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Comprehensive Reproductive Medicine, Regulation of Internal Environment and Reproduction, Systemic Organ Regulation, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
Address all correspondence and requests for reprints to: Naoyuki Yoshiki, M.D., Tokyo Medical and Dental University, Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: n.yoshiki.gyne{at}med.tmd.ac.jp
Abstract
The aim of the present study was to investigate the presence of heme oxygenase (HO)-1 and HO-2 in human endometrium at various stages of the menstrual cycle using RT-PCR, Western blotting, and immunohistochemistry. RT-PCR detected mRNA for HO-1 and HO-2 in human endometrium at all stages of the menstrual cycle. Western blotting also revealed the expression of the two distinct HO proteins throughout the menstrual cycle. HO-1 was constitutively expressed, whereas HO-2 expression was apparently greater in the secretory phase than in the menstrual and proliferative phases. Immunohistochemistry showed that distribution of the two HO isoforms had distinct topographic patterns: HO-1 was observed in endometrial epithelial cells and macrophages, whereas HO-2 was found in endothelial cells and smooth muscle cells of blood vessels in the endometrium. The detection of mRNA and protein for HO-1 and HO-2 in normal human endometrium suggests that the carbon monoxide/HO system may play a role in the local control of endometrial function.
CARBON MONOXIDE (CO) is endogenously synthesized during the conversion of intracellular heme to biliverdin and iron, which is catalyzed by heme oxygenase (HO) in a process that requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen as cofactors, as well as NADPH cytochrome P450 reductase (1). To date, three HO isozymes are identified [i.e. inducible HO-1 (2), constitutive HO-2 (3), and a poor heme catalyst, HO-3 (4)]. HO-1, also known as heat shock protein-32, is induced not only by its substrate heme, but also by various kinds of oxidative stress (5). This information indicates that oxygen concentration may also influence HO-1 gene expression. HO-1 has been shown to be an oxygen-regulated protein 33 (6). This isoform is highly expressed in stimulated liver (1) and the spleen, where it is responsible for the destruction of heme from red blood cells (7). On the other hand, HO-2 is constitutively expressed and is distributed in unstimulated liver (1), the testis (8), and the brain at the highest concentration (9). HO-3 has very low activity, and its physiological function probably involves heme binding (4).
CO, as well as nitric oxide (NO), is a novel gaseous chemical messenger that plays key roles in cell function and cell-cell communication in many organ systems (10, 11). The recognition of NO as an important gaseous mediator of cell signaling has led to a reappraisal of the likely role of endogenous CO (12). NO and CO signaling processes have been demonstrated in the central nervous system (13), and NO and CO induce vasodilatation via the activation of soluble guanylate cyclase and ensuring relaxation of vascular smooth muscle in the cardiovascular system (14).
In female reproductive system, some previous studies in humans have shown that NO synthase (NOS) proteins were localized predominantly in the trophoblast, the endothelium, and vascular smooth muscle of chorionic villi during gestation (15, 16, 17). Recent immunohistochemical studies have revealed the presence of HO proteins in human placenta (18, 19, 20). There have been some studies showing that inducible NOS (iNOS) and endothelial NOS (eNOS) were expressed in human endometrium (17, 21, 22, 23). It has been proposed that NO production in the endometrium is involved in the initiation of successful implantation and placentation through its roles in local vasodilatation and immunosuppression (24). In view of the vasodilatory action of CO, similar to that of NO, CO may be involved in the control of endometrial function. However, to our knowledge, no evidence for the expression of HO in human endometrium has been presented to date. These facts led us to determine HO expression in the endometrium.
We hypothesize that HO isoforms are expressed in human endometrium throughout the menstrual cycle. Therefore, the aim of the present study was to determine whether HO-1 and HO-2 are expressed in human endometrium using RT-PCR and Western blotting and to investigate their distribution patterns in these tissues by immunohistochemistry.
Materials and Methods
Tissue samples
Human endometrial samples were obtained from normally cycling parous women (n = 15) undergoing hysterectomy for benign indications at the Tokyo Medical and Dental University Hospital (Tokyo, Japan). The stage of the cycle was determined based on the date of the last menstrual period and was histologically confirmed according to the standard criteria (25). Human endometrial samples were divided into five groups: menstrual (n = 3), proliferative (n = 3), early secretory (n = 3), midsecretory (n = 3), and late secretory (n = 3). All patients investigated had received no hormonal treatments. Informed consent was obtained before tissue collections, and the study was approved by the local ethics committee. Immediately after collection, the samples were snap-frozen in liquid nitrogen and kept at -80 C until required for RNA and protein extraction or cryostat sectioning. All samples were studied by each method.
