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Unidad de Reproducción y Desarrollo,3 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago 8331010, Chile
Millennium Institute for Fundamental and Applied Biology,4 Santiago 7780272, Chile
Laboratorio de Inmunología de la Reproducción,5 Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 9170022, Chile
ABSTRACT
Estradiol (E2) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E2-accelerated egg transport. Intrabursal application of OR486 prevented E2 from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E2 on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E2 accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E2 nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.
17β-estradiol, catechol-O-methyltransferase, estrogen receptor, methoxyestradiols, oocyte transport, oviduct, ovum pick-up/transport
The duration of oviductal egg transport in the rat is dependent upon ovarian hormone levels and mating-associated signals [1]. A single injection of estradiol (E2) into cyclic or pregnant rats shortens the duration of oviductal transport of eggs from the normal 72–96 h to less than 24 h [2].
E2 binds to estrogen receptors (ER) and modifies gene expression and protein synthesis in its target organs [3]. For example, E2 increases the mRNA and protein levels of aquaporins and connexin 43 in the rat oviduct [4, 5]. In keeping with this notion, RNA and protein synthesis inhibitors suppress accelerated oviductal egg transport induced by E2 in pregnant rats, although they fail to do so in cyclic rats [6, 7]. Furthermore, in cyclic rats, exogenous E2 induces protein phosphorylation in the oviduct when mRNA synthesis is completely suppressed by 
-amanitin [8]. Estradiol-induced phosphorylation is essential for oocyte transport in cycling rats, since intrabursal administration of a broad-spectrum inhibitor of protein kinases abrogates E2-induced acceleration of egg transport [8–11]. Therefore, E2 accelerates egg transport through intraoviductal genomic pathways in pregnant rats and through nongenomic pathways in cyclic rats. Utilizing the ER antagonist ICI 182780, we have demonstrated that both pathways require activation of ER [9, and Orihuela and Parada-Bustamante, unpublished results]. We have provisionally designated this change in pathways as intracellular path shifting (IPS). Among mating-associated signals, mechanical sensory stimulation of the genital area and the presence of spermatozoa in the uterus have been shown to elicit IPS [11, 12]. IPS caused by mating is a novel example of functional plasticity in well-differentiated cells and is probably associated with changes in gene expression and signal transduction cascades.
In order to explore this phenomenon, we first performed microarray analysis, to determine changes in oviductal gene expression induced by mating. The results oriented us towards the possible involvement of the enzyme catechol-O-methyltransferase (COMT) in the nongenomic signaling pathways that operate prior to mating. According to the literature, COMT forms the E2 metabolites 2-methoxyestradiol and 4-methoxyestradiol (2ME and 4ME, respectively) from 2-hydroxyestradiol and 4-hydroxyestradiol (2OHE and 4OHE, respectively) produced from E2 by cytochrome P450 isoforms CYP1A1 and CYP1B1 [13]. Methoxyestradiols activate distinct cascades of signal transduction, including the generation of nitric oxide in cultured bovine carotid artery endothelial cells [14] and cAMP synthesis in MCF-7 cells [15]. Furthermore, these metabolites act through nongenomic pathways in various cell systems, such as the rat uterus and aorta [16, 17] or Chinese hamster V79 cells [18]. Thus, we investigated whether methoxyestradiols and oviductal COMT participate in accelerated egg transport. For this purpose, we examined the effect of E2 on egg transport in cyclic and pregnant rats under conditions in which COMT activity was quenched by a selective inhibitor. The effects of 2ME and 4ME on egg transport in cycling and pregnant rats were determined. In addition, the involvement of COMT, ER, RNA and protein synthesis, and protein phosphorylation in 2ME-induced acceleration of egg transport was assessed in cycling rats.
