels of Hapln1 mRNA began to increase at 4 h, peaked at 8 and 12 h, and then decreased by 24 h (Fig. 1C). High expression of HAPLN1 protein was detected at 8 h, and the expression remained elevated until 24 h after hCG treatment (Fig. 1D).

Regulation of Hapln1 expression in preovulatory granulosa cells in vitro

To determine whether the in vivo induction of Hapln1 expression can be mimicked in vitro by activating LH receptor, granulosa cells isolated from PMSG-primed immature rat ovaries (48 h after PMSG) were cultured in the absence or presence of a luteinizing dose of hCG (1 IU/ml). hCG stimulated Hapln1 expression in cultured granulosa cells in a manner similar to that seen in vivo: the peak expression was observed at 8 and 12 h after hCG (Fig. 2, A and B).

Figure 2.

Figure 2

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Regulation of Hapln1 expression by hCG and hCG-induced mediators in preovulatory granulosa cells in vitro. A and B, The expression of Hapln1 mRNA (A) and HAPLN1 protein (B) in granulosa cells obtained from preovulatory ovaries (48 h after PMSG) and cultured with medium alone (ctrl) or hCG (1 IU/ml) for 0, 4, 8, 12, or 24 h. C and D, The expression of Hapln1 mRNA (C) and HAPLN1 protein (D) in preovulatory granulosa cells cultured with FSK (10 μm) or hCG (1 IU/ml) in the presence of 30 min pretreatment with vehicle (0.1% DMSO) or H89 (20 μm, PKA inhibitor) for 8 h. E and F, The expression of Hapln1 mRNA (E) and HAPLN1 protein (F) in preovulatory granulosa cells cultured with or without hCG (1 IU/ml) in the presence of 30 min pretreatment with 0.1% DMSO, LY (25 μm, PI3K inhibitor), SB (20 μm, p38 MAPK inhibitor), PD (20 μm, MEK inhibitor), CHX (1 μg/ml, protein synthesis inhibitor), RU486 (RU; 1 μm, PGR antagonist), or NS (1 μm, PTGS2 inhibitor) for 8 h. G and H, The expression of Hapln1 mRNA (G) and HAPLN1 protein (H) in preovulatory granulosa cells cultured with hCG (1 IU/ml) or AREG (250 ng/ml) in the presence of 30 min pretreatment with vehicle (0.1% DMSO) or AG1478 (1 μm, EGF receptor inhibitor) for 8 h. Levels of mRNA for Hapln1 were normalized to the L32 in each sample (mean ± sem; n = 3 independent experiments). β-Actin was used as a loading control for Western blot. Bars with no common superscripts are significantly different (P < 0.05).

The protein kinase A (PKA) signaling pathway is known to be activated by hCG in preovulatory granulosa cells (30). To investigate whether PKA pathway is involved in the up-regulation of Hapln1 expression in response to hCG stimulation, preovulatory granulosa cells were cultured with hCG in the absence or presence of the PKA inhibitor (H89; 20 μm), or forskolin (FSK), which is an activator of adenylate cyclase. FSK mimicked the action of hCG to induce Hapln1 expression, whereas hCG-stimulated Hapln1 expression was inhibited by H89 (Fig. 2, C and D).

To further determine the signaling mediators involved in LH-induced Hapln1 expression, granulosa cells were cultured with hCG in the absence or presence of the PI3K inhibitor [LY294002 (LY), 25 μm], p38 MAPK inhibitor [SB2035850 (SB) 20 μm], and MAPK kinase (MEK) inhibitor [PD98059 (PD); 20 μm]. The PI3K and p38 MAPK inhibitors, but not the MEK inhibitor, fully blocked Hapln1 induction (Fig. 2, E and F), indicating that hCG-induced Hapln1 expression requires the activation of PI3K and p38 MAPK signaling molecules.

To further determine whether the hCG-induced increase in Hapln1 expression in periovulatory granulosa cells requires de novo protein synthesis, granulosa cells were incubated with hCG in the absence or presence of cyclohexamide (CHX, 1 μg/μl), an inhibitor of new protein synthesis. CHX completely blocked the hCG-induced increase in levels of Hapln1 mRNA and protein (Fig. 2, E and F), suggesting that hCG-induced de novo protein(s) are involved in Hapln1 expression in periovulatory granulosa cells.

It is well documented that the LH surge or hCG induces progesterone receptor (PGR) and prostaglandin-endoperoxide synthase 2 (PTGS2). These factors were shown to play essential roles in the ovulatory process by regulating the expression of periovulatory genes (34). To determine whether PGR and PTGS2 are involved in the induction of Hapln1 in response to hCG stimulation, preovulatory granulosa cells were treated with the PGR antagonist [RU486 (RU), 1 μm] or PTGS2 inhibitor [NS-398 (NS), 1 μm]. NS-398 inhibited hCG-induced Hapln1 expression, whereas RU486 had no effect on Hapln1 expression (Fig. 2, E and F).

Recent studies have demonstrated crucial roles for LH-induced epidermal growth factor (EGF)-related peptides (e.g. amphiregulin) in the periovulatory process (35). We tested whether the up-regulation of Hapln1 expression is mediated by the activation of LH-induced EGF signaling. We treated granulosa cells with AG1478, which blocks the activation of EGF receptor tyrosine kinase. The hCG-stimulated increase in Hapln1 expression was completely inhibited by AG1478, whereas amphiregulin stimulated Hapln1 expression (Fig. 2, G and H).

