| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,4
* Department of Animal and Veterinary Science, Center for Reproductive Biology, University of Idaho, Moscow 83844;
and
Department of Animal Science, Center for Reproductive Biology, Washington State University, Pullman 99164; and
and
Department of Animal Science, University of Wyoming, Laramie 82071
5 Correspondence:
309 Ag. Biotech. Bldg, Moscow, ID 83844-2330 (phone: 208-885-7370; fax: 208-885-7036; E-mail:
tott{at}uidaho.edu).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
(IFN) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the antiluteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts—animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.
Key Words: Cows • Gene Expression • Horses • Interferon • Pigs • Uterus
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
secreted by the endometrium (Bazer et al., 1998). In each of these animals, the antiluteolytic signal originates from the conceptus and acts locally on the endometrium. The pregnancy recognition signal in cows and ewes is the type-I interferon, interferon- (IFN), whereas in sows, the signal is estrogen (Bazer and Thatcher, 1977; Mirando et al., 1996; Bazer et al., 1998). In mares, this signal has not been identified, and the antiluteolytic mechanism is poorly characterized.
Interferon- regulates expression of a number of proteins in the endometrium (Vallet et al., 1987), but few of these proteins have been characterized, and their functions are largely unknown. One of the earliest intracellular proteins upregulated in response to IFN- or pregnancy is Mx protein (Charleston and Stewart, 1993; Ott et al., 1998).
The Mx proteins were first identified in laboratory mice and are a well-characterized component of the immune response to viral infection (Horisberger and Gunst, 1991). In ewes, Mx is upregulated during early pregnancy (Charleston and Stewart, 1993; Ott et al., 1998), and the temporal and spatial regulation of Mx during early pregnancy in ewes suggests an alternative function (or functions) for Mx outside the immune response (Ott et al., 1998). However, uterine Mx expression has not been characterized in cattle or in other domestic species that utilize different conceptus signals to maintain luteal function.
The present experiments were conducted to determine whether the uterine Mx gene is expressed similarly in cows as was previously shown for ewes. These animals both utilize an IFN as a signal for pregnancy recognition. A further objective was to determine whether Mx is expressed during the estrous cycle and regulated during early pregnancy in the uterus of gilts and mares; animals that apparently do not use IFN as the signal for pregnancy recognition.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Porcine Endometrium.
Prepubertal crossbred gilts were observed daily for estrus in the presence of an intact boar. At estrus (d 0), gilts were assigned randomly to pregnant or cyclic statuses. Gilts assigned to the pregnant group were mated to an intact boar at the onset of estrus and then at 24-h intervals for a maximum of three matings. Pregnancy was confirmed by the presence of apparently normal conceptuses in the uterus at hysterectomy. Pregnant and cyclic gilts were hysterectomized on d 5, 10, 12, 14, 16, or 18 of the estrous cycle or d 10, 12, 14, 16, or 18 of pregnancy (n = 4 gilts/status for each day) for collection of endometrium. Endometrial samples were snap-frozen in liquid nitrogen and stored at -80°C for future protein or RNA extraction. Cross-sections from uterine horns were fixed, embedded, and utilized for in situ localization of Mx mRNA as described below.
Equine Endometrium.
From September to October, mares of mixed light horse breeds, 3 to 15 yr old and weighing 300 to 500 kg, were examined four times weekly by transrectal palpation and ultrasonography (Aloka 210, Wallingford, CT). Estrus was synchronized using one injection of Cloprostenol (250 µg i.m., Bayer Corp., Shawnee Mission, KS), and examined by palpation and ultrasound to monitor the stage of the estrous cycle, growth of the follicles, ovulation, corpus luteum formation, and pregnancy status. Once a preovulatory follicle reached 
35 mm, mares were injected i.v. with hCG (Chorulon, 2,500 IU; Intervet, Millsboro, DE) and assigned randomly to pregnant or cyclic status. Fresh semen was collected from two stallions as needed, and mares assigned to the pregnant group were inseminated with at least 500 x 106 progressively motile sperm. Endometrial samples (0.5 to 1.0 g total weight) were collected by transcervical biopsy on d 0 (ovulation), 8, 11, 14, 17, and 20 from cyclic mares and on d 11, 14, 17, and 20 from pregnant mares (n = 4 mares/status for each day). Each mare was sampled once; endometrial samples were snap-frozen in liquid nitrogen and stored at -80°C for later extraction of protein and mRNA or immediately fixed and embedded for in situ localization of Mx mRNA.
