BIOLOGY OF REPRODUCTION 57, 7-15 (1997)
Expression of Tissue Inhibitor of Metalloproteinase-1 Protein and Messenger Ribonucleic Acid by the Oviduct of Cyclic, Early-Pregnant, and Ovariectomized Steroid-Treated Gilts' W.C. Buhi,2 ,4 I.M. Alvarez, 4 A.R. Pickard, 3 5 E.W. Mclntush,6 A.J. Kouba, 4 C.J. Ashworth, 5 and M.F. Smith6
Department of Obstetrics and Gynecology,4 University of Florida College of Medicine, Gainesville, Florida 32610-0294 The Rowett Research Institute,5 Aberdeen, United Kingdom Department of Animal Science,6 University of Missouri, Columbia, Missouri 6521 1 ABSTRACT
stromelysins, and gelatinases [1, 2]. TIMP-1 and other metalloproteinase inhibitors are synthesized and secreted by a variety of cell types and are thought to be intimately involved in intracellular communication in addition to interactions between cells and between cells and extracellular matrix, i.e., tissue remodeling. TIMPs form a 1:1 stoichiometric complex with specific MMPs and thereby regulate extracellular matrix degradation. Recent studies in reproductive tissues suggest a role for TIMP-1 in uterine endometrium during implantation and placentation [3, 4]; in the ovary, as a regulator of tissue remodeling or steroidogenesis or exhibiting growth activity in the ovary [5]; and in the oviduct, as an enhancer and stimulator of embryo development [6, 7]. While little is known about the expression of TIMPs in reproductive tissues during the estrous or menstrual cycle and early pregnancy, some studies have indicated control or regulation of TIMPs by steroids. In the mouse, an apparent temporal regulation of TIMP-1 mRNA has been shown in the uterus, decidua, and placenta [3]. Hormonal regulation of TIMP-1 by estrogen or progesterone (P4 ) in other studies, however, appears contradictory. In rabbit cervical fibroblasts [8], TIMP-1 mRNA and protein were increased by both estrogen and P4, although the increase was significantly greater with P4. In ovariectomized (OVX) nontreated ewes, endometrial TIMP-1 mRNA was elevated, but this was substantially reduced when animals were treated with estrogen or P4 [4]. In the cyclic and pregnant ewe, the abundance of endometrial TIMP- 1ImRNA was low through Day 10; however, expression increased through Day 16 in cyclic ewes and through Day 20 in pregnant ewes. Because there are many factors influencing fertilization and early cleavage-stage embryonic development in the oviduct that have not been recognized or understood, we designed the present study to establish that TIMP-1, which may participate in such a role [6, 7], was present in the porcine oviduct at the time when fertilization and early embryonic developmental events occurred. Specific objectives were 1) to determine whether TIMP-1 was synthesized and secreted by the oviduct during the estrous cycle and early pregnancy, 2) to determine whether there were synthetic differences between oviductal functional segments, 3) to determine temporal changes in the concentration of TIMP-1 protein in oviductal flushings during the estrous cycle, early pregnancy, and after OVX and treatment with estrogen or P4, 4) to examine differences between standard Western and prolific Chinese breeds, 5) to immunolocalize TIMP-1 within the oviduct, and 6) to determine steady-state levels of TIMP-1 mRNA during the estrous cycle and after OVX and steroid treatment.
