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Ovary; |
Institute of Genetechnology/Microbiology,3 University of Bielefeld, D-33501 Bielefeld, Germany
Department of Biochemistry,4 King's College London, London SE1 9NH, United Kingdom
Biochemical Institute,5 University of Kiel, 24098 Kiel, Germany
N.V. Organon,6 5340 BH Oss, The Netherlands
Department of Molecular and Cellular Biology,7 Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
ADAM8 (a disintegrin and metalloprotease domain 8) is expressed in immune, neuronal, and bone progenitor cells and is thought to be involved in the tissue-remodeling process. Microarray analyses indicate that Adam8 is a potential target of the progesterone receptor (Pgr) in murine ovary. Further studies document that Adam8 mRNA and protein are expressed in granulosa cells and cumulus cells of periovulatory follicles whereas expression is significantly reduced in Pgr null mice that fail to ovulate. There is a reduced expression in granulosa cells from cultured, in vitro ovulated follicles exposed to inhibitors of progesterone or epidermal growth factor signaling while epiregulin induced its expression in the absence of hCG. In vitro studies with primary mouse granulosa cells document that Adam8 is induced in response to forskolin (Fo) and phorbol ester (PMA) or Fo and Amphiregulin treatment. To understand the transcriptional regulation of the Adam8, we amplified 1 kb of the mouse Adam8 promoter by PCR and subcloned it into a pGL3-luciferase reporter construct. The Adam8 promoter-luciferase constructs are induced by Fo and PMA treatment after transfection into rat granulosa cells, and cotransfection with a PGR-A expression vector further augment basal and Fo/PMA inducibility. Site-specific mutations within the –615/+50 promoter document that a GC-rich region, NF-1 (nuclear factor-1) site, and putative TATA box are critical for Adam8 promoter activation by Fo/PMA. Thus, ADAM8 is expressed in a stage-specific manner and is hormonally regulated in ovulating follicles by the coordinate actions of LH and PGR. To our knowledge, ADAM8 is the first member of the ADAM family shown to be hormonally regulated.
luteinizing hormone, ovary, ovulation, progesterone receptor
Ovulation is a complex event in mammalian reproduction and resembles an inflammatory reaction [1]. This process is associated with significant changes in the extracellular matrix of the ovary that control the release of the oocyte from the mature preovulatory follicle [1, 2]. Matrix-modifying enzymes, namely, cathepsins [3, 4], ADAMTSs (a disintegrin and metalloprotease domain with thrombospondin motifs) [5], matrix metalloproteinases [6], and plasmin [7], have been implicated in these remodeling events. The nuclear transcription factor progesterone receptor (Pgr) is critical for ovulation, and mice null for Pgr do not ovulate and are infertile due to severe defects in the ovary, uterus, and mammary gland [8]. The PGR-A isoform is predominantly expressed in granulosa cells, and one prevailing hypothesis is that PGR regulates follicular rupture by induction of matrix-modifying enzymes such as ADAMTS1, cathepsin L (Ctsl) [9], and plasminogen activator system [7]. In our studies of Pgr null mice (PRKO), Adam8 (a disintegrin and metalloprotease domain 8) was identified as a putative PGR target gene, and this suggests that it may be expressed in a stage-specific manner by PGR action.
The ADAMTS1 protein is one of the first members of the metzincin superfamily that was shown to be essential for cumulus expansion [10]. Targeted disruption of the Adamts1 gene leads to subfertility in mice [11]. ADAMTS1 is induced by the progesterone-signaling pathway [9], while the role and control of the other members of the ADAMs family in the ovary remain unknown. ADAM proteins constitute a family of transmembrane glycoproteins with essential physiological roles in fertilization, neovascularization, myogenesis, and neurogenesis [12, 13]. They are composed of several conserved and characteristic structural modules, including a prodomain, metalloprotease domain, disintegrin domain, cysteine-rich region, EGF (epidermal growth factor) repeat transmembrane domain, and cytoplasmic tail [12, 13]. Distinct structural domains within these proteins confer specific functions in cell-cell fusion, cell-cell interactions, or metalloprotease activity [12, 13].
