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Original Studies |
Department of Obstetrics and Gynecology (K.Ko., Y.O., O.T., M.M., A.S., K.Ku., T.F., Ya.T., T.Y., Yu.T.), University of Tokyo, Tokyo, 113-8655; and Core Research for Evolutional Science and Technology (O.T.), Japan Science and Technology, Kawaguchi 332-0012, Japan
Address correspondence and requests for reprints to: Dr. Yuji Taketani, Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan.
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Abstract |
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Introduction |
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Angiogenin, a 14.1-kD single-chain polypeptide, was initially isolated from supernatants of colon carcinoma cells and found to be a member of the pancreatic ribonuclease superfamily (1). Angiogenin is also present in sera of human (2) and other mammals (3), and in bovine milk (4) as well. There is persuasive evidence that angiogenin is a potent inducer of angiogenesis, thus playing a role in the pathogenesis of a variety of cancers (5, 6, 7, 8).
Recently, immunoreactivity and messenger RNA (mRNA) expression for angiogenin were shown in the early corpus luteum of the bovine ovary (9), highlighting angiogenin as an integral player involved in angiogenesis in the process of the formation of the corpus luteum. It may be worthwhile in investigating whether gonadotropin, an inducer of the formation of the corpus luteum, regulates the expression of angiogenin. In addition, it is reasonable to look at the effect of hypoxia on its expression in view of hypoxic milieu of the preovulatory follicle. In this study, we first investigated the presence of angiogenin in human ovarian follicles. Then, in pursuit of its regulatory mechanisms in human ovaries, the effects of human CG (hCG), cAMP, and hypoxia on the expression of angiogenin were studied using human luteinizing GC culture system.
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Materials and Methods |
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Collection of follicular fluid (FF)
To evaluate the relationship between the concentrations of angiogenin and those of steroid hormones and hCG in FF, FF from each follicle was separately aspirated without flushes. After isolation of the oocyte, aspirated FF was immediately centrifuged (300 x g), and the supernatant was divided into several aliquots and stored at -80 C until use. Only follicles in which an oocyte was clearly identified were used for analysis. In total, 145 FF samples were obtained from 29 women.
Isolation of GCs
To obtain GCs for culture, FF was aspirated with repeated flushes. All the follicular aspirates and flush medium from each woman were mixed and centrifuged at 200 x g for 5 min to obtain a cell pellet, which was resuspended in phosphate-buffered saline with 0.1% hyaluronidase and incubated at 37 C in a shaking water bath for 30 min. Suspension was layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden) and centrifuged at 150 x g for 30 min. GCs recovered from the interface were washed with phosphate-buffered saline.
Western blotting
Isolated GCs were homogenized in lysis buffer containing 50 mM TrisHCl (pH7.4), 0.1% SDS, 1 mM ethylenediamine tetraacetic acid, 0.5% Igepal, and 50 mM dithiothreitol and diluted to 1 mg total protein/mL. FF was diluted with the lysis buffer in a 1:50 dilution. Samples were resolved by 10% SDS-PAGE under reducing conditions in the parallel lane with recombinant human angiogenin (Genzyme/Techne, Minneapolis, MN). Proteins were blotted onto nitrocellulose membranes and incubated with an antihuman angiogenin goat antibody (1:500; Genzyme/Techne) as a primary antibody and an antigoat horseradish peroxidase antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) as a secondary antibody. Immune complexes were visualized by ECL Western blotting system (Amersham, Buckinghamshire, UK). For negative controls, the primary antibody was either replaced with a nonimmune goat IgG or preabsorbed with excessive amount of recombinant human angiogenin.
