Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.
Agricola
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.
Biology of Reproduction 66, 574-579 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Detection of Bradykinin and Bradykinin-ß2 Receptors in the Porcine Endometrium During the Estrous Cycle and Early Pregnancy1

Melanie R. Allena, Bing R. Zhanga, Andrea M. Hettingera, David W. Goada, Jerry R. Malayerb, and Rodney D. Geisert2,a

a Department of Animal Science, Oklahoma Agriculture Experiment Station b Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin ß2 receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12–18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12–18 of pregnancy compared with the estrous cycle. Endometrial bradykinin ß2 receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin ß2 receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.

conceptus, female reproductive tract, gene regulation, implantation, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Porcine conceptuses initiate attachment to the uterine lumenal surface following rapid expansion of the trophectoderm on Day 12 of pregnancy [1–3]. During adhesion of the trophoblast to the uterine epithelial apical surface, a localized increase in transcapillary transport [1] as well as an overall increase in uterine blood flow occurs [4] concurrently with the increase in conceptus estrogen release [5, 6] for establishment of pregnancy in the pig.

Many of the endometrial responses evoked by the developing conceptuses resemble the acute-phase response induced during generalized tissue inflammation [3, 7]. The presence of tissue kallikrein in the porcine uterine lumen [8] suggests that bradykinin could be involved with regulation of uterine function through the proteolytic cleavage of kininogen [9]. Kinins are vasoactive peptides that are normally involved with inflammatory-associated effects such as tissue prostaglandin synthesis and release, increased blood flow, histamine release, and induction of smooth muscle contraction [10], which are many of the same alterations that occur in the uterus during establishment of pregnancy in the pig [2, 11]. Components of the kallikrein-kinin system are present in the uterus of the rat [12, 13] and mouse [14], in which blastocyst implantation is activated by ovarian release of estrogens [15]. In addition, bradykinin ß2 receptor has been localized in the uterus of the rat [16, 17] as well as the ewe [18]. The detection of kallikrein enzymatic activity in the porcine uterine lumen and endometrial gene expression [8], as well as identification of endometrial low molecular weight (LMW) kininogen protein and gene expression (unpublished data) indicates the possible presence of an active and functional kallikrein-kinin system in the porcine uterus during early pregnancy.

The objective of the current investigation was to determine the changes in bradykinin content in the porcine uterine lumen and alteration of endometrial bradykinin receptor expression during the estrous cycle and early pregnancy. Although bradykinin can affect tissues through 2 specific receptors, ß1 and ß2, the ß2 receptor mediates the majority of physiological effects of kinins [10]. Therefore, we evaluated the presence and alteration of endometrial bradykinin ß2 receptor protein and gene expression during the estrous cycle and early pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Research was conducted in accordance with the Guiding Principles for Care and Use of Animals promoted by the Society for the Study of Reproduction and approved by the Oklahoma State Institutional Animal Care and Use Committee. Cyclic, Large White gilts of similar age (8–10 mo) and weight (100–130 kg) were checked twice daily for estrous behavior using intact boars. The onset of estrus was considered Day 0 of the estrous cycle. Gilts assigned to be mated were bred by fertile boars at the first detection of estrus and 12 h later.

Evaluation of Uterine Lumenal Content of Bradykinin and Endometrial Bradykinin ß2 Receptor Gene Expression of Cyclic and Pregnant Gilts

Cyclic and pregnant gilts (4 animals/status per day) were hysterectomized through midventral laparotomy on Days 0, 5, 12, 15, or 18 of the estrous cycle and Days 12, 15, or 18 of pregnancy as previously described by Gries et al. [19]. Induction of anesthesia was with 2.5 ml i.m. administration of a cocktail consisting of 2.5 ml Rompun (zylazime; 100 mg/ml; Miles, Inc., Shawnee Mission, KS), 2.5 ml Vetamine (ketamine HCL; 100 mg/ml; Mallickrodt Veterinary, Mundelein, IL) in 500 mg Telazol (tiletamine HCL and zolazepam HCL; Fort Dodge, Syracuse, NE). Anesthesia was maintained with a closed-circuit system of halothane (Halocarbon Laboratories, Riveredge, NJ) and oxygen (1.0 L/min). After exposure by midventral laparotomy, the uterine horns and ovaries were surgically removed. The incision site was closed using routine surgical procedures, and gilts were treated i.m. with penicillin (20 000 IU/kg body weight).

