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Departments of Obstetrics, Gynecology, Reproductive Sciences, and Physiology, Center for Studies in Reproduction (A.L.N., J.S.B., G.W.A., E.D.A.), University of Maryland School of Medicine, Baltimore, Maryland 21201; and Department of Physiological Sciences, Eastern Virginia Medical School (G.J.P.), Norfolk, Virginia 23501
Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11-019, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: ealbrech{at}umaryland.edu.
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In summary, VEG/PF mRNA and protein expression in glandular epithelial and stromal cells were markedly suppressed after ovariectomy, indicating that synthesis of this angiogenic factor in these endometrial cells is dependent upon a product(s) secreted by the ovary. Moreover, endometrial VEG/PF expression remained relatively constant and thus was available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.
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Introduction |
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In vivo studies in the ovariectomized rat (6, 7, 8) and sheep (9, 10) have shown that estradiol rapidly increased uterine VEG/PF mRNA levels and angiogenesis. Moreover, because administration of a specific antagonist to VEGF disrupted both ovarian and endometrial maturation in rats (11), it appears that estrogen-induced VEG/PF is a key regulator of cyclical angiogenesis in the rodent female reproductive tract (8). A similar role for ovarian steroid hormones in human endometrial VEG/PF and angiogenesis has not been established in vivo. Thus, although VEG/PF mRNA and protein have been localized in glandular epithelial and stromal cells of the human endometrium (5, 12, 13), VEGF expression either did not change (14, 15, 16) or was only moderately higher in the secretory than the proliferative phase of the menstrual cycle (5, 12, 17, 18). Moreover, although estrogen increased VEGF expression in cultured human endometrial cells (12, 13), the extent of the increase was considerably lower than that elicited by hypoxia (18). Based on these findings as well as the observation that endometrial vascular endothelial cell proliferation and blood vessel density remained relatively constant during the menstrual cycle (16, 17, 19) despite the cyclical surges in estrogen and progesterone, it has been suggested that there is no relationship between and/or that steroid hormones are not important to the regulation of VEGF expression and angiogenesis in the human endometrium (17, 19).
For obvious ethical considerations, in vivo studies to test the role of ovarian steroid hormones in uterine endometrial VEG/PF expression and angiogenesis cannot be performed in humans, and thus our understanding of the potential requirement for ovarian factors is based on in vitro studies and correlative data obtained during the menstrual cycle. Therefore, in vivo experimental paradigms are needed to ascertain whether ovarian steroid hormones are integral to uterine VEG/PF expression and angiogenesis.
Our laboratories have been instrumental in establishing the baboon as a nonhuman primate model for the study of human reproductive and perinatal endocrinology (20). In the present study as a first step to assess the roles of estrogen and progesterone in vivo on endometrial angiogenesis, the baboon was used to investigate whether VEG/PF expression by specific cells of the endometrium is dependent upon products secreted by the ovary and is altered by the surges in estrogen and progesterone levels that occur during the menstrual cycle. VEG/PF mRNA levels were quantified by competitive RT-PCR in whole endometrium and glandular epithelial and stromal cells isolated by laser capture microdissection (LCM) from baboons during the normal menstrual cycle and after ovariectomy, which removed the principal source of estrogen and progesterone. The levels of VEG/PF mRNA in baboons were correlated with VEG/PF protein localization assessed by immunocytochemistry and blood vessel density determined by image analysis.
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Materials and Methods |
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Only female baboons exhibiting regular menstrual cycles were used for this study. The stage of the menstrual cycle was determined from serum estradiol concentrations, which peak 12–14 h before the midcycle LH surge, and daily records of external sex skin swelling/perineal turgescence, which declines 3 d after the midcycle estrogen surge, designated in this study as d 0 (21). Five baboons underwent ovariectomy to remove the principal source of estrogen and progesterone. Baboons were anesthetized with a mixture of isoflurane (1.0–1.5%), nitrous oxide (0.5 liters/min), and oxygen (2.0 liters/min), and the ovaries were exposed by laparotomy. The connecting stalk was ligated with silk, and both ovaries were surgically excised. Ovariectomized baboons were then left for at least 60 d before being treated with the highly specific aromatase inhibitor CGS 20267 (letrozole, 4,4'-[1,2,3-triazyol-1-yl-methylene]-bis-benzonitrite; Novartis Pharma AG, Basel, Switzerland) at a dosage of 0.5 mg/d, sc, for 5 d to fully suppress potential residual aromatization in nonovarian sources. Peripheral saphenous vein blood samples (2–4 ml) were obtained from baboons after ketamine HCl sedation (10 mg/kg body weight, im), and serum estradiol and progesterone concentrations were determined by RIA as described previously (22).
