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a Department of Animal Science, The University of Tennessee, Knoxville, Tennessee 37901
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retinol is essential to reproduction in both males and females (reviewed in [19]). Deficiencies in vitamin A lead to decreased ovarian size, reproductive senescence, abortion, and congenital fetal malformation. The transport of retinol is accomplished by a specific transport protein, retinol-binding protein (RBP). RBP or its mRNA has been identified in equine and porcine oviducts [20, 21] and has been shown to be steroid responsive in uterine tissues [22, 23]. The objective of this study was to identify RBP in ovine oviducts and determine the role of ovarian steroids in regulating its expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Silicone elastomer tubing and Bouin's fixation fluid were purchased from Baxter Scientific Products (McGaw Park, IL). Silicone sealant was purchased from Dow Corning Corp. (Midland, MI). Cesium trifluoroacetate (CsTFA) was obtained from Pharmacia Biotech (Uppsala, Sweden). Formamide and Random Prime labeling kits were purchased from United States Biochemicals (Cleveland, OH). S & S Nytran membrane was purchased from Schleicher and Schuell (Keene, NH). [
-32]dCTP and [3H]leucine were obtained from ICN Pharmaceuticals (Costa Mesa, CA). Microtiter plates (96 well) were obtained from Corning (Corning, NY). Goat anti-rabbit IgG alkaline phosphatase conjugate was purchased from Bio-Rad Laboratories (Hercules, CA). The p-nitrophenylphosphate (p-NPP) substrate was obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD) and prepared according to manufacturer's directions. Histogen peroxide anti-peroxidase immunostaining system was purchased from BioGenex Laboratories (San Ramon, CA). Microprobe system including slide holder assembly, 30-well Isolon reagent isolators, and ProbeOn microscope slides was purchased from Fisher Scientific (Pittsburgh, PA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Animals
For the first study, 13 sexually mature crossbred ewes that had exhibited at least three normal 16-day estrous cycles were salpingectomized via midventral laparotomy on Day 1 (n = 5), Day 5 (n = 4), or Day 10 (n = 4) of their subsequent estrous cycles (Day 0 = estrus). The oviducts were prepared for RNA analysis, immunocytochemistry (ICC), or organ culture as described below.
In the second study, 25 sexually mature crossbred ewes that had exhibited at least two normal estrous cycles were ovariectomized via midventral laparotomy. Ovariectomies were performed at least 45 days prior to the experiment to allow sufficient time for endogenous ovarian steroids to be cleared from the circulation. Animals assigned to the control group received no exogenous steroids. Steroid treatments were administered according to the method of Homanics and Silvia [24] with a modification of the steroid pretreatment. Steroids that were injected were dissolved in corn oil. Briefly, 20 animals underwent 9 days of progesterone treatments (12 mg/day i.m.) and 2 days of estradiol-17ß treatment (3.5 µg, 7.0 µg, 14.0 µg, 7.0 µg, and 3.5 µg i.m. sequentially, at 8-h intervals) in order to induce behavioral estrus. The day following the last injection was designated Day 0, and animals were checked for estrus with vasectomized rams. All ewes showed estrus behavior and were randomly allotted to one of three treatments: 1) progesterone (P4), 2) estradiol-17ß (E2), or 3) E2+P4 (E2+P4). On the day following estrus, ewes assigned to groups 1 and 3 received 12-mg i.m. injections of P4 once daily. The ewes assigned to treatments 2 and 3 received s.c. silicone elastomer implants (3 cm long, 3.35 mm inner diameter and 4.65 mm outer diameter) containing E2 [25]. The control treatment group received only vehicle (n = 5). Ewes were bled via jugular venipuncture on Days 6, 10, and 13 of treatment. Blood was collected in chilled tubes containing 200 µl of sodium citrate (0.025% solution) and immediately placed on ice. Blood samples were centrifuged for 20 min at 2500 x g. Plasma was recovered and stored at -20°C until RIAs were performed. Three animals were removed from the study due to loss of E2 implant; 2 from the E2 group and 1 from the E2+P4 group. This left 5 animals in the E2 and control groups and 6 animals in the E2+P4 and P4 groups. After 13–15 days of treatment, ewes were salpingectomized and oviducts treated as in experiment 1. All procedures were approved by the University of Tennessee Institutional Animal Care and Use Committee.