Reduction of Hapln1 mRNA expression by Runx1 and Runx2 siRNA as well as dominant-negative RUNX (DNRUNX) in preovulatory granulosa cell cultures

RUNX1 and RUNX2 belong to the RUNX family of nuclear transcription factors, are induced by the LH surge, and are known to regulate gene expression in periovulatory granulosa cells (30,33,36). Our previous work showed that Hapln1 mRNA levels were decreased in cultured granulosa cells treated with Runx1 siRNA, suggesting possible transcriptional regulation by RUNX proteins (30). To determine whether LH-induced RUNX1 and RUNX2 are involved in the expression of the Hapln1 gene, preovulatory granulosa cells were transfected with Runx1 or Runx2 siRNA and cultured in the presence of hCG for 8 h. Real-time PCR and Western blot data revealed that the hCG-stimulated expression of Hapln1 was decreased in Runx1 or Runx2 siRNA-treated cells (Fig. 3, A, B and E). These data indicated that the LH-induced expression of both Runx1 and Runx2 is important for the up-regulation of Hapln1 expression.

Figure 3.

Figure 3

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Reduction of Hapln1 mRNA expression by Runx1 and Runx2 siRNA and DNRUNX in preovulatory granulosa cell cultures. A and B, Granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) were transfected with scrambled siRNA, Runx1 or Runx2 siRNA, treated with hCG (1 IU/ml), and cultured for 8 h. C, Granulosa cells obtained from preovulatory ovaries (48 h after PMSG) were treated with hCG (1 IU/ml) or FSK (10 μm) plus PMA (20 nm) for 8 h. D, The cells were isolated from rat preovulatory ovaries (48 h after PMSG), infected with adenoviral GFP or Ad-DNRUNX at 10 multiplicity of infections for 24 h, and then treated with FSK plus PMA and cultured for 8 h. The expression of each gene was measured by real-time PCR. Levels of mRNA for Runx1, Runx2, and Hapln1 were normalized to the L32 in each sample (mean ± sem; n = 3 independent experiments). E, The expression of RUNX1, RUNX2, and HAPLN1 protein was measured by Western blot. β-Actin was used as a loading control for Western blot. Bars with no common superscripts are significantly different (P < 0.05). ctrl, Control.

To further clarify the role of RUNX proteins in hCG-induced Hapln1 expression, we developed adenoviral DNRUNX (Ad-DNRUNX), which competes with endogenous RUNX transcriptional factors for DNA binding and subsequently inhibits their transcriptional activities (36). Preovulatory granulosa cells were infected with either a control green fluorescent protein (GFP) adenovirus vector (Ad-GFP) or Ad-DNRUNX overnight and then cultured with FSK plus an activator of protein kinase C, phorbol 12 myristate 13-acetate (PMA). Because of LH receptor down-regulation during overnight incubation, respective PKA and protein kinase C activators were used in preovulatory granulosa cell cultures to mimic the action of an ovulatory dose of LH/hCG in inducing Runx1 and Runx2 expression (30,36). The results showed that FSK plus PMA treatment also imitated the action of hCG to stimulate the expression of Hapln1 in fresh preovulatory granulosa cells in vitro (Fig. 3C). Treatment with Ad-DNRUNX inhibited agonist-induced Hapln1 expression (Fig. 3, D and E).

Endogenous RUNX1 and RUNX2 bind to the Hapln1 promoter region in periovulatory granulosa cells

To further investigate the transcriptional regulation of Hapln1 expression, we studied the putative promoter region (0.4 kb upstream of the transcription start site) of the rat Hapln1 gene using a TFSEARCH program (http://www.cbrc.jp/research/db/TFSEARCH.html). The analysis revealed three consensus RUNX-binding sequences within 396 bp of the Hapln1 promoter region (Fig. 4A).

Figure 4.

Figure 4

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Endogenous RUNX1 and RUNX2 bind to the Hapln1 promoter region in periovulatory granulosa cells. A, The rat Hapln1 promoter nucleotide sequence was obtained from the National Center for Biotechnology Information genomic library (http://www.ncbi.nlm.nih.gov/). Putative transcription factor binding sites (boxed sequences) were predicted by a TFSEARCH program. Nucleotide sequences were numbered from transcription start site at +1. B, ChIP detection of RUNX1 and RUNX2 transcription factor binding to the rat Hapln1 promoter region. Periovulatory granulosa cells were isolated from PMSG/hCG-primed immature rats (0 h and 10 h after hCG), and chromatins were incubated with RUNX1 or RUNX2 antibody. IgG was used as an antibody control. The immunoprecipitated chromatins were analyzed by PCR using the primers (arrows) designed to amplify fragments spanning the RUNX motif in the Hapln1 promoter. The experiment was repeated at least three times with different granulosa cell samples.

To determine whether RUNX1 and RUNX2 specifically bind to these candidate sites in the Hapln1 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays using chromatin samples extracted from granulosa cells isolated from periovulatory ovaries obtained before (0 h) and at 10 h after hCG injection, a time point of high expression of RUNX proteins. PCR results revealed that chromatin fragments containing the RUNX-binding sequence were enriched in granulosa cell samples obtained at 10 h after hCG, but not in 0 h samples by immunoprecipitation with both RUNX1 and RUNX2 antibodies. Interestingly, the enrichment of proximal promoter region was much stronger than that of distal region (Fig. 4B). This result indicated that endogenous RUNX1 and RUNX2 proteins induced by hCG associate with the Hapln1 promoter in periovulatory granulosa cells, and RUNX binding appears to be more prominent in the proximal region containing two consensus RUNX-binding sequences.