Immediately upon removal of the uterus, a 7- to 10-cm section anterior to the uterine body was excised and opened along its antimesometrial border. Small segments (2–3 cm) of endometrium were removed from the underlying myometrium with scissors and immersed in 10% buffered formalin for 6 h at room temperature.

Uterine flushings and endometrium were collected immediately following removal of the uterus. The 2 horns were isolated, and 1 horn was flushed with 20 ml of PBS (pH 7.4), while the second horn was flushed with 20 ml of PBS containing 200 µl of a protease inhibitor cocktail solution consisting of 4.5 mM EDTA (Sigma Chemical Company, St. Louis, MO), 4.5 mM 1,10 phenanthroline (Sigma), 2.5 µM chicken-egg-albumin trypsin inhibitor (Boehringer-Mannheim, Mannheim, Germany), 0.8 mM hexadimethrine bromide (Sigma), and 3 µM aprotinin (Boehringer-Mannheim) to prevent the rapid proteolytic cleavage of endogenous bradykinin. Uterine flushings were examined to confirm pregnancy in mated gilts. Conceptuses collected on Day 12 of pregnancy were characterized according to stage of development (spherical, tubular, or filamentous) and then were immediately snap-frozen in liquid nitrogen. Uterine flushings were placed on ice until centrifugation (3000 x g, for 20 min at 4°C) at the laboratory and stored at -80°C. After flushing, the uterine horn was cut along its antimesometrial border, and endometrium was exposed for removal with sterile scissors. Endometrium was collected, snap-frozen in liquid nitrogen, and stored at -80°C until processed for the extraction of total RNA. Endometrium was collected, and total RNA extracted from an additional group of animals (3/status) on Day 10 of the estrous cycle and early pregnancy to evaluate bradykinin ß2 receptor gene expression more completely.

Bradykinin Radioimmunoassay

The content of bradykinin in uterine flushings containing the cocktail of protease inhibitors was quantified through use of a commercial bradykinin RIA kit according to the manufacturer's recommendations (Peninsula Labs, Belmont, CA). Addition of 64, 128, 256, or 512 pg bradykinin to a uterine flushing sample produced a binding curve parallel to the standard curve (Fig. 1), which resulted in a recovery of 61, 138, 226, or 508 pg, respectively. The sensitivity of the bradykinin RIA assay was 2 pg/ml. Uterine flushing samples (100 µl) were assayed in duplicate in a single assay with a 19.18% intraassay coefficient of variation.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Parallel increase between the standard curve (triangle) and uterine flushing (square) following the addition of either 64, 128, 256, or 512 pg of bradykinin

Endometrial RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction

Total RNA was isolated from endometrial tissue using TRIzol reagent (Gibco/Life Sciences, Gaithersburg, MD) as previously described [8]. Approximately 0.5 g of endometrial tissue was homogenized in 5 ml TRIzol reagent using a Virtishear homogenizer (Virtis Company Inc., Gardiner, NY). RNA pellets were rehydrated with 10 mM Tris, 1 mM EDTA pH 7.4, and stored at -80°C until further analysis. Total RNA was quantified spectrophotometrically at an absorbance of 260 nm, while the purity was determined based on 260:280 nm ratios. Integrity of the RNA was determined via gel electrophoresis.

Total RNA (2 µg) was reverse transcribed to cDNA with Moloney murine leukemia virus reverse transcriptase-Rnase H- (M-MLV-RT) (Promega, Madison, WI) using oligo(dT)15 primer in a Perkin Elmer Cetus (Norwalk, CT) DNA thermal cycler model 480 as previously described [20]. Quality and quantity of endometrial cDNA was checked by evaluating polymerase chain reaction (PCR) expression of glyceraldehyde-3-phosphate dehydrogenase as previously described [20].