RNA extraction and RT-PCR
Total RNA was isolated from frozen tissues by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (26). Deoxyribonuclease (DNase) treatment was performed using RQ1 ribonuclease-free DNase (Promega Corp., Madison, WI) at 37 C for 15 min, and RNA was then extracted with phenol/chloroform, followed by extraction with chloroform. DNase was inactivated by heating at 65 C for 10 min before cDNA synthesis. RT-PCR was performed using a RNA PCR kit in accordance with the manufacturer’s instructions (Takara, Otsu, Japan). The primer sequences were designed according to the published cDNA sequences for human HO-1 (2) and HO-2 (3). The forward 5'-CAGGCAGAGAATGCTGAG-3' (79–96) and the reverse 5'-GCTTCACATAGCGCTGCA-3' (349–332) primers for HO-1 were used to amplify a 271-bp fragment, and the forward 5'-ATGTCAGCGGAAGTGGAA-3' (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) and the reverse 5'-GGGAGTTTCAGTGCTCGC-3' () primers for HO-2 were used to amplify a 533-bp fragment. Human HO isoforms were amplified using a duplex PCR technique with 30 cycles of the following sequential steps: 94 C for 30 sec, 58 C for 60 sec, and 72 C for 60 sec in a Perkin-Elmer GeneAmp 2400 Thermal Cycler (Perkin-Elmer Corp., Foster City, CA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was also amplified using the following primers (27): 5'-TGAAGGTCGGAGTCAACGGATTTG-3' (71–94) and 5'-GCGCCAGTAGAGGCAGGGATGATG-3' (698–675), yielding a 628-bp product. PCR amplification was performed as described previously. The amplified products were subjected to 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The sequences of the two HO PCR products were determined using an automated ABI PRISM 310 sequencer (Perkin-Elmer Corp.).
Western blotting
Endometrial tissues were homogenized and fixed in 10% trichloroacetic acid in Dulbecco’s PBS (D-PBS), lysed with lysis buffer (9 M urea/2% Triton X-100/1% dithiothreitol), and then supplemented with lithium dodecyl sulfate. Protein concentration was measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Twenty micrograms of total protein for each sample were loaded on 10% SDS-polyacrylamide gel and separated by electrophoresis, and then transferred onto a polyvinylidene difluoride membrane (Atto, Tokyo, Japan). After blocking with Block Ace (Dainippon, Osaka, Japan), membranes were incubated with either an affinity-purified antihuman HO-1 (1:3000) or HO-2 (1:1000) polyclonal antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), followed by incubation with a horseradish peroxidase-conjugated antigoat IgG antibody (1:5000) (Santa Cruz). Furthermore, membranes were incubated with an antihuman ß-actin polyclonal antibody (Santa Cruz), followed by the same incubation as described above. Bands were detected by the enhanced chemiluminescence detection system according to the recommended procedure (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunohistochemistry
Serial frozen sections (8 µm thick) were mounted onto silane-coated slides. The sections were then fixed in acetone at 4 C for 10 min and washed with D-PBS. Thereafter, endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide in methanol. Nonspecific protein binding sites were blocked with 10% normal rabbit serum in D-PBS at room temperature for 10 min in a humidified chamber. They were then incubated at room temperature for 1 h with either an affinity-purified antihuman HO-1 or HO-2 polyclonal antibody at 1:100 dilution. Primary antibody signals were amplified and detected using a HistoStain-SP kit according to the manufacturer’s instructions (Zymed Laboratories, Inc., San Francisco, CA). 3-Amino-9-ethylcarbazol was used as a chromogen. The immunostained sections were counterstained with Mayer’s hematoxylin and mounted in an aqueous medium. There was no cross-reaction between the HO-1 and HO-2 antibodies. Omission of the primary antibody or substitution of the primary antibody by an appropriately diluted isotype antibody was included as controls and resulted in no immunostaining.
Immunohistochemistry was also performed on serial sections using an
antihuman CD68 (DAKO Corp., Carpinteria, CA), 
-smooth
muscle actin-1 (American Research, Belmont, MA), and factor
VIII-related antigen (Zymed Laboratories, Inc.) monoclonal
antibody. The procedure used in the case of using the each antibody was
the same as that used in the case of using the HO antibodies, except
that 2% normal goat serum in D-PBS was used for blocking
and that a biotinylated antimouse IgG antibody (Nichirei, Tokyo, Japan)
was used as the secondary antibody.
Results
Detection of HO mRNA by RT-PCR
Duplex PCR amplification of endometrial cDNA with the HO-1 and
HO-2 primer sets resulted in the production of each band of the
predicted size (Fig. 1
). Each PCR product
was subsequently verified to be identical to the previously sequenced
product. There was a greater amount of the HO-2 PCR product in the
secretory phase than in the menstrual and proliferative phases,
suggesting more abundant HO-2 mRNA template in the secretory phase.
