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Am J Physiol Endocrinol Metab 278: E1038-E1044, 2000;
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Vol. 278, Issue 6, E1038-E1044, June 2000

The oviduct produces erythropoietin in an estrogen- and oxygen-dependent manner

Seiji Masuda, Toshihiro Kobayashi, Mariko Chikuma, Masaya Nagao, and Ryuzo Sasaki

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we showed that erythropoietin (Epo) is produced in the mouse uterus, where Epo is indispensable for estrogen (E2)-dependent angiogenesis. Expression of uterine Epo mRNA is stimulated by E2 and hypoxia. The hypoxic induction requires the presence of E2. In the present study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E2 and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E2-induced stimulation of Epo mRNA was found only in the oviduct. The E2-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E2. E2-dependent production of Epo and its mRNA expression were also found by use of cultured oviducts. The E2 action is probably mediated through the E2 receptor, and de novo protein synthesis is not required for E2 induction of Epo mRNA. In the oviduct, the ampulla and isthmus regions produce Epo.

hypoxia; ovary; isthmus; tamoxifen; ICI-182780


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A WELL-RECOGNIZED FUNCTION of erythropoietin (Epo) is to increase the production of red blood cells by preventing apoptotic death of Epo-responsive erythroid precursor cells and by stimulating their proliferation and differentiation (reviewed in Refs. 18, 23, and 50). The fetal liver is the site of Epo production, which is essential for fetal erythropoiesis (24, 46). The kidney is the major site of Epo production in adults, and the kidney-derived Epo is responsible for the stimulation of erythropoiesis in adults (reviewed in Refs. 18 and 23). Epo production in these sites is stimulated under hypoxia, mainly through the activation of Epo gene expression and partly through stabilization of mRNA (reviewed in Refs. 5, 14, 36, 37, 40, 45).

The hypoxic activation of Epo gene expression is caused by binding of the hypoxia-inducible factor-1 (HIF-1) to the hypoxia-responsive enhancer that lies in a region 120 bp 3' to the polyadenylation site. HIF-1 is a heterodimer consisting of the subunit HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT), both of which are basic-helix-loop-helix proteins in the PAS (Per-AHR-ARNT-SIM) family of transcription factors. Although ARNT, which is present in high amounts, is not affected by oxygen levels, HIF-1alpha levels are very low under normoxia, because HIF-1alpha is rapidly degraded via the ubiquitin-proteasome pathway. Interaction of HIF-1alpha and the von hippel-Lindau protein appears to be necessary for the degradation of HIF-1alpha (31). Under hypoxia, HIF-1alpha is stabilized by an unknown mechanism to form the active heterodimer with ARNT. Thus HIF-1 activation correlates well with the hypoxia-induced accumulation of the HIF-1alpha subunit.

In addition to liver and kidney, recently two sites (brain and uterus) have been shown to produce Epo with new physiological functions. Neurons express the Epo receptor (EpoR) (8, 29, 32), and astrocytes produce Epo (26, 27, 30). Thus the central nervous system has a paracrine Epo/EpoR system, which is independent of the erythropoietic system (3, 8, 25-30, 32). Epo infusion into the brain prevents ischemia-induced death of cerebrocortical and hippocampal neurons (38, 39). Evidence that endogenous brain Epo plays a critical role in neuron survival under brain ischemia has been presented (39). Consistent with the view that glutamate toxicity is a major cause of ischemia-induced neuron death, Epo has been shown to protect primary cultured cerebrocortical and hippocampal neurons from glutamate toxicity (32). Low oxygen tension elevates Epo mRNA in the brain (43) and enhances Epo production by the cultured astrocytes (26, 30), which may be appropriate for the neuroprotective function of Epo in the ischemic brain.

