Acceleration of Oviductal Transport of Oocytes Induced by Estradiol in ...

BIOLOGY OF REPRODUCTION 65, 1238–1245 (2001)

Acceleration of Oviductal Transport of Oocytes Induced by Estradiol in Cycling Rats Is Mediated by Nongenomic Stimulation of Protein Phosphorylation in the Oviduct1 Pedro A. Orihuela and Horacio B. Croxatto2 Unidad de Reproduccioń y Desarrollo, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile ABSTRACT In order to explore nongenomic actions of estradiol (E 2) and progesterone (P4) in the oviduct, we determined the effect of E 2 and P4 on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E 2, P4, or E 2 1 P4, and 0.5 h or 2.5 h later their oviducts were incubated in medium with 32P-orthophosphate for 2 h. Oviducts were homogenized and proteins were separated by SDS-PAGE. Following autoradiography, protein bands were quantitated by densitometry. The phosphorylation of some proteins was increased by hormonal treatments, exhibiting steroid specificity and different individual time courses. Possible mediation of the E 2 effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E 2 also received an intrabursal (i.b.) injection of a-amanitin (Am), or the PK inhibitors H-89 or GF 109203X, and 0.5 h later their oviducts were incubated as above plus the corresponding inhibitors in the medium. Increased incorporation of 32P into total oviductal protein induced by E 2 was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E 2-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E 2 s.c. and H-89 i.b. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E 2-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E 2 and P4 change the pattern of oviductal protein phosphorylation. Estradiol increases oviductal protein phosphorylation in cyclic rats due to a nongenomic action mediated by PK-A and PK-C. In the abscence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E 2 in the oviduct by waiving this nongenomic action as a requirement for E 2-induced embryo transport acceleration.

estradiol, kinases, mechanisms of hormone action, oviduct, ovum, progesterone, steroid hormones

INTRODUCTION

In the rat, the time at which eggs pass from the oviduct into the uterus is controlled by a balance between estradiol17b (E 2) and progesterone (P4) levels. A single injection of E 2 to cyclic or pregnant rats shortens oviductal transport This work received financial support from grants of FONDECYT nos. 2990007 and 8980008, the Rockefeller Foundation (RF 98024 no. 98), Ca´tedra Presidencial en Ciencias H. Croxatto, Laboratorios Silesia S.A., and MIFAB (Millennium Institute for Fundamental and Applied Biology). 2 Correspondence: H.B. Croxatto, Unidad de Reproduccioń y Desarrollo, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Casilla 114-D, Santiago, Chile. FAX: 56 2 222 5515; e-mail: [email protected] 1

Received: 4 December 2000. First decision: 10 January 2001. Accepted: 21 May 2001. Q 2001 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

of eggs from the normal 72–96 h to less than 24 h [1]. Concomitant treatment with P4 blocks the E 2-induced ovum transport acceleration in cyclic and pregnant rats [2], whereas administration of P4 alone retards oviductal transport in cyclic but not in pregnant rats [3]. Many physiological effects of ovarian steroids are mediated by intracellular receptors that control the production of specific RNAs and proteins [4]. However, some effects are not blocked by inhibitors of transcription or translation [5–7], or by classical antagonists of steroid receptors [8], or are too rapid to be due to changes in gene expression [9–11]. Because these features do not appear compatible with the classical genomic action of steroids, these effects have been termed nongenomic. Although it is well established that the effect of E 2 on oviductal embryo transport in mated rats is mediated in part by de novo RNA and protein synthesis in the oviduct [12], we have presumptive evidence that the effects of E 2 and P4 on oocyte transport in the rat may be mediated also by nongenomic actions of these hormones. Local administration of actinomycin D inhibits to a great extent the effect of E 2 on oviductal transport in mated rats [12] but totally lacks this effects in cycling rats, although in both conditions actinomycin D abolishes protein synthesis in the oviduct ([13] and accompanying paper [14]). Progesterone antagonizes the effect of E 2 on oviductal egg transport in mated rats without inhibiting E 2-induced protein synthesis in the oviduct ([15] and accompanying paper [14]). Furthermore, treatments with E 2, P4, or the combination E 2 1 P4 that alter oviductal egg transport in cyclic rats neither stimulate oviductal total protein synthesis nor change the 35S-methionine incorporation pattern into protein bands of these rats ([15] and accompanying paper [14]). The above observations led us to explore further possible nongenomic actions of E 2 and P4 in the rat oviduct and their association with the effects of these hormones on egg transport. The mechanisms underlying the nongenomic effects of steroids are not well understood but may involve signaling transduction pathways mediated by tyrosine kinases, mitogen-activated protein kinases, protein kinases A (PK-A) or protein kinases C (PK-C) [16–19]. Therefore we examined the effects of E 2 and P4 on protein phosphorylation in the oviduct of pregnant and cyclic rats. The increased phosphorylation induced by E 2 in the oviduct of cyclic rats was assessed under conditions in which the genomic actions of the hormone were blocked by an mRNA synthesis inhibitor. In addition, local administration of a broad-spectrum inhibitor of protein kinases was utilized to determine whether or not E 2-induced egg transport acceleration requires protein phosphorylation in the rat oviduct. Some of the results presented here were previously reported in abstract form by Orihuela and Croxatto [20]. MATERIALS AND METHODS Animals Sprague-Dawley rats weighing 200–260 g were used (bred in house). The animals were kept under controlled temperature (21–248C), and lights