Protein Extraction and Western Blot Analysis
Total protein was isolated from endometrium (0.5 to 1.0 g) using 10 mL of lysis buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 5 mM EDTA; 0.5% Tergitol type NP-40 [Sigma, St. Louis, MO]; 0.1 mM phenylmethylsulfonyl flouride [Sigma]). Tissue was homogenized for 15 to 20 s at 24,000 rpm using a Tissuemizer Ultra Turrax (IKA Works, Inc., Wilmington, NC) and incubated for 30 min on ice. Following incubation, samples were centrifuged at 2,700 x g for 15 min, and the supernatant was transferred to 15-mL tubes and stored at -80°C.
Protein concentration was quantified with the bicinchoninic acid assay (Pierce, Rockford, IL), according to manufacturer’s instructions using BSA as the standard (0 to 2 mg/mL). Following quantification, samples were examined by one-dimensional SDS-PAGE and Western blot analysis for Mx protein expression. Briefly, samples (8 µg) were diluted in 1x sample buffer (4x stock = 7.5 mL distilled water, 760 mg of Tris-base, 2 g of SDS, 10 mL of glycerol, pH adjusted to 6.8 with HCl, 5 mL of 2-mercaptoethanol, and 300 µL of 2% solution of bromphenol blue in 100% ethanol) and distilled water to an equal volume, heated to 95°C for 5 min, and cooled to room temperature before loading. Proteins were separated on duplicate SDS 12%-PAGE gels with 6% stacking gels at a constant current of 70 mA for approximately 45 min. One gel was stained with 25 mL of 0.125% Coomassie brilliant blue R-250 stain (Sigma) in 38% ethanol and 7% glacial acetic acid (vol/vol) in water for 30 min at room temperature. The gel was then destained with 25 mL of 38% ethanol and 7% glacial acetic acid (vol/vol) water and photographed using the Bio-Rad Fluor-S Multi-imager system (40-mm lens, F-stop = 11, exposure = 1 to 5 s; Bio-Rad Laboratories, Hercules, CA).
Proteins on the duplicate gel were transferred to a nitrocellulose membrane (Protran BA83; pore size = 0.2 µm, Schleicher & Schuell, Keene, NH), using a Mini-Protean II Cell apparatus (Bio-Rad Laboratories) with constant stirring at 70 V (constant voltage) for 1 h with an ice pack. Following transfer, nonspecific binding was blocked by incubating the membrane in blocking buffer (20mM Tris, pH 7.5, 137 mM NaCl, 0.05% Tween 20) containing 5% nonfat dried milk with slow rocking at room temperature for 2 h. Membranes were placed in 30 mL of blocking buffer containing 2% nonfat dried milk and 1:1,000 dilution of either a carboxyl (No. 90621-3) or amino terminal (No. 90618-2) rabbit polyclonal ovine Mx peptide antiserum (Multiple Peptide Systems, San Diego, CA), and incubated on a rocking platform at 4°C overnight. Following overnight incubation, the membrane was washed four times with 100 to 200 mL of blocking buffer for 5 min each. Blots were then incubated with 50 mL of blocking buffer containing 2% nonfat milk and 1:200,000 dilution (wt/vol) of goat anti-rabbit IgG-horse radish peroxidase conjugate (product No. 31460, 0.8 mg/mL; Pierce) on a rocking platform at room temperature for 1 h. Following incubation with the secondary antibody, membranes were washed four times with blocking buffer (10 min each) and incubated with Supersignal West Femto Maximum Sensitivity Substrate chemiluminescent kit (Pierce) according to manufacturer’s instructions for 5 min to detect immunoreactive proteins. Signal was quantified using the Bio-Rad Fluor-S Multiimager system (40-mm lens, F-stop = 2.7, exposure = 60 to 600 s) and Quantity One software (Bio-Rad Laboratories). Endometrial samples collected previously (Ott et al., 1998) from ewes on d 11 of the estrous cycle and d 15 of pregnancy were used as positive controls.
