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Female Reproductive Tract |
Department of Animal Science,4 Oklahoma Agriculture Experiment Station,
Department of Physiological Sciences,5 College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conceptus, female reproductive tract, implantation, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following the initial period of maternal recognition of pregnancy, many changes occur within the porcine uterine lumen to permit conceptus attachment and growth to allow formation of the epitheliochorial type of placentation in the pig. Establishment of pregnancy in the pig occurs during rapid elongation of the trophoblastic membrane in Day 12 of gestation. Trophoblastic elongation occurs concurrently with conceptus synthesis and release of estrogens to initiate maintenance of the corpora lutea, stimulate uterine alterations in the uterine epithelial surface, and enhance uterine secretion necessary for conceptus attachment and growth [11]. Kinin production has been associated with increased prostaglandin production, blood flow, and smooth muscle contraction [1], all of which occur during establishment of pregnancy in the pig [11]. Presence of conceptuses in the uterine lumen is temporally associated with an increase in bradykinin release into the uterine lumen of the pig during pregnancy [9]. Increased release of bradykinin into the uterine lumen during early pregnancy indicates that either plasma kallikrein is transported into the uterine lumen where the serine protease can release bradykinin from high molecular weight (H) kininogen or endometrial synthesis of tissue kallikrein may utilize its natural substrate, low molecular weight (L) kininogen, to release the potent vasoactive peptide, lysyl-bradykinin [1]. Plasma prekallikrein circulates as a proenzyme complexed to H-kininogen, which when activated by coagulation Factor XII (Hageman factor) releases bradykinin from H-kininogen [12]. Although tissue kallikrein and bradykinin have been detected in the porcine uterus, information concerning conceptus and endometrial gene expression for plasma kallikrein, Factor XII, and kininogen during the estrous cycle and early pregnancy has not been reported. The current study investigated porcine endometrial L- and H-kininogen gene expression as well as the presence of kininogens in the uterine flushings throughout the estrous cycle and early pregnancy in the pig. Endometrial and conceptus plasma kallikrein and Factor XII gene expression were also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cyclic, crossbred gilts of similar age (8–10 mo) and weight (100–130 kg) were checked twice daily for the onset of estrous behavior by intact boars. Onset of estrus was considered Day 0 of the estrous cycle. Gilts assigned for mating were bred naturally with fertile boars 12 h after the onset of estrus and again 12 h later. Cyclic gilts (n = 20) were hysterectomized on either Day 0, 5, 10, 12, or 15 of the estrous cycle, whereas pregnant gilts (n = 16) were hysterectomized on either Day 10, 12, or 15 as previously described by Gries et al. [13]. Following initial induction of anesthesia with a 1.5 ml i.m. administration of a cocktail consisting of 2.5 ml Rompum (xylazine; 100 mg/ml; Miles, Inc., Shawnee Mission, KS) and 2.5 ml Vetamine (ketamine HCl; 100 mg/ml; Mallickrodt Veterinary, Mundelein, IL) in 500 mg of 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. All animal procedures were in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction and were approved by the Institutional Animal Care and Use Committee (No. AG028).
Collection of Uterine Flushings and Endometrium
Uterine flushings (UTF) and endometrium were obtained by isolating the uterine horns and flushing with 20 ml of phosphate buffered saline (PBS; pH 7.4). Flushings were examined to confirm pregnancy in mated gilts and conceptuses collected were immediately snap frozen in liquid nitrogen. Uterine flushings were placed on ice following centrifugation (2500 x g, 10 min, 4°C) to remove cellular debris stored at -80°C. After flushing, one horn was cut along its antimesometrial border and endometrium was exposed for removal with sterile scissors. Collected tissue was immediately snap frozen in liquid nitrogen and stored at -80°C until RNA extraction was performed. Endometrium was collected from an additional group of gilts on Day 18 of the estrous cycle (n = 4) and pregnancy (n = 4) for analysis of gene expression.
Microconcentration and Protein Determination
Volume recovered from flushing the uterus of gilts averaged between 18 and 19 ml. UTF samples from each animal were prepared for Western blot analysis by concentrating 2 ml of the flushing sample using Centricon 10 microconcentrators (Amicon, Beverly, MA) with a molecular weight cut-off of 10 000 Da. Protein concentration in the 70 µl of concentrated flushing was determined by the method of Lowry et al. [14] prior to loading acrylamide gel.