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NONGENOMIC ACTION OF E2 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. Only rats that showed at least two regular 4-day cycles were used. Females in proestrus were either kept isolated (nonmated) or caged with fertile males (mated). 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 made in accordance with the ethical guidelines of our institution.

Treatments Systemic administration of E 2 and P4. On C1 or P1, E 2 or P4 were injected s.c. as a single dose dissolved in 0.1 ml propylene glycol or olive oil, respectively. Control rats received the vehicle alone. Local administration of inhibitors. a-Amanitin (Am) was used to inhibit mRNA synthesis and therefore the genomic actions of E 2. The protein kinase inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide-dihydrochloride; Calbiochem, La Jolla, CA) was used at a high dose as a broad-spectrum inhibitor of protein kinases and at a low dose as a specific PK-A inhibitor [21, 22]. GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; Calbiochem) was utilized as a specific PK-C inhibitor [23, 24]. The inhibitors were injected only intrabursally (i.b.). Either 18 mg of Am, 0.05, 5, or 15 mg of H-89 or 165 ng of GF 109203X dissolved in 4 ml of saline solution were injected into each ovarian bursa. Control rats received the vehicle alone. In vitro administration of inhibitors. Oviducts were incubated in minimum essential medium (MEM; Sigma Chemical Co., St. Louis, MO) supplemented with 0.1 mM of Am, 450 nM or 45 mM of H-89, or 100 nM of GF 109203X as appropriate to each experiment.

Animal Surgery Intrabursal administration of inhibitors was done in the morning of C1 or P1 using a surgical microscope (OPMI 6-SDFC; Zeiss, Oberkochen, Germany). Oviducts and ovaries 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 immediately 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.

Assessment of Egg Transport Twenty-four hours after treatment the animals were killed by excess ether inhalation, the genital tract was removed, and oviducts and uterine horns were flushed separately with saline. Flushings were examined under low-power magnification. The number and distribution of ova in the genital tract were recorded.

In Vitro Incorporation of Labeled Phosphate into Oviductal Proteins Rats were killed by excess ether inhalation and the oviducts were removed, cleaned from fat tissue, and then flushed to avoid contamination with egg or sperm proteins. Afterward, oviducts were transferred to 0.5 ml of prewarmed MEM supplemented with 600 mCi/ml of 32P-orthophosphate (specific activity 285 Ci/mg; DuPont NEN, Boston, MA) and the corresponding inhibitors or their vehicles. Oviducts in groups of four were incubated separately for different periods at 378C on a rocking platform in an atmosphere of 5% CO2 and 100% relative humidity. At the end of incubation, organs were blotted on filter paper and washed with buffer containing 0.25 mM saccharose, 3.0 mM MgCl2, 25 mM Tris, and 0.5 mM PMSF [25]. Oviducts 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 48C in order to remove particulate material and collect the clarified homogenate that was stored at 2208C until use. In order to avoid proteolytic degradation by freezing-thawing of sample, 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 electrophoresis. 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 908C in a temp-block (Lab-Line Instruments, Inc., Melrose Park, IL) and then placed in an ice bath for 10 min. The insoluble material was collected

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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 32P was calculated as cpm/mg of protein. The protein concentration in the clarified homogenate was determined according to the method of Bradford [26] using BSA as standard.