RNA Isolation and Analysis
Oviducts were obtained surgically and transported to the laboratory on ice. The broad ligament, infundibulum, and any excess uterine tissue were carefully removed. A 5- to 7-mm section was removed from the end of the ampulla and isthmus and reserved for ICC. The remaining tissue was minced and homogenized in 4 M guanidine isothiocyanate (4°C) [26]. Total RNA isolation was performed according to the procedure described by Doré et al. [27]. Briefly, supernatant was layered on CsTFA (prepared according to the manufacturer's instructions to a specific gravity of 1.51) for isopycnic gradient ultracentrifugation. The RNA pellet was resuspended in a solution of 30 mM sodium citrate, 0.1% (w:v) SDS, and 1% (v:v) ß-mercaptoethanol. Ethanol precipitation was performed to remove any residual CsTFA. The RNA pellet was dissolved in a solution of 40 mM morpholinopropane sulfate, 10 mM sodium acetate, 1 mM EDTA [26] with 0.1% (w:v) SDS, and 1% (v:v) ß-mercaptoethanol, quantified by absorbance at 260 nm, and stored at -100°C.
A DNA probe specific for RBP, designated bcRBP-700 and isolated from a bovine conceptus cDNA library, was used for slot-blot analysis [28]. The probe DNA was prepared and random prime labeled with [-32P]dCTP to specific activities of 2.0 x 109 cpm/µg DNA as described by Liu et al. [28]. Total RNA (10 µg) was electrophoresed in 1.5% (w:v) agarose 3-[N-morpholino]propanesulfonic acid/formaldehyde gels [26] and stained with 0.2 µg/ml ethidium bromide to confirm its integrity. Intact RNA (10 µg) was then loaded onto nylon membranes using a Minifold slot-blot apparatus according to the procedure recommended by the manufacturer (Schleicher and Schuell). RNA was cross-linked to membranes by UV irradiation (0.12 J, UVC 1000; Hoefer, San Francisco, CA). Membranes were prehybridized, hybridized, and washed as described previously [28]. Washed, hybridized membranes were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) for 96 h at -100°C. The membrane was stripped of the RBP probe by boiling in 0.1% SDS and rehybridized with rat ß-actin (specific activity 1–1.5 x 109 cpm/µg DNA) to correct for loading inaccuracies as described by Doré et al. [27]. Signal intensities from each exposure were analyzed by integration using an LKB Ultrascan Laser Scanning Densitometer and Gelscan XL version 2.0 software package (Pharmacia LKB Biotechnology, Uppsala, Sweden). ß-Actin signals were used to correct for loading inaccuracy and to allow RBP area to be used to quantify relative RBP expression from each day of the estrous cycle or hormone treatment group.
In Vitro Culture of Oviductal Explants
Oviducts were removed and transported to the lab in 0.9% NaCl at 30–35°C, and excess tissue was removed as described above. Each oviduct was then split into four approximately equal sections and cultured as described by Godkin et al. [29]. Briefly, oviducts were divided into isthmus or ampullar sections and minced to approximately 1-mm3 pieces, and explants were placed in 5 ml of leucine-deficient, high-glucose Dulbecco's Minimum Essential medium (DMEM) containing 50 µCi of L-[3H]leucine. Tissues were cultured for 24 h at 37°C on a rocking platform in a gaseous atmosphere of 45% O2:50% N2:5% CO2. Cultures were terminated by centrifugation at 12 000 x g for 15 min and the supernatant was stored at -80°C.
Immunoprecipitation
Aliquots of dialyzed medium from oviductal cultures were lyophilized, and samples were resuspended in water such that 200 000 cpm of nondialyzable radioactivity was present in 750-µl aliquots. Aliquots were incubated with 5 µl of anti-RBP or normal rabbit serum overnight at 4°C. The immunoprecipitation was performed as described by Lifsey et al. [30] and analyzed by one-dimensional PAGE [31]. Fluorography was performed according to the procedure of Chamberlain [32]. Signal intensities of five autoradiographs, each representing 1 animal from each treatment (6 animals per autoradiograph), were analyzed by integration using LKB Ultrascan Laser Densitometer and GSXL version software and expressed as AU/mm2 (AU is arbitrary optical density units).