EpoR mRNA is expressed in endothelial cells from human umbilical vein, bovine adrenal capillary, and rat brain capillary (2, 47). The angiogenic activity of Epo was shown by the use of in vitro cultured endothelial cells (1, 6, 15), but the in vivo significance of these findings was not demonstrated. In healthy adults, blood vessel formation is repressed, but an exception is the female reproductive organ, where the active angiogenesis cyclically takes place for remodeling of destroyed tissues. We have shown that there is another paracrine Epo/EpoR system in the uterus and that Epo plays an important role in the uterine angiogenesis via EpoR expressed in vascular endothelial cells of the uterine endometrium (28, 49). Furthermore, Epo production in the uterine tissue is stimulated by 17beta -estradiol (or estrogen, E2), an ovarian hormone. Because oxygen concentration was thought to be a major regulator of Epo production, E2 stimulation of uterine Epo production was surprising, but it provided relevance of Epo function in an E2-dependent cyclical angiogenesis in the uterus (49).

Encouraged by these findings, we explored the expression of Epo mRNA and the production of Epo in other female reproductive organs (ovary and oviduct). Here we report changes of Epo mRNA contents in the mouse oviduct upon E2 administration and/or exposure to a hypoxic condition. The production of Epo protein and expression of Epo mRNA by in vitro cultured oviducts were also examined.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Animals were maintained and handled in accordance with the guidelines for the care and use of laboratory animals at Kyoto University. Outbred mice of the ICR strain (Clea) were used for experiments at 3 wk of age.

E2 administration and/or hypoxic exposure. To examine the effect of E2 administration and/or hypoxic exposure on Epo mRNA levels in the ovary, oviduct, and uterus, we divided the mice into three groups. The animals in the first group received intraperitoneal administration of E2 and were left under normoxia, those in the second group were given olive oil and exposed to normobaric hypoxia (7% O2-93% N2), and those in the third group were exposed to hypoxia immediately after receiving E2 administration. E2 (from Research Biochemicals International) was dissolved in olive oil. Mice were given 100 µl of E2 solution per animal (0.5 µg E2/g body wt) by intraperitoneal injection. Olive oil was given to control mice. Hypoxic stimulation was achieved by use of an air-tight cabinet into which a premixed gas was introduced. The gas flow rate was adjusted so that 7% O2 was achieved ~30 min after the animals were placed into the cabinet. At different time points after E2 administration and/or hypoxic exposure, the animals were anesthetized with ether, and then the tissues were quickly removed and frozen in liquid nitrogen until used for RNA extraction.

Standard cDNA fragments of Epo and beta -actin. Sequence coordinates of mouse Epo cDNA are based on the definition of the transcription start site as +1 (42). A 451-bp fragment encompassing 272-722 of the mouse Epo cDNA was ligated into a vector pCR3.1-Uni by use of the Eukaryotic TA Cloning Kit (Invitrogen). The resulting plasmid was used as a standard Epo cDNA for PCR. As a standard beta -actin cDNA, pAL41 (accession no. X03765) was used. These standard cDNA fragments cover the 112-bp (Epo) and 261-bp (beta -actin) nucleotide sequences, which are identical to those that are amplified from reverse transcription (RT)-derived cDNAs by use of the primers described in the next two sections.

RT. Total RNA was prepared from the frozen tissues according to the protocol of the RNA Isolation System kit (Promega). RT was carried out at 45°C for 60 min in 20 µl of RT mixture containing 1 µg total RNA, 200 U SuperScript II (GIBCO BRL), 20 U RNase inhibitor (Takara), 0.5 mM of each dNTP, and 2.5 µM random nonamer primer. One microliter of cDNA product was used for real-time PCR.

