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Female Reproductive Tract |
a Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cyclic adenosine monophosphate, estradiol, estradiol receptor, oviduct, ovum pick-up/transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological effects of E2 are mediated through intracellular receptors that are members of a large superfamily of nuclear receptors that function as ligand-activated transcription factors [5]. The activation of estrogen receptors (ER) regulates the transcriptional activity of specific genes, thus mediating the classical genomic actions [5]. However, there is ample evidence obtained in several cell systems that some E2 effects cannot be explained by the classical model of steroid-target cell interaction [6]. These effects are not blocked by inhibitors of transcription or translation [7, 8] or are too rapid to be due to changes in gene expression [9, 10] and have been called nongenomic [11, 12]. The nongenomic actions of E2 result mainly from activation of cellular signaling systems on binding of this hormone to ER [11]. Signal transduction activation can lead to the modulation of downstream pathways that have discrete cellular actions, including stimulation of adenylyl cyclase in breast and vascular tissues [7, 13], activation of Ca2+ flux in arterial smooth muscle [14], cGMP-dependent protein kinase in pancreatic ß-cells [15], protein kinases A (PK-A) and protein kinases C (PK-C) in ß-endorphin neurons [8], and protein phosphorylation via activation of tyrosine kinases and mitogen-activated protein kinase pathways in MCF-7 cells [16].
We recently demonstrated that RNA and protein synthesis inhibitors suppress E2-induced oviductal embryo transport acceleration in pregnant rats but failed to do so in cyclic rats [17, 18]. Therefore, we conclude that E2 affects egg transport through a genomic action in pregnant rats and through a nongenomic action in cyclic rats. Furthermore, in cyclic rats, exogenous E2 activates PK-A and PK-C in the oviduct also via a nongenomic action because such activation occurs when mRNA synthesis is completely suppressed by 
-Amanitin [19]. The E2-induced phosphorylation is essential for its effect on oviductal egg transport since local administration of a broad-spectrum inhibitor of protein kinases totally blocked E2-induced acceleration of egg transport [19]. Thus, E2 accelerates oviductal transport of oocytes via nongenomic stimulation of protein phosphorylation in the oviduct.
Here we report possible signaling transduction cascades that could be involved in the nongenomic pathway by which E2 accelerates egg transport in cyclic rats. We first examined the role of ER on the increased oviductal protein phosphorylation and oviductal oocyte transport acceleration induced by E2 in cyclic rats. The involvement of adenylyl cyclase, PK-A, PK-C, or tyrosine kinases in the effect of E2 on oocyte transport was then determined. In addition, the effect of E2 on the levels of cAMP in the rat oviduct and a possible direct effect of E2 alone or bound to its receptor on in vitro PK-A activity were also examined. Some of these results were previously reported in abstract form by Orihuela et al. [20].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprague-Dawley rats (bred in-house) weighing 200–260 g were used. The animals were kept under controlled temperature (21–24°C), and lights were on from 0700 to 2100 h. Water and pelleted rat chow 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. The day of estrus was considered Day 1 of the cycle. The care and manipulation of the animals were done in accordance with the ethical guidelines of our institution.
Treatments
Systemic administration of E2 On Day 1 of the cycle, 1 µg E2 was injected s.c. as a single dose in a volume of injection of 0.1 ml. Control rats received propylene glycol as the vehicle.
Local administration of drugs Rats on Day 1 of the cycle were injected into each ovarian bursa with one of the drugs described below. The volume of injection for each drug was 4 µl. Control rats received the appropriate vehicle alone.
Antagonist of ER. ICI 182 780 (kindly donated by W. Elger, Entech, Jena, Germany [21]) was injected as a single dose at a concentration of 6.25 µg/µl in 0.1% dimethyl sulfoxide (DMSO; Sigma Chemical, St. Louis, MO).
Adenylyl cyclase inhibitor. SQ 22536 (9-(tetrahydro-2‘furyl)adenine; Calbiochem, La Jolla, CA [22]) was injected as a single dose at a concentration of 7.5 µg/µl in saline solution.
PK-A inhibitor. Rp-cAMP, TEA (adenosine 3',5'-cyclic phosphorothiolate-Rp; Sigma Chemical or Calbiochem [23]) was injected as a single dose at concentrations of 7.5 or 25 µg/µl in saline solution.