Locally bred Sprague-Dawley rats were used. The animals were kept under controlled temperature (21–24°C) and the lights were on from 0700 h to 2100 h. Water and pelleted rat chow was supplied ad libitum. Females weighing 200–220 g were selected from those that had at least two regular cycles of 4 days immediately before the experiments started. Daily vaginal smears, which were taken between 0800 h and 0900 h, were used to verify cycle regularity [19]. To obtain cyclic and pregnant rats for the experiments described below, females in the evening of proestrus were either kept isolated or caged with fertile males. The next morning, isolated rats that presented cornified cells in the vaginal smear, which is a cell phenotype that is associated with ovulation (estrus day), were designated as rats on Day 1 of the cycle (C1), and the rats caged with fertile males that presented cornified cells and spermatozoa in the vaginal smear were designated as rats on Day 1 of pregnancy (P1). The care and manipulation of the animals was carried out in accordance with the ethical guidelines of the Pontificia Universidad Católica de Chile and the Universidad de Santiago de Chile.
All of the treatments described below were administered at 1200 h on C1 or P1.
Systemic administration of E2 or its metabolites. Rats on C1 or P1 were injected s.c. (subcutaneous) with 1 or 3 µg of E2 as a single dose in an injection volume of 0.1 ml of propylene glycol. Control rats received propylene glycol as the vehicle. On C1 or P1, doses of 1, 10, or 100 µg of 2ME (Sigma, St. Louis, MO) and 4ME (Steraloids, Newport, RI) were injected s.c. as a single dose in an injection volume of 0.1 ml propylene glycol. Control rats received propylene glycol alone.
Local administration of 2ME or inhibitors. Rats on C1 or P1 were injected in the ovarian bursa with one of the drugs described below. Control rats received the appropriate vehicle only. 2ME was injected as a single dose of 0.001, 0.01, 0.1, 1, or 10 µg, dissolved in 4 µl of 0.1% dimethylsulfoxide (DMSO, Sigma). OR486 (Tocris, Langford, United Kingdom) was used to inhibit COMT activity in the oviduct and presumably blocked the local production of 2ME. OR486 at 5, 25, or 125 µg dissolved in 4 µl of 25% ethanol was injected into rats that were treated s.c. with 1 µg E2 on C1, while 125 µg OR486 was administered to rats that were treated s.c. with 3 µg E2 on C1 or P1. The RNA synthesis inhibitor actinomycin D (Act D; Calbiochem, La Jolla, CA) was used to inhibit the potential genomic action of 2ME. Act D at 1 µg dissolved in 2 µl saline was injected into rats on C1. This dosage completely blocks protein synthesis in the rat oviduct [7]. ICI 182780 (kindly donated by W. Elger, Entech, Jena, Germany) was used to block the ER and to determine whether ER are required for the effect of 2ME. ICI 182780 at 25 µg dissolved in 4 µl of 25% DMSO was injected into rats on C1. This dosage completely blocks the effect of E2 on egg transport [9]. H-89 (N-[2-(p-bromcinnamylamino)ethyl]-5-isoquinolinesulfanomide-dihydrochloride; Calbiochem), which is a broad-spectrum inhibitor of protein kinases, was used to block the nongenomic actions of 2ME. H-89 at 15 µg dissolved in 4 µl saline was injected into rats on C1. This dosage completely blocks protein phosphorylation in the rat oviduct [8].
Intrabursal (i.b.) administration, which minimizes the dose needed to affect the oviduct without systemic effects, was performed on rats at C1 and P1 at 1200 h, as described by Orihuela et al. [8]. Briefly, the oviduct and ovary were exposed through flank incisions made under anesthesia and using a surgical microscope (OPMI 6-SDFC; Zeiss, Oberkochen, Germany). The drugs or vehicle alone were injected into the periovarial sac using a Hamilton syringe (Hamilton Co., Reno, NV), and the injection site in the bursa was immediately sealed with an electric coagulator (Codman CMC-1; Codman and Shurleff Inc., Randolph, MA). The organs were returned to the peritoneal cavity, and the muscles and skin were sutured. Since ovulation was completed at this time-point, this treatment did not affect the number of oocytes that ovulated. Furthermore, we have previously demonstrated that drugs that are administered i.b. act locally on the oviduct [9, 10].