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Immunoreactive HO-1 and HO-2 proteins were detectable in
endometrial extracts by Western blotting (Fig. 2). The HO-1 antibody produced a 33-kDa
protein band corresponding to the approximate molecular weight of human
HO-1 protein, and the HO-2 antibody produced a 36-kDa one corresponding
to that of human HO-2 protein. No cross-immunoreactivity was observed
between the two HO isoforms. There was a stronger staining of HO-2
protein band in the secretory phase than in the menstrual and
proliferative phases.
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HO-1 protein was present in endometrial epithelial cells and
macrophages (Fig. 3, A
and B), which expressed CD68 (Fig. 3C). Immunostaining for HO-1 was
stronger in luminal epithelial cells than in glandular epithelial
cells. The number of macrophages that exhibited positive staining for
HO-1 and were also CD68 positive increased in the secretory phase.
There was interindividual variation in the intensity of HO-1 antibody
staining in epithelial cells, which was not related to the stage of the
menstrual cycle. On the other hand, HO-2 protein was localized in
endothelial cells and smooth muscle cells of blood vessels in the
endometrium (Fig. 3, D–G). The number of blood vessels that exhibited
positive staining for HO-2 increased in the secretory phase. No
reaction product was observed in control sections with substitution of
the primary antibody by an appropriately diluted isotype antibody (Fig. 3H).
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This study demonstrates for the first time that the two distinct
HO isoforms are distributed in different kinds of cells in human
endometrium: HO-1 in epithelial cells and macrophages, and HO-2 in
vascular endothelial cells and smooth muscle cells. We have recently
shown that distribution of the two HO isoforms in human placental villi
had distinct topographic patterns: HO-1 was observed in villous
trophoblastic cells, whereas HO-2 was found in endothelial cells and
smooth muscle cells of blood vessels (20). Furthermore,
there has been an immunohistochemical study on rat liver that showed
distinct distribution patterns of HO: HO-1 was observed only in
Kupffer’s cells, whereas HO-2 was distributed to parenchymal cells,
but not to Kupffer’s cells (28). It is well known that
Kupffer’s cells express CD68, so it is reasonable that CD68-positive
macrophages in human endometrium strongly express HO-1. In the present
study, HO-1 was immunohistochemically observed in endometrial
epithelial cells. Although the intensity of immunostaining varied
between individuals, its relationship with the stage of the menstrual
cycle at which tissues were obtained had remained unclear. On the other
hand, the number of macrophages that exhibited positive staining for
HO-1 and were also CD68 positive increased in the secretory phase. It
has been reported that endometrial macrophages increased in number in
late secretory phase and in early pregnancy (29). We have
already reported that the combined administration of IL-1ß and
interferon-
induced mRNA and protein for iNOS in human cultured
endometrial stromal cells (30). Because macrophages are
present in the endometrium and in blood vessels, cytokines produced by
macrophages may activate iNOS in human endometrium. Cytokines are also
known to induce HO-1 (31). Therefore, the synergetic
biological effects of HO-1 and iNOS induced by macrophages are likely
to contribute to some physiological functions of human endometrium.
HO-2 was found in endothelial cells and smooth muscle cells of blood vessels in human endometrium by immunohistochemistry. Because HO-2 has been reported to be expressed in endothelial cells of blood vessels and neurons in their adventitial layers outside the brain (32), HO-2 expression in blood vessels in the endometrium is confirmed. Our results obtained by RT-PCR and Western blotting have revealed that HO-2 expression was lower in the menstrual and proliferative phases than in the secretory phase. In addition, the number of blood vessels that exhibited positive staining for HO-2 increased in the secretory phase. This phenomenon is probably related to the growth of blood vessels in the endometrium throughout the menstrual cycle. It has been recently reviewed that the majority of studies on eNOS expression demonstrated the presence of eNOS in endometrial glandular epithelium and microvascular endothelium, accompanied by its greater expression during the secretory phase (33). These results imply that the increase of HO-2 and eNOS expression may contribute to the regulation of blood flow during endometrial receptivity by causing dilatation of endometrial blood vessels.
The only known function of HO is to catalyze the conversion of heme to CO, iron, and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase (1). CO binds to heme and activates soluble guanylate cyclase. NO also binds to the heme prosthetic moiety of soluble guanylate cyclase, leading to an increase in the level of cyclic guanosine monophosphate. Discrepancies in localization of NOS and guanylate cyclase in the brain indicate that a substantial portion of guanylate cyclase may not serve as a target for NO (12). It has been proposed that the similarity of HO-2 and NOS localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiological roles for CO and NO (32). CO has a similar physiological function in the cardiovascular system, with CO being more chemically stable, but less potent, as a vasorelaxant factor than NO (34). CO production has also been demonstrated in vascular tissues where obvious parallels exist between vasodilatory function of CO and that of NO.