At designated intervals of the menstrual cycle or after ovariectomy and CGS 20267 treatment, baboons were anesthetized with isoflurane, and the uterus was exposed by laparotomy. Two 5-mm diameter core biopsies (Acu-Punch, Acuderm, Inc., Ft. Lauderdale, FL) were then taken from the uterine fundus extending transmurally from outer surface to the lumen. In the first biopsy the endometrium was macroscopically sliced from the myometrium, ensuring that a thin border of endometrium was left behind to prevent myometrial contamination. This segment was immediately frozen and stored in liquid nitrogen for subsequent whole endometrial VEG/PF mRNA analysis by RT-PCR. The entire second uterine biopsy was embedded in a cryomold filled with OCT medium (Miles, Elkhart, IN), frozen on dry ice, and stored at -80 C for subsequent LCM of endometrial cells.
A single 5-mm diameter punch biopsy was also obtained from the uterus of additional baboons, fixed in 10% neutral-buffered formalin for 24 h, washed in 0.5 M potassium phosphate buffer, embedded in paraffin, processed for immunocytochemistry, and analyzed for vessel density.
LCM of endometrial cells
Serial 8-µm sections of the uterine biopsy were cut longitudinally (to include endometrium and myometrium) on a Jung Frigocut 2800E cryostat at -20 C (Leica Corp., Deerfield, IL) and mounted onto SuperFrost Plus glass slides (Fisher Scientific, Suwanee, CA) at room temperature. Sections were immediately fixed in 70% ethanol for 30 sec, washed with distilled water, incubated in 95% ethanol (three incubations, 5 min each), immersed in a filtered solution of Eosin-Y (Richard Allen, Kalamazoo, MI) for 10 sec, dehydrated in four changes of 100% ethanol, and incubated 5, 10, and 15 min in fresh xylene (Fisher Scientific, Pittsburgh, PA). Slides were wrapped in one layer of large Kim wipes, air-dried for at least 15 min, and transferred to a desiccator containing fresh silica gel at room temperature. Immediately thereafter, an Arcturus PixCell II LCM system equipped with Olympus microscope (Arcturus Engineering, Inc., Mountain View, CA) was employed to capture glandular epithelial (but not luminal) and stromal (excluding observable blood vessels) cells from the basalis and functionalis zones of the endometrial sections. A single LCM cap (Capture Transfer Film TF100, Arcturus Engineering, Inc.), stored in a desiccator at room temperature for at least 72 h before capture, was used per tissue section. Optimal conditions for LCM capture of endometrial cells included a laser power of 40 mW, a duration of 1.5–2.5 msec, and a laser spot size of 7.5 or 15 µm for glandular epithelium (depending on gland size) and 15 or 30 µm for stroma. The LCM cap with captured cells was then tightly fitted to an Eppendorf tube containing lysis buffer (RNeasy, QIAGEN, Valencia, CA) and inverted several times at room temperature. Lysates from each tissue section were pooled into a single Eppendorf tube, which was microcentrifuged after addition of the contents of each cap. At completion of the LCM process, samples were stored in lysate buffer overnight at -80 C, and RNA was extracted within 72 h. The entire cell capture process, from tissue sectioning to tissue lysis, was rapidly and sequentially completed to limit RNA degradation.
VEG/PF mRNA competitive RT-PCR
RNA extraction.
Total RNA was extracted from whole endometrium according to the modified method of Chirgwin et al. (23). Briefly, tissues were homogenized in 4 M guanidine isothiocyanate/0.83% 2ß-mercaptoethanol and extracted with chloroform/isoamyl alcohol, and RNA was isolated by 5.7-M cesium chloride gradient centrifugation.