Bradykinin ß2 Receptor Primer, Optimization, and Sequencing

Because a porcine sequence for bradykinin ß2 receptor was not available when the present study was undertaken, primers to bradykinin ß2 receptor cDNA were designed to regions of homology between human [21] and mouse bradykinin ß2 receptor mRNAs [22]. The sequence corresponding to base pairs (bp) 668–1288 of the human bradykinin ß2 receptor mRNA (GenBank accession number M88714) was used to construct the forward 5'-TCTACAGCTTGGTGATCTGGGG and reverse 5'-GTTTGTGAATCTGGCGTTCCAC primers. PCRs were carried out in 25-µl volumes covered with 30 µl of mineral oil. To optimize the PCR conditions, cDNA representing endometrium from all days was pooled, and 2 µl was amplified with 0.6 units of Taq DNA polymerase in MgCl2-free buffer (Promega) and a 3 x 2 x 3 factorial combination of primer (50, 150, 250 nM), deoxynucleotide triphosphates (dNTPs; 50 or 100 µM), and MgCl2 (1.25, 2.50, or 3.75 mM) was evaluated as previously described [20]. The optimal conditions for endometrial bradykinin ß2 receptor gene amplification were 3.75 mM MgCl2, 100 µM dNTPs, and 150 nM primer. The resultant 621-bp PCR product sequenced by the Recombinant DNA/Protein Research Facility at Oklahoma State University was 90% homologous to the mRNA encoding for bovine bradykinin ß2 receptor (GenBank accession number AF207860) and 85% homologous to human bradykinin ß2 receptor [21]. Following completion of this study, the complete mRNA sequence for porcine bradykinin ß2 receptor became available in GenBank. Our endometrial bradykinin ß2 receptor mRNA sequence is 95% homologous to the 682–1302 bp region of porcine bradykinin ß2 receptor mRNA (GenBank accession number AB051422).

Quantitative Reverse Transcriptase-PCR

Bradykinin receptor endometrial gene expression was quantified using the one-step reverse transcriptase (RT)-PCR reaction for the TaqMan Gold RT-PCR kit (P/N N808-0233; PE Applied Biosystems, Foster City, CA). The total reaction volume of 50 µl contained 100 ng of total RNA and 200 nM bradykinin receptor forward primer 5'-GCCTCCTACGTGGCCTACAG, 200 nM bradykinin receptor reverse primer 5'-AGTGCTTGCCCACGATCAC, and 100 nM fluorescent labeled bradykinin receptor probe 5'-AACAGCTGCCTCAACCCGCTGG. The probes were all designed from the 621-bp porcine endometrial bradykinin ß2 receptor cDNA sequence that was obtained from the RT-PCR product that we submitted to the Recombinant DNA/Protein Research Facility. Although a few mismatches exist, the forward primer, reverse primer, and fluorescent probe designed to our endometrial bradykinin ß2 receptor sequence cDNA corresponds to the GenBank porcine cDNA sequence (AB051422) regions of 1104–1123, 1152–1170, and 1125–1146 bp, respectively. The PCR amplification was carried out in the ABI PRISM 7700 sequence detection system (PE Applied Biosystems). Thermal cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by repetitive cycles of 95°C for 15 sec and 60°C for 1 min. Ribosomal 18S RNA (18S, RNA Control Kit, 43108993E; PE Applied Biosystems) was run as a control for RNA loading.

Following RT-PCR, quantitation of gene amplification was made by setting the cycle threshold (CT) in the geometric region of the plot after examining the semilog view of the amplification plot. Relative quantitation of bradykinin ß2 receptor gene expression was evaluated using the comparative CT method described previously by Hettinger et al. [23]. The {Delta}CT value is determined by subtracting the bradykinin ß2 receptor CT of each sample from its ribosomal 18S CT value (Table 1). Calculation of {Delta}{Delta}CT involves using the highest sample {Delta}CT value as an arbitrary constant to subtract from all other {Delta}CT sample values. Fold-changes in gene expression of bradykinin receptor were determined by evaluating the expression 2-{Delta}{Delta}Ct. A validation of the quantitative PCR was performed with 5, 1, 0.2, 0.04, 0.008, and 0.0016 ng RNA from a selected sample of RNA using both bradykinin ß2 receptor and 18S primers within the same PCR well as previously described for our laboratory [23]. Expression of bradykinin ß2 receptor and 18S RNA was parallel across the dilutions of RNA (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of endometrial bradykinin ß2 receptor gene expression across days of the estrous cycle and early pregnancy using the comparative CT method.

Immunohistochemistry

Fixed endometrial tissue sections were dehydrated in graded ethanol changes, cleared with toluene, and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Sections (5 µm) were cut with an AO model 820 rotary microtome (American Optical, Buffalo, NY), placed on poly-L-lysine (Sigma)-coated slides, deparaffinized, and rehydrated for immunostaining with the Vector Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA). Mouse anti-bradykinin ß2 receptor monoclonal antibody (Research Diagnostics, Inc., Flanders, NJ) was incubated with tissue sections at a 1:100 dilution. Biotin-SP-conjugated AffiniPure goat anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), which has minimal cross-reactivity to porcine serum proteins, was used as second antibody. Negative controls for immunostaining included exclusion of the primary antibody from incubation and preabsorption of the primary antibody with rat bradykinin receptor (Research Diagnostics). Photomicrographs were taken with a Zeiss photomicroscope (Carl Zeiss, Thornwood, NY).