Transactivation of Hapln1 promoter reporter constructs was reduced by mutation of RUNX- and CRE-binding sequences in preovulatory granulosa cell cultures

To investigate the role of RUNX proteins in the transcriptional up-regulation of Hapln1, we generated three rat Hapln1 promoter reporter constructs (−42/+35, −86/+35, and −148/+35 bp). These constructs were transfected to preovulatory granulosa cells and stimulated with FSK plus PMA for 8 h. FSK treatments increased the luciferase activities of three reporter constructs compared with that of control cultures. PMA alone had no effect on the transactivation of Hapln1 promoter constructs but significantly enhanced the action of FSK on activities of the −86/+35 and −148/+35 Hapln1 promoter (Fig. 5A). Noticeably, FSK plus PMA-stimulated transactivation of the −86/+35 and −148/+35 Hapln1 promoter constructs that contained RUNX-binding sites was significantly higher than that of the truncated −42/+35 promoter construct, suggesting that the region −148/−43 plays a key role in hormonal regulation of the Hapln1 promoter activity (Fig. 5A).

Figure 5.

Figure 5

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Transcriptional activity of Hapln1 promoter reporter constructs was reduced by mutation in RUNX- and CRE-binding sequences as well as Runx1 and Runx2 siRNA in preovulatory granulosa cell cultures. A, Granulosa cells isolated from preovulatory ovaries (48 h after PMSG) were transiently transfected with empty luciferase reporter vector (LUC), −42/+35 bp, −86/+35 bp, or −148/+35 bp Hapln1-luciferase reporter constructs, treated with FSK (10 μm), PMA (20 nm), or FSK plus PMA and cultured for 8 h. B, Preovulatory granulosa cells were transiently transfected with wild type (−148/+35 bp), mutant A, mutant B, mutant AB, or mutant C Hapln1-luciferase reporter constructs, treated with FSK plus PMA and cultured for 8 h. C, Preovulatory granulosa cells were transiently transfected with wild type (−148/+35 bp) Hapln1-luciferase reporter constructs in the presence of scrambled siRNA, Runx1 or Runx2 siRNA, treated with FSK plus PMA, and cultured for 8 h. Firefly luciferase activities were normalized by Renilla luciferase activities. Each experiment was performed in triplicate, and the experiment was repeated at least three times. Bars with no common superscripts are significantly different (P < 0.05).

Next, to determine the importance of consensus RUNX-binding sites within the −148/−43 bp region of the Hapln1 promoter, site-directed mutants were generated and transfected into cultured preovulatory granulosa cells in the presence of FSK plus PMA (Fig. 5B). The results indicated that mutation of either the −49/−44 RUNX binding site (mutant A) or −115/−110 RUNX binding site (mutant B) markedly decreased FSK plus PMA-induced activity of the Hapln1 promoter (Fig. 5B). The double mutation of both regions of consensus RUNX-binding sites (mutant AB) completely obliterated the agonist-induced Hapln1 promoter activity (Fig. 5B). Because FSK stimulated transcriptional activity of the −42/+35 region of the rat Hapln1 promoter, we investigated the importance of the cAMP response element (CRE) within the region of −25/−18 bp by generating an additional mutation of CRE (mutant C). The mutation of CRE resulted in a decrease in FSK plus PMA-induced transactivation of the Hapln1 promoter (Fig. 5B).

Next, we determined whether knockdown of Runx1 or Runx2 expression affects the transcriptional activity of the Hapln1 promoter. To accomplish this, the −148/+35 wild-type construct and Runx1- or Runx2-specific siRNA were cotransfected into cultured preovulatory granulosa cells stimulated with FSK plus PMA. The luciferase activity assay revealed that the agonist-stimulated transcriptional activity of the Hapln1 promoter reporter construct was reduced by 50% in Runx1 or Runx2 siRNA-treated granulosa cells compared with the cells with scrambled siRNA (Fig. 5C).

RUNX regulation on Hapln1 expression in preovulatory COC cultures

It was shown that HAPLN1 protein was secreted by cumulus cells of expanding COCs (17,18), but the regulatory mechanism of Hapln1 expression in cumulus cells remains to be determined. We investigated whether RUNX proteins are involved in the up-regulation of Hapln1 expression in expanding rat COCs. In our in vitro COC model, compact COCs obtained from preovulatory rat ovaries undergo full expansion by FSK plus amphiregulin (AREG) treatment, whereas AREG or FSK alone induced little to modest expansion at the concentration indicated, respectively (Supplemental Fig. 1 published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). As shown in Fig. 6, A and B, the levels of Hapln1 mRNA and protein were increased in COCs stimulated with FSK or AREG. However, the combinatorial treatment of FSK plus AREG had a synergistic effect on Hapln1 expression. We also found that FSK as well as AREG increased the levels of Runx1 and Runx2 mRNA over vehicle treatment (control). The stimulatory effect on these genes, however, was highest by FSK plus AREG treatment (Fig. 6C), which induced full expansion of COCs in vitro.

Figure 6.

