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Medical Research Council Human Reproductive Sciences Unit (H.M.F., J.B., H.W., P.D.T., K.M., R.A.A.) and Department of Reproductive and Developmental Sciences (W.C.D.), Centre for Reproductive Biology, Edinburgh EH16 4SB, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Hamish M. Fraser, Ph.D., Medical Research Council Human Reproductive Sciences Unit, The University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom. E-mail: h.fraser{at}hrsu.mrc.ac.uk.
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Abstract |
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Introduction |
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A novel angiogenic factor with a degree of specificity for the ovary and endocrine glands, endocrine gland vascular endothelial growth factor (EG-VEGF), has recently been described in the human ovary (6, 7, 8). The protein was designated EG-VEGF because it was also found in the testis, adrenal, and placenta (6), and this has been confirmed by others (9). However, EG-VEGF is also present at much lower levels in other tissues such as the small intestine (10), where it was discovered independently and named prokineticin (PK)-1 (10). EG-VEGF/PK-1 acts via G protein-coupled receptors, termed PK-R1 and PK-R2 (11). EG-VEGF is a member of a class of proteins that includes Bv8, otherwise known as PK-2, which has an 83% homology and appears to be preferentially expressed in the testis (12). Structurally, EG-VEGF/ PK-1 is distinct from VEGF but exhibits functional similarities.
The aim of the current study was to define the localization of EG-VEGF mRNA and determine changes in its expression in dated human corpora lutea from the early, mid-, and late luteal phase using in situ hybridization and quantitative RT-PCR analysis. In addition, to gain more information on the factors regulating EG-VEGF expression, a primary culture system for luteinized granulosa cells was employed in which the cells were stimulated with human chorionic gonadotropin (hCG) in the presence or absence of an inhibitor of progesterone synthesis.
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Subjects and Methods |
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The study was approved by the Lothian Pediatrics and Reproductive Medicine Ethics Committee, and informed consent was obtained from all patients before tissue collection. Corpora lutea were enucleated at the time of hysterectomy as described previously (13). All women were healthy and aged 32–45 yr and with benign gynecological conditions, typically uterine fibroids. Only women with regular menstrual cycles who had not received any form of hormonal treatment during the previous 3 months took part in the study. The date of the preovulatory LH surge was determined by measuring LH concentration in serial early-morning urine samples collected before operation (13). On this basis, eight corpora lutea were classified as early luteal (LH + 1 to + 5 d), 12 as mid-luteal (LH + 6 to + 10 d), and six as late luteal (LH + 11 to + 14 d). There were no differences in the average age between the groups. Serum was collected at the time of surgery for progesterone determination (13).
Corpora lutea were enucleated from the ovary by blunt dissection. The tissue was immediately divided into radial blocks to ensure standard representation of all regions of the corpus luteum, and portions were either frozen on dry ice and stored at –70 C until RNA extraction or fixed in 4% neutral buffered formalin for paraffin embedding. In each case, an endometrial biopsy was obtained, and dating of the luteal-phase endometrium (14) confirmed the luteal-phase classification.
Preparation of cDNA
A potential disadvantage of the RT-PCR approach using whole tissue containing heterogeneous cell types is that specimens have variable degrees of stromal or extracellular matrix component. To reduce this potential problem in the present study, a large proportion of the corpus luteum (between 30 and 50%) was extracted. Thus, total RNA was extracted from 0.2- to 0.5-g frozen corpora lutea samples using Tri Reagent (Sigma-Aldrich Co. Ltd., Gillingham, UK) according to the manufacturer’s instructions. The integrity of the RNA was confirmed from the absorbance 260/280-nm ratio and by ethidium bromide gel electrophoresis. RNA concentration was calculated by absorbance at 260 nm, measured on a Gene Quant-Pro spectrophotometer (Amersham Biosciences, Little Chalfont, UK).