Cell culture and treatment with hCG, dibutyryl adenosine cyclic monophosphate (dbcAMP), and hypoxia
Isolated GCs, were resuspended in DMEM/Ham’s F-12 medium containing 10% FBS, 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B. The cells were plated at a density of 50,000 cells/35-mm plate and kept at 37 C in a humidified 5% CO2/95% air environment. After 24 h, media were replaced, and the cells were cultured for another 24–48 h before treatments. Then, cell preparations were cultured in replenished media without (control) or with hCG (150 ng/mL; Mochida) or dbcAMP (1 mM; Sigma, St. Louis, MO) for 24 h. In an experiment to examine the effect of hypoxia, the cells were incubated as stated above for initial 48–72 h, then further cultured under normoxic or hypoxic conditions for another 24 h. Hypoxic condition (around 0.2% O2, 18% CO2) was produced by placing culture plates in commercially available hypoxia chambers (Anaeocult P; Mikrobiologie, Darmstadt, Germany). The mean partial pressure of O2 in the medium achieved with the chamber was 84.3 mm Hg as measured with a blood gas analyzer (Stat Profile Ultra; Nova Biomedical, Boston, MA), whereas that under normoxic condition was 158.3 mm Hg. Conditioned medium was collected, centrifuged, and stored at -80 C for subsequent analysis. Total mRNA was isolated as described below.
Hormone measurement
Concentrations of progesterone, estradiol, and testosterone in FFs were measured using an SR1 analyzer (Biochem ImmunoSystems Italia, Rome, Italy). The intra-assay and interassay coefficients of variation were less than 10% for these assays.
Angiogenin concentrations in FF and conditioned media were measured using a specific enzyme-linked immunosorbent assay (Quantikine Human Angiogenin Immunoassay; Genzyme/Techne). The limit of sensitivity of this enzyme-linked immunosorbent assay was 6 pg/mL. The intra-assay and interassay coefficients of variation were 4.3% and 8.7%, respectively.
RT and real-time quantitative PCR
Total RNA was extracted from cultured human GCs, using RNeasy Mini Kit (QIAGEN, Hilden, Germany). RT was performed using TaKaRa RNA PCR Kit (Takara Shuzo, Tokyo, Japan), and real-time quantitative PCR and data analysis were carried out using a Light Cycler (Roche Diagnostic, Mannheim, Germany), according to the manufacturer’s instructions. Two hundred nanograms of total RNA were reverse-transcribed in a 20-µL volume. PCR primers for the angiogenin were 5'-CCTGGGCGTTTTGTTGTTGG-3', sense primer corresponding to nucleotides 1820–1839 of the published sequence (11), and 5'-TGTGGCTCGGTACTGGCATG-3', antisense primer corresponding to nucleotides 2152–2171. Amplification was performed in a total volume of 20 µL including 2.5 µL of each RT reaction, 2 µL LightCycler-FastStart Reaction Mix SYBR Green 1 (Roche Diagnostic), 0.5 µmol/L of each primer, 3 mmol/L MgCl2 with 40 cycles of denaturing (95 C, 15 sec), annealing (62 C 10 sec), and extension (72 C, 14 sec), followed by melting curve analysis.
Statistical analysis
Student’s t test was used for the analysis of concentrations of angiogenin in culture media. Correlations between concentrations of angiogenin and that of progesterone, estradiol, and testosterone were calculated by linear regression analysis. P less than 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Western blotting
analysis of the samples of both GC lysate and FF revealed a band of the
same size as recombinant human angiogenin. The apparent molecular
weight of the band was 
14 kD, which corresponds to the reported size
of angiogenin (12). No corresponding band was detected
when a nonimmune IgG or a preabsorbed antibody was used instead of a
primary antibody for angiogenin (data not shown).
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shows angiogenin concentrations
in culture media of GCs treated with hCG or dbcAMP for 24 h. The
addition of hCG or dbcAMP similarly increased angiogenin accumulation
in culture media, an up to 3-fold increase being observed over the
control (P < 0.0005 and P < 0.0001,
respectively).
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. GCs under hypoxic conditions
released an increased amount of angiogenin, which was 5.5-fold
increase over that in the control culture (P < 0.05).