PK-C inhibitors. GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide); Calbiochem or Sigma Chemical [24, 25]) or staurosporine, Streptomyces sp. (Sigma Chemical [26, 27]) were injected as a single dose at a concentration of 0.5 or 1.75 µg/µl, respectively, in 0.1% DMSO.
Tyrosine kinases inhibitor. The phytoestrogen genisteine (4',5',7-trihidroxylisoflavone; Calbiochem [28]) was injected as a single dose at a concentration of 2 µg/µl in 0.1% DMSO.
Adenylyl cyclase activator. Forskolin (7ß-acetoxy-8,13-epoxy-1,6ß,9-trihidroxy-labd-14-ene-11-one; Sigma Chemical [29]) was injected as a single dose at concentrations of 2.5 or 5 µg/µl in 0.1% DMSO.
Animal Surgery
Intrabursal administration of drugs was performed on the morning of Day 1 of the cycle as described by Orihuela et al. [18]. At this time, ovulation had already taken place, so this treatment could not affect the number of oocytes ovulated. Furthermore, we had previously demonstrated that drugs administered intrabursally act locally in the oviduct [18, 19].
Assessment of Egg Transport
Twenty-four hours after treatment, animals were killed via inhalation of excess ether and their oviducts were flushed individually with saline. Flushings were examined under low-power magnification (25x). The number of eggs in the oviduct was recorded. We have previously determined from egg recovery experiments from the uterus and the vagina and from placing ligatures in the uterine horns 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 it as E2-induced oviductal transport acceleration.
Protein Gel Electrophoresis and Immunoblotting
Six hours after treatment, rats were killed by excess ether inhalation and the oviducts were removed and cleaned from fat tissue. Because we wanted to analyze only oviductal proteins, oviducts were flushed with saline in order to remove the cumulus-oocyte complex and thus avoid contamination with their proteins. Then oviducts in groups of four (obtained from two rats) were homogenized on ice in a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) for 10 sec in 1 ml of buffer containing 0.25 mM sucrose, 3.0 mM MgCl2, 25 mM Tris, and 0.5 mM phenylmethylsulfonyl fluoride [30], followed subsequently by centrifugation at 6000 rpm for 10 min at 4°C. The supernatant (clarified homogenate) was harvested and stored at -20°C until used. The protein concentration in the clarified homogenate was determined according to Bradford [31] using BSA as standard. Aliquots of the clarified homogenate containing 10 µg of protein were denatured for 2 min at 90°C in equal volumes 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 [32] utilizing a mini PROTEAN electrophoretic chamber (Bio-Rad, Hercules, CA). Proteins resolved in the gels were stained with 2% (w/v) Coomassie blue R-250 (Bio-Rad) or electroblotted onto nitrocellulose membranes (Bio-Rad [33]). Nitrocellulose blots were blocked by incubation overnight at 4°C in TTBS (100 mM Tris/HCl, pH 7.5, 0.9% v/v 150 mM NaCl, and 0.05% v/v Tween 20) containing 1% BSA and were incubated for 2 h with the antibody blend of rabbit antiphosphoserine/antiphosphothreonine/antiphosphotyrosine polyclonal antibodies (Omni-Phos Blend; Chemicon International, Temecula, CA) in 1:200 dilution for detection of phosphorylated protein bands. Blots were rinsed five times for 5 min each in TBS (100 mM Tris/HCl, pH 7.5, and 0.9% v/v 150 mM NaCl) and were incubated for 2 h in TTBS containing a 1:5000 dilution of goat anti-rabbit IgG alkaline phosphatase conjugate (Chemicon International [34]). The alkaline phosphatase activity was detected by color development during incubation of the blots in 100 mM Tris/HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2, containing BCIP/NBT tablets (1 tablet in 10 ml; Sigma Chemical [34]).
Densitometry of the Immunoblots
Immunoblots were scanned using an Epson model Expression 636 scanner (Epson Co., Santiago, Chile) and each band density was quantitatively analyzed with the NIH Image 1.61 Software (National Institutes of Health, Bethesda, MD). 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 [35].
