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Regular Article |
a Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
ABSTRACT
{Previously, we found that the dose of estradiol (E2) required to accelerate egg transport increases 5- to 10-fold, in mated compared to cyclic rats. Here we examined protein synthesis in the oviduct of mated and cyclic rats following a single injection of E2 known to accelerate oviductal egg transport or after concomitant treatment with progesterone (P4) known to block this acceleration. On Day 1 of the cycle or pregnancy, E2, P4, or E2 + P4 were injected s.c., and 4 h later oviducts were removed and incubated for 8 h in medium with 35S-methionine. Tissue proteins were separated by SDS-PAGE, and protein bands were quantitated by fluorography and densitometry. In mated rats, E2 and P4 increased different protein bands and P4 did not affect the fluorographic pattern induced by E2. In contrast with mated rats, none of these treatments changed the fluorographic pattern of the oviductal proteins in cyclic rats. Estradiol-induced egg transport acceleration was then compared under conditions in which oviductal protein synthesis was suppressed. Mated and cyclic rats treated with equipotent doses of E2 for accelerating egg transport also received actinomycin D (Act D) locally. Estradiol-induced oviductal egg loss was partially blocked by Act D in mated but had no effect in cyclic rats. We conclude that the oviduct of mated and cyclic rats differs in that only the former responds with increased protein synthesis to a pulse of exogenous E2 and P4 and requires an intact protein synthesis machinery in order to accelerate egg transport in response to E2.
estradiol, gene regulation, mechanisms of hormone action, oviduct, progesterone, steroid hormones
INTRODUCTION
It is well established that egg transport through the oviduct in mammals is regulated by ovarian steroids and by embryonic signals [1]. In cyclic rats, the oocytes reach the uterus approximately 72 h after ovulation, whereas in mated rats, the embryos take 96 h. Because oocytes take 96 h to traverse the oviduct in rats made pseudopregnant by mechanical stimulation of the cervix in the evening of proestrus, we concluded that the above difference is due to mating-associated signals and is independent of whether eggs are fertilized or not [2]. Neither the signaling pathway through which mating or cervical stimulation affect the duration of oviductal egg transport nor the physiologic changes that take place in the oviduct in response to mating have been elucidated.
In the rat, a single injection of estradiol-17ß (E2) on Day 1 of the cycle or pregnancy shortens oviductal transport of eggs to less than 24 h [3]. Concomitant treatment with progesterone (P4) blocks this response in cyclic and pregnant rats [4]. Therefore mating could indirectly affect oviductal transport through changes in the E2:P4 ratio in blood. In fact, this ratio becomes different in mated and nonmated rats on the morning of Day 3, at a time when, in cyclic females, E2 levels start rising and P4 levels drop [5]. That the rise in P4 beginning on Day 3 is responsible for the longer duration of oviductal transport in mated rats is supported by the fact that the antiprogestin RU486 given every day following Day 1 of pregnancy advances embryo transport to the uterus in these rats by only 1 day [4]. In contrast, RU486 accelerates oocyte transport in cyclic rats as early as 24 h after the onset of the same treatment schedule. This marked and early difference, between mated and cyclic rats, in their response to RU486 suggested that mating could change the responsiveness of the oviduct to E2 and P4. In support of this interpretation, we found that mating decreases the sensitivity of the oviduct to E2 as 5–10 times higher doses are needed on Day 1 of pregnancy as compared to Day 1 of the cycle to accelerate the same number of eggs [6]. Thereafter, the effectiveness of a single injection of E2 for accelerating oviductal embryo transport increases progressively from Day 1 to Day 3 of pregnancy in parallel with increasing oviductal uptake of the hormone at equal blood concentrations [7]. Taken together, the above antecedents clearly indicate that, as a consequence of mating, a profound change takes place in the responsiveness of the rat oviduct to E2 and P4.