Real-time PCR. The PCR product of Epo mRNA-derived cDNA was quantified on real time, which is accomplished by using a double dye-labeled fluorogenic oligonucleotide probe (16) and an automated fluorescence-based system for detection of PCR products. The probe is labeled at its 5' end with a fluorogenic reporter dye, 6-carboxy-fluorescein (FAM), and at its 3' end with a quencher dye, 6-carboxy-tetramethylrhodamine (TAMRA). The nucleotide sequence in the probe 5'-(FAM)-TGCAGAAGGTCCCAGACTGAGTGAAAATA-3'-(TAMRA) corresponds to fragments 397-425 in the mouse Epo cDNA. The Epo-specific sequences used for PCR were the forward primer 371F, 5'-GAGGCAGAAAATGTCACGATG-3', and the reverse primer 482R, 5'-CTTCCACCTCCATTCTTTTCC-3'. The forward and reverse primers correspond to the nucleotides 371-391 and 462-482 in Epo cDNA, respectively. They span exon/intron boundaries (exons II and III in the 371F, and exons III and IV in the 482R); thus amplification of contaminating genomic DNA is prevented. For PCR, we used TaqMan Universal PCR Master Mix containing dUTP instead of dTTP (PE Applied Biosystems, cat. no. 4304447). This master mixture also contains uracil-N-glycosylase (UNG), which destroys any carryover PCR product that might remain in PCR chambers. Before PCR was started, the complete PCR mixture containing reverse-transcribed cDNA was treated in a PCR chamber at 50°C for 2 min for the action of UNG, and then treated at 95°C for 10 min, resulting in the inactivation of UNG and activation of DNA polymerase. Then PCR, consisting of 50 cycles at 95°C for 15 s and at 60°C for 1 min, was performed. When DNA polymerase engaged in extension of the primer reaches the quencher-labeled nucleotide of the probe hybridized to cDNA, the exonuclease activity of DNA polymerase excises the labeled nucleotide, resulting in the emission of fluorescence. All procedures including data analysis were performed on the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) using the software provided with the instrument. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential amplification. Messenger RNA of beta -actin was also measured, and its level was used as an internal control for normalization of the Epo mRNA level. The beta -actin sequence was amplified and detected using the primers 5'-CTAGGCACCAAGGTGTGAT-3' and 5'-CAAACATGATCTGGGTCATC-3' and the probe 5'-(FAM)-TGGCACCACACCTTCTACAATGAG-3'-(TAMRA). When samples lacking RNA or reverse transcriptase in the reverse transcriptase reactions were subjected to PCR, each of these control reactions yielded no detectable fluorescence, excluding the possibility that the contaminating DNA, including the RNA preparation-derived genomic DNA, was amplified.

Copy numbers of Epo or beta -actin mRNA-derived cDNA were calculated from the standard curves of PCR drawn by the use of the standard Epo or beta -actin cDNA fragments. The lower limit of quantitative detection by the present method was 30 copies of cDNA.

Culture of the oviduct. The oviducts were removed from 3-wk-old mice. The unilateral oviduct was cultured in a phenol red-free DMEM supplemented with 10% charcoal-treated fetal calf serum containing the test substance, and the contralateral oviduct was cultured in the same medium but without the test substance, as a control. These media were incubated in a humid 5% CO2 atmosphere at 37°C. Epo protein secreted in the culture media was measured by a sandwich-type enzyme-linked immunoassay (EIA) by use of two monoclonal antibodies that bind Epo at different epitopes (33), and the tissue was used for total RNA preparation. Recombinant human Epo, produced and isolated as described previously (12, 13), was used as a standard. This assay measures Epo at a concentration as low as 1 pg/ml.

To identify the oviductal Epo production site, the infundibulum, ampulla, and isthmus were removed from the oviduct under a microscope and cultured in the presence or absence of E2 for 8 h. Epo protein secreted in the culture media was measured by EIA.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo induction of Epo mRNA in female reproductive organs by E2 and hypoxia. Previously (unpublished observations), we showed with ovariectomized mice that Epo mRNA in the uterus was E2 inducible but not hypoxia inducible in the absence of E2. In the present experiments, to avoid the effects of the internal E2, we used 3-wk-old mice before commencement of cyclical synthesis of E2 in the ovaries. E2 was given intraperitoneally, and then the ovary and oviduct were removed to measure Epo mRNA at 1 h after E2 injection. Figure 1 shows the results. Epo mRNA was detected in both the ovary and oviduct from E2-uninjected animals, and the Epo mRNA level in the oviduct was threefold greater than that in the ovary. Furthermore, Epo mRNA in the oviduct was highly E2 inducible (4- to 5-fold induction), whereas that in the ovary slightly increased upon E2 injection. Therefore, we examined the Epo expression in the oviduct.