Statistical Analysis

Data were analyzed by least-squares ANOVA using the general linear models of the Statistical Analysis System [24]. Due to nonnormality of the uterine flushing bradykinin concentrations across days of the estrous cycle and pregnancy, bradykinin concentrations were subjected to log transformation. The statistical model tested the effect of day on uterine flushing bradykinin concentration and endometrial gene expression of bradykinin ß2 receptor during the estrous cycle. The model for analysis of reproductive status effects included the effects of day, reproductive status (cyclic and pregnant), and day x reproductive status.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Uterine Lumenal Bradykinin Content

A day effect (P < 0.01) for total bradykinin concentration in uterine flushings during the estrous cycle was detected (Fig. 2). During the estrous cycle, bradykinin in the uterine flushings is greatest on Day 0 (estrus), followed by a 5-fold decrease on Day 5. A moderate 2-fold increase in the concentration of bradykinin occurred between Days 12 and 18 of the estrous cycle. Pregnancy affected (P < 0.001) bradykinin concentrations in uterine flushings when compared to Days 12–18 of the estrous cycle. Effects of day x reproductive status were detected for concentrations of bradykinin in uterine flushings (P < 0.05). Bradykinin concentrations in uterine flushings were 5-fold greater in pregnant gilts than in cyclic gilts on Day 12, which was followed by an 8- to 10-fold greater increase for bradykinin in uterine flushings from pregnant gilts on Days 15 and 18, respectively (Fig. 2).



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Concentration of bradykinin in uterine flushings (4 animals/status per day) collected on Days 0, 5, 12, 15, and 18 from cyclic (gray) and Days 12, 15, and 18 pregnant (black) gilts. Data were subjected to log transformation prior to statistical analysis. During the estrous cycle, an effect of day (P < 0.01) for bradykinin concentration in uterine flushings was detected and there was an effect of status (P < 0.001)

Quantitative RT-PCR of Bradykinin ß2 Receptor with the ABI Prism System

Endometrial gene expression of bradykinin ß2 receptor was detected on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, and 18 of pregnancy. Relative quantitative bradykinin ß2 receptor gene expression was evaluated using the comparative CT (threshold cycle) method (see Table 1). There was an effect of day of the estrous cycle on the {Delta}CT bradykinin ß2 receptor mRNA expression (P < 0.001), with expression being greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy (Table 1), as mRNA expression was lower on Days 5 and 10. In comparison with Day 12 of the estrous cycle, the {Delta}CT bradykinin ß2 receptor mRNA expression increased on Day 12 of pregnancy, but a day x reproductive status effect on bradykinin ß2 receptor mRNA expression only approached significance (P < 0.06; Table 1). Conversion of {Delta}CT to fold differences (Fig. 3) illustrates the decline of bradykinin ß2 receptor mRNA expression on Days 5 and 10, with the 6-fold increase in expression by Days 12–15 of the estrous cycle and pregnancy.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. Fold differences in endometrial bradykinin ß2 receptor mRNA expression during Days 0, 5, 10, 12, 15, and 18 of the estrous cycle (gray) and Days 10, 12, 15, and 18 of pregnancy (black) detected through quantitative RT-PCR analysis (3–4 animals/day within each status group). The fold increases are presented within parenthesis. The fold increases in bradykinin ß2 receptor gene expression were calculated as described in Materials and Methods, and previously published by Hettinger et al. [23]