Total RNA was extracted from LCM-captured glandular epithelial or stromal cells by Nonidet P-40-guanidine isothiocyanate extraction/silica gel spin column centrifugation as described by the manufacturer (RNeasy, QIAGEN), although the protocol was modified to include deoxyribonuclease (DNase) treatment and salt precipitation steps. Briefly, 100 µl column-eluted RNA were mixed with 1 µl (5 mg/ml) carrier linear acrylamide (Ambion, Inc., Austin, TX) and precipitated with 10 µl 3 M sodium acetate (pH 5.2) and 2.5 vol absolute ethanol. Samples were centrifuged at 13,000 x g for 45 min at 4 C, washed in 70% ethanol, and air-dried, and the pellet was resuspended in 16 µl ribonuclease (RNase)-free water. To remove any potential genomic DNA contamination, samples were incubated at room temperature for 15 min with 2 µl (1 U/µl) amplification grade DNase 1 (Invitrogen Life Technologies, Inc., Carlsbad, CA) and 2 µl of 10x DNase I reaction buffer. DNase was inactivated at 65 C for 10 min in the presence of 2 µl 25 mM EDTA. At the end of the incubation, carrier was added, and the sample was sodium acetate/ethanol precipitated, centrifuged, and resuspended in 15 µl RNase-free water.
Although the amount of total RNA in whole endometrial tissue could be quantified by UV absorption spectrophotometry, thereby permitting normalization of VEG/PF mRNA levels, the amount of total RNA obtained from the LCM samples was low. Therefore, 18S rRNA, a cellular RNA whose expression was relatively constant in the follicular and luteal phases of the menstrual cycle (data not shown), was also quantified by competitive RT-PCR, and values were used to normalize VEG/PF mRNA levels determined in uterine cells isolated by LCM.
VEG/PF primers.
Oligonucleotide primers were synthesized by Invitrogen Life Technologies, Inc., and were based on the human VEG/PF cDNA sequence described by Tischer et al. (24). The primers flanked a portion of the sequence spanning exons 1, 2, and 3 and were designed with positions in the nucleotide sequence for VEG/PF as follows: primer 1: downstream, 5'-GGTGAGGTTTGATCCGCATAATCTGCGCATCAGGGGCACACAGGAT-3' (positions 337–312 and 244–225); primer 2: upstream, 5'-AATTTAATACGACTCACTATAGGGACTGCTGTCTTGGGTGCATTGG-3' [T7 polymerase sequence (underlined) and position 10–30]; primer 3: downstream, 5'-GGTTTGATCCGCATAATCTGC-3' (positions 332–312); and primer 4: upstream, 5'-CTGCTGTCTTGGGTGCATTGG-3' (position 10–30). Primers 3 and 4 were located upstream of the alternative splice site that generated the different VEG/PF isoforms. Hence, these primers produced a single 323-bp PCR product.
18S rRNA primers.
The 18S rRNA oligonucleotide primers were synthesized by Invitrogen Life Technologies, Inc., and selected from the human 18S rRNA gene sequence (25). The primers were designed with positions in the sequence for 18S rRNA as follows: primer 5: downstream, 5'-CGGCGTAGGGTAGGCACACGCTGAGCCAGTCAGTGTAGCGCGCGTGCAGCCCCGGACATCTAAGGGCATCACA-3' (positions 1667–1595); primer 6: upstream, 5'-GCGGCGTAATACGACTCAC-TATAGGGAGAGGAGTCAAGAACGAAAGTCGGAGGGCTTCCGGGAAACCAAAGTC-3' [T7 polymerase sequence (underlined) plus 6 extra nucleotides 5', 10 extra nucleotides 3', and positions 1126–1145 and 1235–1254]; primer 7: downstream, 5'-GGACATCTAAGGGCATCACA-3' (positions 1614–1595); and primer 8: upstream, 5'-TCAAGAACGAAAGTCGGAGG-3' (positions 1126–1145).
RNA competitor construction.