Protein Electrophoresis and Autoradiography Aliquots of the clarified homogenate containing the same amount of protein (100 mg) were dissociated for 2 min at 908C in an equal volume of 0.125 M Tris-HCl, pH 6.8, containing 4% SDS, 10% b-mercaptoethanol, 20% glycerol, and 0.04% bromophenol blue. Samples were run on 8% or 12% SDS polyacrylamide slab gels according to the method of Laemmli [27] utilizing a Protean II electrophoretic chamber (Bio-Rad, Hercules, CA). Following the one-dimensional polyacrylamide gel electrophoresis, gels were stained with 2% Coomassie blue R-250 (Bio-Rad) dissolved in a mixture of acetic acid:methanol:distilled water (10:40:50 v/v/v). In order to determine the relative radioactivity of the bands, gels were dried and exposed to radioactivity-sensitive film (Hyperfilm MP; Amersham Life Science, Little Chalfont, Buckinghamshire, UK) with intensifying screen (Hyperscreen; Amersham Life Science) at 2708C for 4 days.

Densitometry of the Autoradiographs Autoradiographs were scanned using an Epson model Expression 636 scanner (Epson Co., Santiago, Chile), and each band density was quantitatively analyzed with the NIH Image 1.6 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 measuring all the bands, but it permits precise measurement of selected bands. The intensity of bands was calculated as pixel2.

In Vitro Incorporation of Labeled Uridine into Oviductal RNA In order to assess the extent of RNA synthesis inhibition caused by Am treatment, oviducts were incubated with 25 mCi/ml of 3H-uridine (specific activity, 15.2 Ci/mM; Sigma) under conditions described above. At the end of incubation, organs were homogenized as already described. Ten microliters of the clarified homogenate were added to numbered tubes. Two milliliters of ice-cold 10% (v/v) TCA were added and mixed on a vortex mixer and then placed in an ice bath for 30 min. The insoluble material was collected on a glass fiber filter GF/C (Whatman), and each tube was rinsed three times with approximately 5 ml of 5% (v/v) TCA and one time with 1 ml of absolute ethanol. Each rinse was poured onto the filter. Filters were dried, placed in counting vials and digested with Protosol (DuPont NEN, Boston, MA), and counted in a toluene-based scintillation cocktail. Incorporation of 3H was calculated as cpm/mg of wet tissue.

Experiment 1 This experiment was designed to determine the effects of E 2 and P4 on protein phosphorylation in the oviduct of mated and cyclic rats after a single injection of E 2 that accelerates oviductal transport or concomitant treatment with P4 that blocks this acceleration. Because E 2 and P4 rapidly regulate protein phosphorylation in other organs, tissues, and cell lines [28–32], 0.5 h was the first point in time selected, and a second point at 2.5 h was considered appropriate for detection of a change along time. A total of 96 rats on C1 and P1 were treated with vehicle, P4 (5 mg), E 2 (1 mg), or E 2 1 P4. In each replicate, two rats for each treatment group were used and their whole oviducts were excised at 0.5 or 2.5 h after treatment. Then, the four oviducts obtained from each treatment group were placed in culture wells and incubated with 32P-orthophosphate for 2 h to determine the incorporation of 32P into total protein as described above. In vitro incorporation took place from 0.5 to 2.5 h and from 2.5 to 4.5 h after treatment. The banding pattern of autoradiographs was also determined as described above. This experiment consisted of three replicates for each treatment group. Oviductal proteins from cyclic rats were separated in 8% SDS-PAGE, however, protein bands were not easily resolved. Therefore, for the experiments with mated rats we used 12% SDS-PAGE that gave better resolution of protein bands.

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ORIHUELA AND CROXATTO

FIG. 1. Autoradiographs of phosphorylated proteins of rat oviducts removed at 0.5 h or 2.5 h after s.c. injection of vehicle (V), 1 mg E 2, 5 mg P4, or E 2 1 P4 on Day 1 of cycle. The oviducts were incubated in the presence of 32P-orthophosphate for 2 h and then homogenized. Aliquots of homogenates containing 100 mg of denatured protein were run on 8% SDS polyacrylamide slab gels. The bands were revealed by exposing gels to radioactivity-sensitive film. Letters indicate bands quantitated by densitometry. Bands without letters were either not present consistently in all replicates or could not be neatly separated for densitometric analysis.