Animals were killed 24 h after different treatments, and their oviducts were flushed individually with saline. Each flushing was examined under low-power magnification (25x). The number of eggs in both oviducts was recorded as a single datum. We have previously determined that the recovery of eggs using this method is close to 100%, comparing the average number of eggs obtained from oviducts with this technique with the number of implanted embryos on Day 12 of pregnancy [2]. Attempts to recover eggs from the uterus and vagina with or without placing ligatures in the uterine horns have shown that the reduction in the number of oviductal oocytes following treatment with E2 corresponds to premature transport to the uterus [2]. Thus, we refer to this phenomenon as E2-induced acceleration of oviductal transport.
Oviduct Collection and Microarray Analysis
Female rats were caged with fertile males at midnight on the day of proestrus (0000 h; pregnant group, N = 5) or kept isolated (cyclic group, N = 5). Two hours later (0200 h), mated rats were isolated, and at 0700 h the animals of both groups were killed and their oviducts were collected and flushed with saline. Total oviductal RNA was isolated from each rat using Trizol reagent (Invitrogen, Gaithersburg, MD) and equivalent quantities were mixed to generate a cyclic group pool and a separate pregnant group pool. Probes prepared from each group were hybridized by Genome Explorations Inc. to the Rat Genome 230 2.0 chips (Affymetrix GeneChip System; Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. The transcriptome profile obtained for the cyclic group was compared with that obtained for the pregnant group. The ‘signal log ratios' were converted to fold-changes. Only those transcripts that increased or decreased on average more than 2-fold were included in the subsequent analysis.
Oviducts from cyclic (N = 5) and pregnant (N = 5) rats were obtained following the same protocol described above for the microarray analysis, and total RNA was isolated using Trizol. One µg of total RNA of each sample (two oviducts from one rat) was treated with DNase I (amplification grade; Invitrogen). The single-strand cDNA was synthesized by reverse transcription using the Superscript III Reverse Transcriptase First Strand System for RT-PCR (Invitrogen), according to the manufacturer's protocol. The Light Cycler instrument (Roche Diagnostics, GmbH Mannheim, Germany) was used to quantify the relative gene expression of Comt in the oviducts of cyclic and pregnant rats; Gapdh was chosen as the housekeeping gene for loading control. The SYBR Green I double-strand DNA binding dye (Roche Diagnostics) was the reagent of choice for these assays. The following primers were used: for Comt, sense 5'-CACCTACTGCACACAGAAGG-3' and antisense 5'-GTTAGTGTGTGCACTCGAAGC-3'; and for Gapdh, sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3'. All real-time PCR assays were performed in duplicate. The thermal cycling conditions included an initial activation step of 95°C for 25 min, followed by 40 cycles of 95°C for 15 sec, 59°C for 30 sec, and 72°C for 30 sec, with an ultimate cycle of melting (95°C to 60°C). To verify the specificity of each product, amplified products were subjected to melting curve analysis as well as electrophoresis, and product sequencing was performed using an ABI Prism 310 sequencer. The expression of Comt was determined using the equation: Y = 2–
Cp, where Y is the relative expression, Cp (crossing point) is the cycle in the amplification reaction in which fluorescence begins to expand exponentially above the background baseline, and -Cp is the result of subtracting the Cp value of Comt from the Cp value of Gapdh for each sample. To simplify the presentation of the data, the relative expression values were multiplied by 103 [20].
Rats were killed on C1 (N = 3) or P1 (N = 3) at 1200 h and their oviducts were collected and flushed with saline. Total RNA was isolated using Trizol. Reverse transcription was performed according to the method of Brañes et al. [4]. In order to detect the transcripts of oviductal Cyp1a1 and Cyp1b1, 500-bp and 376-bp products related to the Cyp1a1 and Cyp1b1 genes, respectively, were amplified by PCR. The PCR reactions for Cyp1a1 and Cyp1b1 were carried out as described previously [21]. Briefly, Taq polymerase buffer that contained 1.5 mM MgCl2, and 0.2 mM dNTP plus 0.4 µM of each primer of rat Cyp1a1 (sense, 5'-GTTCCCAAAGGTCTGAAGAG-3' and antisense, 5'-CATATGGCACAGATGACATTGG-3') or Cyp1b1 (sense, 5'-GCTCAGCCACAACGAGGAGTTC-3 and antisense, 5'-CTGGTAAAGAGGATGAGCAGC-3') were used. The housekeeping gene ribosomal 18S (sense, 5'-GCTCGTCGTCGACAACGGGTC-3' and antisense, 5'-CAAACATGATCTGGGTCATCTTCTC-3') was used as an internal standard. The thermal sequence consisted of 94°C for 30 sec, 65°C for 1 min, and 68°C for 2 min, followed by a final template extension step of 68°C for 7 min. In total, 37 cycles were used for Cyp1a1, 32 cycles were used for Cyp1b1, and 23 cycles were used for 18S. The PCR products were resolved in 12% (v/v) acrylamide gels and subjected to silver staining (kit provided by Winkler Ltda., Santiago, Chile). Total RNA samples of the rat brain and ovary were used as positive controls.