Homologous RNA competitive reference standards (CRS) that shared the same primer binding sites, but contained a shortened internal sequence with respect to the endogenous target RNA for VEG/PF and 18S rRNA, were either prepared by methods established by Riedy et al. (26) as modified by our laboratory (27) or those described in the RT-PCR competitor construction kit (Ambion, Inc., Austin, TX). Primer pairs, 1,2 and 5,6 were designed to generate respective cDNA templates for VEG/PF and 18S rRNA CRS that, when efficiently transcribed using T7 polymerase, incorporate a deleted region (67 and 89 bp, respectively) within the internal sequence of the synthesized CRS. This enables the CRS to be distinguishable from the endogenous target when the respective gene-specific primer pairs (3,4 and 7,8) are used in the RT-PCR reaction. Total RNA (0.5–3.0 µg) from baboon placenta (VEG/PF) or uterus (18S rRNA) was reverse transcribed at 42 C for 60 min in a 20-µl reaction mixture containing 1 mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP (Invitrogen Life Technologies, Inc.); 1 mM dithiothreitol; 200 U Superscript RNase H-reverse transcriptase (RT) or Moloney murine leukemia virus RT (Invitrogen Life Technologies, Inc.); 40 U RNAguard (Amersham Pharmacia Biotech, Piscataway, NJ); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; and 250 ng random primers (Invitrogen Life Technologies, Inc.). After 60 min, the RT mixture was incubated at 70 C for 15 min and cooled to 4 C, and 5 µl of the RT reaction were added to separate PCR reaction mixtures (45 µl) containing 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 1.25 U cloned Thermus aquaticus DNA polymerase (AmpliTaq, Perkin Elmer Corp./Cetus, Norwalk, CT); and 10 pmol of the respective paired primers (1,2 and 5,6) to generate cDNA templates for VEG/PF and 18S rRNA. PCR was performed in a programmable thermal cycler (MJ Research, Inc., Cambridge, MA) for 25 (VEG/PF) and 20 (18S rRNA) sequential cycles, respectively, at 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min. To obtain fully double-stranded product, the sample was further incubated for an additional 5 min at 72 C. PCR products were fractionated in 2% agarose gel containing ethidium bromide, visualized with a UV transilluminator, and gel purified using a DNA gel extraction kit (QIAGEN). The CRS was synthesized from 150 ng of each respective cDNA template using the MEGAscript T7 in vitro transcription kit (Ambion, Inc.). The synthesized VEG/PF and 18S rRNA-CRS were then treated with RNase-free DNase I (Ambion, Inc.) at 37 C for 30 min to digest the cDNA templates. VEG/PF- and 18S rRNA-CRS were extracted with chloroform/isoamyl alcohol and precipitated with ammonium acetate and isopropyl alcohol, and aliquots were quantitated using UV absorption spectrophotometry at an OD of 260 nm.
Competitive RT-PCR.
VEG/PF and 18S rRNA mRNA levels were simultaneously quantified by a competitive RT-PCR assay (26, 27). A constant amount of RNA (1.5 µl of the estimated volume of the LCM sample or 10 ng of whole endometrium) was added to an RT mixture containing 2-fold serial dilutions of both VEG/PF-CRS (25–3.12 attomoles) and 18S rRNA-CRS (5–0.10 fmol). To test for possible pseudogene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. At least four points of the CRS curve were used for both VEG/PF and 18S rRNA analysis.
Upon completion of the RT, 5 and 2 µl of the RT mixture for VEG/PF and 18S rRNA, respectively, were added to separate PCR reaction mixtures (45 and 48 µl, respectively) containing 10 pmol of the respective paired primers (3,4 and 7,8) for VEG/PF and 18S rRNA. VEG/PF total endometrial, VEG/PF LCM, and 18S rRNA LCM samples were amplified for 32, 34, and 22 sequential cycles, respectively; PCR products (323-bp VEG/PF target and 256-bp CRS, or 489-bp 18S rRNA target and 400-bp CRS) were gel fractionated, visualized with ethidium bromide, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA).
Negatives were scanned using a Gel Doc 1000 imaging system and MultiAnalyst software program (Bio-Rad Laboratories, Inc., Hercules, CA). The intensity of the amplified products was represented as the relative area under each product band. A correction factor (28) was used to account for the relative size difference between the target and the CRS cDNAs. The logarithm (log) of the ratio of CRS to target area was plotted as a function of the log concentration of VEG/PF or 18S rRNA CRS added to each PCR reaction. The concentration of VEG/PF or 18S rRNA target mRNA was determined where the ratio of the log of CRS and target area was equal to 0 (i.e. the equivalence point).
VEG/PF isoforms.
To identify which VEG/PF isoforms were expressed in the baboon endometrium, RT-PCR was performed as described above using 1 µg total RNA and VEG/PF primers (7) that spanned the alternative splice site.
Qualitative analysis of RNA.