ADAM8 was originally cloned from mouse macrophages [14] and is expressed in neurons and oligodendrocytes [15], and, with the exception of T-cells, in most human immune cell types [13]. ADAM8 contains the catalytic consensus sequence HEXXHXXGXXHD required for the metalloproteinase activity, and the recombinant soluble ADAM8 possesses catalytic activity [16]. In vitro, it can cleave myelin basic protein (MBP), amyloid polypeptide precursor protein, and FCER2 (Fc fragment of IgE receptor; CD23), a low-affinity IgE receptor that promotes cell motility and leukocyte infiltration [17]. Recently the neural cell adhesion molecule close homologue of L1, termed CHL1 [18], that promotes neurite outgrowth and suppresses neuronal cell death has been shown to be a substrate of ADAM8. Expression of catalytically active ADAM8 is associated with increased invasive activity and/or extracellular matrix (ECM) remodeling [19]. Thus, ADAM8 can influence inflammation, cell adhesion, and migration processes that are relevant to the ovulatory process.
The goal of this study was to understand the hormonal regulation of ADAM8 expression in ovary during follicular development, ovulation, and luteinization, employing a number of different experimental approaches to study control of expression of Adam8. These included analysis of expression of Adam8 in ovary of in vivo ovulated follicles and in hormonally stimulated control and PRKO mice. Furthermore, expression was analyzed in granulosa cells from preantral follicle cultures stimulated by hCG (human chorionic gonadotropin) and EGF to ovulate in vitro, with and without exposure to inhibitors of PGR and EGF receptor during the preovulatory period. Finally, we studied luciferase expression from a transgene carrying different segments of the Adam8 promoter in mammalian primary granulosa cells stimulated by forskolin and phorbol ester that activates protein kinase A and protein kinase C signaling pathways. Although the function of Adam8 in the preovulatory follicle in vivo remains elusive, our studies document the induction of Adam8 mRNA and protein in mammalian granulosa cells and of mRNA in cumulus-oocyte-complexes (COCs) of the preovulatory follicle and a role for LH and PGR in the induction of this gene.
Equine chorionic gonadotropin (eCG) and forskolin (Fo) were purchased from Calbiochem (San Diego, CA) and hCG from American Pharmaceutical Partners Inc. (Schaumburg, IL). Recombinant luteinizing hormone (rLH) and recombinant follicular stimulating hormone (rFSH) were from Serono (Unterschleißheim, Germany, kindly donated), and recombinant epidermal growth factor (rEGF) was from Promega (Mannheim, Germany). Media and cell culture reagents and materials were procured from Life Technologies, Inc. (Grand Island, NY) and Corning Inc. (Corning, NY); fetal bovine serum was procured from Hyclone Laboratories, Inc. (Logan, UT). L15 Leibovitz-glutamax medium, fetal calf serum, and 
-minimal essential medium with glutamax for preantral follicle culture were from GIBCO Invitrogen (Karlsruhe, Germany). Insulin-transferrin-selenium mixture and progesterone receptor inhibitor RU486 were purchased from Sigma (Taufkirchen, Germany), and AG1478 inhibitor of epidermal growth factor receptor (EGFR) was from Calbiochem. Epiregulin came from R & D Systems (Wiesbaden, Germany). Trypsin, soybean trypsin inhibitor, deoxyribonuclease, and PMA (phorbol 12-myristate 13-acetate) were obtained from Sigma Chemical Co. (St. Louis, MO), electrophoresis and molecular biology-grade reagents were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA), and reagents for RT-PCR were obtained from Promega Corp. (Madison, WI). Amphiregulin (AREG) was obtained from R&D Systems, Inc. (Minneapolis, MN). Oligonucleotides were synthesized by Sigma-Genosys (Houston, TX). The generation of rabbit polyclonal antibodies against ADAM8 has been described [15], and its application in Western blots have been demonstrated earlier [15, 16].
Immature (23 days old) C57BL/6 mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed under a 16 h light and 8 h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine with food and water ad libitum. Mice were injected i.p. with 4 IU eCG to stimulate follicular growth, and after 44 h with 5 IU hCG (i.p.), which, like LH, induces ovulation and luteinization. Ovulation occurs approximately 12–16 h after hCG administration in this model [20]. Ovaries were isolated from these hormone-stimulated mice at selected time intervals for extraction of RNA and protein, or fixed for in situ hybridization. Pgr null mice were used in selected experiments because follicles develop normally in response to eCG but fail to ovulate in response to hCG [9]. Cumulus-oocyte-complexes were isolated from eCG treated mice (0 h) as well as from preovulatory and ovulating follicles of eCG- and hCG-stimulated mice (8 and 12 h). Ovulated COCs were collected from oviduct at 16 and 24 h.
C57BL/6 mice for preantral follicle culture were maintained at Bielefeld University under a 12 h light and 12 h dark cycle with food and water ad libitum, and animal experimentation was conducted under a permit from the university authorities.