Experiment 2 This experiment was designed to determine the time course of total protein phosphorylation in oviducts of cyclic rats incubated under the influence of exogenous E 2. A total of 64 rats on C1 were treated with vehicle or E 2 (1 mg). In each replicate, two rats for each treatment group were used, and their whole oviducts were excised at 0.5 h after treatment. Then, the four oviducts obtained from each treatment group were placed in culture wells and incubated with 32P-orthophosphate for 1, 2, 4, or 8 h. The incorporation of 32P into total protein was determined as described above. This experiment consisted of four replicates for each incubation time.

Experiment 3 This experiment was designed to establish the dose-response curve of E 2 on oviductal protein phosphorylation in cyclic rats. A total of 40 rats on C1 were treated with vehicle or 0.3, 1, 3, or 10 mg E 2, and 0.5 h later oviducts were incubated for 8 h to determine the incorporation of 32P into total protein as described above. This experiment consisted of four replicates for each dose.

Experiment 4 This experiment was designed to determine if and to what extent oviductal protein phosphorylation induced by E 2 is dependent on de novo mRNA synthesis. Enhanced phosphorylation induced by E 2 1 mg was assessed under conditions in which Am completely inhibited 3H-uridine incorporation into RNA. A total of 32 rats on C1 were divided into four

FIG. 2. Densitometric analysis of autoradiographs of phosphorylated protein bands from rat oviducts removed at 0.5 or 2.5 h after s.c. injection of vehicle (V), 1 mg E 2, 5 mg P4, or E 2 1 P4 on Day 1 of the cycle. Whole oviducts were incubated for 2 h in medium with 600 mCi of 32P-orthophosphate, and after extraction the proteins were resolved by SDS-PAGE. The autoradiographs prepared as described in Materials and Methods, were scanned to determine band density (pixel2) using NIH Image 1.61 software. Each bar represents the mean of three replicates with four oviducts per treatment group. *P , 0.05 versus corresponding control value.

treatment groups: 1) saline 1 propylene glycol, 2) Am 1 propylene glycol, 3) saline 1 E 2, and 4) Am 1 E 2. Oviducts were excised 0.5 h later and incubated with MEM (groups 1 and 3) or MEM 1 Am (groups 2 and 4) supplemented with 32P-orthophosphate and 3H-uridine for 8 h. The incorporation of 32P into total protein and the banding pattern of autoradiographs were determined, and the incorporation of 3H-uridine into total RNA was also determined as described above. This experiment consisted of four replicates for each treatment group.

Experiments 5–7 These experiments were designed to determine whether H-89 at a high concentration or at a low concentration, as well as GF 109203X, can block the stimulation of oviductal protein phosphorylation induced by E 2 1 mg. For each experiment, 32 rats on C1 were divided into four treatment groups: 1) saline 1 propylene glycol, 2) inhibitor 1 propylene glycol, 3) saline 1 E 2, and 4) inhibitor 1 E 2. Oviducts were excised 0.5 h later and incubated with MEM (groups 1 and 3) or MEM 1 inhibitor (groups 2 and 4) supplemented with 32P-orthophosphate for 8 h to determine the incorporation of 32P into total protein as described above. Each experiment consisted of four replicates for each treatment group.

Experiment 8 This experiment was designed to determine whether i.b. administration of H-89 can inhibit acceleration of oviductal egg transport induced either by E 2 1 mg or 10 mg in cyclic or mated rats, respectively. Previous experiments have shown that 10 mg E 2 given on P1 is roughly equipotent with 1 mg given on Day 1 of the cycle in terms of the number of eggs that leave the oviduct prematurely. Furthermore, in the companion paper we have shown the i.b. injection to be as effective as intraoviductal injec-

NONGENOMIC ACTION OF E2

FIG. 3. Autoradiographs of phosphorylated proteins of rat oviducts removed at 0.5 or 2.5 h after s.c. injection of vehicle (V), 1 mg E 2, 5 mg P4, or E 2 1 P4 on Day 1 of pregnancy. The oviducts were incubated in the presence of 32P-orthophosphate for 2 h and then homogenized. Aliquots of homogenates containing 100 mg of denatured protein were run on 12% SDS polyacrylamide slab gels. The bands were revealed by exposing gels to radioactivity-sensitive film. Letters indicate bands quantitated by densitometry. Bands without letters were either not present consistently in all replicates or could not be neatly separated for densitometric analysis.