During reproductive age, entire endometrium undergoes cyclic changes to assure optimum conditions for implantation. If implantation does not occur, the upper part of the endometrium is shed during menstruation. The endometrium represents a unique tissue with regard to vascular changes during the cycle. Cyclic changes of endometrial blood vessels such as their damage during menstruation, neovascularization, and an increase in growth and number of blood vessels occur within a single month. Vasoactive agents such as prostaglandins, endothelin, vascular endothelial growth factor, platelet-activating factor, and NO may act directly on endometrial blood vessels during implantation and menstruation. Some previous studies have shown that iNOS and eNOS were expressed in epithelial cells, immunocompetent cells, vascular endothelial cells, and smooth muscle cells in human endometrium (17, 21, 22, 23). In addition, the present study has revealed that HO proteins are also detected in the epithelium, macrophages, and blood vessels in human endometrium. Thus, HO and NOS are likely to function as important enzymes in these cells, where endogenous CO and NO may exert vasodilatation in endometrial vascular beds. It has been proposed that NO production may play a role in both endometrial vasodilatation and receptivity during the secretory phase, and that high concentrations of NO may be involved in the initiation and maintenance of menstrual bleeding by inducing tissue breakdown and vascular relaxation as well as by inhibiting platelet aggregation (33). In addition, HO may relate to the physiology of menstruation. With vascular breakdown that occurs at the end of each endometrial cycle, vast quantities of extravasated blood, and hence heme, will come into direct contact with HO-1 released from degenerating endometrial epithelium. This seemingly would have the potential to generate high levels of CO, which might be expected to cause further vasodilatation. Therefore, we speculate that, in keeping with complementary roles of HO and NOS, HO in the endometrium may have overlapping roles with those proposed for NOS in human endometrium.
In addition to its ability to produce vasodilator gas CO, HO may have an additional important function in the defense of the endometrium from toxic oxygen radicals through its role in the generation of biliverdin and its product bilirubin, both of which are powerful antioxidants. In human uterus, reactive oxygen species such as superoxide radicals and lipid peroxides are generated in the endometrium and are increased in late secretory phase (35, 36). Superoxide dismutase, which converts superoxide to hydrogen peroxide and oxygen (37), works protectively by scavenging superoxide radicals. It has been reported that superoxide dismutase was identified in human endometrium and increased immediately before implantation (36). Furthermore, recent evidence has also shown that thioredoxin, which is a cellular redox-active protein and protects cells against oxidative stress, was expressed in human endometrium (38). Therefore, it is suggested that HO expression is important for successful implantation and placentation by protecting the endometrium against damage by toxic oxygen radicals.
Although the present finding shows that the two distinct HO isoforms are distributed in different kinds of cells in human endometrium, it remains unknown which cellular component plays a major role for endogenous CO and biliverdin production. HO-1 is known to be induced by a variety of stressors (5). Therefore, spontaneous expression of HO-1 in epithelial cells and macrophages during the menstrual cycle seems to result from constant exposure of cells to stressors, and it is proposed that produced biliverdin/bilirubin may protect the endometrium against oxidative injury. On the other hand, HO-2 is expressed in endothelial cells and smooth muscle cells of blood vessels in the endometrium. It is suggested that CO derived from HO-2 in vascular endothelium and smooth muscle contributes to endometrial vasodilatation mainly during the secretory phase. Furthermore, a possible cooperative role of these isozymes in heme catabolism in different cellular components should be considered.
In conclusion, the present study has been the first to demonstrate that the two distinct HO isoforms are distributed in different kinds of cells in human endometrium. We have also found that HO-1 is constitutively expressed throughout the menstrual cycle, but that HO-2 expression is apparently greater in the secretory phase than in the menstrual and proliferative phases. It is proposed that the CO/HO system may be involved in the local control of endometrial function. Further study will be required to examine how the CO/HO system, in association with the NO/NOS system, modulates the endometrial milieu throughout the menstrual cycle.
Acknowledgments
We are grateful to Fusako Nishiyama for technical assistance.
Footnotes
This study was supported by a Science Research Grant (11671599) to T.K. from the Ministry of Education, Science, Sports and Culture of Japan.
Abbreviations: CO, carbon monoxide; DNase, deoxyribonuclease; D-PBS, Dulbecco’s PBS; eNOS, endothelial nitric oxide synthase; HO, heme oxygenase; iNOS, inducible nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOS, NO synthase.
Received .
Accepted July 1, 2001.
References
This article has been cited by other articles:
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  H. Dassen, R. Kamps, C. Punyadeera, F. Dijcks, A. de Goeij, A. Ederveen, G. Dunselman, and P. Groothuis Haemoglobin expression in human endometrium Hum. Reprod., March 1, 2008; 23(3): 635 - 641. [Abstract] [Full Text] [PDF]
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