To eliminate genomic DNA contamination, 10 µg RNA was DNase treated (RQ-1 RNase-free DNase, Promega, Southampton, UK) by incubation at 37 C for 30 min and the reaction stopped by the addition of Stop Solution (20 mM EGTA, Promega) and heating to 70 C for 10 min. Reverse transcription (RT) was performed under carefully controlled conditions, using a Taqman Reverse Transcription kit with random hexamers (Applera UK, Warrington, UK). All samples were treated simultaneously from the same reagent mix containing 2 µl RT buffer; 2 µl deoxynucleotide triphosphates (10 mM); 1 µl each of RNase inhibitor (20 U/µl), random hexamers (50 µM), and Multiscribe RT enzyme (50 U/µl); 2.4 µl MgCl2 (25 mM); and 0.2 µg of DNase-treated RNA in a final sample volume of 20 µl. RT was performed at room temperature for 10 min and at 42 C for 60 min and was stopped by incubation at 95 C for 10 min. The resulting cDNA was stored at –20 C until use. An equal amount of human placental RNA (Cambridge Bioscience, Cambridge, UK, 1 mg/ml) was concomitantly reverse transcribed to provide cDNA for the generation of standard calibration curves for each gene examined.
Primer design
Oligonucleotide PCR primers for each gene investigated were designed using Primer3 software (available at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) from DNA sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov). Primers were synthesized by MWG (MWG-AG Biotech, Milton Keynes, UK). For VEGF-A (GenBank accession no. E15157), primers were 5'-TACCTCCACCATGCCAAGTG-3' and 5'-TAGCTGCGCTGATAGACATCCA-3', giving a 103-bp product. Primers for EG-VEGF (GenBank accession no. AF333024) were 5'-GCAAGCGCCTAAAAATTGAT-3' and 5'-CCTTCTTCAGGAAACGCAAG-3' and gave a 124-bp product, and for PK-2 (GenBank accession no. AF333025), the primers were 5'-CCCACTCCTGCTCCTCTTG-3' and 5'-CCAGATACTGACAGCACAGCA-3', and the product was 133 bp.
Optimization of PCRs
Each assay was optimized with regard to anneal temperature and magnesium concentration. Firstly, PCR amplification of human placental cDNA was performed with Thermostart Taq Polymerase (Advanced Biotechnologies, Epsom, UK) at anneal temperatures between 51 and 69 C, using the gradient feature of a DNA Engine gradient cycler (MJ Research Inc., Watertown, MA). All products were examined by gel electrophoresis to confirm the presence of a single band at the correct size and to determine the optimum anneal temperature for each primer set. The identity of each PCR product was further confirmed by sequence analysis.
Successful reactions were then transferred to the LightCycler, and the magnesium concentration was titrated in the range 3–5 mM, using the LightCycler Fast Start DNA Master SYBR Green 1 kit (Roche, Lewes, UK), according to the manufacturer’s instructions. The PCR cycling conditions were denaturation at 95 C for 10 min, followed by 45 cycles of 95 C for 10 sec, annealing at the temperature defined above for 5 sec, and extension at 72 C for 15 sec. A melting curve analysis following the amplification schedule allowed optimization of the temperature used to quantify the level of gene expression by minimizing background fluorescence from nonspecifically amplified DNA. All PCR assays exhibited a single DNA melting curve peak. The optimized PCR conditions were as follows: VEGF-A annealed at 65 C using 3 mM MgCl2, and fluorescence was measured at 83 C; EG-VEGF annealed at 61 C using 5 mM MgCl2, and fluorescence was measured at 84 C; and PK-2 annealed at 66 C using 3 mM MgCl2, and fluorescence was measured at 83 C. The VEGF-A product melted at 85 C, the EG-VEGF product at 87 C, and the PK-2 product at 90 C.
Quantitative real-time PCR
Quantitative real-time PCR was performed in duplicate 10-µl reaction volumes on the LightCycler using the Mastermix supplied with the LightCycler Fast Start DNA Master SYBR Green 1 kit (Roche), at the optimized conditions described above. In each case a standard curve was generated using serial dilutions of human placental cDNA.
Using the second derivative maximum method provided in the LightCycler software (version 3.3), a standard curve was generated by plotting the external standard concentration against threshold cycle. This analysis method ensured interrun consistency. The software automatically calculated PCR product concentration for each tissue sample. All samples were analyzed in duplicate, and assay variation was typically within 10%. Data were normalized according to the expression level of glucose-6-phosphate-dehydrogenase determined in duplicate by reference to a serial dilution calibration curve as above. The generation of calibration curves for both the gene of interest and the normalizing gene allows for any difference in amplification efficiencies between the genes. PCR products were extracted from the LightCycler capillaries and analyzed on 2% agarose gels to confirm product size and reaction specificity.
In situ hybridization
To investigate possible changes in expression patterns of the mRNA for EG-VEGF in the corpus luteum, in situ hybridization was performed. Forward 5'-AGAGGCATCTAAGCAGGC and reverse 5'-AGGTATGTCTGCCTGTGTGC primers were used to generate a cDNA fragment corresponding to nucleotides 10–533 of the human sequence (GenBank accession no. NM032414). This fragment was cloned into a pGEM-T easy vector (Promega Corp., Madison, WI) containing both T7 and SP6 transcription sites. Sense and antisense probes were prepared using a RNA transcription kit (Ambion, Inc., Austin, TX) and were labeled with [35S]UTP (NEN Life Science Products, Boston, MA). The synthesized probes were purified from free bases using Chroma Spin-100 columns (Clontech Laboratories, Inc., Palo Alto, CA).
Paraffin sections (5 µm) were mounted onto SuperFrost Plus glass slides (BDH, Dorset, UK). Sections were deparaffinized in xylene and hydrated through descending concentrations of ethanol. Sections were treated with 0.1 N HCl and then digested in proteinase K (5 µg/ml; Sigma, St. Louis, MO) for 30 min at 37 C. The digestion was stopped by treating the slides with 0.2% glycine for 10 min at 4 C. After acetylation with 0.2% acetic anhydride in triethanolamine buffer (Sigma), the slides were washed in 4x SSC (saline sodium citrate). A prehybridization step was carried out by incubation in prehybridization buffer (50 µl/slide) containing 50% formamide, 4x SSC, 1x Denhardt’s solution, 125 µg/ml salmon testes DNA, 125 µg/ml yeast transfer RNA, and 10 mmol/liter dithiothreitol at 55 C in a moist chamber for 2 h. Hybridization was performed in a moist chamber overnight at 55 C. The hybridization buffer was similar to the prehybridization buffer, but contained 10% dextran sulfate additionally. Two sections per slide were exposed to the antisense or the sense sequences. After hybridization, slides were rinsed in 4x SSC and treated with ribonuclease A (20 µg/ml; Sigma) for 30 min at 37 C to remove all excess probe; desalted in descending concentrations of SSC (2x SSC for 30 min at room temperature, 1x SSC at 65 C, and 0.1x SSC at room temperature); and dehydrated for 2 min each in 50, 84, and 94% ethanol containing 0.3 mol/liter ammonium acetate. Dry slides were dipped in Ilford G5 liquid emulsion (Ilford Imaging, Cheshire, UK), exposed for 5 wk at 4 C, and subsequently developed (Kodak D19 developer, Eastman Kodak Co., Rochester, NY) and fixed (Kodak GBS). All slides were counterstained with hematoxylin, dehydrated, and mounted.
The slides were analyzed under both light- and dark-field conditions. The intensity of the hybridization signal was scored for the various compartments (granulosa-derived lutein cells, theca-derived lutein cells, stroma, and endothelium) as follows: 0, no expression above tissue background; 1, detectable grains above background; 2, low expression; 3, moderate expression; 4, high expression; and 5, intense expression with grain coalescence. Two observers scored the sections "blind." Comparison of the results between the observers showed excellent agreement.
Immunohistochemistry
To determine the origin of the steroid-producing luteal cells, 5-µm paraffin-embedded sections were immunostained for 3ß hydroxysteroid dehydrogenase (3ßHSD), which stains granulosa-derived cells strongly and theca-derived cells weakly, and 17
-hydroxylase, which stains theca-derived cells only. Paraffin-embedded tissue sections were dewaxed in xylene and rehydrated through decreasing grades of alcohol. Endogenous peroxidase activity was blocked by 30-min incubation in 3% hydrogen peroxide in methanol. For the 3ßHSD immunostain, nonspecific binding was reduced by blocking with normal goat serum and diluted 1:5 in Tris-buffered saline (TBS) containing 5% BSA. Sections were incubated overnight at 4 C in rabbit polyclonal 3ßHSD antibody (kindly donated by Prof. J. I. Mason, University of Edinburgh, Edinburgh, UK) diluted 1:1000 in normal goat serum block. Slides were washed three times in TBS. Incubation with the secondary antibody biotinylated goat antirabbit IgG (Dako Ltd, Cambridgeshire, UK), diluted 1:500, was performed for 40 min at room temperature. For the 17-hydroxylase immunostain, a normal porcine serum block was used to reduce nonspecific staining as above. The primary antibody, rabbit anti-pig 17-hydroxylase (CYP17, kindly donated by Prof. J. I. Mason), was applied at a dilution of 1:750 and incubated overnight at 4 C. A biotinylated swine antirabbit secondary antibody (Dako Ltd.) was used in a 40-min incubation as above. Detection in each case involved, after washing in TBS, a 30-min incubation in avidin-biotin horseradish peroxidase complex (Dako Ltd.) followed by incubation in diaminobenzidine (Dako Ltd.) to give a brown color. Sections were lightly counterstained with hematoxylin, dehydrated, and mounted in Pertex (CellPath, Powys, UK). In each case negative controls, with omission of the primary antibody, were included.
Collection of human luteinized granulosa cells
The Reproductive Medicine Subcommittee of the Lothian Medical Ethics Committee approved the collection of luteinized granulosa cells from patients undergoing assisted conception. Follicular fluid was collected from women undergoing transvaginal oocyte retrieval for IVF following ovarian stimulation using a standard procedure. Briefly, a long protocol-stimulated cycle was followed, using nasal naferelin (Pharmacia, Milton Keynes, Bucks, UK) for down-regulation and daily purified gonadotrophins (Menopur, Ferring Pharmaceuticals, Langley, Berks, UK) for ovarian stimulation. When at least three follicles reached 18 mm in diameter, 10,000 IU of hCG was administered. Transvaginal oocyte collection was performed under sonographic guidance 35 h later.
Granulosa cells were obtained from the follicular aspirates after the removal of the oocytes. Individual follicles were not distinguished, and all follicular fluid from the same individual was pooled and centrifuged at 1500 rpm for 10 min. The cells were resuspended in culture medium (DMEM/F12 Ham mixture, Life Technologies, Inc., Gaithersburg, MD), layered over a 45% Percoll/culture medium mixture, and centrifuged at 1200 rpm for 30 min to pellet the blood cells. Luteinized granulosa cells, visible in the interface, were collected by pipette and washed three times in PBS. The cells were resuspended in culture medium, and viable cells were counted using a Trypan blue exclusion test. Eighty thousand viable cells were plated onto each well of 24-well plates precoated with Matrigel (15, 16) and cultured using 1 ml culture medium at 37 C in 5% CO2 in air. Cells were cultured for 7–8 days using culture medium supplemented with glutamine (2 mmol/liter), insulin (6.25 µg/liter), transferrin (6.25 mg/liter), selenious acid (6.25 µg/liter), amphoteracin (2.5 mg/liter), penicillin (50 mg/liter), and streptomycin (60 mg/liter) as described previously (16). Media were changed every 2–3 d over the course of the culture period.
After 7–8 d in culture, fresh serum-free medium was added containing the following treatments: 1) low-density lipoprotein (LDL; 50 mg/liter) (Sigma), 2) hCG (10 ng/ml) (Serono) and LDL (50 mg/liter), 3) hCG (100 ng/ml), and 4) hCG (100 ng/ml) and aminoglutethamide (100 µM) (Sigma). This treatment regime was designed to manipulate progesterone concentrations in the presence of hCG. After 24 h, medium was collected for progesterone measurement, and cells were collected for mRNA extraction. The treatment regimens and doses used had previously been optimized, and the experiments were repeated in triplicate on three different occasions.
Preparation of cDNA from cultured cells
After removal and storage of culture medium, cells were rinsed in PBS and Tri Reagent was added. The resulting solution was stored at –70 C until batch extraction of RNA was carried out. RNA was extracted following the manufacturer’s instructions. To remove contaminating genomic DNA, RNA was treated with DNase I at a concentration of 1 U per µg RNA for 30 min at 37 C. After stopping the reaction with Stop Solution, the samples were heated to 70 C for 10 min. Using random hexamers, 200 µg RNA was reverse transcribed in a solution containing 5.5 mM MgCl2, 2.5 µM random hexamers, 500 µM each deoxynucleotide triphosphates, 0.4 U/µl RNase inhibitor, and 1.25 U/µl Multiscribe reverse transcriptase (PE Applied Biosystems, Warrington, Cheshire, UK). Samples were incubated at room temperature for 10 min, followed by 42 C for 60 min and 95 C for 10 min. Two controls were used; one omitted the Multiscribe enzyme and the other the template RNA. The resulting cDNA was used for PCR.
Measurement of progesterone
Progesterone concentrations in serum and in culture media collected were measured using a plate modification of a standard in-house progesterone RIA (13). This assay has a detection limit of 0.1 nmol/liter and intra- and interassay coefficient of variation of less than 4% and less than 11%, respectively.
Statistical analysis
Differences between groups with respect to level of mRNA expression after in situ hybridization were analyzed using the Kruskal-Wallis test, significance being ascribed at a level of P < 0.05. To determine changes in serum progesterone and differences in expression between stages of the luteal phase after RT-PCR and after cell culture, data were exported into Microsoft Excel, and statistical analyses (ANOVA) with the Bonferroni test for pairwise comparisons were performed with SPSS (Statistical Package for Social Sciences, SPSS Inc, Chicago, IL), version 10.7 for Macintosh, with significance being indicated by P < 0.05.
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Results |
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The serum concentrations of progesterone at the time of surgery were 24.7 ± 6.3 nmol/liter (mean ± SEM) for the early luteal phase, rising to 56.0 ± 8.1 nmol/liter at mid-luteal phase before declining significantly (P < 0.05) to 10.3 ± 4.5 nmol/liter in the late luteal phase group.
Quantitative RT-PCR
The amplification efficiencies of each primer set were similar as detected by generation of standard curves by the LightCycler software. Quantitative RT-PCR showed there was no significant change in the expression of VEGF mRNA during the luteal phase (Fig. 1
). EG-VEGF mRNA progressively increased throughout the luteal phase, showing a significant increase in expression (P < 0.05) between early and late luteal phase (Fig. 1). Expression of PK-2 showed a nonsignificant trend to increase through the luteal phase, in a similar pattern to EG-VEGF mRNA (Fig. 1).
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In situ hybridization and immunohistochemistry
In situ hybridization showed that localized concentrations of silver grains representing EG-VEGF mRNA were present in all 26 specimens studied. Typical examples from the early, mid-, and late luteal phases are shown in Fig. 2. Early luteal phase specimens had relatively low-intensity expression, whereas those of the mid- and late luteal phase showed EG-VEGF mRNA to be moderately to intensely expressed. Grains were clearly predominantly localized to the granulosa-lutein cells over which they were uniformly distributed, and this was confirmed by comparison with sections stained with 3ßHSD and 17-hydroxylase, which localized to hormone-producing and theca-derived cells, respectively (Fig. 2). The sense probe showed absence of tissue localization above background (Fig. 2J). Control sections for 3ßHSD and 17-hydroxylase immunostaining were negative (Fig. 2, K and L).
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). Endothelium showed no hybridization, whereas stromal expression was weak or absent and was unaffected by stage (Fig. 3).
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). There were no stage-specific significant differences in the low levels of stromal grain intensity.
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Incubation of luteinized granulosa cells with hCG significantly increased (P < 0.05) progesterone production and significantly (P < 0.05) increased expression of both VEGF and EG-VEGF mRNA (Fig. 5). However, stimulation of progesterone production further by addition of LDL failed to induce a further rise in either VEGF or EG-VEGF mRNA. In addition, reduction of hCG and LDL-stimulated progesterone synthesis by aminoglutethamide also failed to alter synthesis of either factor.
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Discussion |
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The observation of independent changes in levels of expression between VEGF and EG-VEGF within the corpus luteum also occurs in the human follicle (19) where EG-VEGF, but not VEGF mRNA is found in granulosa cells of primordial and primary follicles. As follicles mature, VEGF expression increases, whereas that of EG-VEGF mRNA declines in the granulosa and increases in the theca (19). In addition, different expression rates between the two factors have also been reported in a human granulosa-lutein cell line (20). The study on the human follicle (19) also described the localization of both mRNAs in the human corpus luteum. Although results on localization of VEGF mRNA to the granulosa lutein cells agree with our results using in situ hybridization (17), they differ in that the theca-lutein cells appeared to be the predominant site of EG-VEGF production (19). In our study, the site of major expression of EG-VEGF was identified as the granulosa-derived cells by visual inspection of hematoxylin-stained slides. To confirm the localization of EG-VEGF mRNA, sections of all corpora lutea were stained with 3ßHSD to localize steroidogenic cells and 17-hydroxylase to localize theca-derived cells specifically. These results established that the predominant site of EG-VEGF expression was the granulosa-lutein cells with some expression in theca-lutein cells in the early luteal specimens and the occasional positive theca-derived cell in the late luteal phase. The previous study on human ovaries (19) was based upon the ovaries of 13 patients collected at random stages of the cycle (assuming six to seven in luteal phase) and staged by histological criteria. This contrasts with the 26 corpora lutea described herein that were staged according to hormonal analysis. Staging from histological criteria alone may result in inaccurate conclusions in some cases, especially with what appears to be the late luteal phase. Both studies agree in that the early corpus luteum exhibits relatively low expression of EG-VEGF. Both studies agree that in the mid-luteal phase, EG-VEGF is highly expressed, but our results provide stronger evidence of the granulosa-lutein cells, rather than the theca-lutein, being the predominant source of EG-VEGF. However, in an ovary estimated to be at d 8 postovulation, a major decrease in VEGF signal with EG-VEGF confined to apparent theca-derived cells was reported (19). At this stage of the luteal phase, VEGF should be highly expressed (2, 17). In the absence of fundamental assessment by progesterone or 3ßHSD immunostaining, it is uncertain whether this sample was correctly timed. In our study, this stage was characterized by moderate to high EG-VEGF in all 12 specimens. Finally, in the other study of a regressing corpus luteum, both VEGF and EG-VEGF expression were virtually absent (19), contrasting markedly with our results, which showed that in all six late luteal specimens EG-VEGF mRNA was either moderately or highly expressed, including in specimens with apoptosis. It may be that for the late luteal phase ovary described (19), the age may have been underestimated, especially as the luteal cells were highly vacuolated and several large antral follicles were present (19).
With respect to the physiological roles of VEGF and EG-VEGF in the corpus luteum, it has been shown that VEGF is essential for normal luteal angiogenesis and function in the nonhuman primate (4, 5). The high level of VEGF mRNA expression during the early luteal phase (2) is likely to be responsible for the intense angiogenesis seen at this time (17). The maintenance of VEGF expression throughout the functional life span of the corpus luteum may serve as a survival factor for endothelial cells and as an ovarian permeability factor (21). Immunoneutralization of VEGF during the mid-luteal phase in the marmoset resulted in suppression of plasma progesterone levels even though the luteal microvascular tree is largely complete by this stage (21), suggesting a continued requirement for VEGF for normal luteal function.
The role of EG-VEGF has been proposed as a regulator of vascular function exhibiting a selectivity for the endocrine glands (8, 22). The current findings support the view that EG-VEGF could have an important role in the functioning of the human corpus luteum. Establishing a physiological role for EG-VEGF must await in vivo studies in which pharmacological inhibitors can be employed to selectively prevent its action or block its receptors as has been carried out for VEGF (5, 23). From the current information, what is of greatest interest is that EG-VEGF mRNA is most highly expressed during the late luteal phase. By this period, the intense angiogenesis associated with the establishment of the luteal microvascular tree has subsided (3). This supports the established role for VEGF in the intense angiogenesis that takes place during the early luteal phase. It has been demonstrated that EG-VEGF enhances the development of fenestrae that allow the passage of molecules across the vascular endothelium. The most likely role for EG-VEGF at the mid- to late luteal phase is the regulation of vascular permeability to enhance transport of LDLs into the luteal cells and secretion of progesterone and other luteal products into the bloodstream (6). This may be of particular importance in allowing the corpus luteum to respond to hCG in early pregnancy.
With respect to the factors regulating the synthesis of EG-VEGF within the corpus luteum, the two most likely candidates are LH and progesterone. LH is an essential trophic factor for luteal function (24), whereas incubation of human luteinized granulosa cells by hCG stimulates VEGF expression (25, 26). Our observation that hCG stimulates the synthesis of both VEGF and EG-VEGF in human luteinized granulosa cells provides evidence for a role of LH in the regulation of this novel factor. Because the distinguishing feature between patterns of VEGF and EG-VEGF synthesis in the corpus luteum was the increased production of EG-VEGF in the mature tissue, it was tempting to believe that synthesis of EG-VEGF could be stimulated as a result of progesterone stimulation. We therefore manipulated progesterone concentrations in the presence of hCG. The addition of LDL significantly increased progesterone concentrations but failed to produce a further rise in either factor. In addition, the reduction of progesterone to basal levels by the addition of aminogluthethamide for 24 h also failed to alter gene expression. Although a role for basal progesterone secretion cannot be excluded, these data suggest that a direct stimulation by progesterone is not a major factor in the regulation of EG-VEGF mRNA in the human corpus luteum. Additional factors regulating ovarian EG-VEGF synthesis such as hypoxia (6, 20), thrombin (20), or local growth factors such as insulin-like growth factors (27) require further investigation.
A clearer picture of the role of EG-VEGF in the ovary may become apparent when the expression patterns of its receptors, PK-R1 and PK-R2, are determined. In our laboratory, we have been unable to detect these receptors on the luteal sections by in situ hybridization, probably as a result of low levels of expression (H.W. and H.M.F., unpublished observations).
In conclusion, a profile of the expression of EG-VEGF within the corpus luteum has been provided by the complementary approach of in situ hybridization and RT-PCR. The results provide support for a role for EG-VEGF within the corpus luteum with respect to the regulation of the luteal vasculature, which is vital for normal function.
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Acknowledgments |
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Footnotes |
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First Published Online October 13, 2004
Abbreviations: EG-VEGF, Endocrine gland VEGF; hCG, human chorionic gonadotropin; 3ßHSD, 3ß hydroxysteroid dehydrogenase; LDL, low-density lipoprotein; PK, prokineticin; RT, reverse transcription; SSC, saline sodium citrate; TBS, Tris-buffered saline; and VEGF, vascular endothelial growth factor.
Received May 5, 2004.
Accepted September 24, 2004.
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S. Begliuomini, E. Casarosa, N. Pluchino, E. Lenzi, M. Centofanti, L. Freschi, M. Pieri, A.D. Genazzani, S. Luisi, and A. R. Genazzani Influence of endogenous and exogenous sex hormones on plasma brain-derived neurotrophic factor Hum. Reprod., April 1, 2007; 22(4): 995 - 1002. [Abstract] [Full Text] [PDF]
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 C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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 Q.-Y. Zhou The Prokineticins: A NOVEL PAIR OF REGULATORY PEPTIDES Mol. Interv., December 1, 2006; 6(6): 330 - 338. [Abstract] [Full Text] [PDF]
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T. Groten, H.M. Fraser, W.C. Duncan, R. Konrad, R. Kreienberg, and C. Wulff Cell junctional proteins in the human corpus luteum: changes during the normal cycle and after HCG treatment Hum. Reprod., December 1, 2006; 21(12): 3096 - 3102. [Abstract] [Full Text] [PDF]
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 A. Tropea, F. Miceli, F. Minici, F. Tiberi, M. Orlando, M. F. Gangale, F. Romani, S. Catino, S. Mancuso, P. Navarra, et al. Regulation of Vascular Endothelial Growth Factor Synthesis and Release by Human Luteal Cells in Vitro J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2303 - 2309. [Abstract] [Full Text] [PDF]
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P. Hoffmann, J.-J. Feige, and N. Alfaidy Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy Endocrinology, April 1, 2006; 147(4): 1675 - 1684. [Abstract] [Full Text] [PDF]
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H. M. Fraser, H. Wilson, K. D. Morris, I. Swanston, and S. J. Wiegand Vascular Endothelial Growth Factor Trap Suppresses Ovarian Function at All Stages of the Luteal Phase in the Macaque J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5811 - 5818. [Abstract] [Full Text] [PDF]
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W. C. Duncan, S. G. Hillier, E. Gay, J. Bell, and H. M. Fraser Connective Tissue Growth Factor Expression in the Human Corpus Luteum: Paracrine Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5366 - 5376. [Abstract] [Full Text] [PDF]
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T. Kisliouk, H. Podlovni, K. Spanel-Borowski, O. Ovadia, Q.-Y. Zhou, and R. Meidan Prokineticins (Endocrine Gland-Derived Vascular Endothelial Growth Factor and BV8) in the Bovine Ovary: Expression and Role as Mitogens and Survival Factors for Corpus Luteum-Derived Endothelial Cells Endocrinology, September 1, 2005; 146(9): 3950 - 3958. [Abstract] [Full Text] [PDF]
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