Ribonucleic Acid Isolation, Northern Blot, and Slot Blot Analysis
Total RNA was isolated from endometrium using Trizol (Gibco BRL, Gaithersburg, MD), as described previously (Ott et al., 1998). The amount and quality of the RNA was assessed by spectrophotometry (260 nm) and by agarose/formaldehyde gel electrophoresis. Northern blot analysis was performed to determine the number and size of Mx transcripts present. Total RNA (5 µg) was fractionated on a 1% agarose, 0.62 M formaldehyde gel and transferred to a nylon membrane (Nitran SuPerCharge; Schleicher & Schuell) by overnight capillary diffusion. The blot was baked for 30 min at 80°C and cross-linked using an ultraviolet illuminator (Stratagene, La Jolla, CA) set on auto cross-link. The blots were hybridized using a North2South chemiluminescent hybridization and detection kit (Pierce). Blots were incubated overnight in hybridization buffer (0.1 mL/cm2 of membrane) containing a biotin-labeled Mx antisense complementary RNA (cRNA) probe (5 ng/mL). Biotin-labeled antisense ovine Mx cRNA riboprobe was made using linearized pCRII plasmid (InVitrogen, Carlsbad, CA), containing an approximately 823-bp ovine Mx partial cDNA (Ott et al. 1998), and the MAXIscript SP6/T7 kit (Ambion Inc., Austin, TX). The MAXIscript kit was used with SP6 RNA polymerase to make an antisense transcript labeled with biotin on the uracil base. Antisense probe was denatured at 95°C for 10 min, cooled on ice for 3 min, added to hybridization buffer, and then incubated overnight at 65°C in a rotating hybridization oven (Fisher Scientific, Pittsburgh, PA). Following overnight hybridization, tubes were cooled to room temperature, and the blots were washed three times for 5 min each in 0.3 M NaCL and 0.03 M Na-citrate (0.2 mL/cm2 of membrane) at room temperature and then washed in 0.3 M NaCL and 0.03 M Na-citrate containing 1 µg/mL of RNase A (Roche Diagnostics Corp., Indianapolis, IN) for 5 min at room temperature. Immediately after RNase A treatment, blots were washed twice for 15 to 20 min at 65°C in 2x stringency wash buffer diluted 1:1 with nuclease-free water (0.3 M NaCL, 0.03 M Na-citrate, and 0.1% SDS; this buffer and other buffers mentioned here were supplied in the Pierce kit) and incubated in blocking solution containing 1:300 dilution (wt/vol) of strepavidin-horse radish peroxidase conjugate. The blots were then washed four times with 4x wash buffer diluted 1:4 and once with equilibrium buffer (5 min/wash with 0.2 mL/cm2 of membrane). The final incubation was with equal volumes of luminol/enhancer and stable peroxide solution to cause light emission.
Following hybridization using the North2South kit, chemiluminescent signal was collected using the Bio-Rad Fluor-S Multiimager system (40-mm zoom lens, f-stop = 2.7, exposure = 60 to 600 s) and quantified using Quantity One software. The blot was then stripped in 0.1% SDS at 90°C for 30 min and reprobed using a biotin-labeled antisense ovine 18s ribosomal RNA (rRNA) cRNA probe as described above. Data for Mx mRNA levels were adjusted for variation in amount of RNA loaded and transferred using the 18s signal as a covariate in the ANOVA. Endometrial samples collected previously (Ott et al., 1998) from ewes on d 11 of the estrous cycle, and on d 15 of pregnancy were used as positive controls.
In situ Hybridization
Cellular distribution of Mx mRNA was determined in a subset (n = 3 animals per status x day) of the endometrial samples using in situ hybridization as described previously (Johnson et al., 1999), using [
-35S]ATP-labeled oMx sense and antisense cRNA probes, prepared as described by Ott et al. (1998). For cows and gilts, endometrium from d 18 of pregnancy was chosen because levels of Mx expression by slot-blot analysis were at or near their highest. For mares, because levels did not seem to change during the cycle or pregnancy, d 14 was chosen because it corresponds to the proposed onset of pregnancy-recognition signaling. Photomicrographs were taken with a Nikon E1000 photomicroscope using a Nikon DXM 1200 digital camera and ACT-1 software (Nikon, Emeryville, NY). Photographic plates were assembled using Adobe Photoshop (Version 6.0, Adobe Systems Inc., San Jose, CA).
Statistical Analysis
Results were analyzed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) and are reported as least squares means ± pooled standard error. The statistical model included day, reproductive status, and their interaction as sources of variation. The 18s rRNA signal was included as a covariate in the analysis to adjust for differences in RNA loading. Error terms used were according to the expectation of mean square for error (Snedecor and Cochran, 1980). Following a significant F-test, changes in Mx mRNA levels across days of the cycle or pregnancy were described using regression analysis. Results from in situ hybridization were evaluated independently by two trained investigators, and sections representing the overall pattern and intensity of hybridization are presented.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
). In general, hybridization intensity was greater in RNA isolated from the endometrium of pregnant vs. cyclic cows and ewes (lanes 4 vs. 5 and lanes 8 vs. 9, respectively), but was not different between pregnant and cyclic gilts and mares (lanes 2 vs. 3 and lanes 6 vs. 7, respectively).