Measurement of cAMP Levels
Three hours after treatment, oviducts in groups of four were homogenized in 0.5 ml of ice cold 10% (v/v) trichloroacetic acid and centrifuged for 15 min at 6000 rpm at 4°C. The pellet was discarded and the supernatant was washed four times with five volumes of water-saturated diethyl ether. The upper layer was discarded after each wash. Following the last wash, the aqueous extract was dried under a stream of nitrogen at 60°C. Levels of cAMP in dried extracts were determined using Biotrak cAMP enzyme immunoassay system (catalog no. RPN 225; Amersham Pharmacia Biotech, Buckinghamshire, England). This kit is based on competition between unlabeled cAMP and a fixed quantity of peroxidase-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody. This allows the construction of a standard curve and the measurement of cAMP levels in unknown samples. Color was developed with 3,3',5,5'-tetramethylbenzidine/hydrogen peroxide as substrate. Optical density was read at 630 nm with a microplate reader (BIO-TEK Instruments, Winooski, VT).
Activity of PK-A In Vitro
PK-A activity was determined using a Protein Kinase Assay Kit, Non-Radioactive (catalog no. 538484, lot B31285; Calbiochem). This kit is based on an enzyme-linked immunosorbent assay that utilizes a synthetic PK-A substrate peptide and a monoclonal biotinylated antibody that recognizes the phosphorylated form of the peptide. Color was developed with peroxidase-conjugated streptavidin and o-phenylenediamine as substrate. Optical density was read at 492 nm with a microplate reader. Estradiol alone or bound to its receptor (estrogen receptor- human, recombinant; Calbiochem) were incubated with 1 mM ATP (Sigma Chemical) and 50 mU bovine heart PK-A (lot 108H7846; Sigma Chemical) and the PK-A activity was determined. Estradiol plus ER was previously incubated at 25°C for 4 h in order to allow E2 binding to its receptor. Bovine heart PK-A with or without 1 or 6 µM cAMP (Sigma Chemical) was used as positive or negative controls, respectively. Although we used bovine heart PK-A instead of rat PK-A, the homology between these enzymes is highly conserved [36, 37]; therefore, we extrapolated data obtained with bovine PK-A to our rat model.
Statistical Analysis
The results are presented as mean ± SEM. Overall analysis was done by Kruskal-Wallis test, followed by the Mann-Whitney test for pairwise comparisons when overall significance was detected. The actual number of replicates (N) in experiments done to determine protein phosphorylation or to measure cAMP levels in the rat oviduct correspond to three replicates. In each replicate, two rats for each treatment group were used and the four oviducts of these two animals were homogenized as a pool to determine protein phosphorylation or cAMP levels. The actual N in experiments done to determine the effect of drugs on oviductal egg transport is the total number of rats used in each experimental group because the total number of oocytes recovered from the two oviducts of a single rat corresponds to a single data point.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This experiment was designed to determine whether the ER antagonist ICI 182 780 blocks the E2-induced oviductal protein phosphorylation in cyclic rats. A total of 24 rats on Day 1 of the cycle were treated with vehicle, ICI 182 780, E2, or ICI 182 780 plus E2.
The gels stained with Coomassie blue showed 39 major protein bands with no differences between treatment groups (Fig. 1). Immunoblots of phosphorylated proteins showed 25 major protein bands (Fig. 1). Only 12 of these bands, whose molecular weights ranged from 116 to 121 kDa, were further analyzed by optical densitometry (Fig. 1). In the control group, protein bands f, i, j, k, l were clearly the most intense with respect to other bands (Fig. 1). Administration of E2 stimulated the phosphorylation of six bands (c, e, f, i, k, l; Fig. 2), whereas ICI 182 780 alone stimulated phosphorylation of bands f and k (Fig. 2). Administration of ICI 182 780 concomitantly with E2 blocked the increase in the phosphorylation of protein bands c, e, i, l (Fig. 2).
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Effect of ICI 182 780 on E2-Induced Egg Transport Acceleration
This experiment was designed to determine whether intrabursal (i.b.) administration of ICI 182 780 can inhibit the acceleration of oviductal egg transport induced by E2 in cyclic rats. A total of 22 animals on Day 1 of the cycle were divided into four treatment groups: 1) DMSO plus propylene glycol, 2) ICI 182 780 plus propylene glycol, 3) DMSO plus E2, and 4) ICI 182 780 plus E2.