It has been suggested that tissue inhibitor of metalloproteinases (TIMP)-1 has a role inreproductive tissues, regulating tissue remodeling or enhancing embryonic development. Oviductal TIMP-1 mRNA levels and protein expression were examined in gilts during the estrous cycle and early pregnancy and insteroidtreated ovariectomized (OVX) gilts by explant culture, two-dimensional SDS-PAGE and fluorography, dot-blot hybridization, immunoblot analysis, RIA, and immunocytochemical studies. TIMP-1 mRNA levels in the oviduct during the estrous cycle were greater (p < 0.02) on Days 2, 15, and 18 than on other days examined, and analysis of oviductal functional segments indicated an effect of day (p < 0.003), an effect of segment (p < 0.007), and a day x segment effect (p < 0.03). The level of TIMP-1 mRNA was greater (p < 0.003) inthe isthmus (I) on Day 2 than in the ampulla (A) or infundibulum (INF) or on other days examined (0 and 12). In steroid-treated OVX gilts, an effect of treatment with estradiol valerate (EV) + progesterone (P4) was shown with increased (p < 0.003) TIMP-1 mRNA levels. De novo synthesis of TIMP-1 protein was found throughout the estrous cycle and early pregnancy in all functional segments, but protein expression was greater in the I and greatest on Day 2. In steroid-treated OVX gilts, TIMP-1 protein synthesis was greatest in the I regardless of treatment, but with increased intensity after EV+P 4 treatment. TIMP-1 protein was found in oviductal flushings during the estrous cycle and early pregnancy, and in steroid-treated OVX gilts regardless of day, status, or treatment. Differences in TIMP-1 concentrations in oviductal fluid were found by day (p < 0.001), with breed differences detected between the Meishan and standard Western breeds. TIMP-1 protein was immunolocalized primarily to luminal epithelium of the INF, A, and I on all days of the estrous cycle and early pregnancy and to some cells in the stroma and blood vessel walls. Staining intensity correlated with TIMP-1 protein levels in oviductal flushings. The role of TIMP-1 in the oviduct remains to be established. INTRODUCTION Tissue inhibitor of metalloproteinases (TIMP)-1 is a specific glycoprotein inhibitor of matrix metalloproteinases (MMP), a group of enzymes consisting of collagenases, Accepted March 11, 1997. Received November 4, 1996. 'Supported by USDA Grant 92-37203-7996 (W.C.B. and F.A. Simmen), 95-37203-2308 (W.C.B.), The Burroughs Wellcome Travel Fund (W.C.B.); Scottish Office, Agricultural, Environmental and Fisheries Department (A.R.P. and C.J.A.); USDA National Needs Fellowship (E.W.M.) 92-38420-7342. 2 Correspondence. FAX: (352) 392-2808; e-mail:
[email protected] 3 Current address: Institute of Zoology, Regent's Park, London, NW1 4RY, UK.
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MATERIALS AND METHODS Materials Acrylamide, N,N'-diallyltartardiamide, urea, and SDS were obtained from Gallard-Schlesinger (Carle Place, NY); X-Omat AR (XAR-5) film was a product of Eastman Kodak (Rochester, NY); amino acids and protein standards were purchased from Sigma Chemical Company (St. Louis, MO); ampholines were from Pharmacia-LKB (Piscataway, NJ); all other supplies and reagents for gel electrophoresis were purchased from either Bio-Rad Laboratories (Richmond, CA) or Fisher Scientific (Orlando, FL); L-[4,53 H]leucine ([ 3 H]leu; specific activity, 120 Ci/mmol) was obtained from Amersham (Arlington Heights, IL); TRIzol, all media, and culture samples were purchased from Gibco (Grand Island, NY). [Gamma- 32 P]dCTP and BioTrans nylon membranes were purchased from ICN Biomedicals (Costa Mesa, CA). Nick-translation kit and Megaprime DNA Labeling systems were obtained from Amersham Life Science (Arlington Heights, IL). ExpressHyb hybridization solution and human G3PDH cDNA probe were purchased from Clontech