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Division of Research, Department of Obstetrics, Gynecology and Women's Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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adenoviral vectors, cAMP signaling, Cox1 and Cox2 mRNA, cyclic adenosine monophosphate, female reproductive tract, gene regulation, LHCGR, Lhcgr gene therapy, LHRKO, luteinizing hormone, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LH released from anterior pituitary gland and hCG released from human placenta are structural and functional homologs that bind to the same G-protein-coupled receptors, which are also present in uterus of a large number of species [6, 7]. The uterine receptor activation results in numerous changes that are considered essential for pregnancy initiation [6, 8]. One of the changes is the upregulation of Cox2 gene expression in endometrial epithelial and stromal cells [8]. This upregulation occurs via an increase in stability of the transcripts rather than an increase in the transcription of the gene [8]. The hCG/LH actions require their receptors, as inhibition of their synthesis by treatment with phosphorothioate antisense receptor oligodeoxynucleotide resulted in a loss of Cox2 response [9]. Cyclic AMP/protein kinase A signaling is required for the hCG/LH actions, as inhibitors of PKA activation could block Cox2 response to hCG [9].
We recently knocked out Lhcgr by gene targeting in embryonic stem cells [10]. The null animals (herein designated LHRKO) have a morphological uterine phenotype, which includes a dramatic decrease in uterine weight, thickness of uterine layers, the number of endometrial glands, luminal epithelial cell height, and vascular space [11]. The molecular phenotype includes decreased expression of 89 genes and increased expression of 66 by 3- to 54-fold as determined by mouse genome U74Av2 Affymetrix genechips [12]. The null uterine phenotype was not entirely due to a decrease in serum estradiol and progesterone levels [12]. In fact, null animals could not implant wild-type donor blastocysts even after estradiol and progesterone therapy [11].
Uterus also contains COX1 and its distinct role from COX2 was revealed by gene-knockout studies. For example, while Cox1 gene inactivation resulted in limited parturitional defects, the Cox2 gene inactivation resulted in an infertility phenotype, which included decidualization failure [5]. Because of the potential importance of uterine LHCGR in implantation through increasing Cox2 gene expression, we used LHRKO animals and adenoviral-directed gene therapy to further investigate the dependence of uterine Cox2 gene expression on LH signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LHRKO mice, generated by gene targeting in embryonic stem cells, were maintained according to the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All studies have been approved by the Animal Care and Use Committee at our institution. The animals were maintained on 12L:12D with food and water provided ad libitum. Adult male and female heterozygous mice were mated to obtain wild-type (+/+), heterozygous (±) and homozygous (–/–) animals. The zygosity was determined by polymerase chain reaction (PCR) of tail genomic DNA. The uteri from 60-day old animals were removed and processed immediately for further experiments. The wild-type animals were sacrificed at random during the cycle. The null animals were placed on 21-day estradiol/progesterone replacement therapy to stimulate uterine growth before sacrifice. Three to six mice were used for each experiment.
Semiquantitative Reverse Transcription-PCR
Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA). Twenty-five microliters total reaction mixture containing 3 µg of total RNA, 1.5 µg of random hexamers, 40 U of RNasin Plus RNase Inhibitor, and 30 U of avian myelablastosis virus reverse transcriptase was incubated for 60 min at 37°C followed by another 5-min incubation at 95°C. Then 32 cycles of PCRs were performed using primer sets for either Cox1 or Cox2 and Rpl19 (also known as ribosomal protein large subunit19) as an internal standard. The reaction mixture contained 1.0 µM of forward and reverse primers, 2 mM MgCl2, 1 µl cDNA, 200 µM dNTP, and 1.5 U of Taq DNA polymerase (Promega, Madison, WI). Each cycle consisted of denaturation for 45 sec at 94°C, annealing for 45 sec at 57°C, extension for 75 sec at 72°C, and the last extension for 5 min at 72°C. The amplified PCR products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide, and analyzed using TotalLab V 2.01 (Nonlinear USA Inc., Durham, NC) image analysis software. Then the ratio of Cox1 or Cox2 to Rpl19 mRNA was calculated and set at 1.0 for wild-type animals or noninfected cells. Procedural controls included omissions of RNA or cDNA templates, reverse transcriptase, or random primers. PCR primers were designed according to the sequences obtained from GenBank using the Vector NTI computer program and synthesized by Qiagen (Valencia, CA). The primer sequences were Cox1: forward, 5'-TGCCCTCACCAGTCAATCCCTG-3', and reverse, 5'-TGGGGATAAGGTTGGACCGCAC-3'; Cox2: forward, 5'-TTTGCCCAGCACTTCACCCATC-3', and reverse, 5'-CTTCCTGCCCCACAGCAAACTG-3'; Rpl19: forward, 5'-CTCAGGCTACAGAAGAGGCTT-3', and reverse, 5'-GGACAGAGTCTTGATGATCTC-3'.
Preparation and Culture of Endometrial Stromal and Myometrial Smooth-Muscle Cells
Dissected fat-free uteri were placed in ice-cold Hanks HEPES solution, slit longitudinally and cut into small pieces. The pieces were immersed in Hanks solution containing 3.7 mg/ml of trypsin type I (Sigma, St. Louis, MO) and incubated for 60 min at 4°C followed by another 60-min incubation at 22°C. Then the tissue pieces were washed and digested for 30 min at 37°C in Hanks solution containing 0.3 mg/ml of trypsin type 1, 0.3 mg/ml of collagenase type I A, and 0.15 mg/ml of DNase I (Sigma) to release endometrial cells. Then the tissue pieces were washed and digested again for 1 h at 37°C with a Hanks solution containing 0.2 mg/ml of trypsin type I, 0.5 mg/ml collagenase type I A, 0.1 mg/ml DNase I, and 0.2 mg/ml of EDTA to release myometrial smooth-muscle cells, which were recovered after passing the digest through 50 mesh filters.
The endometrial stromal and myometrial smooth-muscle cell suspensions were centrifuged for 10 min at 300 x g and washed three times with Hanks solution. The cells were counted in a hemocytometer and the viability was determined by trypan blue exclusion that was greater than 85%. The cell suspensions were seeded into 25-cm2 flasks at a final density of 2–3 x 106 cells in 5 ml of Dulbecco modified Eagle medium/F12 containing fetal bovine serum (10% v/v), penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (10 µg/ml).
Endometrial stromal cells were maintained for 1 h at 37°C in a humidified air atmosphere containing 5% CO2. During this period, most of the cells begin to attach. The attached cells were washed several times to remove the luminal and glandular epithelial cells. The purity of stromal cells was determined by immunostaining with anti-vimentin antibody (Sigma), which was estimated to be greater than 95%. The washed endometrial stromal cells were cultured at 37°C in humidified air containing 5% CO2 to grow until adequate cell numbers were obtained for adenoviral infection.
Myometrial cells were maintained for 16 h at 37°C in humidified air containing 5% CO2, which allowed fibroblasts to adhere to the culture flasks. Unattached smooth-muscle cells were transferred to new flasks and cultured at 37°C in humidified air containing 5% CO2. The purity of smooth-muscle cells was determined by immunostaining with anti-smooth-muscle actin monoclonal antibody (Sigma), which was estimated to be greater than 95%. To obtain adequate cell numbers from null uteri for adenoviral infection, pooled uterine tissue was used for cell dispersion. The dispersed null endometrial stromal and myometrial smooth-muscle cells failed to grow during culture. Therefore, we had to seed them at high density for primary cultures and avoid the multiple washing steps to use cells from primary culture for viral infection.
Preparation of Adeno Viral Particles Containing Lhcgr Gene, Infection, and Treatment of Uterine Cells
Adenoviral stocks containing native or constitutively activated mutant (LhcgrD578H) human Lhcgr were gifts from Dr. Anthony J. Zeleznik (University of Pittsburgh School of Medicine, Pittsburgh, PA) [13]. They were propagated by infecting the HEK293 cells. Monolayers of stromal and smooth-muscle cells in culture were infected either with adeno-native Lhcgr or adeno-LhcgrD578H viruses for 48 h. The infected cells were then incubated for 24 h with either no hCG or with 100 ng/ml of highly purified hCG (CR127; 14 900 IU/mg, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, NIH).
Cyclic AMP Assay Measurement
Media cyclic AMP levels were quantified using an enzyme immunoassay. Samples were assayed according to the manufacturer instructions (Cayman Chemical Co., Ann Arbor, MI). The results were calculated by enzyme immunoassay analysis tools available on the Cayman Chemical web site.
Statistical Analysis
The data were presented as means ± SEM. Significant differences were obtained by the data analysis by ANOVA and Duncan multiple range test [14].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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If the decrease in Cox2 mRNA levels was due to a loss of uterine LH receptors, then gene therapy to restore receptors should bring back to wild-type Cox2 mRNA levels. Lhcgr gene therapy was used to test this possibility. Figure 2 shows that the hCG treatment or the transfer of native or activated Lhcgr genes had no effect on Cox1 mRNA levels in null (Fig. 2A) and wild-type (Fig. 2B) endometrial stromal cells. While hCG treatment resulted in a small but significant increase in Cox2 mRNA levels in wild-type stromal cells (Fig. 2B), the treatment failed in null cells (Fig. 2A) unless native Lhcgr was first introduced by adenoviral-directed gene therapy. While the transfer of native Lhcgr alone had no effect, the transfer of activated Lhcgr resulted in a Cox2 mRNA increase to the levels seen in hCG-treated null stromal cells containing transferred native Lhcgr (Fig. 2A).
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The Cox2 mRNA increase was exaggerated in hCG-treated wild-type stromal cells that contained transferred native Lhcgr (Fig. 2B). The hCG treatment was not needed for wild-type stromal cells that contained activated Lhcgr. In fact, the increase of Cox2 mRNA was the same as in hCG-treated wild-type stromal cells containing transferred native Lhcgr (Fig. 2B).
The results obtained on null and wild-type myometrial smooth-muscle cells paralleled those obtained on stromal cells. Thus, hCG treatment or the transfer of native or activated Lhcgr had no effect on Cox1 mRNA levels (Figs. 3A and 4B). Null cells' Cox2 mRNA levels were not responsive to hCG treatment unless native Lhcgr were first introduced by gene therapy (Fig. 3A). As expected, wild-type cells could modestly respond to hCG treatment, which became exaggerated following gene therapy (Fig. 3B). While transfer of native Lhcgr alone had no effect, the transfer of activated Lhcgr resulted in a significant Cox2 mRNA increase to the same degree as in hCG-treated cells containing introduced native Lhcgr (Fig. 3B).
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The hCG and LH use multiple signaling pathways, with cyclic AMP being the most common among them in nongonadal cells, just as in gonadal cells [9, 15]. We used cAMP measurement in the present study to determine the hCG response in the absence or presence of transferred native or activated Lhcgr in null and wild-type endometrial stromal and myometrial smooth-muscle cells. As shown in Figure 4A, null endometrial stromal and myometrial smooth-muscle cells did not respond to hCG unless gene therapy was used. While the transfer of native Lhcgr alone had no effect, the transfer of activated Lhcgr resulted in an equivalent response to hCG-treated cells containing introduced native Lhcgr.
As expected, the wild-type endometrial stromal cells and myometrial smooth-muscle cells showed a small but significant cAMP increase in response of hCG treatment (Fig. 4B). This increase was several fold higher in hCG-treated cells containing transferred native Lhcgr. While the transfer of native Lhcgr alone did not increase cAMP production, the transfer of activated Lhcgr resulted in a cAMP increase to the levels seen with hCG-treated cells containing introduced native Lhcgr.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Until recently, LH and hCG were not considered among the hormones that regulate uterine Cox2 gene expression. However, the studies from around the world have now demonstrated that uteri of a number of species contains LHCGR and their activation results in numerous changes, one of which is the upregulation of Cox2 gene expression [8, 9, 18–20]. This increase was seen at the mRNA, protein, and enzyme activity levels, and the increases were time and dose dependent and hormone specific [8, 9]. Blocking the cyclic AMP/protein kinase A second messenger pathway resulted in the loss of the LH and hCG's ability to increase Cox2 gene expression, suggesting that cAMP/PKA mediates their action [9]. These findings clearly establish that LH and hCG can activate Cox2 gene expression, which plays an essential role in implantation, which is defective in LHRKO animals even after estradiol and progesterone therapy [11]. The implantation failure in LHRKO animals suggested the possibility of selective Cox2 gene expression impairment in the uterus. Consistent with this possibility, uterine Cox2 mRNA levels were lower in null animals than in wild-type siblings. If these lower levels were due to a loss of uterine LHCGR, then gene therapy to introduce them should restore the hCG's ability to upregulate Cox2 gene expression. Consistent with this expectation, hCG treatment indeed increased Cox2 mRNA levels after the native Lhcgr was introduced into the null uterine cells by gene therapy.
The LHCGR activation is required for the increase of Cox2 mRNA levels. Constitutively activated receptors due to a mutation do not require activation. The concept of receptor activation is also supported by the previous findings, which demonstrated the loss of hCG response following the inhibition of LHCGR synthesis by treatment with antisense phosphorothioate oligodeoxynucleotide [9].
The wild-type uterine cells containing LHCGR served as valuable controls in further ascertaining the necessity of receptors in the LH and hCG upregulation of uterine Cox2 gene expression. Thus, wild-type uterine cells responded to hCG treatment without gene therapy. The gene therapy, however, made the hCG response exaggerated, which was probably due to elevation of cellular receptor levels.
The cAMP increase in uterine cells bearing native or activated LHCGR and after hCG treatment paralleled Cox2 mRNA responses. This finding, along with the previous ones, are consistent with the premise that cAMP is a second messenger in the LH and hCG actions to upregulate uterine Cox2 gene expression [8, 9].
The present results do not eliminate the possible involvement of other hormones, growth factors, and cytokines in the upregulation of uterine Cox2 gene expression. This is supported by the finding that null uterus does express Cox2 mRNA, even though its ability to respond to hCG stimulation was lost. This non-hCG-responsive Cox-2 mRNA could be maintained by other signals that are intact in the LHRKO animals. The continued implantation failure in estradiol/progesterone-replaced null animals could be due not only to the absence of LH responsive expression of Cox2, but also of other genes.
In conclusion, the present results with the inactivation of the Lhcgr gene and adenoviral-directed gene therapy to introduce native or activated Lhcgr add further support to the idea that LH signaling upregulates uterine Cox2 gene expression and the implantation defect in LHRKO animals could at least partly be due to the defective Cox2 gene regulation.
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FOOTNOTES |
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2 Current address: Department of Biochemistry, Fujian Medical University, 88 Jiao Tong Road, Fuzhou, Fujian, 350004, The People's Republic of China
Received: 3 February 2005.
First decision: 28 February 2005.
Accepted: 5 April 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  E.A. Campbell, L. O'Hara, R.D. Catalano, A.M. Sharkey, T.C. Freeman, and M. H. Johnson Temporal expression profiling of the uterine luminal epithelium of the pseudo-pregnant mouse suggests receptivity to the fertilized egg is associated with complex transcriptional changes Hum. Reprod., October 1, 2006; 21(10): 2495 - 2513. [Abstract] [Full Text] [PDF]
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