Rat granulosa cells were employed for transfection studies to analyze Adam8 promoter. Intact immature (23 days old) Holtzman Sprague Dawley females (Harlan Sprague Dawley, Inc., Indianapolis, IN) were housed under a 16 h light and 8 h dark schedule in the Center for Comparative Medicine at Baylor college of Medicine with food and water ad libitum. The rats were injected with estradiol-17β (E2) for 3 days (1.5 mg E2/0.2 ml in propylene glycol), and the ovaries were harvested for isolation of granulosa cells. All Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine (Houston, TX).
RNA Isolation from Whole Ovary, Granulosa Cells, and COCs, and RT-PCR
Total RNA was isolated from whole ovaries, granulosa cells, and COCs of immature (23 days old) untreated mice, and from eCG- and hCG-treated mice, using TRIzol reagent (Life Technologies, Inc.) and purified as described in the manufacturer's instructions. For each RNA sample, whole ovaries, granulosa cells, or COCs were pooled separately from two or three animals. RNA was similarly prepared from cultured cells. To determine the expression of Adam8 in ovary, 2 µg of RNA were reverse transcribed in 20 µl reaction containing oligo-deoxythymidine (Amersham Pharmacia Biotech, Newark, NJ) and AMV (avian myeloblastosis virus) reverse transcriptase (Promega Corp.) at 42°C for 75 min [20]. cDNA (2 µl) obtained from reverse transcription reaction was subjected to labeled PCR with [P32]deoxy-CTP (ICN, Los Angeles, CA) using Taq polymerase [20] and specific primers for Adam8 and Rpl19 (Table 1). All PCRs were carried out within the experimentally determined linear amplification range. The ribosomal protein L19 (Rpl19) was employed as an internal control. PCR products were resolved by a 6% PAGE and visualized by autoradiography [20]. The products were quantified using PhosphoImager (Molecular Dynamics, Inc., Sunnyvale, CA) [20]. All PCR products were cloned in TOPO vector, and their authenticity was confirmed by sequencing at the Baylor College of Medicine Core Facility.
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In situ hybridization was performed as described previously and as reported by our laboratory [20]. The experiments were performed with four ovarian serial sections obtained from three separate hormonal treatments for each time point. Eight to ten follicles at each stage were assessed for hybridization. The riboprobe in vitro transcription system kit (Promega Corp.) was used to make [S35] uridine triphosphate-labeled antisense and sense probes from the mouse Adam8 cDNA. The cDNA probe were generated by RT-PCR amplification and TOPO TA cloning (Invitrogen, Carlsbad, CA); their identity was verified by sequencing, and their specificity was verified by basic local alignment and search tool analyses. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm onto Fisher Brand Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were rehydrated, treated with 20 µg/ml proteinase K and 0.1 M triethanolamine/acetic anhydride, dehydrated, and incubated with radiolabeled riboprobe overnight at 55°C. The next day, slides were washed at high stringency, dried, and exposed to X-OMAT film (Kodak, Rochester, NY) overnight to determine the specificity and intensity of the probe. Slides were dipped in photographic NTB-2 emulsion, exposed at 4°C for an appropriate length of time, developed with D-19 developer and fixer (Kodak), and stained with hematoxylin. Tissue histology was observed by both light-field illumination and dark-field illumination to visualize the regions of hybridization.
Protein extracts from mouse ovaries were prepared by homogenization in 6 M urea and 0.1% Triton buffers containing a protease inhibitor cocktail (Sigma, St. Louis, MO) and 0.05 M EDTA [21]. Protein concentrations were determined by the Bradford method, and for Western Blot analyses of ADAM8, 20 µg protein were loaded onto gels. Samples were resolved in 10% acrylamide gels under reducing SDS-PAGE conditions and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Membranes were blocked with 5% nonfat milk for 2 h, followed by overnight incubation with ADAM8 antiserum, total ERK (extracellular signal-related kinase) antibody (Cell Signaling Technology, Danvers, MA), or preimmune serum obtained before immunization of ADAM8 peptide at 1:1000 dilution and then washed with TBST (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20). Subsequently, blots were incubated with 1:12,500 diluted horseradish peroxidase linked to anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) and then washed with TBST. Immunoreactive bands were detected using enhanced chemiluminescence according to the manufacturer's specifications (Pierce Chemical Co., Rockford, IL). The blots that were probed with ADAM8 antibody were also probed -actin antibody (Sigma, St. Louis, MO). The assessment of -actin and total ERK expression served as a loading control. The immunoreactive bands were quantitated by densitometer (Molecular Dynamics, Inc., Sunnyvale, CA) and Image quant 5.0 software.