Rats were killed on C1 (N = 3) or P1 (N = 3) at 1200 h and their oviducts were collected and flushed with saline. Total proteins were isolated as described previously [9]. Proteins (100 µg) were separated on 12% SDS-PAGE slab gels in a Mini PROTEAN electrophoretic chamber (Bio-Rad, Hercules, CA). Proteins resolved in the gels were electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked overnight at 4°C in TTBS (100 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% [v/v] Tween-20) that contained 5% nonfat dry milk and incubated for 2 h with rabbit anti-CYP1A1, goat anti-CYP1B1 or mouse anti-β-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000, 1:1500 or 1:1000 dilution, respectively. The blots were rinsed five times for 5 min each in TBS (100 mM Tris-HCl [pH 7.5], 150 mM NaCl) and incubated for 2 h in TTBS that contained horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (1:5000 dilution; Chemicon) or HRP-conjugated chicken anti-goat IgG (Chemicon). HRP activity was detected by enhanced chemiluminescence using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA). Oviductal samples without anti-CYP1A1, anti-CYP1B1 or anti-β-tubulin antibody were included as negative controls. Protein extracts of rat brain or ovary were used as positive controls.
Preparation of Oviductal Extracts for Measuring Rate of O-Methylation
All procedures were carried out at 0–4°C. Oviducts from cyclic (N = 3) or pregnant rats (N = 3) were obtained as described above for the microarray analysis. The preparation of oviductal extracts was carried out as described previously [22], with some modifications. Briefly, three cyclic or three pregnant rats provided each a total of six oviducts, which were pooled separately and homogenized in 10 mM EDTA, 1.4% KCl (pH 7.4). Tissue homogenates were first centrifuged at 9000 x g for 10 min, and the supernatants were recentrifuged at 105 000 x g (4°C) for 60 min. The supernatant fractions were cleared by passage through a 0.45-µm filter. The proteins in the filtrate were precipitated with six volumes of cold acetone overnight, collected by centrifugation at 9000 x g for 10 min, and resuspended in 0.01 M Tris-HCl (pH 7.4) to a final concentration of 2 mg/ml. Aliquots of these preparations were stored at –80°C until use.
O-Methylation of 2OHE by Oviductal Protein Extracts
O-Methylation of 2OHE was carried out as described previously [22], with some modifications. Briefly, the reaction mixture consisted of 0.01 M Tris-HCl buffer (pH 7.9), 1.2 mM MgCl2, 1 mM dithiothreitol, 200 µM AdoMet iodide (containing 0.5 µCi [methyl-3H] AdoMet; PerkinElmer) and 0, 30, and 80 µM 2OHE (Sigma) in a final volume of 1 ml. The reaction was started by the addition of porcine liver COMT (Sigma) or oviductal protein extracts at a final concentration of 25 U/ml or 125 µg protein/ml, respectively. Incubations were carried out at 37°C for 0, 20, 40, and 80 min. The O-Methylation reaction was stopped by the addition of 200 µl of concentrated HCl. The reaction mixtures were immediately extracted with 2 ml of toluene. After a short centrifugation, 3-ml aliquots of the organic extracts were assayed for radioactivity in a toluene-based scintillation cocktail. All samples were assayed in duplicate and three replicates of this experiment were performed.