ICC
Ampullar and isthmus sections from each ewe were immersion fixed in Bouin's fixation fluid for 4–6 h and washed in 70% ethanol, which was changed twice daily until the yellow color had dissipated. Tissues were dehydrated and embedded in paraffin, and ICC was performed according to Liu and Godkin [33]. The ICC procedure was performed on oviducts (ampulla and isthmus section) such that 1 animal from each treatment was represented in each analysis. In each replication, all slides were processed in the microprobe slide assembly so that all tissue sections received identical treatment. Similar conditions were applied to each replication, and a representative sample is shown.
ELISA
Explant medium from the second experiment was analyzed for RBP by ELISA according to the procedure of MacKenzie et al. [34]. Briefly, microtiter plates were coated with purified serum RBP [35]. Samples and standards (ranging from 0.1–10 ng/well) were mixed with bovine placental RBP antibody [35] and incubated overnight at 4°C. The sample-antibody mixture was then transferred to the antigen-coated plates and allowed to incubate for 2 h in a humidified chamber at 30°C. Plates were then washed (Dynatech Ultrawash II Microplate Washer, Chantilly, VA) with a solution of PBS with 0.05 (v:v) Tween 20. Goat anti-rabbit IgG alkaline phosphatase conjugate (diluted 1:3000) was added to each well, and the plate was then incubated for 2 h at 30°C. The plate was washed as before and p-NPP phosphatase substrate was added to the wells. Plates were read (absorbance 405 nm; BioTek Automated Microplate Reader, Winooski, VT) every 15 min until the 100% antibody wells (antigen-coated wells with anti-RBP antibody only) reached an optical density of 0.8–0.9. The reaction was then stopped with 5% Na2 EDTA and the plate was read to calculate the results. The intraassay variation was < 5%, and the interassay variation was < 10%.
RIAs
Circulating E2 concentrations were determined in plasma samples by a validated RIA according to the method of Moura and Erickson [36]. Circulating P4 levels were determined by a RIA validated by Seals et al. [37]. All samples were run in a single assay, and intraassay coefficient of variation for E2 and P4 was 9% and 4%, respectively. Minimum sensitivity per tube was 1.5 pg/ml (E2) and 0.05 ng/ml (P4).
Statistical Analyses
Data were analyzed using the Statistical Analysis System (SAS Institute, Cary, NC) [38]. ANOVA was performed using the General Linear Models Procedure (PROC GLM) to detect differences due to days (experiment 1) and differences due to the E2 and P4 hormone treatment factorial (experiment 2). Differences among days/hormone treatments were tested utilizing protected least-significant difference. Interpretation focused on the interaction means, because the treatment combinations were of primary interest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Temporal changes in RBP mRNA expression in oviductal tissue prepared from cyclic animals were determined by quantitative slot-blot analysis. Mean areas for each day were normalized to ß-actin levels and plotted (Fig. 1A). RBP mRNA expression on Day 1 of the cycle was dramatically higher (p < 0.01) than on either Day 5 or Day 10. There were no significant differences (p > 0.95) in RBP mRNA expression between oviducts collected on Days 5 and 10.
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Relative changes in RBP mRNA expression in oviductal tissue from ewes treated with steroid hormones were also determined by quantitative slot-blot analysis, and the normalized plots are illustrated in Figure 1B. RBP mRNA expression was affected by P4 (p < 0.05) but not E2 (p > 0.70) treatment, and an interaction between the two was suggested (p < 0.09). Comparison of treatment-combination means showed no difference between ovariectomized controls and E2-treated animals (p > 0.10). P4 treatment significantly decreased RBP mRNA expression levels in comparison to the E2 animals but not the control-treated animals (p < 0.05). The E2+P4 treatment significantly reduced RBP mRNA expression levels in comparison to those in control or E2 animals (p < 0.05), but levels after E2+P4 treatment were not significantly different (p > 0.15) from those in the P4-treated animals.
Immunolocalization of RBP
RBP was immunolocalized to the luminal epithelia of oviductal mucosa with diffuse staining in stroma and muscularis in both the ampulla and isthmus of all animals. For brevity, only isthmus sections are shown. Intense RBP staining was noted in the epithelium on Day 5 of the cycle; it was somewhat decreased on Day 10 and noticeably diminished on Day 1 (Fig. 2). In the hormone-treated animals, ICC analysis revealed intense RBP staining in the oviductal epithelium of control and P4-treated animals, whereas E2 treatments alone or in combination with P4 resulted in diminished staining (Fig. 2).