Western Blot Analysis
UTFs were analyzed by Western blotting for the presence of immunoreactivity to antisera against human L-kininogen (Biogenesis, England) and H-kininogen (Novus Biologicals, Inc., Littleton, CO). Loading of concentrated UTF from each animal was standardized to 25 µg of total protein. Proteins separated by a 10% or 12% 1D SDS-PAGE were immediately transferred to PVDF membrane (Millipore Corporation, Bedford, MA) at 350 mA constant current for 90 min. Following electroblotting, the membranes were washed in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) and incubated for 1 h with the first blocking solution (3% gelatin in TBS). After washing in Tween-TBS (TTBS; 20 mM Tris, 500 mM NaCl, 0.05% Tween 20, pH 7.5) for 10 min, membranes were incubated overnight with either a 1:200 dilution of primary rabbit polyclonal antiserum raised against human L-kininogen (catalog 5575–6007; Biogenesis) or a 1:500 dilution of a primary sheep polyclonal antiserum to H-kininogen (product ab89994, Novus Biologicals) in 1% gelatin TTBS. Membranes were washed twice in TBS following incubation with antiserum. Immunoreactive polypeptides were detected using the Bio-Rad Immuno-Blot kit (Bio-Rad, Hercules, CA) as specified by the manufacturer's recommendations.
RNA Extraction
Total RNA was extracted from endometrium and porcine liver, which served as a positive control for gene expression of the acute phase proteins. TRIzol reagent (Gibco/Life Sciences, Gaithersburg, MD) was utilized to extract total RNA from the tissues according to manufacturer's instructions. RNA was rehydrated with 10 mM Tris 1 mM EDTA (pH 7.4) and stored at -80°C until further analyzed. Total RNA was extracted from conceptus tissue as previously described by Ross et al. [15]. Total RNA was estimated spectrophotometrically by absorbance at 260 nm. RNA purity was determined from calculations of 260/280 ratios. Integrity of the RNA was evaluated via gel electrophoresis.
Determination of Porcine L- and H-Kininogen cDNA Sequence
Endometrial and conceptus total RNA were reverse transcribed into cDNA using Superscript II RNAseH reverse transcriptase (Invitrogen, Carlsbad, CA) as specified by manufacturer's instructions.
Primers for L-kininogen were designed from the porcine L-kininogen cDNA sequence, and H-kininogen primers were designed from the conserved region of the bovine and human H-kininogen sequence (Table 1). All PCR reactions and optimization of the PCR conditions were conducted with pooled cDNA from endometrium representing various days of the estrous cycle and early pregnancy as previously described by Yelich et al. [16]. The PCR amplicons were extracted from the agarose using Qiaquick gel purification columns (Qiagen, Santa Clarita, CA) and sequenced by the Recombinant DNA/Protein Research Facility at Oklahoma State University. Sequence of the endometrial 339 bp product of L-kininogen had a 100% identity to porcine L-kininogen (GenBank accession AY321363), and the 316 bp PCR amplified fragment of the porcine H-kininogen (GenBank accession AY322570) had a 91% and 82% identity to bovine and human H-kininogen, respectively.
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Endometrial, Conceptus, and Liver RT-PCR Gene Amplification
Porcine endometrial, conceptus, and liver gene expression of H-kininogen, L-kininogen, plasma kallikrein, and Factor XII were evaluated through RT-PCR using primers presented in Table 1. Since endometrial expression of plasma kallikrein and Factor XII produced only faint bands in the initial RT-PCR, a second round of amplification was performed. The endometrial 785 bp product produced with primers to plasma kallikrein and 376 bp product to primers for Factor XII were sequenced and verified to be 100% identical to the porcine sequences of plasma kallikrein and Factor XII, respectively. PCR products generated by endometrial plasma kallikrein and Factor XII were verified to be from cDNA and not through genomic contamination as amplicons generated from genomic DNA are larger in size (unpublished data). Primers for ß-actin gene expression (Table 1) were utilized to verify RNA quality. Absence of genomic DNA contamination in samples was verified through the ß-actin primers, which produce two bands in the presence of genomic DNA. cDNA PCR reactions were performed at 20-µl volume in a MJ Dyad thermal cycler (MJ Research, Inc., Waltham, MA).