Mouse Preantral Follicle Culture
Preantral follicles were manually isolated from 16 to 18 day old C57BL/6 mice. Cell culture was essentially as previously described [22–25]. Briefly, preantral follicles of 100–130 µm diameter with two to three layers of granulosa cells and some theca cells (3b follicles) [26] were mechanically isolated in L15 Leibovitz-glutamax medium with foetal calf serum and antibiotics. Individual follicles were placed into 50 µl of culture medium consisting of -minimal essential medium with glutamax, supplemented with 10 µIU/ml rLH, 10 µIU/ml rFSH, foetal calf serum, and an insulin-transferrin-selenium mixture in 96 microtiter plates (Costar, Schiphol-Rijk, Netherlands) covered by a layer of 30 µl mineral oil. At each fourth day of culture, 20 µl medium containing 10 µIU of rFSH but no rLH was replenished. At Day 12 when antral-like cavities had developed and oocytes had grown and reached maturational competence, oocyte resumption of maturation and in vitro ovulation was induced by adding 1.5 IU/ml recombinant hCG (rhCG) and 5ng/ml rEGF. Four and 18 h later, when oocytes had undergone germinal vesicle breakdown (GVBD) or reached metaphase II and emitted a first polar body (PB) and when in vitro ovulation had occurred, respectively, the mural granulosa cells were collected for RNA preparation and analysis of expression of Adam8 by quantitative RT-PCR.
In order to study the influence of PGR- or EGFR-mediated signaling on Adam8 expression in follicles stimulated for in vitro ovulation, 1 µM of RU486 (PGR inhibitor) or 10 µM AG1478 (EGFR inhibitor) were added to the antral follicles together with rhCG and rEGF on Day 12 of culture. In one set of experiments, 100 ng/ml epiregulin was added on Day 12 instead of rhCG and rEGF to induce in vitro ovulation.
Oocytes retrieved from in vitro ovulated follicles were vital stained for chromatin with Hoechst 33342 (Sigma, Germany; 20 min in M2 medium) and analyzed for induction of GVBD or PB formation with a Zeiss (Axiovert 10). Images of in vitro ovulated COCs or isolated oocytes were obtained using a Canon PC1099 camera attached to the Zeiss Axiovert microscope.
RNA Isolation and Quantitative RT-PCR from Granulosa Cells of In Vitro Grown Follicles
About 100 ng of total RNA isolated from follicles were transcribed with Expand Reverse Transcriptase (Roche, Mannheim, Germany) in a volume of 20 µl; 2 µl of the RT-product was used for PCR in a 20 µl reaction volume with primers indicated in Table 1. Real-time PCR consisted of a 20 µl reaction with QuantiTecti SYBR Green PCR Kit (Qiagen, Hilden, Germany), and 2 µl of the RT product was used as the template. Specific Adam8 transcript numbers were normalized to the ribosomal L7 gene (Rpl7) as described elsewhere [19] and presented as normalized values relative to controls (control = 1). Experiments with control and inhibitors were performed in triplicate with isolation of follicles for RNA isolation from control and treatment group of each experimental group. Granulosa cells were pooled from 15 to 20 follicles from each plate. Relative increase or decease in Adam8 mRNA/amplification product was calculated relative to the untreated controls in the studies with inhibitor or in the epiregulin study, comparing data from triplicate RT-PCR assays.
sAdam8 Promoter Constructs and Mutants
The mouse Adam8 promoter (–1065/+50) was amplified using specific primers containing Nhe and XhoI sites and ligated to the pGL3 luciferase reporter plasmid using PCR cloning techniques. PCR primers were designed based on previous studies that characterized the Adam8 gene and putative transcription factor binding sites. In addition, three other Adam8 promoter regions that extend from –615 /+50. –240/+50, and –106/+50 that span the transcription start site +1 were amplified and subcloned. The authenticity of each construct was verified by sequence analysis in both directions. Site-specific mutations in the Adam8 promoter constructs were generated using the gene editor site-directed mutagenesis kit from Promega Corp. (Madison, WI). Oligonucleotides used for creating site-specific mutations at the critical nucleotides necessary for transcription factor binding to the GC boxes, NF-1 (nuclear factor-1) site, and the TATA box are listed in Table 1. The mutants obtained were confirmed by sequencing.