Laboratories (Palo Alto, CA). All other biotechnology grade reagents and supplies were obtained from either Fisher Scientific, Sigma, Qiagen (Chatsworth, CA), or Life Technologies (Grand Island, NY). Tissue Collection Cyclic studies. Sexually mature Florida crossbred (FLA) gilts (Yorkshire x Duroc x Hampshire) were observed for behavioral estrus for at least two estrous cycles in the presence of intact boars, and the first day of standing estrus was designated as Day 0. On the appropriate day, i.e., Day 0, 1, 2, 4, 8, 10, 12, 15, or 18, cyclic gilts (n = 27) were taken to the abattoir for slaughter or were anesthetized. The reproductive tracts of the anesthetized gilts were exposed by a midline laparotomy, and tissues were collected aseptically. Oviductal functional segments, the infundibulum (INF), ampulla (A), and isthmus (I), were identified by gross examination and separated by dissection; one oviduct from each animal was frozen immediately in liquid nitrogen and stored at -80 0C for subsequent RNA extraction. The second oviduct, similarly dissected, was fixed for immunocytochemical analysis or placed in culture. OVX studies. Gilts (FLA) to be treated with various regimens of steroids (n = 12) were bilaterally OVX on the fourth day following a natural estrus and randomly assigned to one of five treatment groups. Groups 1-4 were injected (i.m.) daily for 11 consecutive days [9] with the following regimens. Group 1 (control) received vehicle (2 ml), which was corn oil and ethanol (9:1, v:v; n = 3); group 2 received 100 Ig estradiol valerate (EV; n = 3); group 3 received 200 mg P4 (n = 3); and group 4 received 100 pIg EV + 200 mg P4 (n = 3). Group 5 were OVX without regard to day of cycle and remained untreated for a time equivalent to three estrous cycles (> 60 days; n = 2). OVX gilts were anesthetized 24 h after the last treatment, 76 days after OVX for untreated gilts, and subjected to surgery [9]. Pregnancy studies. For pregnancy studies, FLA (n = 18), Large White x Landrace (LW) (n = 18), and highly prolific Chinese Meishan (MS) (n = 18) gilts were observed for behavioral estrus for five estrous cycles, and all experiments were performed at the same time of year. On Day 0 of the fifth cycle, gilts were bred with a boar of the respective breed at 0 and 24 h after detection of estrus, with the exception of those gilts assigned to Day 0, which were
bred only at 0 h. Gilts (n = 3 per day) were taken to the abattoir on Days 0, 2, 5, 8, 10, and 12 of pregnancy for slaughter, and tissues were collected as described above. Oviductal Flushing Oviducts from gilts in all treatment groups, i.e., cyclic, pregnant, and OVX, were flushed. Before oviducts were dissected, a surgical needle with a cutting edge was used to make a small incision in an avascular area of the I above the tubo-uterine junction. A polyvinyl catheter (i.d., 1.25 mm) was inserted 1-2 cm toward the A. The INF was clamped, and 5 ml of leucine (leu)-deficient minimal essential medium (MEM) [10] was introduced at the INF with a 5-ml syringe (20-gauge needle), massaged toward the isthmic catheter, and collected in a sterile tube. The fluids were centrifuged (2200 x g, 10 min, 4°C), lyophilized, and stored (-20°C) until analyzed [10]. Pregnancy was verified in the pregnant group, except in Day 0 gilts, by the presence of embryos in the oviductal flush or in a subsequent uterine flush. Explant Culture, Electrophoresis, and Western Blotting Oviductal functional segments (INF, A, and I) from cyclic, steroid-treated OVX, or early-pregnant gilts were cultured as previously described [10] using leu-deficient MEM and [3H]leu. Secretion of proteins synthesized de novo by explant-cultured tissue was determined by measuring the incorporation of [3 H]leu into nondialyzable macromolecules [10]. Distribution of proteins in culture medium