In mated rats, the response of egg transport to E2 requires an intact protein synthesis machinery in the oviduct [8]. Because differences in protein synthesis may explain the differences in sensitivity of egg transport to E2 and P4 caused by mating, we compared protein synthesis in the oviduct of mated and cyclic rats to see whether any difference was detectable. This was done following a single injection of E2 known to accelerate transport or after concomitant treatment with P4 known to block this acceleration. In view of the remarkable disparity in protein synthesis found under these conditions, we also compared E2-induced egg transport acceleration in mated and cyclic rats while oviductal protein synthesis was inhibited by local administration of actinomycin D (Act D). Part of this work was presented in abstract form by Orihuela and Croxatto [9].
MATERIALS AND METHODS
Animals
Sprague-Dawley rats weighing 200–260 g were used (bred in house). The animals were kept under controlled temperature (21–24°C), and lights were on from 0700 to 2100 h. Water and pelleted food were supplied ad libitum. The phases of the estrous cycle were determined by daily vaginal smears, and all females were used after they had shown at least two regular 4-day cycles. Females in proestrus were either kept isolated or caged with fertile males. The following day (estrus) was designated as Day 1 of the cycle (C1) in the first instance and Day 1 of pregnancy (P1) in the second, provided spermatozoa were found in the vaginal smear. The care and manipulation of the animals was done in accordance with the ethical guidelines of our institution.
Treatments
Systemic administration of E2 and P4 For experiment 1, rats on C1 and P1 were injected s.c. with 1 µg of E2 and/or 5 mg of P4 as a single dose dissolved in 0.1 ml propylene glycol or olive oil, respectively. For experiments 2 and 3, animals on C1 or Day 2 of pregnancy (P2) received 1 and 10 µg of E2, respectively. In previous experiments we found that Act D given on P1 inhibits cumulus dispersion thus creating a mechanical hindrance that prevents eggs from passing from the ampulla to the isthmus, where E2 exerts its transport-accelerating effect [8, 10]. To avoid this problem mated rats were treated on P2, at a time when embryos are denuded and already in the isthmic segment. Furthermore, 1 µg of E2 given on C1 is more effective to accelerate egg transport than the same dose given on P1 (mean ± SEM: 2 ± 1 oviductal oocytes versus 8.4 ± 0.8 oviductal embryos, n = 10) [6]. For this reason, mated rats were treated with 10 µg of E2, which given on P2 is roughly equipotent with 1 µg given on C1 in terms of the number of eggs that leave the oviduct prematurely. Control rats received the vehicle alone.
Local administration of Act D Mated and cyclic rats were injected intraoviductally or intrabursally, respectively, with 1 µg of Act D dissolved in 2 µl of saline solution. While intraoviductal injection did not disturb normal embryo transport, this procedure was found to disturb normal oocyte transport in cyclic rats. Intrabursal injection proved to be effective in cyclic rats without affecting normal oocyte transport. As shown in the companion paper [11] intrabursal administration works in mated rats as well, therefore the use of different routes of administration of Act D was considered an acceptable imperfection in this experiment. Control rats received the vehicle alone.
Animal Surgery
Intrabursal administration of Act D was done in the morning of C1 using a surgical microscope (OPMI 6-SDFC; Zeiss, Oberkochen, Germany). The oviduct and ovary were exposed through flank incisions made under ether anesthesia. Drugs or vehicle alone were injected into the periovarial sac using a Hamilton syringe (Hamilton Company, Reno, NV), and inmediately the injection site was closed with an electric coagulator (Codman CMC-1; Codman and Shurleff, Inc., Randolph, MA). The organs were returned to the peritoneal cavity, and muscles and skin were sutured. Intraoviductal administration was done in the morning of P2 as described by Ríos et al. [8].
Assessment of Egg Transport
Twenty-four hours after treatment the animals were killed by excess ether inhalation, the genital tract was removed, and the oviducts and uterine horns were flushed separately with saline. The flushings were examined under low-power magnification. The number and distribution of ova in the genital tract were recorded.