The mean number (±SEM) of eggs recovered from the oviducts of the control group was 10.2 ± 0.7, while in the groups treated with E2, it was 2.5 ± 1.0. Intrabursal administration of ICI 182 780 alone did not affect oviductal egg recovery (8.0 ± 0.7), although it blocked the E2-induced egg transport acceleration (8.2 ± 0.6; Fig. 3).
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Effect of Specific Signaling Pathway Inhibitors on E2-Induced Egg Transport Acceleration
Five experiments were performed to determine whether i.b. administration of selective adenylyl cyclase (SQ 22536), PK-A (Rp-cAMP, TEA), PK-C (GF 109203X or staurosporine), or tyrosine kinase (genistein) inhibitors can prevent the acceleration of oviductal egg transport induced by E2 in cyclic rats. A total of 164 animals on Day 1 of the cycle were used and, for each experiment, they were divided into four treatment groups: 1) vehicle plus propylene glycol, 2) inhibitor plus propylene glycol, 3) vehicle plus E2, and 4) inhibitor plus E2. Twenty-four hours after treatment, egg transport was assessed as described.
The results are shown in Figure 4. The mean number (± SEM) of eggs recovered from the oviducts of control groups ranged from 8.8 ± 0.6 to 12.2 ± 0.5, while in groups treated with E2, it ranged from 1.8 ± 0.6 to 5.0 ± 0.8. Local administration of inhibitors alone did not affect oviductal egg recovery (range 9.0 ± 0.7 to 10.0 ± 0.7). The adenylyl cyclase inhibitor blocked completely the E2-induced oviductal egg transport acceleration (7.3 ± 1.0), while the PK-A inhibitor blocked it only partially (7.5 µg/µl, 5.4 ± 0.9; 25 µg/µl, 5.0 ± 0.8). Administration of PK-C or tyrosine kinase inhibitors did not block the effect of E2 on egg transport. Although it has been reported that genistein has estrogenic activity in reproductive organs [38], administration of this drug alone did not accelerate oviductal egg transport, suggesting that the concentration used had no estrogenic effects in the rat oviduct.
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Effect of Forskolin on Oviductal Egg Transport
This experiment was designed to determine whether i.b. administration of the selective adenylyl cyclase activator forskolin can mimic the effect of E2 on oviductal egg transport. A total of 25 animals on Day 1 of the cycle were divided into two treatment groups: 1) DMSO and 2) forskolin.
The mean number (±SEM) of eggs recovered from the control group was 11.3 ± 0.5, while forskolin at 2.5 or 5 µg/µl significantly decreased the number of eggs recovered from the oviduct (Fig. 5).
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Effect of E2 on cAMP Levels in the Oviduct
A total of 12 rats on Day 1 of the cycle were treated with vehicle or E2. In the vehicle control, the basal level of cAMP in the oviduct was 1.8 ± 0.4 pmol/oviduct, while E2 administration significantly increased the level of cAMP 3 h after treatment to 4.8 ± 1.4 pmol/oviduct.
Effect of E2 on PK-A Activity In Vitro
This experiment was designed to determine whether E2 at 0, 0.1, 1, or 10 nM can directly activate PK-A in vitro.
The optical density at the end of incubation was the same at all E2 concentrations and similar to the negative control. Therefore, none of the E2 concentrations activated PK-A in vitro (Table 1).
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Effect of E2-ER Complex on PK-A Activity In Vitro
Here we tested whether E2 bound with its receptor can directly activate PK-A in vitro. The 2 nM of E2 plus vehicle of ER, vehicle of E2 plus 2 µg of ER, or E2 plus ER were allowed to interact with PK-A.