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Oviductal RBP Synthesis In Vitro
Oviductal tissues from both experiments were incubated for 24 h in DMEM containing L-[3H]leucine. Immunocomplex precipitation with anti-bovine placental RBP serum, followed by one-dimensional PAGE and fluorography, was used to show oviductal RBP synthesis. No differences in patterns of RBP synthesis were observed between ampulla and isthmus tissue. Ampulla is shown in the cycling ewe fluorograph, and isthmus is shown in the steroid-treated ewe fluorograph (Fig. 3).
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Relative changes in metabolically labeled RBP production by oviductal tissue collected on three different days of the estrous cycle are illustrated in Figure 4A. RBP production by oviducts collected on Day 1 was strikingly higher (p < 0.05) than production on either Day 5 or Day 10. However, no differences in oviductal RBP production were noted between Day 5 and Day 10 of the cycle (p > 0.60).
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Temporal changes in metabolically labeled RBP production by oviductal tissue collected from ovariectomized, steroid-treated ewes were quantified and are shown in Figure 4B. RBP production was affected by E2 treatment (p < 0.01) but not by P4 treatment or an interaction between the two (p > 0.40). When treatment-combination means were considered, RBP production by oviducts collected from ewes treated with E2 alone or in combination with P4 was dramatically higher (p < 0.05) than that produced by P4-treated or control animals. No differences were noted between E2 and E2+P4 treatments or between P4-treated and control animals (p > 0.75).
ELISA
Total RBP in the medium conditioned by oviductal explants from steroid-treated ewes was determined by ELISA and expressed as nanograms of RBP per milligram of oviductal tissue (Fig. 5). No differences in patterns of concentrations were observed between ampulla and isthmus tissue, so samples were combined in final analysis. RBP concentration was affected by E2 treatment (p < 0.05) but not P4 treatment (p > 0.70), and an interaction between the two was suggested (p < 0.08). E2 treatment increased RBP production 3- to 5-fold over P4 or control treatments (p < 0.05). The E2+P4 treatment also significantly increased RBP production over P4 and control treatments (p < 0.07 and p < 0.05, respectively). There were no differences between either the E2 and E2+P4 treatments (p > 0.40) or the P4 and control treatments (p > 0.50).
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Plasma Steroid Concentrations
Concentrations of exogenous steroids in the peripheral circulation were determined by RIA. The range of P4 (in P4-treated animals) was 1–3 ng/ml, which is within the range of normal luteal phase production. E2-treated animals had more than 10 pg/ml of estradiol, which is above the normal cyclic estradiol production [25]. The reason for this is unknown, since the silicone elastomer implants were the same in size and type as those used by Homanics and Silvia [24] and the steroid treatments were identical except for a 9-day P4 pretreatment period instead of a 6-day pretreatment period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Results from the steroid hormone replacement study provide insight into the mechanisms regulating RBP production. Analyses of steady-state RBP mRNA expression levels, as well as RBP synthesis and release by oviductal explants from ovariectomized ewes, demonstrate that RBP is constitutively expressed in the absence of ovarian steroids. Progesterone administered on a background of estradiol appears to negatively regulate RBP gene expression, whereas estradiol stimulates RBP synthesis and secretion. We point out that although progesterone diminished RBP mRNA steady-state levels compared to those in controls, this decrease was not statistically significant, whereas the progesterone plus estradiol treatment significantly reduced RBP mRNA expression compared to control or estradiol treatments. These results may be explained by the regulation of progesterone receptors by the ovarian steroids. Spencer and Bazer [40] showed that treatment of ewes with progesterone for 12 days resulted in down-regulation of progesterone receptor expression in the uterus. On the other hand, treatment with progesterone and estradiol increased progesterone receptor expression by about 2-fold compared to that in progesterone-treated animals. In our study, down-regulation of the progesterone receptor in progesterone-treated animals could account for the similarity between RBP mRNA levels in these animals and levels in controls. Conversely, in ewes treated with progesterone plus estradiol, it is likely that progesterone receptors were up-regulated, allowing progesterone to exert negative control over RBP mRNA expression. Steady-state levels of RBP mRNA were highest in estradiol-treated ewes, and this level of expression was significantly different from that in animals treated with progesterone or progesterone plus estradiol but not from that in controls. Oviductal explants from estradiol-treated animals synthesized and released significantly more RBP than explants from control or progesterone-treated animals. It is possible that estradiol treatment stimulated both transcription and mRNA degradation. This would explain the dramatic effects of estradiol on RBP synthesis and release but only modest effects on steady-state RBP mRNA levels. Unfortunately, we cannot confirm this suggestion, since RNA turnover studies were not performed.