In Vitro Incorporation of Labeled Methionine into Oviductal Proteins
Rats were killed by excess ether inhalation 4 or 8 h after treatment, and the oviducts were removed, cleaned of fat tissue, and then flushed to avoid contamination with egg or sperm proteins. Afterward, oviducts were transferred to 0.5 ml of prewarmed methionine-deficient minimum essential medium (MEM; Sigma Chemical Co., St. Louis, MO) supplemented with 25 µCi of 35S-methionine (specific activity, >1000 Ci mM; Dupont NEN, Boston, MA). The oviducts in groups of four were incubated for 8 h at 37°C on a rocking platform in an atmosphere of 5% CO2 and 100% relative humidity. We chose 8 h of incubation because in pilot experiments this time was found to be optimal for obtaining high incorporation of the radiolabeled amino acid. At the end of incubation organs were blotted on filter paper and washed with buffer containing 0.25 M saccharose, 3.0 mM MgCl2, 25 mM Tris, and 0.5 mM PMSF [11]. Organs in groups of four were homogenized on ice in a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) for 10 sec in 1 ml of the same buffer and centrifuged for 10 min at 6000 rpm at 4°C in order to remove particulate material and collect the clarified homogenate that was stored at -20°C until use. In order to avoid proteolytic degradation by freezing-thawing of the samples, the clarified homogenates were divided in two aliquots: one for precipitation of proteins with trichloroacetic acid (TCA) and determination of protein concentration, and the other for electrophoretic running. Ten microliters of the clarified homogenate was added to numbered tubes. One milliliter of ice-cold 10% (v/v) TCA was added and mixed on a vortex mixer. Afterward, tubes were incubated for 30 min at 90°C in a temp-blok (Lab-Line Instruments Inc., Melrose Park, IL) and then placed in an ice bath for 10 min. The insoluble materials were collected on a glass fiber filter GF/C (Whatman, Maidstone, England), and each tube was rinsed three times with approximately 5 ml of 5% (v/v) TCA. Each rinse was poured onto the filter. Filters were dried, placed in counting vials, and counted in a toluene-based scintillation cocktail. Incorporation of 35S-methionine was calculated as cpm/µg of protein. The protein concentration in the clarified homogenate was determined according to Bradford [13] using BSA as standard.
Protein Electrophoresis and Fluorography
Aliquots of the clarified homogenate containing the same amount of protein (125 µg) were dissociated for 2 min at 90°C in equal volume of 0.125 M Tris-HCl, pH 6.8, containing 4% SDS, 10% ß-mercaptoethanol, 20% glycerol, and 0.04% bromophenol blue. Samples were run on 12% SDS polyacrylamide slab gels according to the method of Laemmli [14], utilizing a PROTEAN II electrophoretic chamber (Bio-Rad Laboratories, Hercules, CA). Following the one-dimensional polyacrylamide gel electrophoresis, the gels were stained with 2% Coomassie blue R-250 (Bio-Rad) dissolved in a mixture of acetic acid:methanol:distilled water (10:40:50%). To determine the relative radioactivity of the bands gels were treated with an enhancer (Entensify; DuPont NEN) for 90 min, washed with water, dried, and exposed to a radioactivity-sensitive film (Reflection; DuPont NEN) at -70°C for 10 days.
Densitometry of the Gels and Fluorographs
The gels stained with Coomassie blue and the fluorographs were scanned using a Scitex model Smart 320 scanner, and each band density was quantitatively analyzed with the NIH Image 1.61 software. Only major bands that were present consistently in all the replicates and that were neatly separated were subjected to densitometric analysis. This method has the limitation of not allowing the measurement of all the bands but it permits precise measurement of them. The intensity of bands was calculated as pixels2.
Experiment 1
This experiment was designed to examine the pattern of protein synthesis in the oviduct after a single injection of E2 that accelerates oviductal transport, or concomitant treatment with P4 that blocks this acceleration. A total of 64 rats on C1 or P1 were treated with vehicle, P4, E2, or E2 + P4. In each replicate, two rats per treatment group were used and their whole oviducts were excised 4 h after treatment. Then, the four oviducts of these two rats were placed in culture wells and incubated to determine the incorporation of 35S-methionine as described. In vitro incorporation of 35S-methionine took place from 4 to 12 h after treatment in vivo. The pattern of Coomassie blue-stained and radioactive bands was determined as described above. This experiment consisted of four replicates.