|
. The Mx mRNA levels increased approximately 15-fold (P < 0.05) between d 12 and 15 of pregnancy and remained high at d 18 of pregnancy, whereas there was no change in steady-state levels of Mx mRNA in cyclic cows. Western blot analysis of Mx protein levels was conducted on endometrial protein isolated from randomly selected d-12 cyclic and d-18 pregnant cows to confirm that changes in Mx mRNA levels resulted in similar changes in levels of Mx protein expression (Figure 3, lanes 3 and 4). Incubation of blots with a carboxyl terminus oMx antiserum identified two bands of appropriate size for Mx (approximately 72 and 75 kDa) in d-18 pregnant cows (Figure 3, lane 5). The brighter of the two bands (approximately 75 kDa) was not detected in d-12 cyclic cows (Figure 3, lane 4), although it was detected in proteins isolated from d-15 cyclic and pregnant ewes (lane 8). The pregnancy-specific bands detected in the cow and ewe comigrated with rhuMxA (lane 9).
|
|
) revealed a spatial distribution of Mx mRNA at d 18 of the cycle or pregnancy in cows similar to that reported for sheep (Ott et al., 1998). The Mx mRNA levels were low in cyclic cows at d 18 (Figure 4, top panel), whereas hybridization signal was dramatically elevated in the lumenal epithelium, glandular epithelium, and stroma at d 18 of pregnancy (Figure 4, middle panel). Control (sense) hybridization was low to undetectable (Figure 4, bottom panel).
|
. Analysis of steady-state levels of Mx mRNA showed that a greater than twofold increase (P = 0.06) in Mx mRNA levels occurred in d-16 pregnant gilts only. Western blot analysis of endometrial protein (Figure 3) identified multiple bands from representative d-12 cyclic (Lane 1) and d-18 pregnant (Lane 2) gilts. When incubated with a carboxyl terminus oMx antiserum, multiple bands were detected (approximately 40, 60, 72, and 80 kDa), one of which (50 kDa band) appeared brighter in pregnant gilts than in cyclic gilts.
Results from in situ hybridization analysis (Figure 5) revealed that Mx mRNA hybridization occurs predominantly in the lumenal and glandular epithelium at d 18 in cyclic gilts (Figure 5, top panel), whereas at d 18 of pregnancy, hybridization was reduced to low levels in the lumenal and glandular epithelium, but had increased dramatically in the stroma (Figure 5, middle panel).
|
. Overall, Mx mRNA was very low, and no difference was detected between cyclic and pregnant mares in steady-state levels of endometrial Mx mRNA. However, Northern blot hybridization detected a band of appropriate size (approximately 2.5 kb) for Mx mRNA (Figure 1, lanes 6 and 7), and Western blot analysis using the carboxyl terminus oMx antiserum (Figure 3, lanes 6 and 7) detected multiple faint bands (predominant band at approximately 60 kDa) of the approximate size for Mx in horse endometrial samples. There were no apparent differences in levels of protein expression between cyclic and pregnant mares.
Results from in situ hybridization analysis of cyclic and pregnant mare endometria (Figure 6) were similar to each other and revealed low levels of Mx hybridization, primarily localized to the lumenal epithelium. It should be noted that the mare endometrial sections were exposed three and six times longer than cow and gilt tissue, respectively.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Results presented here show that the uterine Mx gene is expressed in cows, gilts, and mares during the estrous cycle. Further, in cows and gilts, like ewes, Mx gene expression is regulated during early pregnancy. Interestingly, the temporal and spatial pattern of Mx expression differs among the different species. Taken together with results that showed uterine Mx expression in sheep (Charleston and Stewart, 1993; Ott et al., 1998), mice (Chang et al., 1990), and humans (T. L. Ott, unpublished observations), we suggest that uterine Mx expression may be a general phenomenon during early pregnancy. In light of the results presented here and elsewhere (Chang et al., 1990; Charleston and Stewart, 1993; Ott et al, 1998, 1999; Yankey et al., 2001), it is becoming increasingly clear that uterine Mx expression is likely not regulated solely by viral infection and that Mx may possess functions in addition to its classically described antiviral activity (Staeheli et al., 1993).
Currently, the function of Mx during early pregnancy is not clear, and its only described function is in the immune response to viral infection (Horisberger and Gunst, 1991). Even though Mx likely functions as an antiviral protein in the uterine mucosal immune system, the temporal and spatial patterns of uterine Mx gene expression during early pregnancy suggest alternative functions. For example, given that Mx is strongly upregulated in the stroma, myometrium (Ott et al., 1998), and peripheral blood lymphocytes (Yankey et al., 2001) during early pregnancy, and that intrauterine injections of recombinant ovine IFN resulted in increased Mx expression in the corpus luteum (Spencer et al., 1999) it is possible that Mx possesses additional pregnancy-specific functions.