The quality of total RNA extracted from glandular epithelial cells isolated by LCM was analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA, isolated via the RNeasy kit, was resuspended in 5 µl RNase-free water, and a 2-µl aliquot was added to a single well of an RNA 6000 Nano LabChip (Agilent) containing a gel-dye mix and marker. The sample components were separated by electrophoresis, and 28S and 18S rRNA bands were detected by fluorescence and translated into a gel-like image (bands) and electropherogram (peak profiles).
Immunocytochemistry of VEG/PF, cytokeratin, vimentin, and factor VIII
Immunocytochemistry of VEG/PF and other factors was carried out as described previously (29). Paraffin blocks of uterine tissue were serially sectioned (4 µm), deparaffinized, and rehydrated in graded alcohols. Tissue sections were untreated (cytokeratin and vimentin) or boiled in 0.01 M sodium citrate (pH 6.0) for 10 min (VEG/PF and factor VIII), incubated in H2O2 to quench endogenous peroxide, and blocked by incubation in 10% normal goat serum (Protein Block Serum, DAKO Corp., Carpinteria, CA). Tissue sections were incubated overnight at 4 C with goat antihuman primary antibody to VEG/PF (AF-293-NA, 1:25 dilution, specified for the 121, 165 and 189 splice variants, R&D Systems, Minneapolis, MN), human polyclonal rabbit antibody to Von Willebrand factor (Factor VIII, DAKO Corp.), mouse antihuman cytokeratin (DAKO Corp.), or mouse antihuman vimentin (DAKO Corp.). Tissues were then incubated with biotinylated antigoat, antirabbit, or antimouse Igs (Vector Laboratories, Inc., Burlingame, CA) for 60 min at room temperature, then immersed in an avidin-biotin complex solution (Elite Vectastain ABC Kit, Vector Laboratories, Inc.). Sections were incubated with 3,3'-diaminobenzidine (0.2 mg/ml; Sigma, St. Louis, MO) for VEG/PF, cytokeratin, and vimentin to produce a brown reaction product or with 2.5% nickel sulfate and diaminobenzidine for factor VIII to produce a black reaction product. Negative controls included omission of the primary antibody, substitution of Igs (DAKO Corp.) for primary antibody, and/or preabsorption of primary antibody with a 10-fold excess of control peptide for human recombinant VEG/PF (R\|[amp ]\|D Systems, Inc.). Sections were counterstained with eosin (factor VIII) or Harris hematoxylin (VEG/PF, cytokeratin, and vimentin).
Vessel quantification by image analysis
Vessel counts were performed using a Nikon Eclipse E 1000M microscope (Nikon, Tokyo, Japan), as described previously (29). Images were digitized with a Power Macintosh G3 computer and assessed using an image analysis software package (IP Lab Scientific Image Processing, Scanalytics, Fairfax, VA). Lumens of factor VIII-immunoreactive vessels, borders of endometrial glands, and regions of endometrium analyzed were recorded in square microns, and data were imported into Excel files. The maximum microscopic field size was 0.142 mm2, and final values were converted to vessels per square millimeter. At a x100 objective, 2 fields of view per slide were randomly selected for analysis of the basalis (not further than 1 field of view from the myometrium) and functionalis (close proximity to luminal surface epithelium). A total of 15 slides (30 field of views of endometrium), represented equally from multiple levels of the tissue block at least 50 µm apart, were used for determination of the following parameters:
Vessel density, i.e. number of vessels per square millimeter of stroma, was calculated as the sum total of the number of vessels analyzed (Vn) multiplied by a conversion factor (CF; changed square micrometers to square millimeters) divided by the sum total of the endometrial field of view area (Ea) minus the sum total of the glandular area (Ga): vessel density = [
(Vn) x CF]/[(Ea) - (Ga)].
The percent vascularized area, i.e. the proportion of endometrial stroma occupied by vessels, was calculated as the sum total of the vessel area (Va) divided by the sum total of the endometrial area (Ea) minus the sum total of the glandular area (Ga) multiplied by 100: % vascularized area = [(Va) x 100]/[(Ea) - (Ga)]. Vessel lumen areas were grouped according to size, and the distribution of vessels within the endometrial stroma was determined as a sum percentage of vessels for each category.
The percent glandular area, i.e. the proportion of the endometrium occupied by glands, was calculated by dividing the sum total of the glandular epithelial area (Ga) by the sum total of the endometrial area (Ea) multiplied by 100: % glandular area = [(Ga) x 100]/(Ea).