Experiment 2
This experiment was designed to determine whether or not local administration of Act D [15] could block oviductal protein synthesis in mated and cyclic rats with or without E2 treatment. Estradiol accelerates oviductal egg transport with a latency period of 9–11 h after treatment; therefore synthesis of some proteins that will be instrumental for accelerating oviductal transport should precede and/or accompany the occurrence of accelerated transport. A total of 128 rats on C1 or P2 were divided into four treatment groups: 1) saline + propylene glycol, 2) Act D + propylene glycol, 3) saline + E2, and 4) Act D + E2. In each replicate, two rats for treatment group were used and their whole oviducts were excised 4 or 12 h after treatment. Then, the four oviducts of these two rats were placed in culture wells and incubated to determine the incorporation of 35S-methionine as described. In vitro incorporation of 35S-methionine took place from 4 to 12 h or 12 to 20 h after treatment in vivo. This experiment consisted of four replicates.
Experiment 3
This experiment was designed to compare the effects of local administration of Act D on E2-induced egg transport acceleration in mated and nonmated rats. A total of 80 animals on C1 or P2 were divided into four treatment groups: 1) saline + propylene glycol, 2) Act D + propylene glycol, 3) saline + E2, and 4) Act D + E2. Twenty-four hours after treatment the animals were autopsied and the number and distribution of the eggs was determined as described above.
Data Analyses
Where considered appropriate, results are presented as cpm/µg of protein, but in most instances cpm/µg of protein or pixel2 in each group were converted to percentage using the corresponding control value as 100%. This was done because the rapid age-associated loss of specific activity of the radiolabeled amino acid introduces an artifactual decrease in its incorporation into protein from one replicate experiment to the next. It has the disadvantage of totally eliminating the variability of control data but it allows the detection of differences between treated and control data in successive replicate experiments and to obtain mean values and standard errors for graphical representation and statistical analyses from replicate animal pools. Differences between the means of these percentages were analyzed using the Mann-Whitney U-test. Differences were considered significant when P < 0.05.
RESULTS
Experiment 1
In control vehicle, the basal incorporation of 35S-methionine into oviductal proteins did not differ significantly in cyclic and pregnant rats (mean ± SEM, n = 4; 2478 ± 962 and 2879 ± 1273 cpm/µg of oviductal protein). Furthermore, none of the treatments changed the incorporation of 35S-methionine into total oviductal protein in mated and cyclic rats (not shown). In order to characterize the effects of E2 and P4 on the banding pattern, proteins were subjected to SDS-PAGE followed by fluorography after Coomassie blue staining. In mated rats, approximately 37 major protein bands were visualized with Coomassie blue with no differences between control and treated groups (data not shown). Only 16 protein bands visualized in fluorographs, whose molecular weight ranged from 200 to 20 kDa were quantitatively analyzed (Fig. 1). Administration of P4 stimulated incorporation of 35S-methionine into two protein bands (B and D). Four other protein bands (E, N, O, P) had increased radioactivity following E2 administration alone or concomitant with P4 (Fig. 2). In cyclic rats approximately 38 major protein bands were visualized with Coomassie blue with no differences between control and treated groups (data not shown). Only 16 protein bands, whose molecular weight ranged from 200 to 20 kDa, were quantitatively analyzed in the fluorographs (Fig. 1). None of the treatments changed the incorporation of 35S-methionine into these protein bands (Fig. 3).
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Experiments 2 and 3
Although local administration of Act D alone or concomitant with E2 completely suppressed protein synthesis in the oviduct of both mated and cyclic rats at 4–12 h or 12–20 h after treatment (data not shown), it blocked the E2-induced oviductal egg loss in pregnant but not in cyclic rats (Fig. 4).
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DISCUSSION
This study shows disparate effects of E2 and P4 on oviductal protein synthesis in mated and cyclic rats. Estradiol and P4 changed the protein synthesis pattern in mated rats but not in cyclic rats. The difference was clear cut in one-dimensional PAGE and warrants a more detailed analysis. Furthermore, E2-induced egg transport acceleration requires protein synthesis in mated but not cyclic rats. Thus, mating greatly influences the response of the oviduct to E2 in terms of protein synthesis and oviductal egg transport.