Immunocytochemistry of Endometrial Bradykinin ß2 Receptor

No staining was detected when primary antibody to bradykinin ß2 receptor was deleted from the incubation or when primary antibody was preabsorbed with bradykinin receptor (Fig. 4). Positive immunostaining was detected in the uterine surface and glandular epithelium on Day 0 (Fig. 4). However, only faint staining was observed on Day 5 of the estrous cycle. Immunostaining in the surface and glandular epithelia became most intense from Days 12 through 18 of the estrous cycle and pregnancy. Bradykinin ß2 receptors were also detected in uterine capillaries.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 4. Immunocytochemical localization of bradykinin ß2 receptor during various days of the porcine estrous cycle (C) and pregnancy (P). Negative controls for bradykinin ß2 receptor included exclusion of primary antibody from incubation and preabsorption of primary antibody with excess rat bradykinin ß2 receptor. Staining intensity declined in the epithelium on Day 5 of the estrous cycle but increased in both cyclic and pregnant gilts on Day 12. No differences in staining intensity were detected between cyclic and pregnant tissues between Days 12 and 15. Note immunostaining in the surface and glandular epithelia of the endometrium (x40)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Developing porcine conceptuses produce estrogens that alter uterine cellular morphology [5], vascular permeability [1, 25, 26], uterine blood flow [4, 6], secretion of endometrial-derived proteins [27], and prostaglandin release into the uterine lumen [28].

Trophoblastic attachment to the uterine surface epithelium begins on Day 13 and continues to Day 18 of pregnancy in the pig. This noninvasive type of implantation leads to formation of the diffuse epitheliochorial placenta of the pig [2, 25], in which embryo development is dependent upon the efficient transport of nutrients from the intact endometrium, across the placenta, and into the fetal-placental circulation. Many of the events associated with conceptus development and implantation in the mouse and rat are associated with uterine activation by estrogen and uterine changes that mirror the inflammatory process [9]. Although porcine conceptuses do not implant into the uterine tissues, they release estrogens that affect attachment of trophoectoderm to the uterine epithelium and induce the inflammatory reaction that occurs during the implantation period [1, 26].

Identification of kallikrein in the porcine uterine lumen [8] and the substrate for kallikrein, LMW-kininogen (unpublished data) suggest that the kallikrein-kinin system is active during the period of placentation. The presence of bradykinin in the uterine flushings from pregnant gilts supports our hypothesis that kallikrein mediates release of the vasoactive nonapeptide from LMW-kininogen during early pregnancy in the pig. Because there is a clear temporal association with conceptus estrogen release and an increase in uterine blood flow during early pregnancy in the pig [29], the release of bradykinin during early pregnancy could be involved with the increase in uterine blood flow, vascular permeability, prostaglandin release, and changes in uterine tone that occurs during early pregnancy in the pig [26, 29].

In cyclic gilts, the uterine lumenal content of bradykinin was greatest during estrus, whereas after estrus, the large increase in uterine lumenal bradykinin content occurred only during pregnancy, when the porcine conceptuses initiate estrogen synthesis on Day 12 of gestation [5]. Estrogen receptors in the uterine endometrial surface and glandular epithelium of the pig are abundant on Days 10 and 12 of the estrous cycle and pregnancy [30], suggesting that estrogen plays a regulatory role in endometrial release of bradykinin in the pig. Kallikrein is the key regulatory enzyme in the liberation of bradykinin from LMW-kininogen. An increase in kallikrein enzyme activity was detected in the porcine uterine lumen after Day 10 of the estrous cycle and pregnancy [8]. However, the increase in tissue kallikrein activity on Days 12 and 15 reported by Vonnahme et al. [8] was not significantly different between cyclic and pregnancy gilts. Uterine tissue kallikrein is involved with cleavage of insulin-like binding proteins on Day 12 in both cyclic and pregnant gilts [31], which indicates that several different kallikreins may be present in the uterine lumen during early pregnancy of the pig. Kallikrein belongs to a multigene family of serine proteases, which consists of approximately 15 related genes in the rat [32] and human [33]. The kallikrein family has diverse substrate specificity and variable sensitivity to inhibitors such as aprotinin [32], however, the genes have extensive homology to each other within and among species [10, 33]. Chan et al. [14] identified and characterized expression of 5 kallikrein genes in the embryo and uterus during implantation in the mouse, many of which were expressed in a tissue- and time-specific manner. The specific porcine uterine kallikrein involved with bradykinin release or its source was not identified in the present study. Association between kallikrein activation, release of bradykinin, and estrogen release by the conceptuses during early pregnancy suggests either an indirect stimulation of endometrial release of a different tissue kallikrein, release of a conceptus kallikrein, or both. It is possible that several different uterine kallikreins are involved with alteration of the extracellular matrix on the uterine surface during implantation in pigs as well as degradation of insulin-like growth factor binding proteins after Day 10 of the estrous cycle and the release of bradykinin from LMW-kininogen during pregnancy [31, 34]. However, we cannot overlook a direct role for conceptus synthesis of kallikrein to release of bradykinin into the uterine lumen that is different from the uterine kallikrein. Vonnahme et al. [8] reported expression of a tissue kallikrein gene by early porcine conceptuses. Expression by the pig conceptus of a kallikrein that is specific for LMW-kininogen would provide local control of bradykinin release into the uterine lumen during early pregnancy to increase prostaglandin synthesis, close the uterine lumen, increased blood flow, and alter vascular permeability [10], all of which occur in the porcine uterus during early pregnancy [11].