Results from the immunocytochemical analysis of oviduct tissues support the in vitro data, which demonstrated that estradiol stimulates RBP secretion. Oviducts from all animals under estradiol influence (i.e., intact ewes on Day 1, and ovariectomized ewes that received estradiol alone or in combination with progesterone) exhibited diminished immunostaining of RBP in the secretory epithelium compared to ovariectomized controls, progesterone-treated ewes, and luteal phase, intact animals. Apparently, in the absence of estradiol stimulation, the oviductal epithelium accumulates RBP.
Uterine production of RBP is also regulated, in part, by ovarian steroids. It has been shown in sheep [4, 23, 41] and cattle [23, 34] that RBP mRNA levels are elevated in endometrium during proestrus and estrus. Conversely, RBP concentrations in uterine flushings from cattle are low and are declining at these times [34]. In addition, it was observed that estradiol stimulated RBP mRNA expression in bovine uterine endometrial explants and epithelial cells [42]. Hence, it appears that estradiol stimulates RBP transcription, synthesis, and secretion by the oviduct (present study), whereas it may stimulate only RBP transcription in the uterus and may be inhibitory to secretion. In both the oviduct (present study) and uterus [34], rising progesterone levels appear to negatively regulate RBP and the mRNA that encodes it during late metestrus and early diestrus; but in the uterus, RBP secretion and mRNA levels rise from mid to late diestrus, possibly due to progesterone receptor down-regulation [34]. Conversely, in the oviduct, RBP expression remains depressed throughout diestrus. Differential regulation of RBP expression by the oviduct and uterus may be explained by the fact that the degenerative morphological alterations of the oviductal secretory epithelium are initiated as progesterone levels begin to rise about 4 days after estrus [43]; these include dedifferentiation of secretory apparatus and reduced numbers of secretory granules. Similar changes were observed in ovariectomized ewes that received exogenous progesterone on an estradiol background [5].
In summary, results from these studies demonstrate that RBP and the mRNA that encodes it are produced by the epithelium of the ampulla and isthmus of the ovine oviduct. In addition, RBP is expressed throughout the estrous cycle and in the absence of ovarian steroids. These findings are in contrast to data on expression of the well-characterized 92-kDa oviduct-specific glycoprotein [2–4], which has been shown to be estradiol dependent and to be expressed only in the ampullary end of the oviduct [5]. The ovarian steroids modulate RBP synthesis, secretion, and gene expression. Production of RBP by the oviduct is not limited to the ovine species. The protein has been identified in the equine oviduct on Day 1 and Day 4, and the mRNA has been identified in the Day 0 porcine oviduct [20, 21]. The high level of RBP expression at the time when the ovum and early embryo are present in the oviduct may indicate an important role for retinol in early development.
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FOOTNOTES |
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2 Correspondence. FAX: 423 974 4359; jgodkin{at}utk.edu
Accepted: October 9, 1998.
Received: June 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in response to oxytocin in ovariectomized ewes. Biol Reprod 1988; 38:804–811.[Abstract] on pregnancy rates in progestogen supplemented cattle. Prostaglandins 1998; (in press).
regulation of endometrial retinol binding protein. Biol Reprod 1997; 56(suppl 1):86.This article has been cited by other articles:
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  J. M. Naciff, M. L. Jump, S. M. Torontali, G. J. Carr, J. P. Tiesman, G. J. Overmann, and G. P. Daston Gene Expression Profile Induced by 17{alpha}-Ethynyl Estradiol, Bisphenol A, and Genistein in the Developing Female Reproductive System of the Rat Toxicol. Sci., July 1, 2002; 68(1): 184 - 199. [Abstract] [Full Text] [PDF]
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 D. M. Eberhardt, W. A. Will, and J. D. Godkin Retinol Administration to Superovulated Ewes Improves In Vitro Embryonic Viability Biol Reprod, June 1, 1999; 60(6): 1483 - 1487. [Abstract] [Full Text]
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