Granulosa Cell Culture and Transfection
Rat granulosa cells were harvested by needle puncture of the ovaries and processed described previously [3]. Briefly, cells were cultured in 12-well culture plates at a density of 0.5 x 106 cells/1.5 ml in 5% serum containing medium (DMEM:F12 containing penicillin and streptomycin). Two hours after plating, cells were transiently transfected with 500 ng of the respective promoter-reporter constructs with Fugene 6 (Roche Molecular Biochemicals) overnight. On the next day, cells were washed and cultured in fresh, serum-free medium containing 10 µM Fo, 20 nM PMA, 100 ng/ml AREG, Fo/PMA, or Fo/AREG, and after 4 h harvested with lysis buffer (0.2 M Tris, pH 8.0, containing 0.1% Triton X-100). An additional group of cells were transfected with pSCT empty vector or the PGR-A expressing pSCT-PGR-A construct (10 ng). Fo and PMA have been used previously to mimic the effects of the LH surge and optimal induction of Ptgs2 and Pgr in cultured rat granulosa cells [27, 28]. Luciferase activity in the extracts was analyzed according to a standard protocol [29]. In brief, a 40 µl aliquot of the cell lysate was mixed automatically with 100 µl luciferase assay reagent (20 mM Tris, pH 8.0, containing 4 mM MgSO4, 0.1 mM EDTA, 30 mM dithiothreitol, 0.5 mM ATP, 0.5 mM luciferin, and 0.25 mM coenzyme A), and each reaction was monitored 20 sec in a luminometer. The protein concentrations were determined by mini-Bradford assay (Bio-Rad Laboratories, Inc.). Data are expressed based on the amount of protein in each sample: light-specific units (LSUs) per microgram of protein (mean ± SD). Transfection of empty pGL3 vector in granulosa cells showed basal values that were less than unstimulated wild-type pGL3–1065/+50 construct. The inducibility of pGL3 by Fo/PMA was never more than 1.5-fold in each experiment.
Primary mouse granulosa cells isolated from eCG-primed mice ovaries by needle puncture was employed to investigate agonist-induced Adam8 expression. The isolated cells were cultured overnight in DMEM:F-12 containing 1% serum, and on the next day cells were treated with Fo/PMA or Fo/AREG for 4 h. At the end of the treatment, the cells were harvested for total RNA isolation and semiquantitative RT-PCR analyses.
Results are presented as means ± SD from at least three different experiments. Significant differences between groups were analyzed by ANOVA followed by the Neuman-Keuls test employing GraphPad Prism software (San Diego, CA).
Adam8 mRNA Is Hormonally Regulated and Differentially Expressed in Ovaries of Wild-Type and Pgr Null Mice
To analyze Adam8 expression, total RNA was prepared from whole ovaries of immature mice at specific times prior to and after hormone treatments to stimulate preovulatory follicle development and ovulation. Semiquantitative RT-PCR analysis revealed that Adam8 mRNA was expressed in ovaries of immature mice and that treatment with eCG did not cause a significant change in Adam8 mRNA levels. Upon administration of an ovulatory dose of hCG, the levels of Adam8 mRNA increased rapidly at 2 h and reached a maximum level by 4 h (Fig. 1A). Adam8 expression remained elevated until 16 h but decreased markedly by 24 h. Changes in the ovarian levels of ADAM8 protein followed a similar expression pattern (Fig. 1B). Western blot analyses of ADAM8 in other tissues typically reveal three immunoreactive bands at 120, 90, and 60 kDa, representing the unprocessed, processed, and remnant forms of the protein, respectively [16]. Identical bands were observed and were found to increase in ovarian samples 4–8 h post-hCG (Figure 1B). Processing of the 
120 kDa proform of ADAM8 by autocatalysis to 90 kDa is required for proteolytic activity [16], and the 60 kDa remnant form is abundant in all tissues and cell types that express ADAM8 [15]. -Actin and total ERK served as a loading control for the Western blots. No specific bands were observed on Western blot analysis with preimmune serum.
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In situ hybridization confirmed induction of Adam8 mRNA at 8 h post-hCG and localized its expression to granulosa cells of preovulatory follicles. Little or no signal was observed in eCG-treated ovaries (Fig. 2A, upper left). To verify and quantify Adam8 expression, semiquantitative RT-PCR analyses were carried out using RNA prepared from granulosa cells and COCs isolated at different time intervals after eCG-hCG treatment. The results confirmed that induced expression of Adam8 mRNA occurred in granulosa cells and COCs 8 h after hCG treatment (Fig. 2B). The expression of Adam8 mRNA was also analyzed in ovaries of Pgr null mice that exhibit an anovulatory phenotype. RT-PCR analyses of total RNA samples prepared from Pgr null homozygote (–/–) and heterozygote (+/–) mouse ovaries at 8, 12, and 16 h after hCG treatment documented significantly reduced levels of Adam8 mRNA in PRKO samples compared to samples from heterozygote at 8 and 12 h but not at 16 h (Fig. 3A). Interestingly, the decline in Adam8 mRNA at 8 h post-hCG correlated with the decrease in number of Pgr alleles (Fig. 3B) relative to wild type mice.