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FIG. 4. Densitometric analysis of autoradiograph of phosphorylated proteins from rat oviducts removed at 0.5 or 2.5 after s.c. injection of vehicle, 1 mg E 2, 5 mg P4, or E 2 1 P4 on Day 1 of pregnancy. The autoradiographs prepared as described in Materials and Methods were scanned to determine band density (pixel2) using NIH Image 1.61 software. Each bar represents the mean of three replicates with four oviducts per treatment group. *P , 0.05 versus corresponding control value.

tion in order to administer drugs into the oviduct. A total of 89 animals on C1 and P1 were divided into four treatment groups: 1) saline 1 propylene glycol, 2) H-89 1 propylene glycol, 3) saline 1 E 2, and 4) H-89 1 E 2. Twenty-four hours after treatment egg transport was assessed as described.

Statistical Analysis The results are presented as mean 6 SEM. Overall analysis was done by Kruskal-Wallis test, followed by Mann-Whitney test for pairwise comparisons when overall significance was detected.

RESULTS

Experiment 1

The basal incorporation of 32P into oviductal total protein did not differ significantly between the two periods of time in cyclic and mated rats. Furthermore, none of the treatments changed the incorporation of 32P into total oviductal protein (not shown). In order to characterize the pattern of phosphorylated protein bands regulated by E 2 and P4 in the oviduct, they were subjected to SDS-PAGE and autoradiography. In cycling rats, the autoradiographic pattern of phosphorylated proteins showed approximately 18

FIG. 5. Incorporation of 32P into into total rat oviductal protein on Day 1 of the cycle. At 0.5 h after s.c. injection of vehicle (V) or 1 mg E 2, oviducts were excised and incubated with 600 mCi of 32P-orthophosphate in MEM for 1, 2, 4, or 8 h. Following incubation, oviducts were homogenized and proteins were precipitated with TCA to assess their radioactivity. Each point represents the mean of four replicates with four oviducts per treatment group. *P , 0.05 versus corresponding control value.

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FIG. 6. Incorporation of 32P into total oviductal protein of rats on Day 1 of the cycle. At 0.5 h after s.c. injection of vehicle or estradiol 0.3, 1, 3, or 10 mg, oviducts were excised and incubated with 600 mCi of 32Porthophosphate in MEM for 8 h. Following incubation, oviducts were homogenized and proteins were precipitated with TCA to assess their radioactivity. Each bar represents the mean of four replicates with four oviducts per treatment group. a ± b ± c ± d, P , 0.05.

major protein bands. Only 8, with molecular weights ranging from 98 to 43 kDa, were quantitatively analyzed. Administration of P4 stimulated the phosphorylation of two protein bands (B and G) in oviducts incubated from 0.5 to 2.5 h after treatment in vivo (at 0.5–2.5 h). Two other protein bands (D and E) were stimulated in oviducts incubated from 2.5 to 4.5 h after treatment in vivo (at 2.5–4.5 h). Protein bands A and H were stimulated at both periods. Estradiol administration stimulated phosphorylation of protein bands G and H at 0.5–2.5 h and D and E at 2.5–4.5 h. Concomitant administration of E 2 and P4 stimulated phosphorylation of band G at 0.5–2.5 h and of A, C, E, and H at 2.5 h. Only protein band D was stimulated at both periods (Figs. 1 and 2). In mated rats the autoradiographic pattern of phosphorylated proteins showed approximately 29 major protein bands with molecular weights ranging from 68 to 29 kDa. Because these were resolved with 12% SDS-PAGE, equivalence with bands from cycling rats cannot be established and they were designated by roman numbers. Only nine of these protein bands were quantitatively analyzed. Administration of P4 stimulated the phosphorylation of protein bands VI and IX only at early periods and of VII and VIII at both periods. Estradiol stimulated phosphorylation of protein bands VII and VIII only at 2.5–4.5 h. Concomitant administration of E 2 and P4 stimulated the phosphorylation of band IX at 0.5–2.5 h and VIII at 2.5– 4.5 h. Protein band VII was stimulated at both periods (Figs. 3 and 4). Experiment 2

In the vehicle control, the basal rate of incorporation of into total oviductal protein remained stable in oviducts incubated up to 8 h. Following E 2 administration the incorporation of 32P exhibited two phases. The first was a latency period of 2.5 h after treatment in which the rate of incorporation was the same as the control group. After this period, the rate augmented leading to a two- to threefold increase in cpm/mg of protein at 4 and 8 h of incubation with respect to the control group (Fig. 5). 32P