O-Methylation of 2OHE in Whole Oviducts
Rats at C1 and P1 were killed at 1200 h and their oviducts were removed, cleaned of fat tissue, and flushed to avoid contamination with egg or sperm proteins. The oviducts were then transferred to 0.5 ml of prewarmed reaction mixture as described above. The oviducts in groups of two were incubated for 80 min at 37°C on a rocking platform at 100% relative humidity. At the end of the incubation period, the organs were blotted on filter paper and homogenized in 0.5 ml PBS on ice using a Polytron homogenizer (Kinematica GmbH, Switzerland) for 10 sec. The homogenates were immediately extracted with 2 ml of toluene. After a short centrifugation, 3-ml aliquots of the organic extracts were assayed for radioactivity in a toluene-based scintillation cocktail. All samples were assayed in duplicate and three replicates of this experiment were performed.
The results are presented as mean ± SEM. Overall analysis was carried out using the Kruskal-Wallis test, followed by the Mann-Whitney test for pairwise comparisons when overall significance was detected. The actual N value in experiments to determine the effects of drugs on oviductal egg transport is the total number of rats used in each experimental group.
Mating Changes Gene Expression in Rat Oviduct; Decreased Comt Expression
A total of 17 transcripts out of 31 100 genes analyzed showed greater than 2-fold changes in level 6 h after mating (Table 1). Four of these 17 transcripts corresponded to expressed sequence tags (EST). Of the well-known gene transcripts, 11 were up-regulated and 2 were down-regulated. One of the three down-regulated transcripts encodes Comt and was decreased 6-fold, as confirmed by real-time PCR (N = 5; 279.5 ± 61.9 Comt-relative copies on C1, and 97.0 ± 38.1 Comt-relative copies on P1, P < 0.05). The remaining 16 transcripts were not submitted to confirmation by real-time PCR.
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Expression of CYP1A1 and CYP1B1 in Rat Oviduct
Since CYP1A1 and CYP1B1 convert E2 into substrates for COMT, we determined whether the corresponding mRNAs and proteins are expressed in the rat oviduct. The mRNA and protein levels for both cytochromes were similar in C1 and P1 rat oviducts (Fig. 1, A and B).
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Mating Decreases O-Methylation Rate of 2OHE in Rat Oviduct
An enzymatic kinetic assay was conducted to obtain a double-reciprocal plot of O-methylation of 2OHE catalyzed by COMT that was purified from porcine liver and by protein extracts of cyclic and pregnant rat oviducts (Fig. 2). The Michaelis-Menten constant (Km) obtained was similar for the three groups (Table 2), whereas the maximum enzyme velocity (Vmax) values obtained for equivalent amounts of protein from cyclic and pregnant rat oviducts were 14.9 and 3.3 pmol (mg protein)–1 min–1, respectively (Table 2). This indicates that the oviducts from pregnant rats form 4-times less product per minute than the oviducts from cyclic rats.
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COMT Inhibitor Blocks O-Methylation of 2OHE in the Oviduct
In order to block COMT activity in the oviduct, the COMT inhibitor OR486, was administered locally by i.b. injection. Six C1 rats were injected in each ovarian bursa with 125 µg OR486, and 3 h later the oviducts were excised, incubated with the reaction mixture, and processed to determine O-methylation of 2OHE. The values for O-methylation of 2OHE were significantly lower in oviducts treated with OR486 than in the control oviducts (2.4 ± 0.8 versus 12.6 ± 1.7 nmol [mg protein]–1 min–1, respectively). Thus, OR486 given i.b. almost completely inhibited oviductal COMT activity. Therefore, we evaluated whether E2 could accelerate egg transport under this condition. C1 and P1 rats were injected at 1200 h with 1 µg or 3 µg E2 s.c. and with OR486 i.b. (Fig. 3). As expected, E2 reduced the number of oviductal eggs in rats at C1 at the two doses tested (for 1 µg E2, 1.0 ± 0.3 eggs; for 3 µg E2, 0.4 ± 0.2 eggs; for the control, 7.0 ± 0.5 eggs), while in rats at P1, E2 reduced the number of eggs but only at the dose of 3 µg (for 1 µg E2, 9.2 ± 0.4 eggs; for 3 µg E2, 4.8 ± 1.5 eggs; for the control, 9.4 ± 0.5 eggs).