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  		Fig. 1.   In vivo effect of estradiol (E2) on expression of erythropoietin (Epo) mRNA in ovary and oviduct. Animals were administered 0.5 µg E2/g body wt or olive oil. At 1 h after injection, ovary and oviduct were removed for quantitative measurement of Epo and beta -actin mRNAs by real-time RT-PCR (see MATERIALS AND METHODS). The ordinate indicates multiple increases in induction of Epo mRNA over control levels (olive oil), which were 29,600 copies/2 oviducts and 11,700 copies/2 ovaries, and defined as 1. Bars are means ± SE (n = 3). Statistical significance of differences was determined with Student's t-test. * P < 0.05, significantly different from values of controls (values in the absence of E2).

To examine the effects of E2 and hypoxia on Epo mRNA levels in the oviduct, mice were given E2 or the vehicle and immediately exposed to hypoxia (7% O2) or kept under normoxia. At different time points the oviducts were removed to measure Epo mRNA. Figure 2A shows the results. When animals received E2 and were kept under normoxia, Epo mRNA was increased eightfold at 2 h and decreased at 4 h. When animals were exposed to hypoxia without E2 injection, a significant induction was seen at 1 and 2 h. Administration of E2 potentiated the induction of Epo mRNA by hypoxia. The oviduct of animals that received both stimuli showed the highest induction (30-fold) of Epo mRNA at 4 h, but the level was markedly decreased at 8 h.


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  		Fig. 2.   Temporal patterns of induction of Epo mRNA in oviduct (A) and uterus (B) by E2 and/or hypoxia. , Animals were exposed to hypoxia (7% O2) immediately after administration of 0.5 µg E2/g body weight (E2/Hx). open circle , Animals were exposed to hypoxia after administration of olive oil (-E2/Hx). , Animals were left under normoxia after administration of 0.5 µg E2/g body wt (E2/Nx). At indicated time points, tissues were removed for measurement of Epo mRNA. The ordinate shows multiples of increase in (fold) induction over control level (at time 0). Bars are means ± SE (n = 4). Statistical significance of differences was determined with ANOVA followed by Dunnett's test. * P < 0.05, ** P < 0.01, significantly different from values of controls (time 0), respectively.

For comparison, Epo mRNA in the uterus was also assayed (Fig. 2B). There was a marked increase under normoxia at 2 h after E2 injection, but at 4 h the level markedly decreased. Without E2 injection, the uterine Epo mRNA level was unchanged despite the continuous hypoxia. However, uterine Epo mRNA became hypoxia inducible in the presence of E2. These results, obtained by the use of prepuberal young mice, were similar to those obtained by the use of ovariectomized adult animals (unpublished observations). A remarkable difference between uterus and oviduct was that the oviductal Epo mRNA of E2-uninjected animals is hypoxia inducible, whereas the uterus does not respond to hypoxia unless E2 is given.

Induction of Epo mRNA and production of Epo by cultured oviducts. To examine Epo production in the in vitro cultured oviducts and its induction by E2, the unilateral oviduct was cultured with E2 for 8 h. The contralateral oviduct was cultured without E2 as a control. Epo protein was detectable in the medium after culture without E2, and E2 stimulated Epo production in a dose-dependent manner (Fig. 3A). Figure 3B shows the production of Epo with culture over time.


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  		Fig. 3.   E2-dependent production of Epo in cultured oviduct. A: dose dependency; B: time dependency. In (A), oviducts were cultured for 8 h with E2 at concentrations indicated. In (B), oviducts were cultured with 100 nM E2 for times indicated. Epo secreted in culture media was measured. Bars represent means ± SE (n = 4). Statistical significance of differences was determined with ANOVA followed by Dunnett's test. * P < 0.05 and ** P < 0.01, significantly different from values of controls, respectively.

Figure 4 shows the effects of the E2 receptor (ER) antagonist and of inhibitors of protein and RNA synthesis on the E2-induced production of Epo. Addition of ICI-182780, a specific antagonist of the ER, decreased Epo production. Cycloheximide and actinomycin D completely inhibited Epo production, demonstrating that Epo secreted into the culture media was newly synthesized during culture. Tamoxifen, which is an E2 antagonist in breast tissues but acts as an agonist in female reproductive organs (10a), stimulated Epo production with a potency similar to E2 (data not shown). ICI-182780 repressed tamoxifen-induced Epo production.