FIG. 7. Incorporation of 32P into total oviductal protein (A) and 3H-uridine into total oviductal RNA (B) from rats on Day 1 of the cycle. Animals were concomitantly injected s.c. with the vehicle (V) of E 2 and i.b. with the vehicle Am, 1 mg E 2 and the vehicle Am, 1 mg E 2, and 18 mg Am, or the vehicle E 2 and 18 mg Am. At 0.5 h after treatment, oviducts were excised and incubated with 600 mCi of 32P-orthophosphate and 25 mCi of 3H-uridine in MEM 1 vehicle Am or MEM 1 0.1 mM of Am for 8 h as appropriate. Following incubation, oviducts were homogenized and proteins and RNA were precipitated with TCA to assess their radioactivity. Each bar represents the mean of four replicates with four oviducts per treatment group. a ± b, P , 0.05.

Experiment 3

A stimulatory effect of E 2 on oviductal protein phosphorylation was observed with the dose of 0.3 mg reaching a maximum with 3 mg (Fig. 6). Experiment 4

Estradiol increased the incorporation of 32P into total oviductal protein. Administration of Am affected neither the basal nor the E 2-stimulated incorporation of 32P (Fig. 7A). However, Am administered alone or concomitant with E 2 totally suppressed the incorporation of 3H-uridine into total RNA (Fig. 7B). The effect of Am on the banding pattern of phosphorylated proteins was also investigated. The autoradiographs showed approximately 21 major protein bands. Only 8 of these, whose molecular weight ranged from 96 to 43 kDa, were quantitatively analyzed. Administration of E 2 stimulated the phosphorylation of six bands (A–D, G, and H). The pattern of phosphorylated proteins was similar with and without Am except for bands A and

NONGENOMIC ACTION OF E2

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FIG. 8. Densitometric analysis of autoradiographs of phosphorylated protein bands from rat oviducts treated as in Figure 7. The autoradiographs prepared as described in Materials and Methods were scanned to determine band density (pixel2) using NIH Image 1.61 software. Each bar represents the mean of three replicates with four oviducts per treatment group. *P , 0.05 and &P , 0.05 versus corresponding control and E 2 groups, respectively.

E whose phosphorylation was inhibited with Am 1 E 2 and/ or Am alone (Fig. 8). Experiments 5–7

Local administration of H-89, both at high and low concentrations, inhibited the basal and the E 2-stimulated incorporation of 32P into total oviductal protein in the rat oviduct while GF 109203X only inhibited 32P incorporation into total oviductal protein after E 2 stimulation (Fig. 9). Experiment 8

The mean number (6SEM) of eggs recovered from the oviducts of the control group was of 7.8 6 0.9 and 10.1 6 0.9 in cyclic and pregnant rats, respectively, while in the groups treated with E 2 it was 3.1 6 0.8 and 1.3 6 0.5. Intrabursal administration of H-89 alone did not affect oviductal egg recovery in cyclic (7.5 6 0.7) or pregnant rats (9.3 6 0.7), although it blocked the E 2-induced oviductal egg loss in cyclic (7.9 6 1.1) but not in pregnant rats (0.6 6 0.3) (Fig. 10).

FIG. 9. Incorporation of 32P into into total oviductal protein on Day 1 of the cycle following s.c. treatment with vehicle (V), 1 mg E 2 alone, or with protein kinase inhibitor i.b. H-89 5 mg (A), 0.5 mg i.b. H-89 (B), or 165 ng i.b. GF 109203X (GF) (C). At 0.5 h after injections, oviducts were excised and incubated with 600 mCi of 32P-orthophosphate in MEM with or without 45 mM H-89 (A) or 450 nM (B) or 100 nM GF (C) for 8 h. Following incubation, oviducts were homogenized and proteins were precipitated with TCA to assess their radioactivity. Each bar represents the mean of four replicates with four oviducts per treatment group. a ± b ± c, P , 0.05.