or in pooled column fractions was examined by two-dimensional (2D)-SDS-PAGE and fluorography [10]. For immunoblot analysis, proteins were separated by 2D-SDS-PAGE, transferred to Immobilon polyvinylidiene fluoride (PVDF) membrane [11], and stored at 40C in deionized water containing 0.02% (w:v) NaN 3. Protein Fractionation Aliquots of explant culture medium were subjected to gel filtration chromatography on Sepharose CL-6B as previously described [10] and ion-exchange chromatography. A DEAE-Sepharose column was prepared and equilibrated with 10 mM Tris-HCl buffer (pH 8.2). Pooled A-explant culture medium (Day 2) was dialyzed against equilibration buffer (4 changes of 4 L within 48 h at 4C), and 54 mg protein was loaded on the DEAE column. After washing with equilibration buffer, protein was eluted (4-ml fractions) with a linear NaCl gradient (0.01-0.25 M) in 10 mM Tris-HCI (pH 8.2). Elution profiles were generated by determining radioactivity in 4 0 0 -1l aliquots of each fraction and spectrophotometric readings at A280. Immunoblot Analysis PVDF membranes were rinsed, blocked, and treated with primary antibody, either rabbit anti-ovine luteal TIMP- I1(M 17 W11) or rabbit nonimmune serum, each at a dilution of 1:500 [12]. Blots were then washed and incubated with secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin. Proteins were visualized by diaminobenzidine/metal ion enhancement of the color reaction. TIMP RIA Ovine (o) TIMP-I for iodination and standards in the assay were purified to homogeneity from ovine luteal cell-
TIMP-I mRNA EXPRESSION AND PROTEIN SYNTHESIS BY THE GILT OVIDUCT conditioned media by conventional lectin affinity, gel filtration, and ion-exchange column chromatography. The identity of this preparation was confirmed by N-terminal amino acid sequencing (N-terminal sequence identical to that predicted [5]), SDS-PAGE (a single band), reversephase fast protein liquid chromatography (a single peak), and inhibition of enzyme activity of recombinant human stromelysin (inhibition stoichiometry of 1:1). Aliquots (200-250 Il) of oviductal flushings were prepared in duplicate by lyophilization followed by resuspension in a volume of phosphate azide buffer (PAB; 0.01 M P0 4, 0.02% [w:v] NaN 3, and 0.1% [w:v] gelatin, pH 7.2) equivalent to the original sample volume for analysis of TIMP-1 concentration. Concentration of TIMP-1 within oviductal flushings was determined using a heterologous competitive equilibrium RIA established and validated in our laboratory (E.W. McIntush and M.F Smith). [ 25 I]oTIMP-1 was prepared by diluting oTIMP-1 (0.4 mg/ml) tenfold in distilled H 20. Two micrograms of solubilized oTIMP-1, 10 RI of 0.05 M P0 4 , and 500 LiCi of [1251]Na (Amersham) were added to 100 Rig of Iodogen (Pierce Chemical, Rockford, IL). The reaction was allowed to proceed for 1.5 min, and the reaction mixture was then separated on a 25-cm Sephadex G25 column saturated with BSA by elution with 0.01 M P0 4 -buffered saline (PBS). Fractions (1 ml) were collected in 1 ml of PAB containing 1% (w:v) Tween 20. [ 2 5I]oTIMP-1 (specific activity, 180 jiCi/RIg) was prepared in PAB containing 0.5% (v:v) Tween 20 to deliver 16 000 cpm/100 Ixl to each assay tube. Purified monoclonal antibody (100 Il) to bovine TIMP-1 (7-6C1; Fuji Chemical, Toyama, Japan) [13], 25 ng/ml in PAB, was added to reconstituted oviductal flushings or standard concentrations of oTIMP-1 and brought to a final volume of 500 LAIwith PAB. Reagents were incubated for 24 h at 4°C, and then antibody-antigen was precipitated with a preprecipitated goat anti-mouse antiserum (27°C, 15 min) and pelleted by centrifugation (3000 x g, 30 min, 4C); the supernatant was discarded, and residual radioactivity was determined. Total specific binding averaged 45%; standard concentrations of 0.25-25 ng oTIMP-1 paralleled serial