The optical density of these three groups at the end of incubation was similar to the negative control. Therefore, E2 bound to its receptor isoform did not activate PK-A in vitro (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or ER-ß because inhibition of ER by ICI 182 780 may vary in different tissues [39–41]. However, ER isoform is more likely to be involved in the effects we are describing because Mowa and Iwanaga [42] and Wang et al. [43] have shown by in situ hybridization and immunohistochemistry that is the predominant isoform in the endosalpynx and myosalpynx of the rat oviduct. Therefore, a major portion of E2-induced oviductal protein phosphorylation requires activation of ER-, and this is in keeping with other nongenomic effects of this hormone [44–48]. The cellular localization of ER- that mediates the nongenomic action of E2 in the rat oviduct remains to be determined. We assume that differences in the intensity of the bands were due to changes in phosphorylation rather than changes in protein expression because the Coomassie blue staining showed no differences between groups. This lack of change in the oviductal protein pattern in gels stained with Coomassie blue is in agreement with previous work indicating that E2 neither increased the incorporation of 35S-methionine nor changed the fluorographic pattern of the oviductal proteins in cyclic rats [18]. On the other hand, further analyses are necessary in order to confirm that the antiestrogen ICI 182 780 does not affect oviductal protein expression in cyclic rats.
Blockade of ER by ICI 182 780 and inhibition of adenylyl cyclase by SQ 22536 totally suppressed the E2-induced egg transport acceleration while inhibition of PK-A by Rp-cAMP, TEA blocked it only partially. Activation of adenylyl cyclase by forskolin mimicked the effect of E2 on egg transport, and cAMP levels were increased in rat oviducts following E2 treatment. These findings suggest that the nongenomic pathway involved in the acceleration of egg transport induced by E2 requires binding of the hormone to its classical receptor and activation of cAMP and PK-A signaling pathways. Activation of the cAMP pathway appears to be an absolute requirement, while activation of the PK-A signaling appears to be a partial requirement. Several lines of evidence have implicated cAMP and PK-A as mediators of the nongenomic actions of E2. Estradiol activates adenylyl cyclase in vascular smooth muscle, breast cancer, and uterine cells by a mechanism that does not require RNA and protein synthesis [6, 13, 49]. In addition, acute stimulation of Ca2+ uptake induced by E2 is accompanied by increased cAMP content in rat duodenal cells and preosteoclastic cells [50, 51]. On the other hand, E2-induced relaxation of porcine coronary arteries was mimicked by the cAMP analogue 8-bromo-cyclic AMP and inhibited by the PK-A inhibitor Rp-cAMP, TEA. Similarly, E2 reduced the potency of the µ-opioid receptor agonist DAMGO in guinea-pig hypothalamic neurons. This effect was mimicked by the PK-A activator Sp-cAMP, while the PK-A inhibitor Rp-cAMP, TEA reversed the steroid-induced effect [7]. Our findings provide the first evidence of a nongenomic action of E2 mediated by cAMP and PK-A signaling pathway in the mammalian oviduct.
The participation of other signaling pathways in the nongenomic action of E2 in the rat oviduct cannot be excluded because PK-A inhibition only partially blocked E2-induced egg transport acceleration. Although cAMP and PK-A have long been shown to mediate specific intracellular signaling events, recent observations have indicated that PK-A does not account for all of the intracellular targets of cAMP [52]. For example, cAMP regulates proliferation in thyroid cells by mechanisms independent of PK-A [53, 54]. Furthermore, it has been shown that cAMP can bind to proteins that exhibit guanine nucleotide exchange activity (GEF). The cAMP-GEF complex can activate small GTPases, Rap 1, Rap 2, and Ras, leading to additional activation of kinase cascades, such as extracellular regulated kinases 1/2, p38 mitogen-activated protein kinase, or phosphoinositol 3 kinase/phosphoinositide-dependent kinase (PI3K/PDK1) pathways [52]. Interestingly, PI3K/PDK1 pathway activates nitric oxide synthase (NOS) in endothelial cells [55], and it has been shown that NOS participates in the regulation of egg transport in the rat [56]. Further studies are needed in order to determine whether some of these kinase cascades are involved in the nongenomic pathway, which mediates the effect of E2 on egg transport in the rat.
Estradiol-bound ER- directly associates with the regulatory subunit of phosphoinositol 3 kinase (PI3K), resulting in the activation of the AKT serine/threonine kinase in human endothelial cells [55]. This finding provided evidence of an important signaling pathway involving the direct interaction of E2-ER- with a specific kinase. Our results showed that E2 alone or bound to its receptor isoform did not stimulate PK-A activity in vitro. This suggests that there is no direct interaction between E2 or E2-ER with PK-A and indicates participation of other mediators (i.e., cAMP) for the effect of E2 on PK-A activity in the rat oviduct.