Quantitative Reverse Transcriptase-Polymerase Chain Reaction
Endometrial gene expression for L-kininogen, H-kininogen, plasma kallikrein, and Factor XII was quantified by using the one-step reverse transcriptase-polymerase chain reaction (RT-PCR) following the manufacturer's recommendations for the QuantiTect Probe RT-PCR kit (catalog 204443; Qiagen, Valencia, CA). Primers and probes for L-kininogen, H-kininogen, plasma kallikrein, and Factor XII developed based on sequence information from porcine sequences are presented in Table 1. All dual-labeled probes were designed to have a 5' reporter dye (6-FAM) and a 3' quenching dye (TAMRA). Depending on optimization for each individual gene of interest, the total reaction volume of 25 µl contained 200–800 nM forward primer, 200–800 nM reverse primer, 100–300 nM fluorescent-labeled probe, and 100–200 ng of total RNA. The PCR amplification was carried out in the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). Thermal cycling conditions were 50°C for 30 min, 95°C for 15 min, followed by 40 repetitive cycles of denaturation (95°C for 15 sec) and annealing/extension (60°C for 1 min). Ribosomal 18S RNA (18S, RNA Control Kit, 43108993E, Applied Biosystems) was assayed for each sample as a control for RNA loading.
Following RT-PCR, quantification of gene amplification was made by setting the threshold cycle (CT) in the geometric region of the plot after examining the semilog view of the amplification plot. Relative quantification of gene expression was evaluated using the comparative CT method as described previously by Hettinger et al. [17]. The 
CT value is determined by subtracting the gene CT of each sample from the corresponding sample ribosomal 18S CT value. Calculation of CT involves using the highest mean CT value as an arbitrary constant to subtract from all other CT mean values. Fold changes in gene expression are calculated from the CT values using the formula 2-Ct.
Statistical Analysis
Endometrial gene expression (CT value) was analyzed with least square means using the PROC MIXED of SAS [18]. The statistical model tested the effect of day, reproductive status (cyclic and pregnant), and the day x reproductive status interaction. Results are presented as arithmetic means ± standard error of the mean.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A Coomassie-stained 1D-gel of proteins in UTF and Western blot analysis of L-kininogen in UTF of cyclic and pregnant gilts is presented in Figure 1. Antiserum to human L-kininogen specifically recognizes the 66 kDa plasma glycoprotein [19]. Western blot analysis detected a 
66 kD immunoreactive product for L-kininogen in UTF across all days of the estrous cycle and early pregnancy in the pig (Fig. 1B).
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A distinct immunoproduct of approximately 47 kDa, corresponding to the H-kininogen light chain, was present in the UTF across all days of the estrous cycle and early pregnancy in the pig (Fig. 2).
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Endometrial, Conceptus, and Liver RT-PCR Gene Amplification
A 339 base pair (bp) RT-PCR L-kininogen product was amplified from liver, endometrial, and conceptus total RNA, whereas the primers generated a larger, single amplicon with genomic DNA (Fig. 3A). The appropriate-sized gene product (Fig. 3B) was detected following amplification of liver and endometrial H-kininogen (316 bp), Factor XII (376 bp), and plasma kallikrein (785 bp). Intensity of endometrial H-kininogen gene expression was similar to that observed with porcine liver. Although endometrial gene expression of Factor XII and plasma kallikrein was detected, the expression of the genes is lower than in liver as clear amplification was only achieved following a secondary PCR amplification using the initial endometrial PCR product as a template. Conceptus gene expression for H-kininogen, Factor XII, and plasma kallikrein remained undetectable even following secondary PCR amplification.
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Quantitative Analysis of Endometrial Gene Expression
The endometrial gene expression of L-kininogen, H-kininogen, plasma kallikrein, and Factor XII were quantified using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA).
Endometrial gene expression for H-kininogen was detected across all days of the estrous cycle and early pregnancy. No significant day or status difference was detected for endometrial H-kininogen gene expression (data not presented). A status by day effect (P < 0.01) was detected for endometrial L-kininogen gene expression (Table 2). During the estrous cycle, endometrial L-kininogen expression was sevenfold greater at estrus compared with Day 5 of the estrous cycle (Fig. 4). Gene expression for L-kininogen increased slightly on Day 10 of the estrous cycle and early pregnancy. There was approximately a three- to fourfold increase in gene expression on Days 15 and 18 in pregnant gilts when compared with the same days of the estrous cycle (Fig. 4).
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Endometrial Factor XII gene expression was detected across all of the days of the estrous cycle and early pregnancy. However, endometrial Factor XII gene expression was similar across the days of the estrous cycle and early pregnancy (data not presented). Although endometrial plasma kallikrein gene expression was low, a significant (P < 0.02) status x day effect was detected (Table 3). Endometrial plasma kallikrein gene expression was elevated on estrus (Day 0) followed by decline on Day 5, which was followed by a threefold increase on Day 15 of the estrous cycle (Fig. 5). Plasma kallikrein gene expression did not significantly change across the days of early pregnancy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-globulin. Difference in the size of L-kininogen compared with H-kininogen results from alternative splicing of exon 10 and 11 of the kininogen gene to produce a 4-kDa light chain in L-kininogen compared with 56-kDa in H-kininogen. Activation of plasma kallikrein releases the nonapeptide bradykinin from H-kininogen, whereas tissue kallikrein releases the decapeptide lysyl-bradykinin from L-kininogen that can be processed to bradykinin by aminopeptidase [12]. Release of the vasoactive kinins from L- and H-kininogen are responsible for many physiological events such as lowering blood pressure, increasing endothelial permeability, smooth muscle contraction, and generalized tissue inflammation [1].
Events involved with implantation/placental attachment mimic inflammatory and immunological responses for which the kallikrein-kininogen-kinin system effects on vasodilation, prostaglandin synthesis, and proteolytic tissue remodeling are well established [2]. The kallikrein-kininogen-kinin system has been proposed to be involved with establishment of pregnancy in the rat [2]. During the time of maternal recognition of pregnancy until the time of conceptus attachment, many alternations in the porcine uterine lumen are similar to an acute inflammatory response. With rapid elongation and growth of the conceptus, estrogen synthesis by conceptuses can alter uterine secretion of proteins [21], prostaglandins [22], uterine blood flow [23], uterine cellular morphology [24], and vascular permeability [25] as the placenta expands over the uterine surface. Endometrial L- and H-kininogen gene expression and detection of both glycoproteins in UTF of cyclic and pregnant gilts in the present study adds additional evidence for the presence of an active kallikrein-kininogen-kinin system during establishment of pregnancy in the pig. Previously, our laboratory demonstrated endometrial tissue kallikrein gene expression and kallikrein enzyme activity increased in the uterine lumen of the pig after Day 10 of the estrous cycle and pregnancy [8]. Tissue kallikrein regulates the activation of many growth factors [26, 27] and the cleavage of inter--trypsin inhibitor heavy chain 4, which has been identified in the pig uterus [28]. Detection of glandular kallikrein (KLK-1) and its gene expression in the porcine endometrium and the conceptuses [8] suggests that the serine protease may be also involved with release of lysl-bradykinin from L-kininogen. However, content of bradykinin in UTF of pregnant gilts increases between Days 12 and 18 of gestation, but not in cyclic gilts [9]. Detection of the 47 kDa light chain of H-kininogen in the UTF of gilts indicates that both L- and H-kininogen are present within the uterine lumen of cyclic and pregnant gilts. Interestingly, although the liver is considered to be the major source of H-kininogen, plasma kallikrein, and Factor XII, gene expression was detected in porcine endometrium. These results suggest that endometrium can exert a local effect on bradykinin release through endometrial synthesis of plasma kallikrein and Factor XII. However, although endometrial gene expression was established for plasma kallikrein and Factor XII in the present study, presence and activity of plasma kallikrein and Factor XII in the uterine lumen needs to be investigated. Vascular transfer of the kallikrein-kininogen-kinin components cannot be completely ruled out as it has been reported to occur in the porcine follicle [29]. The kallikrein-kininogen-kinin system has been implicated in the follicular development and ovulation in pig [6]. However, although plasma kallikrein, Factor XII, and H-kininogen were present in the follicular fluid, ovarian gene expression was undetectable by Northern blot analysis [6]. Thus the porcine endometrium may be unique in its capacity to independently generate the components for the kallikrein-kininogen-kinin system during pregnancy.
Developmental stage of the porcine conceptuses affected endometrial L-kininogen gene expression, which increased fourfold on Days 15 and 18 of gestation. Kininogen gene expression is enhanced by estrogen [30], and therefore it is possible that endometrial L-kininogen gene expression is stimulated by the short increase in conceptus estrogen release on Day 12 and the prolonged estrogen release that occurs after Day 15 of gestation [11]. With the enhancement of L-kininogen gene expression, one would expect an increase in protein within the uterine lumen. However, although Western blot analysis identified L- and H-kininogen in the uterine lumen during the estrous cycle and early pregnancy, the total amount of the kininogens present in the uterine lumen cannot be evaluated through loading a standardized amount of UTF protein. The various types of uterine proteins increase and decrease during the estrous cycle and early pregnancy, and there is a four- to fivefold increase in total protein following Day 12 of the estrous cycle and pregnancy [24]. In pregnant gilts, conceptus proteins would also contribute to the UTF prohibiting any conclusion to be drawn for pregnancy-induced changes in total kininogen content. Uterine content of L- and H-kininogen needs to be evaluated with specific assays in the future. The increase in endometrial kininogen gene expression in pregnant gilts during placental attachment is consistent with the pregnancy-specific eight- to tenfold rise in bradykinin content [9] temporally associated with increased uterine blood flow [31] and vascular permeability [25] that occurs during early porcine pregnancy. These data indicate that activation for L- and H-kininogen cleavage is regulated by the porcine conceptuses. Conceptus stimulation that activates the release of bradykinin from H-kininogen is unknown at present. It is possible that during trophoblastic attachment to the uterine surface, Factor XII can bind and be enzymatically activated to interface with plasma kallikrein. Alternatively, plasmin has been demonstrated to cleave and activate Factor XII [12]. The possibility that conceptus synthesis and release of plasminogen activator and subsequent increase in plasmin within the uterine lumen during rapid trophoblastic elongation and placental attachment [32] could play a role in activation of bradykinin release during early pregnancy needs to be evaluated. Many of the biological effects of bradykinin release during pregnancy may be mediated through bradykinin ß2 receptor activation to release prostaglandins, histamine, and other cytokines involved with the inflammatory response [12]. Bradykinin increases the release of arachidonic acid from human decidua cells in vitro [33]. The release of arachidonic acid by bradykinin is increased by pretreatment of the cultured cells with interleukin 1ß (IL-1ß), which enhances bradykinin ß2 receptor. Interestingly, there is an increase in endometrial bradykinin ß2 receptor on Day 12 of gestation [9] when porcine conceptuses release IL-1ß during trophoblastic elongation [34]. It is possible that the release of bradykinin and IL-1ß play important roles in conceptus remodeling during trophoblast elongation and alteration of uterine prostaglandin secretion needed to establish pregnancy in the pig.
The present study has established the presence of L- and H-kininogen in the porcine uterus. Combined with our previous studies that have demonstrated the porcine uterus secretes tissue kallikrein [8] and bradykinin increases during pregnancy indicates the kallikrein-kininogen-kinin system is active in the pig. Although endometrial gene expression of plasma kallikrein and Factor XII have been detected, further studies are necessary to determine if this branch of the pathway is biologically active during establishment of pregnancy in the pig. The role that the kallikrein-kininogen-kinin system serves in the porcine uterus during early pregnancy may initiate numerous events that are necessary for conceptus attachment and survival. Future studies will establish how the conceptus activates the kallikrein-kininogen-kinin system to establish pregnancy and provide an environment for continued conceptus growth and survival.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rodney D. Geisert, Oklahoma State University, Department of Animal Science, Animal Science Building, Room 114, Stillwater, OK 74078. FAX: 405 744 7390; geisert{at}okstate.edu
3 Current address: University of Wyoming, Department of Animal Science, Laramie, WY 82071
Received: 16 June 2003.
First decision: 7 July 2003.
Accepted: 9 September 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by the endometrium. Prostaglandins 1977 14:397-401[CrossRef][Medline]-trypsin inhibitor heavy chain like protein in the pig endometrium during the estrous cycle and early pregnancy. J Reprod Fertil 1998 114:35-43This article has been cited by other articles:
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  M. Maruyama, A. Hattori, Y. Goto, M. Ueda, M. Maeda, H. Fujiwara, and M. Tsujimoto Laeverin/Aminopeptidase Q, a Novel Bestatin-sensitive Leucine Aminopeptidase Belonging to the M1 Family of Aminopeptidases J. Biol. Chem., July 13, 2007; 282(28): 20088 - 20096. [Abstract] [Full Text] [PDF]
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 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]
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 J. A. MacLean II, R. M. Roberts, and J. A. Green Atypical Kunitz-Type Serine Proteinase Inhibitors Produced by the Ruminant Placenta Biol Reprod, August 1, 2004; 71(2): 455 - 463. [Abstract] [Full Text] [PDF]
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