dilutions of reconstituted oviductal flushings from 1- to 0.05-ml equivalents with a slope of -3.1 (log/logit); and the interand intraassay coefficients of variation were 6.4% and 3.2%, respectively. The monoclonal antibody directed against bovine TIMP-1 has previously been shown to be specific for bovine TIMP-1 [13] and to cross-react with human [13], porcine [14], and ovine [15] TIMP-1. The sensitivity of the assay was 250 pg per tube. Dot-Blot Hybridization Ovine TIMP-1 recombinant clone was prepared by Dr. M. Smith, University of Missouri [5]. Plasmid DNA was purified and digested with EcoRI and Xho I restriction enzymes to generate a cDNA probe approximately 900 base pairs in length. TIMP-1 mRNA was detected on dot blots hybridized with an [ot- 32 P]dCTP-labeled cDNA [16] probe as previously described [17]. Briefly, total RNA was isolated from frozen tissues using TRIzol according to the manufacturer's protocol. For dot-blot analysis, 2 pIg of total RNA was loaded into each well and then UV cross-linked to the nylon membrane. The membrane was prehybridized (0.5 M Na2HPO 4 [pH 7.4], 0.01 M EDTA, 7% [w:v] SDS, 1% [w: v] BSA containing yeast RNA [0.5 mg/ml]) for 2 h at 65 0C. The 3 2P-labeled cDNA probe (106 cpm/ml) was added to the
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TUBE NUMBER FIG. 1. Representative Sepharose CL-6B fractionation of explant culture medium proteins labeled with [3Hlleu. Medium conditioned by AMP tissue taken from Day 1 cyclic FLA gilts was fractionated on a Sepharose CL-6B column, fractions were counted, and tubes 35-43 (peak 2A) were pooled.
fresh solution, and the membrane was hybridized overnight at 65°C. The membrane was then washed twice (40 mM Na 2HPO4 [pH 7.4], 0.1% [w:v] SDS) for 15 min at 65°C with constant agitation, briefly air dried, and wrapped in plastic film. An autoradiograph was prepared by exposing the dot blot to Kodak XAR-5 film at -85 0C for 14 days. Hybridization was quantitated by phosphorimaging, and data were analyzed by ImageQuant (Molecular Dynamics, Sunnyvale, CA). The dot blot was stripped and reprobed with a human GAPDH cDNA probe, 3 2 P-labeled (specific activity, 1 x 109 cpm/jg) using random primer labeling (Megaprime DNA Labeling kit), for data normalization. Immunocytochemistry Rabbit polyclonal anti-ovine luteal TIMP-1 (M 17 W1) was used to immunolocalize TIMP-1 in porcine oviductal tissue. Portions of oviductal tissue (INF, A, and I) were collected from cyclic and pregnant FLA gilts and pregnant LW and MS gilts on various days as indicated above. Tissues were cut into small portions, fixed in Bouin's solution, embedded in paraffin, sectioned, and mounted on glass slides. Immunocytochemistry was carried out as previously described [18]. Antibody was used at a dilution of 1:300 prepared in PBS (pH 7.4). Vectastain ABC Elite kits were purchased from Vector Laboratories (Burlingame, CA) and used according to the manufacturer's instructions. Control experiments included incubation of sections in the absence of primary antibody and replacement with normal sera. Statistical Analysis TIMP-1 mRNA levels in the oviduct were examined by least-squares analysis of variance using the General Linear Models procedure of the Statistical Analysis System [19]. Values with a p < 0.05 were considered significant. The model for levels of TIMP-1 mRNA in FLA cyclic gilts included the main effects of day. The model for TIMP-1 mRNA levels in oviductal sections (INF, A, I) of FLA cyclic gilts included the main effects of day, gilts nested within day, section, and day by section. The model for TIMP-1 mRNA levels in the oviduct of steroid-treated OVX gilts included the main effects of treatment: corn oil, EV, P4, and EV+P4 . Relative concentrations of TIMP- 1 protein within the oviduct were examined by least-squares analysis of variance.