Although PK-C activation is necessary for E2-induced oviductal protein phosphorylation [19], inhibition of this enzyme did not block the effect of E2 on egg transport. Probably other oviductal functions (e.g., secretion, ciliary beat, sperm binding to the oviductal epithelium), involving activation of PK-C signaling, are controlled by nongenomic actions of E2.
In summary, this study shows that ER participates in the nongenomic action of E2 in the rat oviduct. Furthermore, the nongenomic pathway by which E2 accelerates oviductal egg transport in the rat requires full participation of cAMP and partial participation of PK-A signaling pathways.
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FOOTNOTES |
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2 Correspondence. FAX: 56 2 222 5515; hbcroxat{at}genes.bio.puc.cl
Received: 15 September 2002.
First decision: 8 October 2002.
Accepted: 17 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by synthetic and natural estrogenic compound using a promoter containing three estrogen-responsive elements. J Steroid Biochem Molec Biol 2001 78:25-32[CrossRef][Medline] and ERß exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 2001 142:2336-2342 and -ß mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. J Endocrinol 2000 165:59-66[Abstract] and ß in the female reproductive tract of the rat during the estrous cycle. Biol Reprod 2000 63:1331-1340 mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 1999 103:401-406[Medline]1. J Biol Chem 2001 276:27071-27076 detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. Endocrinology 1999 140:3805-3814 protein in neuronal process of cultured hippocampal neurons. Neuroendocrinology 2000 71:34-42[CrossRef][Medline]This article has been cited by other articles:
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  A. Parada-Bustamante, P. A Orihuela, M. Rios, P. A Navarrete-Gomez, C. A Cuevas, L. A Velasquez, M. J Villalon, and H. B Croxatto Catechol-O-Methyltransferase and Methoxyestradiols Participate in the Intraoviductal Nongenomic Pathway Through Which Estradiol Accelerates Egg Transport in Cycling Rats Biol Reprod, December 1, 2007; 77(6): 934 - 941. [Abstract] [Full Text] [PDF]
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 R. Shao, B. Weijdegard, J. Fernandez-Rodriguez, E. Egecioglu, C. Zhu, N. Andersson, A. Thurin-Kjellberg, C. Bergh, and H. Billig Ciliated epithelial-specific and regional-specific expression and regulation of the estrogen receptor-beta2 in the fallopian tubes of immature rats: a possible mechanism for estrogen-mediated transport process in vivo Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E147 - E158. [Abstract] [Full Text] [PDF]
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 J. Lapointe, M. Roy, I. St-Pierre, S. Kimmins, D. Gauvreau, L. A. MacLaren, and J.-F. Bilodeau Hormonal and Spatial Regulation of Nitric Oxide Synthases (NOS) (Neuronal NOS, Inducible NOS, and Endothelial NOS) in the Oviducts Endocrinology, December 1, 2006; 147(12): 5600 - 5610. [Abstract] [Full Text] [PDF]
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 P. A Orihuela, A. Parada-Bustamante, L. M Zuniga, and H. B Croxatto Inositol triphosphate participates in an oestradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats. J. Endocrinol., March 1, 2006; 188(3): 579 - 588. [Abstract] [Full Text] [PDF]
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J. Lapointe, S. Kimmins, L. A. MacLaren, and J.-F. Bilodeau Estrogen Selectively Up-Regulates the Phospholipid Hydroperoxide Glutathione Peroxidase in the Oviducts Endocrinology, June 1, 2005; 146(6): 2583 - 2592. [Abstract] [Full Text] [PDF]
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P < 0.05 from corresponding V and AE groups; 
P < 0.05 from corresponding V and E2 groups; ¶P < 0.05 from corresponding V, AE, and AE + E2 groups; and &P < 0.05 from corresponding V, E2, and AE + E2 groups. Note that increased density induced by E2 in bands c (molecular weight, 82.2 kDa), e (molecular weight, 78.7 kDa), i (molecular weight, 38.6 kDa), and l (molecular weight, 21.5 kDa) is suppressed by concomitant treatment with the AE