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  		Fig. 4.   Effect of an E2 antagonist and inhibitors of protein and RNA synthesis on Epo production by cultured oviducts. Oviducts were cultured for 8 h with 100 nM E2, 100 µM ICI-182780 (ICI, an E2 antagonist), 10 µM actinomycin D (Act D), or 200 µM cycloheximide (CHX). Epo secreted in culture media was measured. Bars represent means ± SE (n = 4). Statistical significance of differences was determined with ANOVA followed by Tukey-Kramer's test. ** P < 0.01, significantly different from each other.

In addition to oxygen and E2, Epo production has been shown to be influenced by some other substances. Thyroid hormones enhance hypoxia-induced Epo production by HepG2 and the perfused rat kidney (10); retinoic acid promotes Epo production in HepG2 and vitamin A-depleted rats (33); and insulin, insulin-like growth factor (IGF) I, and IGF-II stimulate Epo production by cultured astrocytes (27). We examined the stimulation of Epo production in the cultured oviducts by various ligands for nuclear receptors, including progesterone, testosterone, 3,3',5-triiodo-L-thyronine, L-thyroxine, all-trans retinoic acid, and vitamin D3. The oviducts were cultured for 8 h in the presence of individual compounds at 100 nM. The addition of these compounds had no significant effect on Epo production (data not shown), indicating that low oxygen and E2 are specific stimulators for Epo production in the oviduct.

Next, we investigated the expression of Epo mRNA in cultured oviducts. As Fig. 5 shows, Epo mRNA was clearly induced by E2 at 2 h after culture, reached the maximum at 4 h, and markedly decreased at 8 h. The E2-induced expression of Epo mRNA in cultured oviducts was repressed by ICI-182780 and actinomycin D (Fig. 6). In contrast, Epo mRNA was superinduced by cycloheximide in both the presence and absence of E2, indicating that de novo protein synthesis is not needed for induction of Epo mRNA.


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  		Fig. 5.   Expression of Epo mRNA in cultured oviducts is induced by E2. Oviducts were cultured with or without 100 nM E2 for indicated times, and Epo mRNA was measured. Open and filled columns, Epo mRNA in oviducts cultured without (-) and with E2 (+), respectively. Bars are means ± SE (n = 3). Statistical significance of differences was determined with ANOVA followed by Tukey-Kramer's test. * P < 0.05 and ** P < 0.01, respectively.



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  		Fig. 6.   Effect of an E2 antagonist and inhibitors of protein and RNA synthesis on E2-induced expression of Epo mRNA in cultured oviducts. Oviducts were cultured for 8 h with 100 nM E2, 100 µM ICI, 10 µM Act D, or 200 µM CHX. Epo mRNA in culture oviducts was measured. Bars represent means ± SE (n = 3). Statistical significance of differences was determined with ANOVA followed by Tukey-Kramer's test. ** P < 0.01, significantly different from each other.

The oviduct is composed of three morphologically distinguishable segments. The funnel-shaped abdominal end of the oviduct is called the infundibulum. The expanded intermediate segment below the infundibulum is the ampulla. The isthmus is the slender medial third near the uterine wall. To clarify which segment is responsible for the oviductal Epo production, oviducts were separated into the three segments, and they were cultured with or without E2. Epo protein in the culture media was assayed (Fig. 7). The ampulla and isthmus produced Epo in the absence of E2, and the production was stimulated by E2. Epo in the culture media of the infundibulum was almost undetectable in the presence and absence of E2.


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  		Fig. 7.   Ampulla and isthmus produce Epo in an E2-dependent manner. Each part of the oviduct was dissected and cultured in the presence or absence of E2 for 8 h. Secreted Epo was measured by enzyme immunoassay. Filled columns, Epo protein secreted into media cultured with 100 nM E2. Open columns, Epo protein when cultured without E2. Bars represent means ± SE (n = 4). Statistical significance of differences was determined with Student's t -test. * P < 0.05 and *** P < 0.001, significantly different from values of controls (values in absence of E2), respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There are at least four sites (liver, kidney, brain, and uterus) of Epo production (28). Epo produced by the liver and kidney stimulates erythropoiesis, brain Epo prevents ischemic neuron death, and uterine Epo plays a critical role in the E2-dependent cyclical angiogenesis in the uterus. Whereas Epo production in the kidney, liver, and brain is hypoxia inducible, that in the uterus is E2 inducible. These findings of Epo production and a new function in the uterus prompted us to explore the possibility that other female reproductive organs (ovary and oviduct) may produce Epo. Although both the ovary and oviduct expressed Epo mRNA, expression of Epo mRNA in the oviduct is highly E2 inducible; therefore, we examined the properties of Epo production by the oviduct. The in vitro cultured oviducts were also found to express Epo mRNA and secrete Epo into the culture media, both of which were E2 inducible.