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FIG. 3. DEAE-Sepharose fractionation of explant culture medum and separation of proteins by 2D-SDS-PAGE. A) Pooled A-conditioned medium from Day 1 cyclic gilts was loaded on a DEAE-Sepharose column and eluted with a linear NaCI gradient (0.01-0.25 M) (cpm/400 Iil [circles], A280 [triangles]). B) Fluorograph of peak 1 (0.015-0.07 M NaCI) proteins after analysis by 2D-SDS-PAGE. Multiple isoforms of TIMP-1 are shown (arrow); one molecular weight standard is indicated (x10-3); and the pH gradient runs from left (pH 8) to right (pH 4).
FIG. 2. Representative fluorograph and immunoblot analysis of pooled peak 2A from the Sepharose CL-6B fractionation of A-conditioned medium. A) Proteins in pooled peak 2A were separated by 2D-SDS-PAGE, and distribution of labeled proteins was determined by fluorography. Parallel specimens were transferred to PVDF membrane, rinsed, blocked, and treated with B) anti-TIMP-1 or C) nonimmune serum. Brackets in A and B indicate the location of TIMP-1. Molecular weight standards are indicated (x 10-). The pH gradient runs from left (pH 8) to right (pH 4).
The model for steroid-treated OVX FLA gilts included the main effects of treatment: corn oil, EV, and P4. The model for the three breeds of pregnant gilts (FLA, LW, and MS) included the main effects of day, breed, and day by breed. RESULTS Electrophoresis and Western Blotting Explant culture medium conditioned by the A of Day I cyclic FLA gilts was fractionated by Sepharose CL-6B chromatography (Fig. 1). Peak 2A was collected, pooled, dialyzed, and analyzed by 2D-SDS-PAGE and fluorography. In this peak, a major radiolabeled basic 29 000 Mr
protein family, previously described after fractionation of porcine oviductal explant culture medium as protein family 6 [9] or family 8 [10], was again detected (Fig. 2A, bracket) and subjected to further analysis. Duplicate samples, separated by 2D-PAGE, were transferred to Immobilon PVDF membrane and incubated with either rabbit anti-ovine luteal TIMP-1 or nonimmune rabbit serum (Fig. 2, B and C, respectively). These results established that the 29 000 M, family, synthesized and secreted by the porcine A and cross-reacting with specific antiserum, was TIMP-1. The molecular weight and isoelectric point further confirmed that the oviductal protein was TIMP-1. Pooled A-explant culture medium was also fractionated by DEAE-Sepharose (Fig. 3A) and eluted with a linear NaCl gradient (0.01-0.25 M). Peak 1, eluted between 0.015 and 0.07 M NaCl, revealed at least eight radioactive isoelectric species in the TIMP-1 family by 2D-PAGE and fluorography (Fig. 3B). Culture Media Conditioned by INF, A, and I during the Estrous Cycle and Early Pregnancy, and after OVX and Steroid Treatment To determine whether different functional segments (INE A, and I) showed differences in synthesis and secre-
TIMP-1 mRNA EXPRESSION AND PROTEIN SYNTHESIS BY THE GILT OVIDUCT
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FIG. 4. Representative fluorographs of the 2D-SDS-PAGE analysis of oviductal functional segment (INF, A, or I)-conditioned explant culture medium (100 000 cpm [H]leu) from pregnant (Days 0 to 12) LW and MS gilts. A) Distribution of 3H-labeled proteins from the INF, A, and I of a Day 2 pregnant LW gilt. Brackets indicate the location of TIMP-1 in the . B and C represent the [3Hlleu-labeled TIMP-1 distribution excised from representative fluorographs of I cultures of LW (B) and MS (C)gilts on Days 0, 2, 5, and 12 of pregnancy. Molecular weight standards are indicated (x10-3), and the pH gradient runs from left (pH 8) to right (pH 4).