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In C1 rats, OR486 at 5, 25, or 125 µg blocked the effect of 1 µg E2 in a dose-response manner (oviductal egg range, 1.5 ± 0.4 to 6.4 ± 0.4), while the effect of 3 µg E2 was blocked only by 125 µg OR486 (2.8 ± 1.0 eggs, and data not shown). In P1 rats, 125 µg OR486 did not prevent accelerated egg transport induced by 3 µg E2 (6.0 ± 0.8 eggs). These results indicate that E2 requires an operational COMT to accelerate egg transport in cyclic rats but not in pregnant rats.
2ME Accelerates Egg Transport in Cyclic but Not in Pregnant Rats
In order to examine the apparent participation of E2 metabolites generated by COMT in accelerated transport induced by E2, we evaluated the effect of 2ME on oviductal egg transport. C1 or P1 rats were treated s.c. or i.b with different doses of 2ME. 2ME given i.b. or s.c. to rats at C1 accelerated ovum transport. When 2ME was administered i.b., maximum acceleration was obtained within the dose range of 0.1–10 µg but was incomplete (Fig. 4A). When 2ME was given s.c., egg transport was accelerated at a dosage of 10 µg and the effect was maximal at a dosage of 100 µg (Fig. 4B). 2ME applied i.b. or s.c. to P1 rats at the same doses as in cyclic rats had no effect on transport (Fig. 4, C and D). Thus, 2ME accelerates egg transport in cyclic rats but is ineffective in pregnant rats.
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Given that 2ME accelerates ovum transport in cyclic rats, we confirmed that COMT inhibitor OR486 has no effect downstream of 2ME. C1 rats were injected i.b. with 125 µg OR486, a dose that blocks the effect of E2, and 2ME was given s.c. at a dose that accelerates ovum transport. No differences in the mean numbers of eggs recovered were found between the groups treated with 2ME and 2ME plus OR486 (2.5 ± 0.7 versus 2.0 ± 0.4, data not shown). These results indicate that OR486 does not block the effect of E2 downstream of 2ME production.
To examine the effects of other E2 metabolites on egg transport, C1 and P1 rats received a single s.c. injection of 4ME. Accelerated egg transport was obtained with 100 µg 4ME (N = 5; 8.3 ± 0.4 oocytes in the control group versus 1.4 ± 0.8 oocytes in the 100 µg 4ME-treated group) and no effect was obtained with 10 µg 4ME (N = 5; 8.0 ± 0.4 oocytes in the control group versus 7.14 ± 1.0 in the 10 µg 4ME-treated group). No effect of 10 µg or 100 µg 4ME on egg transport was observed in the P1 rats (N = 5; 7.3 ± 0.5 eggs in the control group versus 7.7 ± 1.4 in the 10 µg 4ME-treated group or 8.8 ± 1.6 in the 100 µg 4ME-treated group).
2ME Acceleration of Egg Transport Through Nongenomic Pathways Requires Participation of ER and Protein Phosphorylation in the Oviduct
To investigate whether the effect of 2ME on egg transport occurs when RNA synthesis is inhibited in the oviduct and whether it is mediated by the activation of ER and protein kinases, Act D, which is an inhibitor of RNA synthesis, ICI 182780, which is an estrogen receptor antagonist, and H-89, a broad-spectrum protein kinase inhibitor, were administered i.b. to C1 rats that were injected s.c. with 2ME. We have previously determined that Act D, ICI 182780, and H-89 given individually at the stated doses have no effect on egg transport [8, 9].
Following treatment with 2ME, on average 70% of the oocytes exited the oviduct prematurely (2.4 ± 0.7 versus 8.0 ± 0.8 oviductal oocytes in the control group) (Fig. 5). ICI 182780 and H-89 completely blocked this effect of 2ME (6.9 ± 0.5 and 5.5 ± 1.2 oocytes, respectively), whereas Act D was ineffective (2.9 ± 0.8 oocytes) (Fig. 5). These results indicate that 2ME requires ER and protein phosphorylation for the acceleration of oviductal egg transport in cyclic rats, while genomic pathways are not involved in this effect.