In mated rats, the fluorographic analysis disclosed a group of protein bands in which the incorporation of 35S-methionine was increased by E2. Administration of P4 also stimulated the incorporation of 35S-methionine into a few oviductal proteins, but this hormone did not affect the fluorographic pattern induced by E2. Thus, these data do not explain the antagonism of P4 on E2-induced embryo transport acceleration by a reversal of the oviductal protein expression induced by E2. Because of the limited analysis performed on the proteins we may have missed such changes, if they do take place. In contrast with mated rats, and using the same analytic procedure, we found no change in the pattern of protein synthesis in the oviduct of cyclic rats following administration of E2 and P4 alone or in combination. This suggests that the effects of E2 and P4 on oocyte transport may be not explained by changes in oviductal protein expression. In order to corroborate this assumption we also investigated oviductal egg transport in cyclic and mated rats treated with 1 µg or 10 µg of E2, respectively, under conditions in which protein synthesis was inhibited by local administration of Act D. The recovery of oviductal eggs in cyclic rats treated with 1 µg E2 was similar to mated rats given 10 µg E2, confirming previous observations that cyclic rats are more sensitive to the effect of E2 than mated rats [6]. Complete suppression of oviductal protein synthesis by Act D partially prevented the effect of E2 on egg transport in mated rats, but it was totally ineffective in cyclic rats. Thus, mating or factors associated with it profoundly change the mechanism through which E2 accelerates oviductal egg transport.
The number and distribution of eggs in the genital tract were not affected by Act D within the first 24 h after treatment. If anything, we would expect a delay in the oviductal transport due to lack of protein synthesis. In order to detect such an effect, autopsies should be performed on Day 4 or 5 of cycle or pregnancy, respectively, but this was not done.
Previous studies have identified glycoproteins secreted into the oviductal lumen in a number of mammalian species, including the mouse [16], hamster [17], rabbit [18], sheep [19], cow [20], baboon [21], human [22], and pig [23] that are temporally associated with elevated levels of E2 and P4. Because we examined total protein—secretory, soluble cell, and structural protein—it is difficult to determine if those reported in this study to be up-regulated by E2 and P4 correspond to the secretory glycoproteins found by others. Western blotting could reveal whether any of the E2- or P4-induced proteins correspond to those described previously, but that question was beyond the scope of this work. On the other hand, we did not detect proteins that have been reported by other groups to be down-regulated by exogenous E2 [24, 25]. In those studies immature animals were treated with E2 to simulate the estrogen basal level of a mature animal, so that the comparison was done between a hypoestrogenic and a normoestrogenic condition, whereas we compared a normoestrogenic with a hyperestrogenic condition. Furthermore, they used Northern blot or immunohistochemistry. Another factor is that our fluorographic analyses only included approximately 40% of the bands so that the chance to find a down-regulated protein was considerably reduced.
Effects of ovarian steroids on their target cells are mediated by intracellular receptors that regulate the synthesis of specific RNAs and proteins [26]. However, some effects are not blocked by inhibitors of transcription or translation, or by classical antagonists of steroid receptors, or are too rapid to be due to changes in gene expression. These features do not appear compatible with the classical genomic action and are termed nongenomic [27]. Our findings suggest that in cyclic rats, E2 accelerates oviductal egg transport by a nongenomic action, while in mated rats E2 affects embryo transport through a genomic action. Further studies are needed in order to confirm this assumption.
In summary, different groups of proteins are upregulated by exogenous E2 and P4 in the oviduct of mated but not in cyclic rats, and E2-induced egg transport acceleration requires protein synthesis in the oviduct of mated but not in cyclic rats. The results provide clear evidence that unidentified factors associated with or consequent to mating have a profound effect on the mechanism of action of E2 in the rat oviduct.
FOOTNOTES
First decision: 10 January 2001.
1 This work received financial support from grants from the Rockefeller Foundation (RF 94025 no. 15), World Health Organization (WHO CHI-LID-2), and FONDECYT 2990007 and 8980008, Cátedra Presidencial en Ciencias H Croxatto and MIFAB (Millennium Institute for Fundamental and Applied Biology). 
2 Correspondence: H.B. Croxatto, Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. FAX: 56 2 222 5515;hbcroxat{at}genes.bio.puc.cl
Accepted: May 21, 2001.
Received: December 4, 2000.
REFERENCES
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