tion of TIMP- 1 during the estrous cycle (FLA gilts) or early pregnancy (LW, FLA, and MS gilts), oviductal tissue was collected on Days 0, 2, 5, 8, 10, and 12 and divided into segments; then each segment was cultured separately. Representative fluorographs of proteins synthesized and secreted by the INF, A, and I from a Day 2 pregnant LW gilt are shown in Figure 4. Only trace amounts of TIMP-1 were found in media from the INF and A cultures of all cyclic and pregnant gilts regardless of day (only Day 2 is shown) or breed, while TIMP-1 appeared to be a major labeled protein from the I regardless of day or breed (INF and A data not shown). The intensity of TIMP- 1 in the I, however, appeared to be greatest on Day 2 of the estrous cycle (data for FLA not shown) and pregnancy (Fig. 4B, LW; Fig. 4C, MS). There did not appear to be differences in the intensity of TIMP-1 between breeds (FLA, LW, and MS). These results suggested both temporal and functional segment differences in synthesis and secretion of TIMP-1 by the oviduct. To examine hormonal control of TIMP- 1 de novo protein synthesis in functional segments, oviducts from bilaterally OVX steroid-treated gilts were examined after explant culture by 2D-SDS-PAGE and fluorography. Similar to results from cyclic and pregnant gilts, TIMP-1 appeared to be a major labeled protein in the I regardless of treatment; however, its intensity was increased with EV+P 4 treatment (data not shown). Further, only traces of TIMP-1 were found in media from the INF and A, regardless of treatment, although slight increases were noted with EV+P 4 treatments.
RIA TIMP-1 concentration in oviductal flushings was measured to determine whether TIMP-1 was present, whether this concentration changed during the estrous cycle and early pregnancy, whether there were breed differences during early pregnancy between the highly prolific Chinese and standard Western breeds, and whether steroids given after OVX would modulate TIMP-1 concentration in oviductal fluid. This study established that TIMP-1 was indeed present in oviductal flushings from all breeds, was present throughout the estrous cycle (data not shown) and early pregnancy, and was present after OVX regardless of treatment. Least-squares means of TIMP-1 in oviductal fluid during early pregnancy for FLA, LW, and MS gilts are shown in Figure 5. Analysis revealed that there were differences in TIMP-1 concentration by day (p < 0.001), with the concentration greatest at estrus (Day 0) and declining as pregnancy continued to Day 12. In cyclic FLA gilts, TIMP-1 fluid concentration was similar to that in pregnant gilts, was greater at estrus, and declined toward Day 12 (data not shown). Breed differences were also detected between MS and LW gilts during early pregnancy (p < 0.04), with greater concentrations found in LW (p < 0.04) than in the MS at Day 0. Although breed and location (Florida versus Scotland) are confounded in this experiment, breed differences were similarly detected between MS and FLA gilts (p < 0.04). However, there were no breed X day differences, nor were there any breed differences between LW and FLA gilts.
BUHI ET AL.
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TIMP-1 concentration (least-squares means) in oviductal flushes from gilts OVX and treated with corn oil, EV, or P4 are shown in Figure 6. TIMP-1 concentrations were only numerically greater in flushes from gilts treated with EV (9.09 + 2.25 ng/ml) compared to gilts treated with corn oil (1.59 ± 2.25 ng/ml, p < 0.057) or P4 (2.30 + 2.25 ng/ml, p = 0.077). This was most likely due to large variability among gilts and the small numbers of animals in each group. There was no difference in concentrations of TIMP-1 between corn oil and P4 treatment. TIMP-1 mRNA Steady-State Levels during the Estrous Cycle and after OVX and Steroid Treatment Steady-state levels of TIMP-1 mRNA during the estrous cycle (Days 0 to 18) are shown in Figure 7. An effect of day was determined (p