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The data reported here clearly show that mating has a strong impact on oviductal physiology by shifting intracellular E2 signaling in the oviduct from nongenomic to genomic pathways [8–11]. Previously, it has been shown that mechanical sensory stimulation of the genital area, as well as the presence of spermatozoa in the uterine horns can independently elicit this shift in signaling pathways [11, 12], although the precise signals that impinge directly on oviductal cells to elicit this phenomenon remain to be disclosed. It is now well established that both E2 and progesterone act on target cells through genomic and nongenomic signaling pathways. However, shifting from one pathway to another in response to a physiologic stimulus (mating) to achieve the same organ response (oviductal transport acceleration) is, to our knowledge, a novel and hitherto unrecognized phenomenon.
In order to explore how the nongenomic pathway is silenced and the genomic pathway is opened after mating, we conducted a microarray analysis and found that several transcripts changed their expression in the oviduct within 6 h after mating. Among the transcripts that showed altered levels, the mRNA for Comt decreased several-fold.
The mechanism by which mating decreases the level of Comt mRNA was not established in the present study. Fazeli et al. [23] have reported changes in gene expression in the mouse oviduct following artificial insemination, suggesting that the mere presence of spermatozoa in the genital tract may be involved in this phenomenon. We have previously reported that IPS can be elicited by artificial insemination and is likely mediated by an immune response, since IPS elicited by the presence of spermatozoa in the genital tract is blocked by cyclosporin A [11, and Parada-Bustamante and Orihuela, unpublished results]. Interestingly, COMT levels can be altered by prostaglandin J2, which is generated under inflammatory conditions in neuronal cells [24]. Taking into account these data, we speculate that mating decreases the Comt levels activating immune system responses to the presence of spermatozoa in the genital tract; however, this needs to be investigated further.
Oviducts from cyclic and pregnant rats were capable of catalyzing O-methylation of 2OHE, and this activity was blocked significantly by the COMT inhibitor OR486 [25, 26], which indicates that this enzyme is responsible to a large extent for this reaction. Oviductal extracts from pregnant rats formed fewer products per minute than those from cyclic rats. The manner in which mating reduces this enzymatic activity needs to be investigated. The lower level of Comt transcript detected after mating is just one of several other possibilities.
COMT forms methoxyestradiols from 2OHE and 4OHE, which in turn are produced from E2 by CYP1A1 and CYP1B1. The mRNAs and proteins of these enzymes are expressed in the oviduct at similar levels in cyclic and pregnant rats, which indicates that the full enzymatic machinery for generating methoxyestradiols is present in the rat oviduct and is subject to regulation, since mating decreases the level of transcript for at least one component (Comt). COMT is localized in epithelial cells of the rat oviduct [27] rather than in muscle cells, where catecholamines are released by nerve terminals, which suggests that oviductal COMT is more important for the metabolism of E2 than for the metabolism of catecholamines.
Inhibition of COMT activity by OR486 antagonized completely the acceleration of oviductal egg transport induced by E2 in cyclic rats but not in pregnant rats. Since OR486 blocked E2 but not 2ME-induced accelerated transport, it appears that E2 regulates egg transport in the oviducts of cyclic rats, acting as a prohormone that is transformed in the oviduct, first into hydroxyestradiols (catecholestrogens) and then into methoxyestradiols. This supports the idea that catecholestrogens also accelerate ovum transport, although this was not confirmed in the present study. Interestingly, important effects of catecholestrogens on reproductive events have been reported; for example, the conversion of E2 to catecholestrogen is an important step in mediating the effect of estrogen on embryo implantation in the rat [28], and the catecholestrogen, 4OHE, which is produced from E2 in the uterus, mediates mouse blastocyst activation by increasing cAMP levels through nongenomic pathways [29].