Further supporting evidence for a role of bradykinin in the establishment of early pregnancy is the presence of bradykinin ß2 receptors in the uterine surface and glandular epithelia and the identification of endometrial mRNA expression for bradykinin ß2 receptor during the estrous cycle and early pregnancy in the pig. Endometrial expression of bradykinin ß2 receptor mRNA changed throughout the estrous cycle and increased expression after Day 10 of the estrous cycle and early pregnancy. Higher levels of bradykinin ß2 receptor protein and mRNA expression during estrus suggests that estrogens stimulate bradykinin ß2 receptor gene expression in the endometrium, because an increase in expression is also detected on Day 12 of pregnancy when conceptus estrogens are released to signal maternal recognition of pregnancy [3] and estrogen receptor is high in the uterine surface and glandular epithelia [30]. However, bradykinin ß2 receptors were also increased during similar days of the estrous cycle, indicating that the change in receptor number is not pregnancy-specific. It is possible that bradykinin ß2 receptors within the uterine surface and glandular epithelium may be negatively regulated by progesterone, because the decrease in both gene and protein expression on Day 5 is temporally associated with elevated plasma progesterone concentrations and the presence of endometrial epithelial progesterone receptors [35]. The increase in both bradykinin ß2 receptor protein and gene expression in cyclic and pregnant gilts after Day 10 is associated with progesterone receptor down-regulation within the uterine epithelia [35]. Although estrogen may influence bradykinin ß2 receptor expression, we hypothesize that the changes in uterine epithelial progesterone receptor modulate the timing for bradykinin ß2 receptor expression in the uterine epithelium to coincide with conceptus attachment. Clearly, further studies are needed to determine the factors that regulate endometrial bradykinin ß2 receptors in the pig.

Estrogen regulation of the kallikrein-kinin system appears to play an important role during implantation in the rat [17, 36, 37]. Results of the present study with gilts are consistent with evidence for modulation of bradykinin receptor expression during the period of implantation [16, 17]. It is clear that porcine endometrial bradykinin ß2 receptor gene expression increases close to the period of conceptus estrogen synthesis, trophoblast elongation, and attachment of trophectoderm to the uterine lumenal epithelium in pigs. The increased expression of bradykinin ß2 receptor and subsequent increase in kallikrein and bradykinin during the peri-implantation period in the present study suggests that the kallikrein-kinin system is important for the establishment of pregnancy in the pig.


   ACKNOWLEDGMENTS
 
The authors thank Mr. Steve Welty for the care and feeding of the animals used in the study. Appreciation is expressed to Clay Lents, Connie Chamberlain, and Anita Ferrell for their assistance with surgeries, and Thea Pratt for immunohistochemical analysis of the endometrial samples. The authors thank the Oklahoma State University Recombinant DNA/Protein Resource Facility for synthesis of oligonucleotides and DNA sequencing.


   FOOTNOTES
 
First decision: 30 July 2001.

1 This research was supported by NRICGP/USDA grant 98-35203-6224 awarded to R.D.G. and approved for publication by the director of the Oklahoma Agriculture Experiment Station. Back

2 Correspondence: Rodney D. Geisert, Department of Animal Science, Animal Science Building, Rm 114, Oklahoma State University, Stillwater, OK 75078-6051. FAX: 405 744 7390; geisert{at}okstate.edu Back

Accepted: October 8, 2001.