Figure 6

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Evidence of RUNX involvement in Hapln1 expression in expanding COCs in vitro. COCs obtained from preovulatory ovaries (48 h after PMSG) were cultured with defined medium alone (control), FSK (10 μm), AREG (250 ng/ml) or FSK plus AREG for 12 h. The expression of mRNA for Hapln1, Runx1 and Runx2 (A and C), and HAPLN1 protein (B) were measured by real-time PCR and Western blot, respectively, as described in Materials and Methods. The expression of each gene was measured by real-time PCR. Levels of mRNA for Hapln1, Runx1, and Runx2 were normalized to the L32 in each sample (mean ± sem; n = 3 independent experiments). D, COCs isolated from rat preovulatory ovaries (48 h after PMSG) were transfected with empty luciferase reporter vector (LUC), wild-type (−148/+35 bp) and mutant AB Hapln1-luciferase reporter constructs, treated with FSK plus AREG and cultured for 10 h. Firefly luciferase activities were normalized by Renilla luciferase activities. Each experiment was performed in triplicate, and the experiment was repeated at least three times. Bars with no common superscripts are significantly different (P < 0.05).

To further investigate the transcriptional regulation of Hapln1 by RUNX proteins in cumulus cells, COCs isolated from preovulatory ovaries were transfected with −148/+35 wild-type or mutant AB constructs and then stimulated with FSK plus AREG. The luciferase assay showed that FSK plus AREG-induced transactivation of the Hapln1 promoter was reduced by the double mutation of RUNX-binding sites (Fig. 6D).

Effects of HAPLN1 on granulosa cell viability

To determine whether HAPLN1 affects the viability of granulosa cells in vitro, preovulatory granulosa cells were cultured with hCG (1 IU/ml) or the native peptide of HAPLN1 (0.01, 0.1, 0.5, 1, and 2 μg/ml) for 24 h. At the end of culture, the cells were subjected to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. The MTS assay measures the reductase activity of mitochondria and is widely used as a measure of cell viability (37). After 24 h of culture, there was a higher viability in cells treated with hCG compared with that of control. HAPLN1 treatment also increased cell viability in a dose-dependent manner (Fig. 7A).

Figure 7.

Figure 7

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Effects of HAPLN1 on granulosa cell viability. A, Preovulatory granulosa cells were cultured with vehicle (0.02% BSA), hCG (1 IU/ml), or HAPLN1 (0.01, 0.1, 0.5, 1, or 2 μg/ml) for 24 h. Granulosa cell viability was measured using a MTS kit (mean ± sem; n = 3 independent culture experiments). B, Preovulatory granulosa cells were cultured with vehicle (0.02% BSA), hCG (1 IU/ml), or HAPLN1 (2 μg/ml) for 24 h, and cell cytotoxicity was measured using a CytoTox-ONE Kit (mean ± sem; n = 3 independent culture experiments). C, Preovulatory granulosa cells were transfected with scrambled siRNA or Hapln1 siRNA, treated with hCG (1 IU/ml), and cultured for 8 h. The expression of Hapln1 mRNA was measured by real-time PCR. D, Preovulatory granulosa cells were cultured with hCG (1 IU/ml) in the presence of Hapln1 siRNA for 24 h, and cell viability was measured using a MTS kit (mean ± sem; n = 3 independent culture experiments). E, Preovulatory granulosa cells were cultured with vehicle (0.02% BSA), hCG (1 IU/ml), or HAPLN1 (2 μg/ml) for 24 h, and the concentration of progesterone in the culture medium was measured using an Immulite kit (mean ± sem; n = 3 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

To further clarify the effects of HAPLN1 on the increase in cell viability, cytotoxicity of cultured granulosa cells was determined using a CytoTox-ONE assay kit (Promega). This kit utilizes a fluorescent measure of the release of lactate dehydrogenase from nonviable cells with a damaged membrane. hCG treatment decreased the percent of dead granulosa cells compared with the control cultures. HAPLN1 treatment mimicked the effects of hCG on the decrease in granulosa cell cytotoxicity (Fig. 7B).

Next, we determined whether knockdown of endogenous Hapln1 expression affects the viability of granulosa cells. As shown in Fig. 7C, Hapln1-specific siRNA effectively attenuated hCG-induced expression of Hapln1 mRNA compared with that of hCG plus scrambled siRNA-treated cells. The MTS assay data showed that Hapln1 siRNA treatment decreased the viability of granulosa cells stimulated with hCG (Fig. 7D).

Due to the ability of progesterone to prevent granulosa cell apoptosis (38,39,40), we investigated whether HAPLN1 affects progesterone production in granulosa cells. As expected, hCG treatment resulted in progesterone accumulation in the conditioned culture media. However, HAPLN1 treatment had no effect on progesterone production (Fig. 7E).

Discussion

Proper ECM formation surrounding COCs and granulosa cell compartment in a periovulatory follicle is requisite for successful ovulation, corpus luteum formation, and fertilization (41). As a component of follicular ECM, HAPLN1 was previously shown to enhance volumetric COC expansion by acting as a stabilizer of HA-rich matrix in vitro (17,20). In the present study, we demonstrated that 1) Hapln1 expression was highly up-regulated both in intact ovaries and granulosa cells by hCG injection; 2) Hapln1 expression was mediated through hCG-induced/activated mediators, including prostaglandins, EGF-related peptides/EGF receptor, PKA, PI3K, and p38 MAPK; and 3) transcriptional activation of the Hapln1 gene was dependent on the action of RUNX transcription factors and CRE-binding protein(s). Importantly, we also found that HAPLN1 can increase the viability of preovulatory granulosa cells in vitro, suggesting a potential novel role for HAPLN1 as an antiapoptotic/survival factor in luteinizing granulosa cells.

Previous studies using immunohistochemical analysis revealed the expression of HAPLN1 not only in expanded COCs but also in the mural granulosa layer of large periovulatory follicles (17,18,42). In agreement with those observations, our results showed that the levels of Hapln1 mRNA and protein were highly induced in intact ovaries and granulosa cells by hCG. Noticeably, the levels of Hapln1 mRNA decreased at 24 h after hCG, whereas the HAPLN1 protein maintained elevated levels. This observation is also consistent with a previous report that the high level of immunoreactive HAPLN1 was present in the newly formed corpus luteum (18), suggesting a potential role for this protein, not only in COC expansion but also in luteal transformation.

In the present study, we showed that hCG-induced expression of Hapln1 mRNA and protein in vivo can be mimicked by stimulating preovulatory granulosa cells with an ovulatory dose of hCG in vitro. These data indicated that the periovulatory up-regulation of Hapln1 expression results from the activation of LH receptors on preovulatory granulosa cells. Using this in vitro experimental model, we further investigated the regulatory mechanism(s) by which ovulatory LH/hCG induces Hapln1 expression in periovulatory granulosa cells. We found that LH/hCG use PKA, PI3K, and p38 MAPK, but not MEK, as their dominant signaling mediators in inducing Hapln1 expression. The inducible effect of LH/hCG on Hapln1 expression was found to be dependent on de novo protein(s). Several of the key downstream proteins induced by LH/hCG in periovulatory granulosa cells have been shown to be essential for the successful ovulation and/or COC expansion, such as PGR (43), PTGS2 (44), and EGF-related peptides (35). Therefore, we further investigated the impact of progesterone, prostaglandins, and the EGF-signaling pathway on the LH-induced up-regulation of Hapln1 expression. Our results indicated that the LH-induced Hapln1 expression was mediated by EGF-related peptides/EGF receptors and prostaglandin(s) but independently of the activation of PGR. Considering that both EGF signaling and prostaglandins are essential for proper COC expansion (44,45,46), the Hapln1 induced by these mediators likely plays an important role in COC matrix formation, which is crucial for successful COC expansion and/or ovulation.

In addition to these mediators, we also investigated the role for RUNX transcription factor(s) in up-regulation of the Hapln1 gene. The rationale for this study stems from our previous finding showing reduction of Hapln1 mRNA levels by Runx1 knockdown (30) and the sequence analysis of the Hapln1 promoter showing multiple RUNX-binding sites. Moreover, recent studies have demonstrated the LH-dependent rapid induction of both RUNX1 and RUNX2 in periovulatory granulosa cells and COCs (33,36) where Hapln1 is also expressed, suggesting that Hapln1 may be a direct target of these transcription factors. We found that knockdown of either Runx1 or Runx2 expression resulted in only partial reduction of Hapln1 expression, whereas inhibition of endogenous RUNX activities by adenoviral DNRUNX expression reduced levels of Hapln1 mRNA and protein to that of the control. These results suggest that the full induction of Hapln1 expression requires both RUNX1 and RUNX2. ChIP analyses further verified the binding of RUNX1 and RUNX2 in the Hapln1 promoter region. These data also suggest that these two RUNX factors are functionally redundant in regulating Hapln1 expression in periovulatory granulosa cells.

Functional promoter analysis of the Hapln1 gene revealed increases in Hapln1 promoter activity by agonists (FSK plus PMA) that can mimic the action of LH/hCG in preovulatory granulosa cells. More importantly, the agonist-induced Hapln1 promoter activity was dramatically decreased by mutation of the RUNX-binding sequence, demonstrating that Hapln1 is a direct transcriptional target of RUNX proteins. RUNX regulation on Hapln1 was not limited to periovulatory granulosa cells. The rapid increase in levels of Runx1 and Runx2 mRNA was previously documented in expanding COCs of rat and mouse (33,36). HAPLN1 staining was also observed in cumulus cells of expanding COCs but not in undifferentiated, compact COCs of rats stimulated with PMSG/hCG (17,18). In the present study, this in vivo COC expression of Hapln1, Runx1, and Runx2 was recapitulated in vitro by treatment with FSK plus AREG, which were used to induce the expansion of rat COCs. Luciferase reporter assay data using preovulatory COCs further confirmed the functional involvement of RUNX transcription factors in Hapln1 expression. Similar to that of RUNX-binding sites, mutation in CRE-binding motifs resulted in complete inhibition of the agonist-induced promoter activity, indicating that CRE-binding protein (CREB) transcription factor(s) also plays an important role in Hapln1 expression. CCAAT enhancer binding protein-β (C/EBPβ), a member of CRE-binding proteins (CREBs), was rapidly induced by the ovulatory LH stimulation in periovulatory granulosa cells and shown to be crucial for successful ovulation and COC expansion (47,48). Interestingly, C/EBPβ was shown to interact with RUNX transcription factors to enhance the transcription of specific genes in nonovarian cells (49,50). Therefore, whether C/EBPβ is indeed a CREB protein that binds to the Hapln1 promoter and RUNX1 or RUNX2 interacts with a CREB protein (e.g. C/EBPβ) to synergistically enhance Hapln1 expression needs to be further investigated. In any case, our present findings indicated that the LH-dependent up-regulation of Hapln1 expression requires cooperative action(s) of multiple transcription factors, including RUNX1, RUNX2, and CREB protein(s).

One of the important findings in this study is the stimulatory effect of exogenous HAPLN1 on granulosa cell viability. There are a couple of recent studies that linked HAPLN1 with cell proliferation and survival (24,25). In the present study, we showed for the first time that exogenous HAPLN1 treatment decreased the number of preovulatory granulosa cells undergoing apoptosis in vitro, and knockdown of endogenous Halpn1 expression decreased the viability of granulosa cells. It was well documented that preovulatory gonadotropin stimulation is crucial for the survival of periovulatory follicles, thus allowing them to complete the ovulatory process and subsequent transformation into CL. Several key studies using in vivo and in vitro models have supported this hypothesis. For instance, if the preovulatory gonadotropin surge is blocked by hypophysectomy (51) or injections of sodium pentobarbital (52), preovulatory follicles failed to ovulate and degenerated by apoptosis. Preovulatory follicles isolated before hCG stimulation in rat ovaries undergo massive apoptosis in vitro, whereas this spontaneous progression of apoptosis is suppressed by an ovulatory dose of LH/or hCG (53,54). Granulosa cells isolated from a periovulatory follicle after the LH surge or hCG stimulation were less susceptible to apoptotic stimuli in vitro than the cells isolated before hCG stimulation (39,40,55). Consistent with these observations, we showed that hCG treatment increased granulosa cell viability compared with the control culture. More importantly, this survival/antiapoptotic effect of hCG on luteinizing granulosa cells was mimicked by HAPLN1 treatment, suggesting that HAPLN1 may play a role in promoting the survival of luteinizing granulosa cells. As one of the major steroids synthesized and secreted by the ovary, progesterone has been postulated to prevent granulosa cells from undergoing apoptosis (38,39,40). Our data showed, however, that exogenous HAPLN1 did not affect progesterone production by granulosa cells, suggesting that the mechanism by which HAPLN1 can impact granulosa cell survival is not mediated through progesterone production. Recently, HA was reported to inhibit apoptosis in human cumulus and mural granulosa cells via the interaction with CD44 receptor (56,57), which can transactivate the ErbB2 receptor in tumor cells and subsequently trigger the cell survival pathway (58,59). Cattaruzza et al. (60) demonstrated that versican, a proteoglycan that binds to HA and HAPLN1, could modulate the proliferation-apoptosis equilibrium of tumor cells through its globular domains. Therefore, it is possible that HAPLN1 indirectly contributes to the increase in granulosa cell viability through the interaction with HA and/or versican.

As summarized in Fig. 8, we demonstrated that the LH-dependent induction of Hapln1 expression was mediated through the activation of key ovulatory mediators, prostaglandins and EGF signaling. The present study also identified RUNX1 and RUNX2 as important transcriptional regulators involved in Hapln1 expression. Our novel findings suggest that HAPLN1 may promote granulosa cell survival, which would facilitate their differentiation into luteal cells. Further studies will be needed to identify the mechanism of HAPLN1 action in promoting granulosa cell survival, thus providing new insight into the periovulatory survival programming necessary for successful ovulation and luteal formation.

Figure 8.

Figure 8

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A hypothetical model of regulatory pathways involved in Hapln1 expression and potential function.

Materials and Methods

Animals

All animal procedures were approved by the University of Kentucky Animal Care and Use Committee. Sprague Dawley rats were obtained from Harlan, Inc. (Indianapolis, IN) and provided with water and chow ad libitum. Animals were maintained on a 12-h light, 12-h dark cycle. Immature female rats (25 or 26 d old) were injected with PMSG (10 IU) sc to stimulate follicular development. The animals were injected 48 h later with hCG (5 IU) sc to induce ovulation. Animals were killed at 0 h (48 h after PMSG) or defined times after hCG administration. Ovaries were collected and processed for culture experiments or isolation of total RNA or protein.

Isolation and culture of rat granulosa cells and COCs

Ovaries were collected from immature rats 48 h after PMSG administration and punctured to isolate granulosa cells as previously described (30,36). The granulosa cells were filtered through a 40-μm cell strainer to remove COCs and pelleted by centrifugation at 200 × g for 5 min. The cells were cultured in Opti-MEM (Life Technologies, Gaithersburg, MD) media supplemented with 0.05 mg/ml of gentamycin, and 1× ITS (insulin, transferin, and selenium). The unexpanded COCs were collected from the ovaries of PMSG-primed rats (48 h after PMSG), plated in MEM (Life Technologies) supplemented with 0.05 mg/ml of gentamycin, 25 mm HEPES, 0.25 mm sodium pyruvate, 3 mm l-glutamine and 1 mg/ml BSA (collection media). COCs were cultured in collection media plus 5% fetal bovine serum. The cells were cultured in the absence or presence of various reagents for time points outlined below for each experiment at 37 C in a humidified atmosphere of 5% CO2. When reagents were dissolved in dimethylsulfoxide (DMSO), the same concentrations of DMSO were added to medium for the control cells. The final concentration of DMSO was less than 0.1%.

Quantification of mRNA

The levels of mRNA for Hapln1, Runx1, Runx2, and L32 were measured by real-time PCR. Briefly, total RNA was isolated from granulosa cells using Trizol (Invitrogen, Carlsbad, CA). Synthesis of first-strand cDNA was performed by reverse transcription of 1.0 μg total RNA using superScript III with Oligo(dT)20 primer according to the manufacturer's protocol (Invitrogen). Oligonucleotide primers corresponding to cDNA for rat L32 (forward, 5′-GAA GCC CAA GAT CGT CAA AA-3′; reverse, 5′-AGG ATC TGG CCC TGG CCC TTG AAT CT-3′); Hapln1 (forward, 5′-AAT TTT ATC GAG ACC CTA CAG C-3′; reverse, 5′-AAA CGA AGA CAT CCA CTT CC-3′); Runx1 (forward, 5′-AAC CCT CAG CCT CAA AGT CA-3′; reverse, 5′-GGG TGC ACA GAA GAG GTG AT-3′); Runx2 (forward, 5′-CTC ACT ACC ACA CGT ACC TGC-3′; reverse, 5′-ATA GGA CGC TGA CGA AGT ACC-3′) were designed using PRIMER3 software. The specificity for each primer set was confirmed by both electrophoresis of the PCR products on a 2.0% agarose gel and analyzing the melting (dissociation) curve using a MxPro real-time PCR analysis program (Stratagene, La Jolla, CA) after each real-time PCR. The real-time PCRs were carried out as previously described (36). The relative amount of each transcript was calculated using the 2−ΔΔCT method (61) and normalized to the endogenous reference gene L32.

Western blot analysis

The protein was isolated from granulosa cells using RIPA buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein (30 μg)was separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with antibodies against HAPLN1 (1:100; Santa Cruz Biotechnology, Inc.) or β-actin (1:1000; Cell Signaling Technology, Danvers, MA), overnight at 4 C. On the next day, the membrane was incubated with respective secondary antibodies linked to horseradish peroxidase for 1 h. Blots were analyzed using an enhanced chemiluminescence detection system (Pierce Chemical Co., Rockford, IL) and exposed to x-ray film.

Knockdown of Runx1, Runx2, and Hapln1 expression by siRNA in granulosa cell cultures

Granulosa cells were collected from immature rats 48 h after PMSG administration as described above and seeded in 24-well plates at approximately 50–60% confluence. A small interfering RNA (siRNA) specific for Runx1 (5′-CAA ACC UGA GGU CGU UGA AUC UCG C-3′; Invitrogen), Runx2 (sense, 5′-GCA CGC UAU UAA AUC CAA Att-3′; antisense, 5′-UUU GGA UUU AAU AGC GUG Ctg-3′; Ambion, Inc., Austin, TX), Hapln1 (5′-CCG CAU UAA GUG GAC CAA GCU AAC U-3′; Invitrogen) or scrambled siRNA (Stealth RNAi Negative Control; Invitrogen) was transfected into granulosa cells using the Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). Transfection media were replaced 4 h later with fresh culture media containing hCG (1 IU/ml), and the cells were further cultured for 8 h. The cells were collected for isolation of total RNA or protein.

Overexpression of DNRUNX in granulosa cells by adenoviral infection in vitro

The DNRUNX recombinant adenovirus was constructed using an AdEasy XL Adenoviral Vector System kit (Stratagene) and produced as described previously (36). Granulosa cells were isolated from immature rats (48 h after PMSG) as described above. The cells were plated and subsequently incubated with adenoviral DNRUNX (AdEasy-Runt homology domain) or green fluorescent protein (AdEasy-GFP) at 10 multiplicity of infections for 24 h. The next day, cells were treated with FSK (10 μm) plus PMA (20 nm) for 8 h. The cells were collected for isolation of total RNA and used for real-time PCR.

ChIP analysis

ChIP assay was performed on RUNX1 and RUNX2 in the Hapln1 promoter region using a ChIP kit (Upstate Biotechnology, Inc., Lake Placid, NY) as described previously (62). Briefly, periovulatory granulosa cells were isolated from PMSG/hCG-stimulated immature rats (0 h and 10 h after hCG). The cells were treated with 1% formaldehyde and lysed to release nuclei. The nuclei were sonicated with a Fisher Sonic Dismembrator model 550 to obtain DNA fragments of an average length of approximately 100–500 bp. Chromatin was immunoprecipitated overnight at 4 C with RUNX1 antibody (5 μg/reaction; Calbiochem) or RUNX2 antibody (5 μg/reaction; Calbiochem), using normal rabbit IgG (5 μg/reaction; Santa Cruz Biotechnology) as a negative control. The immunoprecipitated chromatin and 1:10 dilution of input chromatin were analyzed by PCR using the primers designed to amplify fragments spanning the RUNX motif in the Hapln1 promoter (Fig. 4B; distal forward, 5′-TGA GAA GTG CCT CCT TCA TCC-3′; distal reverse, 5′-GAA GCT AAT GCT CCT CCT GAC C-3′; and proximal forward, 5′-CAA GGT GAC AAC TGT GTA GG-3′; proximal reverse, 5′-AGC AGT CTC TTA GAT GAC TCG-3′). After 25–30 cycles of amplification, PCR products were run on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light.

Cloning of the rat Hapln1 promoter and generation of Hapln1 promoter-reporter plasmid constructs

Genomic DNA was isolated from tail samples from rats using an easy-DNA kit (Invitrogen). The 78-bp (−42/+35), 122-bp (−86/+35), and 184-bp (−148/+35) fragments of the Hapln1 gene were amplified using the primers attached with restriction enzyme sites (KpnI and HindIII) and cloned into a multiple cloning site of the pGL3 basic vector (Promega Corp., Madison, WI). Site-directed point mutations of the Hapln1 promoter were generated using a QuikChange II Site-Directed Mutagenesis kit according to the manufacturer's protocol (Stratagene). The sequences of the oligonucleotide primers used to generate respective Hapln1 promoters containing mutations (shown in lowercase) for RUNX or CRE motifs (underlined) are as follows: mutant A (5′-CCA CAC CCA CAC CCAC cttGCA CTC GCC CAG AGA C-3′); mutant B (5′-CAG GAC CTC TGC CAT CCA GC cttACA AAG AGA CAT TCT GCA CA -3′); mutant C (5′-CGC ACT CGC CCA GAG ACA AACT TacatgCA GGA GGA GCA TTA GCT TC-3′). All constructs cloned into the expression vector were sequenced commercially to verify their authenticity (MWG Biotech, Inc., High Point, NC).

Transient transfection and luciferase reporter assay

Granulosa cells or COCs were isolated from immature rats (48 h after PMSG) as described above. The cells were transfected with respective firefly luciferase reporter plasmids (pGL3-basic vector or pGL3-Hapln1 promoter constructs) and Renilla luciferase vector (pRL-TK vector) using a Lipofectamine 2000 reagent (Invitrogen). The next day, cells were treated with FSK (10 μm), PMA (20 nm), FSK plus PMA, or FSK plus amphiregulin (250 ng/ml) for 8 h. The cells were harvested to measure firefly and Renilla luciferase activities using a Dual-luciferase reporter assay system (Promega), and each reaction was monitored for 10 sec by Luminescence System in the Infinite F200 plate reader (Tecan USA, Durham, NC). Firefly luciferase activities were normalized by Renilla luciferase activities, and each experiment was performed in triplicate at least three times.

MTS cell viability assay

Granulosa cells were collected from immature rat ovaries (48 h after PMSG) and seeded in 96-well plates (5000 cells per well). Cells were incubated with vehicle (0.02% BSA), hCG (1 IU/ml), or human recombinant HAPLN1 peptide (0.01, 0.1, 0.5, 1, and 2 μg/ml; R&D Systems, Minneapolis, MN) for 24 h. HAPLN1 peptide was dissolved in PBS with 1% BSA, and the same concentration of BSA was added to medium for the control cells. The final concentration of BSA was 0.02%. Cell viability was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer's protocol (Promega). Briefly, at end of culture, 20 μl of reagent were pipetted into each well containing the cells in 100 μl of culture medium, and cells were then returned to the incubator for an additional 2 h. The absorbance was measured at 492 nm in the Infinite F200 plate reader (Tecan USA) to determine the formazan concentration, which is proportional to the number of live cells (37).

CytoTox-ONE homogeneous membrane integrity assay

Granulosa cells were collected from rat ovaries (48 h after PMSG) and seeded in 96-well plates (5000 cells per well). Cells were incubated with vehicle (0.02% BSA), hCG (1 IU/ml), or human recombinant HAPLN protein (2 μg/ml) for 24 h. Cytotoxicity was measured using CytoTox-ONE Homogeneous Membrane Integrity Assay according to the manufacturer's protocol (Promega). Briefly, at end of culture, 100 μl of CytoTox-ONE Reagent were added to 100 μl of culture medium containing cells in each well. After incubation for 10 min, 50 μl of Stop Solution were added to each well. The fluorescence was measured at an excitation wavelength of 595 nm and an emission wavelength of 635 nm in the Infinite F200 plate reader (Tecan USA) to determine the release of lactate dehydrogenase, which is proportional to the number of dead cells.

Progesterone measurement

Concentrations of progesterone in culture media were measured using an Immulite kit (Diagnostic Products, Los Angeles, CA). Assay sensitivity was 0.02 ng/ml. The intraassay and interassay coefficients of variation were 9.6% and 10%, respectively.

Statistical analyses

All data are presented as means ± sem. One-way ANOVA was used to test differences in levels of Runx1, Runx2, and Hapln1 mRNA, luciferase activities of respective Hapln1 promoter constructs, cell viability, and progesterone concentration among treatments. If ANOVA revealed significant effects of treatments, the means were compared by Tukey's test, with P < 0.05 considered significant.

Supplementary Material

[Supplemental Data]
me.2009-0325_index.html (794B, html)

Footnotes

This work was supported by National Institutes of Health Grant P20 RR 15592.

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 25, 2010

Abbreviations: Ad-DNRUNX, Adenoviral DNRUNX; AREG, amphiregulin; C/EBPβ, CCAAT enhancer binding protein-β; ChIP, chromatin immunoprecipitation; CHX, cyclohexamide; COC, cumulus-oocyte complex; CRE, cAMP response element; CREB, CRE-binding protein; DMSO, dimethylsulfoxide; DNRUNX, dominant-negative RUNX; ECM, extracellular matrix; EGF, epidermal growth factor; FSK, forskolin; GFP, green fluorescent protein; HA, hyaluronic acid; HAPLN1, hyaluronan and proteoglycan link protein 1; hCG, human chorionic gonadotropin; IαI, inter-α-trypsin inhibitor; LY, LY294002; MEK, MAPK kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NS, NS-398; PD, PD98059; PGR, progesterone receptor; PI3K, phosphatidylinositol-dependent kinase; PKA, protein kinase A; PMA, phorbol 12 myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; PTGS2, prostaglandin-endoperoxide synthase 2; SB, SB2035850; siRNA, small interfering RNA.

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