The effect of 2ME was not suppressed by Act D and was sensitive to ICI 182780 and H-89, which suggests that the effect of 2ME must be exerted through nongenomic pathways that involve ER and protein phosphorylation. On the other hand, 2ME or 4ME given to pregnant rats had no effect on oviductal transport, which indicates that mating-associated signals are able to shut down the nongenomic pathway of E2 downstream of methoxyestradiols. Taken together, these findings accord with the concept that E2 metabolites formed in the oviduct by COMT are essential for the nongenomic pathway through which E2 accelerates egg transport in cyclic rats. We cannot discount the notion that methoxyestradiols alter gene expression in the oviduct; even if this is true, such changes are not relevant for the acceleration of egg transport.
When 2ME was given i.b. to cyclic rats, oviductal transport was only partially accelerated within a wide range of doses (0.1–10 µg). This was surprising, since most of the reported effects of 2ME in vivo and in vitro show a dose-dependent curve [30–32]. Biphasic effects of 2ME have also been reported; for example, 1 µM 2ME exerts a proliferative effect on MCF-7 breast tumor cells, whereas higher concentrations inhibit cell proliferation [32, 33]. However, this is not the case, since 2ME accelerated egg transport at all of the doses tested. One possible explanation for this result is that concentrations higher than 0.1 µg in 5 µl DMSO were saturating, and at these concentrations, the compound was not completely dissolved. Alternatively, 2ME may need the synergistic co-operation of other E2 metabolites in order to exert its full effect on egg transport. However, we do not rule out that this result represents a chance event. When 2ME was given s.c. to cyclic rats, the effect was dose-dependent. 2ME at pharmacologically relevant dosages (5–25 mg) inhibits angiogenesis and cell proliferation in several cancer cell lines [34]. In the present study, we report that 2ME at lower doses than those previously reported to suppress solid tumor growth [35–36] is able to regulate a complex physiologic process accomplished by an entire organ composed of diverse cell phenotypes.
2ME given systemically at a dosage of 100 µg was slightly less effective than E2 at 1 µg, possibly due to the rapid turnover of 2ME (t1/2 = 20.2 min) in male rats [37] or its rapid conversion to 2-methoxyestrone followed by conjugation, as shown in humans [38]. Since we could not find any reports on the turnover of methoxyestradiol in the female rat and we did not measure the concentration of 2ME in the oviduct after E2 or 2ME injection, this explanation is unproven. On the other hand, E2 itself or other metabolites of E2 may contribute to the acceleration of egg transport. This latter assumption is supported by the fact that 4ME given s.c. also accelerated egg transport.
Data on the presence and activity of COMT in the oviduct are scarce. To our knowledge, this is the first report of COMT activity and the direct actions of methoxyestradiols in the rat oviduct. Previously, Inoue et al. [27] assessed COMT expression by immunohistochemistry in rat oviducts obtained at estrus and diestrus. Casimiri et al. [39] have determined that COMT is active in the rat uterus throughout the estrous cycle. We show that COMT catalyzes O-methylation of 2-hydroxyestradiol in oviducts obtained from rats at estrus (C1) or on Day 1 of pregnancy (P1), and that this activity is lower on P1. The decrease in COMT activity in the oviduct after mating probably protects the embryos from the deleterious effect that methoxyestradiols exert during the first stages of development [40].
In summary, our results show that in the absence of mating, local formation of methoxyestradiols mediated by COMT is essential for the intraoviductal nongenomic pathway utilized by E2 to accelerate egg transport in the rat oviduct. Mating-associated signals, which remain to be identified, shut down this signaling pathway upstream and downstream of methoxyestradiols. These data provide evidence for an early onset of intracellular path shifting in the rat oviduct following mating and provide a partial mechanistic explanation for its occurrence.
FOOTNOTES
1Suppported by FONDECYT (#8980008, 1030315, 1040804) and PROGRESAR (PRE 004/2003). 
Correspondence: 2P.A. Orihuela, Laboratorio de Inmunología de la Reproducción, Facultad de Química y Biología, Universidad de Santiago de Chile, Alameda 3363, Casilla 40, Correo 33, Santiago 9170022, Chile. FAX: 56 2 681 2808; e-mail: porihuela{at}usach.cl
Received: 16 March 2007.
First decision: 16 April 2007.
Accepted: 9 August 2007.
REFERENCES
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