Received: June 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Keys JL, King GJ. Morphological evidence for increased uterine vascular permeability at the time of embryonic attachment in the pig. Biol Reprod 1988; 39:473-487[Abstract]
  2. Stroband HWJ, Van der Lende T. Embryonic and uterine development during early pregnancy in pigs. J Reprod Fertil Suppl 1990; 40:261-277[Medline]
  3. Geisert RD, Yelich JV. Regulation of conceptus development and attachment in pigs. J Reprod Fertil Suppl 1997; 52:133-149[Medline]
  4. Ford SP, Christenson RK, Ford JJ. Uterine blood flow and uterine arterial, venous and lumenal concentrations of oestrogens on days 11, 13, and 15 after oestrus in pregnant and non-pregnant sows. J Reprod Fertil 1982; 64:185-190[Abstract/Free Full Text]
  5. Geisert RD, Renegar RH, Thatcher WW, Roberts RM, Bazer FW. Establishment of pregnancy in the pig: I. Interrelationships between preimplantation development of the pig blastocyst and uterine endometrial secretions. Biol Reprod 1982; 27:925-939[CrossRef][Medline]
  6. Ford SP, Christensen RK. Blood flow to uteri of sows during the estrous cycle and early pregnancy: local effect of the conceptus on the uterine blood supply. Biol Reprod 1979; 21:617-624[Abstract]
  7. Salier J-P, Rouet P, Raguenez G, Daveau M. The inter-{alpha}-inhibitor family: from structure to regulation. Biochem J 1996; 315:1-9
  8. Vonnahme KA, Malayer JR, Spivey HO, Ford SP, Clutter A, Geisert RD. Detection of kallikrein gene expression and enzymatic activity in porcine endometrium during the estrous cycle and early pregnancy. Biol Reprod 1999; 61:1235-1241[Abstract/Free Full Text]
  9. Clements J. The molecular biology of the kallikreins and their roles in inflammation. In: Farmer S (ed.), The Kinin System. Great Britain: Academic Press; 1997: 71–97
  10. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kinases. Pharmacol Rev 1992; 44:1-80[Medline]
  11. Geisert RD, Zavy MT, Moffatt RJ, Blair RM, Yellin T. Embryonic steroids and the establishment of pregnancy in pigs. J Reprod Fertil Suppl 1990; 40:293-305[Medline]
  12. Brann DW, Greenbaum L, Mahesh VB, Gao X. Changes in kininogens and kallikrein in the plasma brain, and uterus during pregnancy in the rat. Endocrinology 1995; 136:46-51[Abstract]
  13. Valdes G, Figueroa CD, Corthorn J. Temporospatial changes of kallikrein-like enzymes during the estrous cycle and pregnancy in the rat uterus. Biol Reprod 1996; 55:236-245[Abstract]
  14. Chan CS, Harvey MB, Clements JA. Temporal and tissue-specific expression of kallikrein (Klk) genes and identification of a novel Klk messenger ribonucleic acid transcript during early development in the mouse. Biol Reprod 1999; 61:621-628[Abstract/Free Full Text]
  15. Dey SK, Johnson DC. Embryonic signals in pregnancy. Ann N Y Acad Sci 1986; 476:49-62[Medline]
  16. Murone C, Chai SY, Muller-Esterl W, Mendelsohn FAO, Clements J. Localization of bradykinin ß2 receptors in the endometrium and myometrium of rat uterus and effects of estrogen and progesterone. Endocrinology 1999; 140:3372-3382[Abstract/Free Full Text]
  17. Figueroa CD, Chacon C, Corthorn J, Ehrenfeld P, Muller-Esterl W, Valdes G. Temporospatial changes of kinin B2 receptors during the estrous cycle and pregnancy in the rat uterus. Biol Reprod 2001; 64::1590-1599[Abstract/Free Full Text]
  18. Murone C, Perich RB, Schlawe I, Muller-Esterl W, Mendelsohn FAO, Chai SY. Localization of bradykinin ß2 receptors in sheep uterus. Immunopharmacology 1996; 33:108-112[CrossRef][Medline]
  19. Gries LK, Geisert RD, Zavy MT, Garrett JE, Morgan GL. Uterine secretory alterations coincident with embryonic mortality in the gilt after exogenous estrogen administration. J Anim Sci 1989; 67:276-284
  20. Yelich JV, Pomp D, Geisert RD. Detection of transcripts for retinoic acid receptors, retinal-binding protein, and transforming growth factors during rapid trophoblastic elongation in the porcine conceptus. Biol Reprod 1997; 57:286-294[Abstract]
  21. Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW. Cloning and pharmacological characterization of human bradykinin (BK-2) receptor. Biochem Biophys Res Commun 1992; 184:260-268[CrossRef][Medline]