Expression of angiogenin mRNA was detected in cultured GCs by RT-PCR.
The sequence of the product was confirmed to coincide with the human
angiogenin coding sequence (data not shown). Additional amplification
cycles (3.64 ± 0.27, mean ± SEM) were required
before fluorescence reached the threshold level for samples in control
condition as compared with that in hypoxic conditions, which means the
expression level of angiogenin mRNA in GCs under hypoxic conditions was
12.5-fold higher than that under control conditions.
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, simple regression
analysis showed that the concentrations of angiogenin in FFs were
positively correlated with those of progesterone (r = 0.366,
P < 0.0001). In contrast, there was no significant
correlation between the concentrations of angiogenin and those of
estradiol and testosterone.
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Discussion |
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An in vitro experiment showed that hCG and cAMP treatment increased the angiogenin secretion by GCs. Treatment with hCG is known to enhance luteinization of GCs. Considering that luteinization of GCs occurs in synchrony with angiogenesis during the process of corpus luteum formation, it makes teleological sense that hCG stimulates luteinization of GCs, on one hand, and accelerates the production of angiogenin, on the other hand.
Exogenous administration of hCG in combination with FSH preparations is well known to pose an increased risk of developing ovarian hyperstimulation syndrome (OHSS) (16). Intriguingly, angiogenin concentrations in serum and ascitic fluid are increased in patients with OHSS (17). Our findings that hCG stimulates the production of angiogenin by GCs might explain increased angiogenin levels in patients with OHSS.
cAMP is an important mediator of gonadotropins in both folliculogenesis and luteogenesis. Although regulatory mechanisms for gene transcription of angiogenin are, at present, poorly understood, our findings suggest cAMP as a possible mediator of the production of angiogenin in the follicle.
Hypoxia-induced up-regulation of angiogenin is hitherto known only in
human malignant melanoma cells (18). In this study,
hypoxia stimulated the release of angiogenin from GCs, thus providing
the first evidence for the stimulatory effect of hypoxia in
nonmalignant cells. Hypoxia is a well known inducer of angiogenesis.
The preovulatory ovarian follicle is a relatively large avascular
multicellular structure (19) and, therefore, assumed to be
underoxygenated based on mathematical models (20). Indeed,
the oxygen contents of FF in mature follicles have been documented to
range from less than 1% to 5.5% (21). Given the
angiogenic activity of angiogenin, it is tempting to speculate that
hypoxia induces angiogenesis in the follicle possibly by stimulating
the expression of angiogenin.
Regarding angiogenin levels in FF, we demonstrated that they positively correlated with progesterone levels in FF obtained from patients undergoing an IVF-embryo transfer program. In view of the finding that hCG stimulates the angiogenin production in cultured GCs, an observed positive correlation of angiogenin levels with those of progesterone in FF is understandable because both substances are locally produced by hCG. Interestingly, the concentrations of VEGF, another angiogenic protein, have been shown to correlate positively with those of progesterone in FF (22). VEGF is also considered to be produced in the ovarian follicles under the stimulation of gonadotropins (23). Collectively, angiogenin and VEGF may play a role in a coordinated manner in angiogenesis occurring in the corpus luteum formation.
It might be argued whether we can generalize data presented here since FF and GCs used in this study derive from IVF patients who receive pharmacological doses of gonadotropins. However, development of each follicle and its subsequent luteinization in association with angiogenesis seem to proceed through essentially the same mechanisms as are seen in natural cycles, even though the number of follicles are increased in IVF settings. Thus, the observed findings may provide a framework for understanding of a physiological role of angiogenin in human ovaries.
In summary, the present study demonstrated the presence of angiogenin in human ovarian follicles and the up-regulation of its production by hCG, cAMP, and hypoxia, thus highlighting angiogenin as a possible local regulator of angiogenesis in the human ovary.
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Acknowledgments |
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Footnotes |
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Received February 10, 2000.
Revised May 31, 2000.
Accepted June 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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