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Expression of Adam8 Is Induced in Granulosa Cells from Preantral Follicle Culture upon Stimulation by hCG and EGF
To further examine the control of Adam8 expression in periovulatory follicles, a follicle culture system was used (Fig. 4A). Preantral follicles with two to three layers of granulosa cells (100–130 µm diameter) were manually dissected from the ovaries of prepubertal mice and cultured for 12 days in the presence of rFSH to promote follicle and oocyte growth, antrum formation, and granulosa cell differentiation in vitro. At Day 12, medium was replenished, and in vitro ovulation was induced by rhCG and rEGF. Within 18 h of culture to Day 13, the majority (on average >95%) of the antral follicles undergo in vitro ovulation. Granulosa cells were harvested to analyze expression of Adam8 by RT-PCR prior to (0 h) and following (4 and 18 h) follicle exposure to rhCG/rEGF (Fig. 4H). There was a low level of expression at Day 12. The relative abundance of transcript increased dramatically at 4 h and remained high at 18 h after stimulation of in vitro ovulation by rhCG/rEGF. Expression was also compared between the 18 h rhCG/rEGF-stimulated follicles cultured in the absence or presence of 10 µM AG1478 (EGFR inhibitor) or 1 µM RU486 (PGR inhibitor) (Fig. 5A) or stimulated by 100 ng/ml epiregulin instead of rhCG/rEGF (Fig. 5B). AG1478 as well as RU486 significantly reduced the expression of Adam8 mRNA whereas the relative abundance of message was significantly increased over the rhCG/rEGF controls by stimulation with epiregulin.
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Hormonal Regulation of the Adam8 Promoter Activity in Granulosa Cells Depends on Multiple Promoter Elements
To understand the transcriptional regulation of Adam8, we confirmed the role of LH signaling in induction of Adam8 mRNA by the addition of Fo/PMA or Fo/AREG to primary granulosa cell cultures. Treatment of primary mouse granulosa cells with Fo/PMA for 4 h induced the Adam8 expression 4–6 fold over the basal levels (Fig. 6A). AREG (100 ng/ml) increased the expression of Adam8 1.4 fold. However, when AREG was combined with Fo, Adam 8 mRNA levels were increased 3–4 fold over basal levels (Fig. 6B).
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To further determine the molecular mechanisms by which Adam8 is induced in granulosa cells, mouse Adam8 promoter-luciferase constructs were generated based on a previous study by Kataoka et al. [30]; they documented that 1 kb of the Adam8 promoter contained the sites that are necessary to confer lipopolysaccharide and interferon-
inducibility. In addition, their studies determined the transcription start site and predicted several putative transcription factor binding sites. Therefore, we cloned 1 kb (–1065/+50) of the murine Adam8 promoter along with 50 bases of exon I. Based on the predicted transcription factor binding sites [30], we also cloned shorter constructs (–615/+50, –240/+50, and –106/+50) by a PCR-based approach. Each promoter fragment was subcloned into pGL3-luciferase reporter constructs. Transfection assays were carried out in primary rat granulosa cells (that can be easily transfected but lack LH-receptor) to determine the inducibility of the promoter by Fo/PMA. The –240 Adam8 promoter-luc construct exhibited the highest basal activity as well as the greatest inducible (15–20 fold) activity (Fig. 7A) that decreased with deletion to –106 Adam8-luc. These results indicated that the –240/+50 region of the promoter contained elements that are critical for the inducibility of the promoter whereas the regions distal to it have putative repressor elements. All the promoter regions were responsive to Fo, PMA, or Fo/PMA, with the latter showing an additive effect for the –106 Adam8 promoter-luc construct (Fig. 7B). Thus, the –106 Adam8 promoter-luc construct appeared to contain the minimal elements that are required for Fo/PMA-induced activation of the promoter.
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Adam8 expression is reduced in ovaries of the Pgr null mice, indicating that it is regulated in part by PGR. Therefore, a PGR-A expression vector was cotransfected with the –106 Adam8-luc promoter construct in granulosa cells. Expression of PGR-A increased the basal, Fo, PMA, and Fo/PMA inducibility 2–3 fold compared to the empty vector controls showing a stimulatory role of PGR for induction of this gene (Fig. 7B). Similar results were obtained with cotransfection of PGR-A and –240 Adam8-luc promoter-reporter construct (data now shown). AREG (100 ng/ml) alone also consistently increased –106 Adam8-luc promoter activity 1.7 fold over the basal levels and exerted an additive effect when combined with Fo (14 fold) (Fig. 7C). A similar trend of induction was observed with the –240 Adam8-luc promoter-reporter construct on AREG or Fo treatment (data now shown).
Multiple Regions of the Adam8 Promoter Are Critical for Its Inducibility in Granulosa Cells
Our previous studies with PGR target genes, namely, Adamts1 and Ctsl, documented that GC-rich Sp1/Sp3 binding sites are critical for mediating Fo/PMA and PGR effects [3, 31]. Therefore, when computer analyses of the Adam8 promoter identified the presence of three GC boxes—II, III, and IV (–296, –277, and –231, respectively)—within the –615 to –220 region (Fig. 8A), these GC boxes were mutated to generate a triple mutant in the –615/+50 version of the Adam8 promoter. However, these mutations did not alter the basal and Fo/PMA inducibility of the promoter (Fig. 8B). Since the region extending between the –106 bp and the transcription start site was enough to confer Fo/PMA responsiveness, we reasoned that this region might contain the key regulatory sites for the induction of the promoter. Studies by Katoaka et al. [30] predicted the presence of binding sites for NF-1 and IRF-1 as well as a TATA box in this regions (Fig. 8B); however the interferon regulatory factor-1 (IRF-1) site did not match with the IRF-1 consensus sequence [32]. In addition to the above-mentioned sites, we noted another GC box adjacent to the putative NF-1 site in the proximal promoter (GC box I; Fig, 8, A and B). The GC box I (GGAGG) is identical to SP1/SP3 binding sites found in the promoters of the Pgr and Adamts1 genes [28, 31] (Fig. 8C). Mutation of the GC box I and TATA box individually did not alter the basal or Fo/PMA inducibility of the promoter; however, a double mutant that disrupted the GC box I and TATA box exhibited 25% reduction in basal and Fo/PMA inducibility of the promoter (Fig. 8B). Mutation of NF-1 site reduced the inducibility of the promoter by 25%. Subsequently, when all three sites (GC box I, NF-1, and TATA) were mutated, the basal and Fo/PMA response declined by more than 75% (Fig. 8B). Thus, transcription activation of Adam8 is uniquely regulated by a combinatorial set of factors distinct from other LH- and PGR-regulated genes that are primarily regulated by SP1/SP3 [33].
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Our results document for the first time that the metalloprotease ADAM8 is expressed in granulosa cells of small follicles and is selectively increased in cumulus cells and granulosa cells of preovulatory follicles of the murine ovary. Specifically, Adam8 mRNA is induced in vivo by the ovulatory surge of LH/hCG. The effects of LH appear to be mediated, in part, by its ability to induce PGR and EGF-like growth factors [34, 35]. In mice null for Pgr, the levels of Adam8 mRNA were reduced markedly prior to ovulation and at 8 and 12 h post-hCG, but not following ovulation at 16 h, indicating that Adam8 is present in granulosa cells but is reduced as the cells luteinize.
As observed in vivo, low levels of Adam8 mRNA were detected in murine granulosa cells of periovulatory follicles from preantral follicle culture. Likewise, within 4 h after the addition of rhCG/rEGF on Day 12 (to mimic the LH surge in vivo), there was a dramatic increase in Adam8 mRNA levels, and these high levels persisted until 18 h, when in vitro ovulation occurred. Moreover, as observed in the Pgr null mice, a PGR inhibitor reduced Adam8 mRNA in granulosa cells from antral follicles cultured in the presence of rhCG/rEGF. Lastly, the positive effects of epiregulin and the inhibitory effects of EGFR blockers on Adam8 expression in the in vitro ovulation model support the notion that the EGFR pathway affect the expression of this protease both in vitro and in vivo. However, the fact that AREG alone did not markedly induce Adam8 in primary murine or rat granulosa cell cultures indicates that the follicle milieu provides additional critical regulatory molecules.
Analyses of Adam8 promoter-luciferase constructs provided additional support that factors downstream of LH, including PGR and EGF-like factors, regulate transcription of the Adam8 gene in vivo. Specifically, transient transfection analyses of various promoter regions from –1 to 0.1 kb were responsive to Fo/PMA, suggesting that this minimal region is sufficient to confer PRG, AREG, and Fo/PMA responsiveness in mammalian granulosa cells. Since many of the genes that are induced during the periovulatory period, including Pgr [28], Sgk1 [36], Ctsl [3], Egr1 [37], Adamts1 [31], and Areg [38], have SP1/SP3 sites that are critical for induction, we originally predicted that a triple mutant of the three GC boxes at –296, –277, and –231bp in Adam8 promoter would abolish promoter activity. However, mutation of these GC boxes did not alter the basal or Fo/PMA inducibility of the Adam8 promoter. Within the 100 bp region, the predicted IRF-1 site [30] did not match with reported consensus AANNGAA [32]; hence, we focused on the NF-1 and TATA site. Mutation of the putative NF-1 site alone reduced the Fo/PMA inducibility of the promoter, suggesting that NF-1 or an NF-1-like factor [31] may be involved. The Adam8 promoter TATA box (TATAGAG) did not match the consensus TATA box TATA(A/T)A(A/T) [39], suggesting that the Adam8 TATA is an unusual or weak TATA box. Moreover, mutation of the TATA box alone did not alter the Fo/PMA inducibility of the promoter. Although the GC box (I) had a consensus SP1/SP3 site binding sequence similar to that characterized in the Pgr and Adamts1 promoters [28, 31], mutation of this region alone also did not alter Adam8 promoter activity (Fig. 8B). However, a double mutation of GC box I and the TATA box and a triple mutant (GC box I, NF-1 site, and TATA) reduced basal and agonist-regulated Adam8 promoter activity 20% and 80%, respectively. Thus, transcription factors binding to these three regions exert a cooperative effect, and any two of the three sites appear sufficient for Adam8 promoter activation. In conclusion, the interaction of PGR with NF-1 and/or SP1/SP3 in these regions of the Adam8 promoter provides a novel regulatory mechanism of induction of a gene in the metzincin family in addition to that observed with the Adamts1 promoter [31].
Although the precise function of ADAM8 in the ovary remains to be determined, its expression in the embryonic gonad [40, 41] and its induction in periovulatory follicles in vivo and in vitro as well as in the proestrous uterus [42] indicate that ADAM8 may have more than one function in the developing ovarian follicles and in fertility. As a metalloprotease, ADAM8 could influence the ovulatory process by modifying the extracellular matrix. Specifically within the follicle, ADAM8 may be critical for the proteolytic release of matrix-associated factors (ectodomain shedding) [12, 13, 43] involved in the expansion of the cumulus oocyte complex and cell migration, which would be similar to its activities in glioma cells [19]. Ectodomain shedding that causes the release of EGF-like growth factors from the matrix following LH surge may also facilitate LH action during ovulation [35]. Preliminary studies document the expression of FCER2 (CD23) and CHL1, known substrates of ADAM8, in the ovary (data now shown). Cumulus cells of COCs have been shown to express genes that are highly relevant to neuronal and immune function, including MBP, another known ADAM8 substrate [44]. Since MBP is known to stabilize membrane structures [45], cleavage of MBP by ADAM8 may alter cumulus cell functions. Despite these putative roles for ADAM8, mice null for Adam8 do not exhibit an overt anovulatory phenotype and are fertile, possibly because of potential functional redundancies with many other proteases expressed and/or activated during ovulation. Collectively, our results document for the first time that Adam8 is expressed in granulosa cells of primary/small preantral follicles within the adult murine ovary and is induced selectively in granulosa/cumulus cells during the periovulatory period in vivo and in culture by the combinatorial effects of LH, PGR, and EGF-like factors. To our knowledge ADAM8 is the first member of the ADAM family that has been shown to be hormonally regulated.
FOOTNOTES
1Supported by NIH grants HD16229 and HD07495 (SCCPIR) (J.S.R.) and by the King's College (J.W.B.). V.S. is supported in part by the NICHD and Office of Research on Women's Health as a Building Interdisciplinary Research Careers in Women's Health Scholar K12HD052023 and ASRM/Organon grant. 
Correspondence: 2Venkataraman Sriraman, Department of Internal Medicine,3 Division of Endocrinology, University of Texas Medical Branch, Galveston, Texas 77555. FAX: 409 772 8709; e-mail: vesriram{at}utmb.edu
Received: 25 October 2007.
First decision: 5 November 2007.
Accepted: 1 February 2008.
REFERENCES
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  J. Kim, I. C. Bagchi, and M. K. Bagchi Signaling by Hypoxia-Inducible Factors Is Critical for Ovulation In Mice Endocrinology, July 1, 2009; 150(7): 3392 - 3400. [Abstract] [Full Text] [PDF]
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