Statistical analysis
Data were expressed as the mean ± SE and were analyzed by ANOVA, with post hoc comparisons of means by Newman-Keuls multiple comparison test or unpaired t test. Slopes of the log of CRS and target ratio vs. the log concentration of CRS were determined by linear regression, and correlation coefficients were analyzed by ANOVA.
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Results |
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. Serum estradiol concentrations increased from 30–60 pg/ml in the follicular phase to a peak of 250–300 pg/ml at midcycle, then abruptly declined before increasing to a plateau of approximately 50 pg/ml in the luteal phase. Approximately 3–4 d after the midcycle surge in estradiol there was a marked increase in progesterone to maximal levels of 10–12 ng/ml, which reflect the formation and function of the corpus luteum. With regression of the corpus luteum there was a precipitous decline in serum progesterone concentrations. After ovariectomy and CGS 20267 treatment, estradiol and progesterone were not detectable in peripheral serum, i.e. less than 10 pg/ml and 0.10 ng/ml, respectively.
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), a pattern similar to that reported in the human (5).
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illustrates a representative analysis of VEG/PF mRNA levels determined by competitive RT-PCR, using primers upstream from the alternative splice site to yield a 323-bp product that reflected collective expression of all of the VEG/PF isoforms, in whole endometrial tissue obtained in the late follicular and midluteal phases of the baboon menstrual cycle. The 323-bp VEG/PF target and 256-bp VEG/PF CRS products generated by PCR appear in Fig. 3A. PCR products were not obtained when RNA or RT enzyme was omitted from the reaction. The slopes of the log of CRS and target areas plotted as a function of the log of increasing CRS concentrations are shown in Fig. 3B; the correlation coefficient was 0.92 (P < 0.03) for follicular samples and 0.96 (P < 0.02) for luteal samples.
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). Mean (±SE) VEG/PF mRNA levels in whole endometrium were 785 and 727 ± 158 attomoles/µg total RNA in the mid and late follicular phase, respectively, and 1108 ± 320 attomoles/µg total RNA during the midcycle surge in estradiol. VEG/PF mRNA levels then declined briefly before increasing to 1029 ± 365 attomoles/µg RNA during the late luteal phase of the menstrual cycle (Fig. 1). Whole endometrial VEG/PF mRNA levels were similar in baboons during the midcycle estradiol surge (1108 ± 320 attomol/µg RNA; n = 6) and after ovariectomy (603 ± 66 attomol/µg total RNA; n = 5).
Figure 4 illustrates the isolation of glandular epithelial and stromal cells by LCM from the endometrium at the midluteal stage of the baboon menstrual cycle. The endometrium is shown before (Fig. 4A) and after (Fig. 4B) LCM isolation of cytokeratin-positive glandular epithelial cells. A homogenous population of glandular epithelial cells devoid of stroma was obtained, as evident by extensive immunocytochemical staining for epithelial cell-specific cytokeratin, by cells captured on the LCM cap to be used for mRNA analysis (Fig. 4C). The endometrium is also shown before (Fig. 4D) and after (Fig. 4E) LCM isolation of vimentin-positive cells, but not blood vessels, from the stroma. A homogenous population of stromal cells devoid of epithelium was obtained, as evident by the intense immunoreactivity for vimentin by cells captured on the LCM cap (Fig. 4F).
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A representative competitive RT-PCR analysis of VEG/PF mRNA levels in glandular epithelial cells isolated by LCM from baboons after ovariectomy and during the midcycle estradiol surge appears in Fig. 5. Despite the degradation of some of the RNA after LCM, PCR amplification was linear (ovariectomy: r2 = 0.95; P < 0.05; estradiol surge: r2 = 0.99, P < 0.001; Fig. 5B), apparently because RNA was intact within the region spanned by our sets of primers. Moreover, the relative VEG/PF mRNA level, determined from the equivalence points, was much less in glands of ovariectomized baboons compared with those of intact baboons.