DISCUSSION

The present results provide unequivocal evidence of a nongenomic action of E 2 in the rat oviduct. This action of E 2 results in increased phosphorylation of some proteins that seem to mediate the acceleration of oocyte transport induced by the hormone in cyclic rats. Although we could not detect changes in the incorporation of 32P into total protein when incubation was limited to 2 h, changes in the pattern of phosphorylated proteins were detected by SDS-PAGE followed by autoradiography. To our knowledge this is the first report on changes in protein phosphorylation induced by E 2 and P4 in the mammalian oviduct. Increased phosphorylation of some protein bands was steroid specific and exhibited individual time courses, suggesting that the two hormones may stimulate phosphorylation through different pathways in cyclic and pregnant rats. However, because different electrophoretic conditions between mated and cyclic rats were used we could not establish whether steroid hormones regulate phos-

phorylation of the same proteins in both physiological conditions. Because E 2 did not affect the incorporation of 32P into total oviductal proteins when oviducts were incubated for 2 h, we explored the effects of E 2 on total protein phosphorylation in oviducts incubated for longer time periods. The basal level of 32P per mg of oviductal total protein increased steadily over an 8-h period, probably reflecting a stable balance between protein kinase and phosphatase activities. In E 2-treated oviducts this balance was shifted toward a relative increase in the rate of incorporation of 32P into total protein, resulting in cpm per mg protein severalfold higher after the first 2 h of incubation. Crucial kinases may be translated, relocated, or activated during the latency period. Phosphorylation is likely to be involved in these processes as suggested by the banding pattern shown in experiment 1. Once these processes reach momentum, massive phosphorylation occurs during the ensuing 4 h. Be-

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The effect of the phosphorylation inhibitor H-89 on E 2induced accelerated transport in cycling and mated rats was also investigated. Local administration of H-89 totally blunted the effect of E 2 on oviductal egg transport in nonmated but not in mated rats. This clearly shows that protein phosphorylation in the oviduct is essential for E 2-induced acceleration of oocyte transport in cycling rats, and that mating with a fertile male completely abolishes this requirement. This effect of mating confirms our previous observations ([15] and accompanying paper [14]) that unidentified factors associated with or consequent to mating have a remarkably effect on the mechanism of action of E 2 in the rat oviduct. We cannot assure that protein kinase inhibitors did not affect normal egg transport because the number and distribution of the eggs were evaluated only within the first 24 h after treatment. If anything, we would expect that inhibitors delay oviductal egg transport. In order to detect such an effect autopsies should be performed on Day 4 or 5 but this was not done. Nongenomic actions of E 2 may involve signal transduction pathways mediated by G-protein-coupled receptors and activation of PK-A, PK-C, or tyrosine kinases [16, 33–35]. Inhibitors of PK-A or PK-C blocked the effect of E 2 on oviductal protein phosphorylation, suggesting that activation of both enzymes was involved in this nongenomic action of E 2. Whether E 2 effects upon these protein kinases are direct or receptor mediated remains to be determined. In summary, E 2 and P4 change the pattern of oviductal protein phosphorylation in cyclic and mated rats. The effect of E 2 on oviductal protein phosphorylation in cyclic rats is due to a nongenomic action and is mediated at least by PKA and PK-C. This nongenomic action of E 2 is essential for its oviductal transport accelerating effect in cyclic rats. REFERENCES FIG. 10. Number of eggs recovered from rat oviducts on Day 2 of the cycle or pregnancy following s.c. and i.b. treatments with estradiol (E 2) alone or with H-89. V, Vehicle of drugs (s.c. and i.b.); E 2, 1 mg (nonmated) or 10 mg (mated); H-89, 15 mg i.b. All treatments were given 24 h before autopsy. Numbers inside the bars indicate the number of animals used. a ± b, P , 0.05.

cause this change was suppressed by PK inhibitors, it must be due to increased phosphorylation rather than decreased phosphatase activity. This effect of E 2 was dose dependent in the dose range 0.3–3 mg. Most importantly, it was totally independent of de novo mRNA synthesis as it remained unchanged when mRNA synthesis was suppressed by Am. Thus, it is a genuine nongenomic, post-transcriptional action of E 2. The densitometric analysis of the oviductal protein bands showed that E 2 stimulated phosphorylation of 75% of the bands examined, while concomitant treatment with Am decreased only one of the bands whose phosphorylation is stimulated by E 2 (band A). We assume this is a protein with high turnover rate whose tissue level decreased as a consequence of impeded replenishment by the action of Am. Band shrinkage probably represents, in this particular case, decreased protein substrate rather than decreased phosphorylation activity. Thus, E 2 stimulates oviductal protein phosphorylation in cycling rats between 0.5 and 8 h after treatment in a dose-dependent manner, and in most of these proteins phosphorylation is increased by a nongenomic action of E 2.

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