Tamoxifen (an ER agonist in female reproductive organs) elevates the expression of oviductal Epo, and ICI-182780 (a specific ER antagonist) counteracts the E2-induced expression. These results have made it very likely that E2 acts through ER. E2-induced accumulation of the oviductal Epo mRNA does not require de novo protein synthesis, and the accumulation is inhibited by actinomycin D, suggesting that this accumulation is largely due to the transcriptional activation of the Epo gene by E2. In the 5' flanking region of both mouse and human Epo genes, the palindromic consensus sequence of the E2 response element is not present, but some consensus half-sites, and the sequences homologous to the consensus half-site, exist. Four half-sites separated from each other by >100 bp have been shown to confer E2 responsiveness to target genes synergistically (20). Expression of the reporter gene flanked by a 5' flanking region of the Epo gene was activated by E2, and this activation required ER-alpha (unpublished observations). Thus E2-induced accumulation of oviductal Epo mRNA is at least partly attributable to the transcriptional activation of the Epo gene.

Downregulation of the oviductal Epo mRNA that occurs shortly after injection of E2 is due to the loss of the cellular response to E2 but not to the metabolic depletion of E2. One possibility to account for this loss is that ER was rapidly degraded by the addition of E2. In fact, stimulation of proteolytic degradation of ER by E2 was seen in mouse uterus and breast cancer cell lines (11, 17, 35). It is also possible that the downregulation of E2-induced transcriptional activation of the Epo gene is caused by E2-induced modulation of transcriptional factors associated with ER. Co-activators such as SRC1/NCoA1, TIF2/GRIP1, AIBI/pCIP/ACTR, p300/CBP, NcoA62, PBP/TRAP220, and/or co-repressors such as TIF-1 and RIP140 (4, 7, 19, 22, 34, 41, 44) have been shown to be involved in E2-induced regulation of gene transcription through ER. In addition, BRCA1 inhibits E2 signaling by blocking the transcriptional activation function of AF-2 in ER-alpha (9). Thus a variety of modes are possible for the downregulation of E2-induced transcriptional activation. Although the mechanism of the downregulation remains to be studied, the short-lived stimulatory effect of E2 on expression of the oviductal Epo gene may be critical for ensuring a yet unknown function of oviductal Epo without significant perturbation of erythropoiesis.

The oviduct provides the appropriate environment for fertilization of the ovum released from the ovary and embryonic development, and it transports the embryo to the uterus. The oviduct can be divided into three segments, infundibulum, ampulla, and isthmus. The infundibulum secures oocytes extruded from the ovary. The ampulla is the site of fertilization. Ciliated cells in the isthmus propel embryos toward the uterus. The isthmus is thought to be an important region for the capacitation of spermatozoa. The ampulla and isthmus are the Epo production sites in the oviduct. Papers have appeared (43, 48) suggesting that Epo might be involved in sperm formation. Epo mRNA has been detected in the rat testis under normoxia, and its level is increased by hypoxia (43). Epo stimulates epididymal sperm maturation and sperm-fertilizing activity in rats (48). We speculate that Epo in the oviduct plays a role in fertilization, including sperm capacitation. Further studies are clearly needed to elucidate the physiological function of Epo in the oviduct.


    ACKNOWLEDGEMENTS

This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and from the "Research for the Future" program in The Japan Society for the Promotion of Science.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Sasaki, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto Univ., Kyoto 606-8502, Japan (E-mail: rsasaki{at}kais.kyoto-u.ac.jp).

Received 7 September 1999; accepted in final form 14 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 278(6):E1038-E1044
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