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shows the expected VEG/PF 323-bp target and 256-bp CRS products (Fig. 6A) and 18S rRNA 489-bp target and 400-bp CRS products (Fig. 6B) generated from glandular epithelial and stromal cells isolated by LCM from the endometrium during the midcycle estradiol surge. Mean (±SE) VEG/PF mRNA levels corrected for 18S rRNA levels (attomoles of VEG/PF mRNA per femtomole of 18S rRNA) were similar in glandular epithelial (2.27 ± 1.11) and stromal (2.54 ± 0.70) cells at the time of the midcycle estradiol peak (Fig. 6C, left panel). Moreover, VEG/PF mRNA levels in glandular epithelium and stroma were similar at the midcycle estrogen surge and mid to late luteal phases, although VEG/PF mRNA expression in the stroma appeared lower in the luteal phase compared with the estrogen surge stage of the menstrual cycle. After ovariectomy, VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) in the glands (0.52 ± 0.21; Fig. 6C, right panel) and stroma (0.22 ± 11) were decreased to values approximately 20% and 10% (P < 0.05), respectively, of those in intact baboons during the midcycle estradiol surge phase of the menstrual cycle.
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). Typically, VEG/PF localization was most intense at the apical border of the endometrial glands. VEG/PF staining in glandular epithelium and stroma appeared similar in the functionalis and basalis zones of the endometrium. However, the intensity of VEG/PF immunostaining, particularly in the stroma, was lower during the luteal (Fig. 7, B and C) compared with midcycle estrogen surge (Fig. 7A) phases of the menstrual cycle. After ovariectomy of baboons, the endometrial glands were relatively small and were comprised of simple cuboidal/columnar epithelial cells, whereas the stroma was densely populated, with highly basophilic cells containing little cytoplasm (Fig. 7, D and E). Moreover, there was relatively little immunostaining for VEG/PF protein in the glands, stroma, and vascular endothelium after ovariectomy. The specificity of VEG/PF immunocytochemistry was evident by the absence of staining when primary antibody was preabsorbed with VEG/PF (Fig. 7F).
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), and were not significantly different during the follicular, midcycle estrogen surge, and luteal phases of the baboon menstrual cycle. The level of vascularization, whether determined as vascular density or area, appeared greater in the basalis than in the functionalis (Table 1), where values also were not significantly different during advancing stages of the menstrual cycle.
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) and functionalis (data not shown) had lumen areas less than 100 µm2, and more than half of all vessels had areas less than 40 µm2. However, there was no apparent difference in the distribution of vessel areas in the endometrial basalis among the follicular, estrogen surge, and luteal stages of the menstrual cycle (Fig. 8).
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Discussion |
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The current study also shows that homogenous populations of glandular epithelial and stromal cells can be isolated by LCM from the primate endometrium and used to quantify mRNA levels of regulatory factors such as VEG/PF. Furthermore, we show that glandular epithelial and stromal cells expressed comparable levels of VEG/PF mRNA, and that VEG/PF mRNA levels in glandular epithelial cells as well as in whole endometrium were relatively similar in the proliferative, midcycle estradiol surge, and secretory phases of the baboon menstrual cycle. However, VEG/PF mRNA expression in stromal cells appeared to decrease, but not significantly, in the secretory phase of the menstrual cycle in the baboon, as previously reported in the human (13, 19). Consistent with our observations in the baboon, glandular epithelial and stromal VEG/PF mRNA and protein expression determined by quite different approaches in the human endometrium were either similar (14, 15, 16, 19) or moderately greater (5, 12, 17, 18) in the secretory compared with the early proliferative phase of the menstrual cycle. The Fms-like tyrosine kinase (flt-1) and kinase-insert domain-containing (KDR/flk-1) VEG/PF receptors are localized in vascular endothelial cells of the human (16, 32), macaque (32), and baboon (Albrecht, E. D., and G. J. Pepe, unpublished observations) endometrium. Thus, VEG/PF expressed by the glandular epithelial and stromal compartments and VEG/PF receptors within the respective vascular endothelium are available as components of the angiogenic system throughout the menstrual cycle to promote vascular reconstruction of the endometrium.
Coinciding with the maintenance of VEG/PF expression during the follicular and luteal phases of the baboon menstrual cycle, the current study further shows that vessel density and percent vascularized area of both the basalis and functionalis zones of the endometrium remained relatively constant. Endometrial microvascular density (16, 33) and endothelial cell proliferation (19) and density (17) also were unchanged during the human menstrual cycle. It appears, therefore, that VEG/PF expression by and neovascularization of the endometrium represent sustained and ongoing processes designed to gradually and continually support and promote the growth and development of the endometrium with advancing stages of the primate menstrual cycle.
Because endometrial VEG/PF expression and vessel density did not change during the human menstrual cycle despite the surges in estrogen and progesterone, it has been concluded that VEG/PF formation and vascularization are not regulated by steroid hormones in the endometrium (17, 19). Considering the marked decline in VEG/PF expression induced by ovariectomy of baboons and the rapid stimulatory effect of estrogen on uterine VEG/PF expression in vivo in the rat (7, 8) and sheep (10), it seems unlikely that this is the case. Rather, we propose that the levels of estrogen, albeit low preceding and following the midcycle estrogen surge, nevertheless are necessary and sufficient to sustain VEG/PF formation. We believe that the nonhuman primate baboon model, in which invasive experimental approaches such as ovariectomy can be performed, provides a unique opportunity to study this important process in vivo.
Although VEG/PF mRNA levels in glandular epithelial and stromal cells were markedly suppressed by ovariectomy, mRNA levels for this angiogenic factor were only decreased by approximately 50% in whole endometrial tissue of ovariectomized baboons. Although VEG/PF protein immunostaining appeared to be markedly decreased throughout the endometrium after ovariectomy, it is possible that VEG/PF mRNA synthesis by those tissues not removed by LCM from the endometrium of animals of the present study was less dependent upon the ovary. For example, blood vessels, which express/synthesize VEG/PF within their vascular endothelial cells (34) and luminal epithelium, were not captured from the baboon endometrium by LCM for the purposes of the present study. Therefore, in contrast to the glandular epithelium and stroma, VEG/PF expression by other cells in the primate uterine endometrium may be constitutive. Consequently, the ovarian-dependent regulation of angiogenic factors in the primate uterus may be cell specific. Collectively, these data point to the value of isolating specific cell types, e.g. by LCM, when studying the expression of factors in heterogeneous tissues such as endometrium.
Although there was some RNA degradation in cells isolated from endometrium by LCM, the RNA was apparently intact within the region spanned by our sets of primers and therefore did not compromise quantitative analysis. Thus, PCR amplification proceeded linearly in all RNA samples obtained by LCM. Moreover, all tissues underwent exactly the same LCM and competitive RT-PCR processing, yet VEG/PF mRNA levels were distinctly different between certain experimental groups, i.e. much greater in LCM-isolated endometrial cells of intact compared with ovariectomized baboons. Finally, VEG/PF mRNA values in all LCM samples were corrected for 18S rRNA, a constitutively expressed large weight molecule.
In the present study the expression of a 323-bp PCR product that reflected collective expression of all of the VEG/PF isomers and immunoreactivity with a VEG/PF antibody that recognized the 121, 165, and 189 splice variants were markedly decreased in endometrial glandular epithelial and stromal cells after ovariectomy of baboons. Certain VEG/PF isoforms, e.g. VEG/PF 121 and 165, appear to induce particular aspects of angiogenesis, such as permeabilization and proliferation of vascular endothelial cells (35), and/or respond to stimuli. Therefore, additional studies, e.g. using a third primer to generate different sized products for each of the VEG/PF isoforms in the RT-PCR or RNase protection assay, are needed to ascertain whether one or more of the specific VEG/PF species was responsible for the changes observed after ovariectomy in animals of this study.
In summary, the results of the present study show that the expression of VEG/PF mRNA and protein in glandular epithelium and stroma was markedly suppressed after ovariectomy, indicating that the synthesis of this angiogenic factor in certain cells of the endometrium was dependent upon a product(s) secreted by the ovary. Moreover, glandular epithelial and stromal cells expressed comparable levels of VEG/PF, which remained relatively constant and thus available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.
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Abbreviations: CRS, Competitive reference standard; d, deoxy; DNase, deoxyribonuclease; LCM, laser capture microdissection; PF, permeability factor; RNase, ribonuclease; RT, reverse transcriptase; VEG, vascular endothelial growth.
Received April 8, 2002.
Accepted for publication June 7, 2002.
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) and luteal (
) phases of the menstrual cycle were plotted as a function of the log of the concentration of CRS added to each PCR reaction. Lines were constructed by linear regression analysis, and VEG/PF mRNA levels were determined from the equivalence points (i.e. intersection of vertical with regression lines). The correlation coefficient (r2) determined by linear regression was 0.92 (P < 0.03) for follicular samples and 0.96 (P < 0.02) for luteal samples.