  22. Yokoyama S, Kimura Y, Taketo M, Black JA, Ransom BR, Gigashida H. ß2 bradykinin receptors in NG108-15 cells: cDNA cloning and functional expression. Biochem Biophys Res Commun 1994; 200::634-631[CrossRef][Medline]
  23. Hettinger AM, Allen MR, Zhang BR, Goad DW, Malayer J, Geisert RD. Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy. Biol Reprod 2001; 65:507-513
  24. SAS. SAS User's Guide: Statistics (version 5). Cary, NC: Statistical Analysis System Institute Inc.; 1985
  25. Keys JL, King GJ, Kennedy TG. Increased uterine vascular permeability at the time of embryonic attachment in the pig. Biol Reprod 1986; 34:405-411[Abstract]
  26. Keys JL, King GJ. Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy. Am J Anat 1990; 188:221-238[CrossRef][Medline]
  27. Roberts RM, Xie S, Trout WE. Embryo-uterine interactions in pigs during week 2 of pregnancy. J Reprod Fertil Suppl 1993; 48:171-186[Medline]
  28. Bazer FW. Mediators of maternal recognition of pregnancy in mammals. Proc Soc Exp Biol Med 1992; 199:373-384[Medline]
  29. Ford SP. Control of blood flow to the gravid uterus of domestic livestock species. J Anim Sci 1995; 73:1852-1860[Abstract]
  30. Geisert RD, Brenner RM, Moffatt RJ, Harney JP, Yellin T, Bazer FW. Changes in estrogen receptor protein, mRNA expression and localization in the endometrium of cyclic and pregnant gilts. Reprod Fertil Dev 1993; 5:247-260[CrossRef][Medline]
  31. Geisert RD, Chamberlain CS, Vonnahme KA, Malayer JR, Spicer LJ. Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the estrous cycle and early pregnancy in pigs. Reproduction 2001; 121:719-728[Abstract]
  32. Gauthier F, Moreau T, Gutman N, EL Moujahed A, Brillard-Bourdet M. Functional diversity of proteinases encoded by genes of the rat tissue kallikrein family. Agents Actions Suppl 1992; 38:42-50
  33. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev 2000; 22:184-204[Abstract/Free Full Text]
  34. Geisert RD, Yelich JV, Pratt T, Pomp D. Expression of an inter-{alpha}-trypsin inhibitor heavy chain-like protein in the pig endometrium during the oestrous cycle and early pregnancy. J Reprod Fertil 1998; 114::35-43[Abstract/Free Full Text]
  35. Geisert RD, Pratt TN, Bazer FW, Mayes JS, Watson HG. Immunocytochemical localization and changes in endometrial progestin receptor protein during the porcine oestrous cycle and early pregnancy. Reprod Fertil Dev 1994; 6:749-760[CrossRef][Medline]
  36. Corthorn J, Valdes G. Variations in uterine kallikrein during cycle and early pregnancy in the rat. Biol Reprod 1994; 50:1261-1264[Abstract]
  37. Corthorn J, Figueroa C, Valdes G. Estrogen and luminal stimulation of rat uterine kallikrein. Biol Reprod 1997; 56:1432-1438[Abstract]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
K. A. Berg, A. M. Patwardhan, T. A. Sanchez, Y. M. Silva, K. M. Hargreaves, and W. P. Clarke
Rapid Modulation of {micro}-Opioid Receptor Signaling in Primary Sensory Neurons
J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 839 - 847.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
S C Fernando, J S Buck, M D Ashworth, J W Ross, R D Geisert, and U DeSilva
Porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 gene expression.
Reproduction, December 1, 2006; 132(6): 939 - 947.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. M. Patwardhan, K. A. Berg, A. N. Akopain, N. A. Jeske, N. Gamper, W. P. Clarke, and K. M. Hargreaves
Bradykinin-Induced Functional Competence and Trafficking of the {delta}-Opioid Receptor in Trigeminal Nociceptors
J. Neurosci., September 28, 2005; 25(39): 8825 - 8832.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K.A. Vonnahme, S.C. Fernando, J.W. Ross, M.D. Ashworth, U. DeSilva, J.R. Malayer, and R.D. Geisert
Porcine Endometrial Expression of Kininogen, Factor XII, and Plasma Kallikrein in Cyclic and Pregnant Gilts
Biol Reprod, January 1, 2004; 70(1): 132 - 138.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.
Agricola
Right arrow Articles by Allen, M. R.
Right arrow Articles by Geisert, R. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS