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Female Reproductive Tract |
Departments of Obstetrics, Gynecology, Reproductive Sciences and Physiology, Center for Studies in Reproduction,3 University of Maryland School of Medicine, Baltimore, Maryland 21201
Department of Physiological Sciences,4 Eastern Virginia Medical School, Norfolk, Virginia 23501
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, estradiol, menstrual cycle, progesterone, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The VEG/PF mRNA levels were elevated by estrogen in vivo in the mouse [8], rat [9, 10], and ovine [11] uterus. Disruption of the estrogen-receptor gene prevented the induction of angiogenesis by estrogen in transgenic mice [12]. Therefore, evidence supports the concept that tropic effects of estrogen on vascularization of the uterus involve increased expression of, and are mediated by, VEG/PF. However, translation of these findings to the human remains to be established, particularly because major differences in vascular redevelopment of the endometrium exist between the rodent and the human. Although in vitro studies with human endometrial cells [5, 13] support a role for estrogen in VEG/PF expression, for ethical reasons no in vivo studies have tested the cause-and-effect roles of estrogen and progesterone on VEG/PF expression by the different cell types comprising the endometrium during the human menstrual cycle.
Using the baboon as a nonhuman primate model to study human reproductive endocrinology, we recently established the cellular and temporal expression of VEG/PF mRNA and protein levels in endometrial glandular epithelial and stromal cells during the menstrual cycle, and we showed that VEG/PF expression was decreased to very low levels by ovariectomy [14]. Based on these results, we proposed that VEG/PF expression by glandular epithelial and/or stromal cells of the primate uterus is regulated by the levels of estrogen and progesterone exhibited during the menstrual cycle. To test this hypothesis in the present study, VEG/PF mRNA levels were determined in glandular epithelial and stromal cells isolated by laser-capture microdissection (LCM) from, and VEG/PF protein was determined by immunocytochemistry in the endometrium of baboons after ovariectomy and administration of estradiol and progesterone in levels designed to replicate the hormonal profiles that are characteristic of the proliferative and secretory phases of the menstrual cycle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female baboons (Papio anubis) obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX) and weighing 12–15 kg, were housed individually in large primate cages and maintained in a controlled environment (12L:12D). Animals were fed twice daily with a commercial primate chow and fresh fruit, and they received water ad libitum. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 86-23, 1985). The experiments conducted were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Five baboons exhibiting regular menstrual cycles as determined by serum estradiol profiles and daily records of perineal turgescence [15], underwent ovariectomy and were then studied longitudinally after ovariectomy alone, ovariectomy and estradiol, and ovariectomy and estradiol plus progesterone treatment. Baboons were anesthetized with a mixture of isoflurane (1.0–1.5%):nitrous oxide (0.5 L/min):oxygen (2.0 L/min), bilaterally ovariectomized via a 5- to 6-cm midline abdominal incision, and then left for at least 60 days before being studied. Endometrial biopsies were obtained from these baboons after 1) ovariectomy alone; 2) ovariectomy and administration s.c. of silastic capsules (outer diameter, 4.65 mm; length 6 cm) containing 17ß-estradiol (one capsule inserted on Day 0 and left in for 14 days, two additional capsules inserted on Day 7 and left in for 7 days, to replicate proliferative/estrogen surge phases of menstrual cycle); or 3) ovariectomy and administration s.c. of one estradiol implant on Day 0 and left in for 14 days, two additional estradiol implants on Day 7 and left in for 7 days, and, after the latter three capsules were removed, one new estradiol and four progesterone-containing capsules inserted for another 7 days (to mimic the normal proliferative and early to midsecretory phases of the menstrual cycle). A 2-mo interval was left between each of the treatment regiments, the sequence of which was alternately assigned for the same five ovariectomized animals. During the final 5 days of steroid treatment, ovariectomized baboons were injected s.c. daily with 0.5 mg of the highly specific aromatase inhibitor CGS 20267 (Letrozole, 4,4'-(1,2,3-triazyol-1-yl-methylene)-bis-benzonitrite; Novartis Pharma AG, Basel, Switzerland), in 0.25 ml of sesame oil, to suppress potential aromatization in nonovarian sites during the interval immediately preceding endometrial sampling.
Peripheral saphenous vein blood samples (2–4 ml) were obtained from baboons daily during the study period after sedation with ketamine HCl (10 mg/kg body wt, i.m.), and serum estradiol and progesterone concentrations were determined by radioimmunoassay as described previously [16].
At the end of each treatment regimen, baboons were anesthetized with isoflurane, and the uterus was exposed by laparotomy for purposes of uterine biopsy. Two core biopsy specimens (diameter, 5 mm; Acu-Punch; Acuderm, Inc., Ft. Lauderdale, FL) were obtained from the uterine fundus extending transmurally from the 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 VEG/PF mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) collectively in all cells of the endometrium (i.e., whole endometrium). The entire second uterine biopsy specimen from three of the longitudinally studied animals was embedded in a cryomold filled with O.C.T. medium (Sakura Finetek, Inc., Torrance, CA), frozen on dry ice, and then stored at -80°C for subsequent RT-PCR of VEG/PF mRNA in specific endometrial cells isolated by LCM. The second uterine biopsy specimen from the remaining two baboons was fixed in 10% neutral-buffered formalin for 24 h, washed in 0.5 M potassium phosphate buffer, embedded in paraffin, and then processed for VEG/PF immunocytochemistry.
LCM of Endometrial Cells
Glandular epithelial and stromal cells were isolated from the endometrium by LCM as described previously [14]. Briefly, serial sections (thickness, 8 µm) of the uterine biopsy specimen were cut longitudinally (to include endometrium and myometrium) on a Jung Frigocut 2800E cryostat at -20°C (Leica, Inc., 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, immersed in Eosin-Y (Richard Allen, Kalamazoo, MI) for 10 sec, dehydrated in 100% ethanol, and incubated for 5, 10, and 15 min in xylene. Slides were air-dried and transferred to a dessicator at room temperature. An Arcturus PixCell II LCM system equipped with an Olympus microscope (Arcturus Engineering, Inc., Mountain View, CA) was used to capture glandular (but not luminal) epithelial and stromal cells (but not observable blood vessels) collectively from the basalis and functionalis zones of the endometrial sections. A single LCM cap (Capture Transfer Film TF100; Arcturus Engineering) was used per tissue section, and optimal conditions for LCM included a laser power of 40 mW, duration of 1.5–2.5 msec, and laser spot-size of 7.5 or 15 µm for glandular epithelium (depending on gland size) and 15 or 30 µm for stroma. Captured cells were then mixed with lysis buffer (RNeasy; Qiagen, Valencia, CA) in a single Eppendorf tube, microcentrifuged, and stored in lysate buffer overnight at -80°C; RNA was extracted within 72 h. The entire cell-capture process, from tissue sectioning to tissue lysis, was rapidly completed to limit RNA degradation.
Quantification of Endometrial Width and Glandular Area
The thickness (i.e., width) of the endometrial layer was measured in 8 to 10 randomly selected sections collected for LCM via image analysis using a Nikon Eclipse E1000M microscope (Nikon, Tokyo, Japan). The percentage glandular area in the basalis and functionalis zones was also determined on tissue sections obtained for LCM by image analysis (IP Lab Scientific Image Processing; Scanalytics, Fairfax, VA) as detailed previously [14]. The percentage glandular area (i.e., proportion of endometrium comprised of glands) was quantified by dividing the sum of the glandular epithelial area by the sum of the endometrial area multiplied by 100.
Competitive RT-PCR of VEG/PF mRNA
RNA extraction and primers The RNA extraction and competitive RT-PCR were performed as detailed previously [14, 17]. Total RNA was extracted from whole endometrium via 4 M guanidine isothiocyanate-cesium chloride gradient centrifugation [18] and from LCM-captured glandular epithelial and stromal cells by Nonidet P-40-guanidine isothiocyanate silica gel spin-column centrifugation (RNeasy). To remove potential genomic DNA contamination, LCM samples were incubated with amplification-grade DNase 1 (Invitrogen Life Technologies, Inc., Carlsbad, CA), then sodium acetate/ethanol precipitated and resuspended in 10 µl of RNase-free water.
Although total RNA in whole-endometrial tissue could be quantified by ultraviolet (UV) absorption spectrophotometry to permit normalization of VEG/PF mRNA levels, the amount of total RNA obtained from LCM samples was low. Therefore, 18S rRNA, a cellular RNA whose expression was relatively constant during the menstrual cycle (data not shown), was also quantified by competitive RT-PCR to normalize VEG/PF mRNA levels determined in uterine cells isolated by LCM.
Oligonucleotide primers were synthesized by Invitrogen Life Technologies and based on the human VEG/PF [19] and 18S rRNA [20] cDNA sequences as detailed previously [14].
Competitive reference standard Homologous RNA competitive reference standards (CRS) that contain the same primer-binding sites but shortened internal sequence with respect to the endogenous target RNA for VEG/PF and 18S rRNA were prepared as described previously [14]. 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 reaction mixture containing 1 mM dNTPs (Invitrogen), 200 U of Superscript ribonuclease (RNase) H-RT or MMLV RT (Invitrogen), 1x RT buffer, 40 U of RNAguard (Amersham Pharmacia Biotech, Piscataway, NJ), and 250 ng of random primers (Invitrogen). On completion of the RT reaction, the RT enzyme was heat-inactivated at 70°C for 15 min and the reaction cooled to 4°C, after which 5 µl of the RT reaction were added to separate PCR reaction mixtures (45 µl) containing 0.2 mM dNTPs, 1.25 U of cloned Thermus aquaticus DNA polymerase (Amplitaq; Perkin-Elmer Corp/Cetus, Norwalk, CT), 1x PCR buffer, and 10 pmol of the respective paired primers to generate cDNA templates for VEG/PF and 18S rRNA. The 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, with a final extension at 72°C for 5 min. The PCR products were gel purified (Qiagen DNA extraction kit; Qiagen) and the CRS synthesized from cDNA template using the MEGAscript T7 in vitro transcription kit (Ambion, Inc., Austin, TX). The VEG/PF and 18S rRNA-CRS were treated with RNase-free DNase 1 (Ambion) to digest the cDNA templates and extracted with chloroform:isoamyl alcohol, after which aliquots were quantitated via UV absorption spectrophotometry at an optical density of 260 nm.
RT-PCR assay The VEG/PF and 18S rRNA mRNA levels were simultaneously quantified by competitive RT-PCR assay [17, 21]. A constant amount of RNA (1.5 µl of LCM sample or 10 ng of whole endometrium) was added to an RT mixture containing 2-fold serial dilutions of both VEG/PF-CRS (25–0.02 amol) and 18S rRNA-CRS (5–0.04 fmol). To test for possible pseudogene or genomic DNA contamination, either the RT enzyme or the 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.
For VEG/PF and 18S rRNA, 5 and 2 µl of the RT mixture, respectively, were added to separate PCR reaction mixtures containing 10 pmol of the respective paired primers for VEG/PF and 18S rRNA. Total endometrial, LCM VEG/PF, and LCM 18S rRNA samples were amplified for 32, 34, and 24 sequential cycles, respectively. The PCR products were then 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 Multi-Analyst software program (Bio-Rad Laboratories, Hercules, CA). The intensity of amplified products was represented as the relative area under each product band. A correction factor [22] was used to account for the relative size difference between target and 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 when the ratio of the log of CRS and target area was equal to zero (i.e., the equivalence point).
Qualitative analysis of RNA The integrity of total RNA extracted from cells isolated by LCM was analyzed via an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was resuspended in 5 µl of RNase-free water and then added to a single well of an RNA 6000 Nano LabChip (Agilent) containing 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
Immunocytochemistry of VEG/PF was performed as described previously [23]. Paraffin blocks of uterine tissue were serially sectioned (thickness, 4 µm), deparaffinized, and rehydrated in graded alcohols. Tissue sections were boiled in 0.01 M sodium citrate for 10 min, incubated in H202, and blocked in 10% normal goat serum (Protein Block Serum; DAKO Corp, Carpinteria, CA). Tissues were incubated overnight at 4°C with goat anti-human primary antibody to VEG/PF (AF-293-NA, diluted 1:25 in 5% goat serum, specific for the 121, 165, and 189 splice variants; R&D Systems, Minneapolis, MN). Following incubation with biotinylated anti-goat immunoglobulin (Vector Laboratories, Inc., Burlingame, CA), sections were immersed in an avidin-biotin complex solution (Elite Vectastain ABC Kit; Vector Laboratories) and incubated with 3,3'-diaminobenzidine (0.2 mg/ml; Sigma Chemical, St. Louis, MO) to produce a brown reaction product. Negative controls included omission of the primary antibody, substitution of immunoglobulins (DAKO) for primary antibody, and/or preabsorption of primary antibody with 10-fold excess of human recombinant VEG/PF peptide (R&D Systems). Sections were counterstained with Harris hematoxylin.
Statistical Analysis
The experimental design consisted of a crossover in which endometrial VEG/PF levels and endometrial width were assessed in all baboons after ovariectomy alone and after estradiol and then estradiol and progesterone treatment. Data were expressed as the mean ± SEM and were analyzed by repeated-measures ANOVA using a model that accounted for within-subject variation. When significant differences were observed across time periods, the Wilcoxon signed-rank test was used to compare the means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum estradiol and progesterone were not detectable (i.e., <20 pg/ml and <0.20 ng/ml, respectively) in ovariectomized baboons treated with CGS 20267 alone during the 5 days immediately preceding endometrial biopsy (Table 1, None). In ovariectomized baboons treated with s.c. implants of estradiol, serum estradiol concentration (209 ± 40 pg/ml) for the 5 days before endometrial sampling approximated the midcycle estradiol surge level observed in intact animals from our primate colony (Table 1), whereas serum progesterone was not detectable. In ovariectomized baboons treated with implants of estradiol and progesterone, serum estradiol (44 ± 15 pg/ml) (Table 1) and progesterone (9.8 ± 2.2 ng/ml) levels immediately before endometrial sampling approximated those exhibited during the early to midsecretory phase of the menstrual cycle.
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Endometrial VEG/PF mRNA Levels
We previously showed [14] that homogeneous populations of glandular epithelial and stromal cells were isolated by LCM from the endometrium of the baboon uterus. Moreover, RNA isolated from LCM-captured endometrial cells exhibited distinct 28S and 18S rRNA bands (Fig. 1A), although the ratio of 28S and 18S rRNA fluorescent intensities was less than 2.0 (Fig. 1B), suggesting that some RNA degradation had occurred.
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Figure 2 illustrates a representative competitive RT-PCR analysis of VEG/PF mRNA levels using primers upstream from the alternative splice site to yield a single PCR product, reflecting collective expression of all VEG/PF isoforms, in endometrial stromal cells obtained by LCM from ovariectomized baboons left untreated or treated with estradiol. The expected 323-base pair (bp) VEG/PF target and 256-bp VEG/PF CRS products generated by PCR are shown in Figure 2A. The PCR products were not obtained when RNA or RT enzyme was omitted from the reaction. Despite the partial degradation of RNA after LCM of endometrial cells, PCR amplification, as shown in Figure 2B, was linear (r2 = 0.97, P < 0.01, ovariectomy; r2 = 0.96, P < 0.02, ovariectomy and estradiol treatment), indicating that RNA integrity was maintained within the region spanned by the primers and that quantitative analysis was not compromised. Moreover, slopes of the log of the ratio of CRS and target areas plotted as a function of the log of increasing CRS for RNA obtained from stromal cells were alike in untreated and estradiol-treated ovariectomized baboons, indicating similar amplification efficiency (Fig. 2B). Similar results for PCR amplification were observed in glandular epithelial cells (data not shown).
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As shown previously [14], glandular epithelial (2.31 ± 0.82 amol/fmol 18S rRNA) and stromal (2.02 ± 0.45 amol/fmol 18S RNA) VEG/PF mRNA levels, corrected for 18S rRNA, in intact baboons during the combined midcycle estradiol surge and secretory phases of the menstrual cycle were decreased after ovariectomy by approximately 80% (0.52 ± 0.21 amol/fmol 18S RNA) and 90% (0.22 ± 0.11 amol/fmol 18S RNA), respectively. The administration of estradiol for 14 days to ovariectomized animals increased/restored VEG/PF mRNA to levels in the glands (5.57 ± 1.53 amol/fmol 18S RNA, P < 0.01) (Fig. 3) and stroma (2.61 ± 1.57 amol/fmol 18S RNA, P < 0.02) (Fig. 4) that were approximately 10-fold greater than those observed after ovariectomy alone and similar to those previously shown in intact baboons. Concomitant administration of estradiol and progesterone resulted in glandular epithelial (3.65 ± 1.42 amol/fmol 18S RNA, P < 0.06) (Fig. 3) and stromal (1.25 ± 0.77 amol/fmol 18S RNA, P < 0.07) (Fig. 4) VEG/PF mRNA levels, which were not significantly different from those observed after ovariectomy or ovariectomy and estradiol treatment.
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Figure 5 shows the levels of VEG/PF mRNA in whole endometrium. The administration of estradiol to ovariectomized baboons increased whole-endometrial VEG/PF mRNA to a level (1088 ± 185 amol/µg total RNA) that was approximately 2-fold greater (P < 0.03) than that after ovariectomy alone (592 ± 62 amol/µg total RNA) and similar to that previously reported in intact animals (911 ± 112 amol/µg total RNA) [14]. Simultaneous administration of estradiol and progesterone also resulted in an endometrial VEG/PF mRNA level (1062 ± 120 amol/µg total RNA) that was higher (P < 0.01) than that obtained in ovariectomized baboons and similar to that after estradiol administration.
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Endometrial Histology and VEG/PF Immunocytochemistry
The width of the endometrial layer after ovariectomy (0.98 ± 0.09 mm) (Table 2) was approximately one-third of that in baboons during the midcycle estradiol surge (3.58 ± 0.32 mm). Administration of estradiol or estradiol and progesterone after ovariectomy increased (P < 0.01) and restored endometrial thickness to normal (3.00 ± 0.12 and 2.70 ± 0.48 mm, respectively). The area of endometrium occupied by glands (i.e., % glandular area) (Table 2) in the basalis (8.1% ± 1.5%) and functionalis (11.7% ± 1.8%) zones was 2- to 4-fold lower after ovariectomy than previously shown [14] in the combined proliferative and secretory phases of the menstrual cycle (32.7% ± 3.3%, basalis; 22.0% ± 2.1%, functionalis). Administration of estradiol and progesterone to ovariectomized animals increased (P < 0.01) glandular area to normal.
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After ovariectomy, the stroma was compact, and relatively little endometrial VEG/PF protein expression was observed (Fig. 6A). Estradiol administration to ovariectomized baboons restored the loosely arranged stroma, and VEG/PF protein expression (Fig. 6B), to what is typically observed in intact animals [14]. Concomitant administration of estradiol and progesterone to ovariectomized baboons resulted in pseudostratified glandular epithelium and loosely arranged stroma, both of which exhibited VEG/PF immunostaining (Fig. 6C). Specificity of VEG/PF immunocytochemistry was demonstrated by the absence of staining when primary antibody was preabsorbed with VEG/PF (Fig. 6D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Considerable data suggest that the action of estrogen on endometrial VEG/PF expression is mediated via the estrogen receptor. Thus, estrogen receptor is expressed in endometrial glandular epithelial and stromal cells of the human [25], baboon [26], and rhesus monkey [27, 28] in an endometrial zone- and menstrual cycle stage-specific manner. Although classical estrogen response elements have not been identified in the 5'-flanking region of the VEG/PF gene, consensus half-palindromic sequences that bound estrogen receptor 
in band-shift assays and that confer estrogen inducibility to reporter constructs have been identified in two regions of the VEG/PF gene, including the 5'-untranslated region [13, 29]. Moreover, the induction of VEG/PF mRNA expression in the rat uterus was blocked by antiestrogens and inhibited by actinomycin D, but not puromycin or cycloheximide [29]. Additional study is needed to determine the specific estrogen-receptor activation and transcriptional mechanisms that mediate the estrogen-dependent upregulation of VEG/PF expression in the glands and stroma of the primate endometrium.
In ovariectomized baboons of the present study, which were synchronously treated with estradiol and progesterone to replicate the steroid hormone profile of the midsecretory phase, the level of endometrial VEG/PF mRNA expression was not significantly different from that observed with estradiol administration alone. Thus, concomitant administration of estradiol and progesterone did not further increase the level of endometrial VEG/PF mRNA observed with estradiol alone, although serum estradiol concentrations were lower in the former case. Thus, it is not possible to determine whether the absence of an effect on VEG/PF expression was caused by introduction of progesterone or reduction in the amount of estradiol administered. Other studies have shown that progesterone alone increased VEG/PF mRNA expression in vivo in the uterus of the rat [9, 29] and in the uterus of rhesus monkeys suppressed with gonadotropin-releasing hormone agonist [30] and in vitro in human endometrial cells [5]. However, the present study was not conducted to assess the potential effect of progesterone alone on endometrial VEG/PF expression.
Previous studies in the human have shown that endometrial VEG/PF mRNA and protein levels were either slightly greater in the secretory than in the proliferative phase [4, 5, 31, 32] or were similar during the course of the menstrual cycle [7, 33–35], despite the temporal cyclical surges in estrogen and progesterone. We previously showed that endometrial VEG/PF mRNA levels also were not significantly different during the midcycle estrogen surge and midsecretory phase of the baboon menstrual cycle [14]. We propose, therefore, that the relatively low levels of estrogen that precede and follow the midcycle estrogen surge are nevertheless sufficient as well as necessary to sustain VEG/PF expression and that only when estrogen is decreased to undetectable values (e.g., after ovariectomy) does VEG/PF formation, particularly when examined in isolated glands and stroma, substantially decline. Thus, it is possible that endometrial VEG/PF expression is maximally stimulated during the normal menstrual cycle. Sustained VEG/PF synthesis throughout the menstrual cycle would seem to be necessary to promote angiogenesis at menstruation for vascular repair, throughout the proliferative phase for rapid expansion of the vessel bed, and during the secretory phase for growth and elongation of the vascular tree. In the present study, ovariectomy and systemic administration of an aromatase inhibitor were employed to suppress estrogen levels in baboons, and this resulted in regression of endometrial mass and function in general. Furthermore, normal cyclical changes in steroid hormone concentrations do not result in significant changes in endometrial VEG/PF expression. Consequently, additional study (e.g., using an acute temporal experimental approach) is needed to confirm that ovarian estradiol has a direct and specific effect on VEG/PF expression by the primate endometrium.
Other factors, notably hypoxia, also upregulate VEG/PF expression in various tissues, including the endometrium [32]. However, the physiological role of hypoxia on the induction of VEG/PF expression within the primate endometrium may be most important at menstruation, when both oxygen tension and steroid hormone levels become very low [35]. Indeed, shortly preceding menstruation is a striking increase in expression of VEG/PF mRNA in the baboon (unpublished observation) and VEG/PF receptor in the rhesus monkey [36] endometrium, which may be important for vessel repair and reconstruction. Under these circumstances, hypoxia may overcome the absence of estrogen and induce VEG/PF synthesis. Nevertheless, in the present study, VEG/PF mRNA and protein were decreased to low levels in isolated glandular epithelial and stromal cells by ovariectomy of baboons, an effect that was prevented by estradiol. These results point to the importance of estrogen in maintaining VEG/PF synthesis in the primate endometrium.
The relatively less marked decrease [14] and increase in VEG/PF mRNA expression observed in whole-endometrial tissue after ovariectomy or ovariectomy and estrogen treatment of baboons, respectively, point to the value of isolating glandular epithelial and stromal cells (e.g., by LCM) when studying the regulation of factors such as VEG/PF in the heterogeneous endometrium. Moreover, because the relative increase in VEG/PF expression induced by estrogen in ovariectomized baboons appeared to be less marked in mixed endometrial cells than in isolated glandular epithelial and stromal cells, it is possible that VEG/PF mRNA expression is not regulated by estrogen in those endometrial cells that were removed by LCM and that express VEG/PF, such as vascular endothelial cells [37] and pericytes [38], or intravascular cells, such as leukocytes [39]. Additional study is needed to further define the role of steroid hormones on VEG/PF expression by these other cell types in the primate endometrium.
In summary, the results of the present in vivo study show that estrogen, administered chronically to ovariectomized baboons to replicate the late-proliferative phase of the menstrual cycle, increased and reversed the low level of VEG/PF mRNA and protein expression in and decreased size of the endometrium induced by ovariectomy. Therefore, we propose that estrogen has a significant role in stimulating VEG/PF expression by glandular epithelial and stromal cells of the endometrium to promote angiogenesis within and, consequently, growth of the endometrium during the menstrual cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Eugene D. Albrecht, Department of Obstetrics, Gynecology and Reproductive Sciences, The University of Maryland School of Medicine, Bressler Research Laboratories 11-019, 655 West Baltimore Street, Baltimore, Maryland 21201. FAX: 410 706 5747; ealbrech{at}umaryland.edu
Received: 25 September 2002.
First decision: 19 October 2002.
Accepted: 27 December 2002.
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E. D. Albrecht, J. S. Babischkin, Y. Lidor, L. D. Anderson, L. C. Udoff, and G. J. Pepe Effect of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells Hum. Reprod., October 1, 2003; 18(10): 2039 - 2047. [Abstract] [Full Text] [PDF]
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) and treated with estradiol (
) were plotted as a function of the log of CRS concentration added to each PCR reaction. Lines were constructed by linear regression analysis and VEG/PF mRNA levels determined from the equivalence points (i.e., intersection of vertical with regression lines). Correlation coefficients (r2) determined by linear regression were 0.97 (P < 0.01) for untreated and 0.96 (P < 0.02) for estradiol-treated ovariectomized baboons