Insight into potential novel functions for Mx in the uterus during the estrous cycle or pregnancy may be derived from analysis of its structure. The Mx protein is a large monomeric guanine triphosphatase that is homologous to members of the mechanochemical enzyme family including the dynamins (Charleston and Stewart, 1993; Staeheli et al., 1993; Melen et al., 1996). In general, proteins in this family are involved in intracellular protein or vesicle trafficking. Based on this structural homology, we hypothesize that Mx functions outside the immune response to viral infection during early pregnancy, perhaps in the processes of endometrial secretion or uterine remodeling accompanying pregnancy recognition (Horisberger, 1992). In support of this, Accola et al. (2002) showed recently that human MxA binds to and tubulates phosphatidylserine droplets in vitro. Futhermore, inhibition of MxA function resulted in accumulation of smooth endoplasmic reticulum. Both of these observations are consistent with a role for Mx in intracellular vesicle trafficking.
This study examined Mx mRNA and protein expression in the endometrium of pregnant and cyclic cows, gilts, and mares to determine if uterine Mx expression differed during early pregnancy in species possessing different pregnancy recognition mechanisms. Slot-blot analysis of steady-state levels of Mx mRNA indicated levels increased approximately 15-fold in pregnant cows as early as d 15 after estrus. This increase occurred approximately 2 d later than in sheep (Ott et al., 1998), but is consistent with the later production of IFN by bovine (compared with ovine) conceptuses (Bazer et al., 1998). Results from in situ localization of Mx mRNA revealed a pattern of expression of Mx similar to that shown for the sheep. The Mx expression was low at d 18 of the cycle and was strongly upregulated at d 18 of pregnancy, particularly in the luminal and glandular epithelium and in the stratum compactum stroma.
Western blot analysis of Mx protein was conducted to confirm previous results from sheep that indicated that changes in Mx protein levels occurred in concert with changes in Mx mRNA levels (Ott et al., 1998). Western blot analysis using a carboxyl terminus antiserum revealed two bands in pregnant cow endometrium (approximately 72 and 75 kDa). The brighter of the two bands (approximately 75 kDa) was specific to pregnancy, and the second band was also detected in cyclic cows. These results indicate that in cows, as in ewes, the abundance of Mx mRNA and protein is increased markedly during early pregnancy.
Pig conceptuses do not produce IFN (Pontzer et al., 1988; Leaman et al., 1992) as the signal for pregnancy recognition. In pigs, conceptus-produced estrogen, secreted between d 11 and 12 and again on d 15, is responsible for redirecting secretion of PGF2 away from the uterine vasculature (endocrine) and towards the uterine lumen (exocrine), thereby protecting the CL from the luteolytic effects of PGF2 (Bazer and Thatcher, 1977; Mirando et al., 1996; Bazer et al., 1998). Interestingly, pig conceptuses express both -IFN and a unique 
-IFN during early pregnancy (La Bonnardiere et al., 1991; Lafevre et al., 1998a,b). The roles of these IFN have not been established, but results of the present study indicate that they may be involved in the activation of some of the same IFN-induced genes previously shown to be upregulated during early pregnancy in ruminants.
In the endometrium of gilts, Western blot analysis using the carboxyl terminus oMx antiserum revealed multiple bands (approximately 45, 60, 72, and 80 kDa), one of which (60 kDa band) was apparently brighter in pregnant than in cyclic gilts. Slot-blot analysis of steady-state levels of Mx mRNA showed a greater than twofold increase in Mx gene expression in d-16 pregnant vs. d-16 cyclic gilts. This increase occurred around the time that markedly elevated levels of antiviral activity were detectable in porcine uterine flushes and conceptus cultures (Mirando et al., 1990; Short et al., 1992). These results indicate that Mx protein and mRNA are present in the swine uterus during early pregnancy and are upregulated in d-16 pregnant gilts at the time that conceptus IFN secretion peaks (Short et al., 1992). Interestingly, whereas steady-state levels of Mx mRNA were not different between cyclic and pregnant gilts on d 18, as determined by slot-blot analysis, results from in situ hybridization analysis revealed a markedly different pattern of Mx mRNA localization. On d 18 of the cycle, Mx was primarily in the luminal and superficial glandular epithelium, whereas on d 18 of pregnancy, hybridization was increased in the stroma and reduced in the luminal epithelium. This pattern is distinct from that reported previously for ewes (Ott et al., 1998) and for cows in the present study.
In mares, the signal for pregnancy recognition is poorly defined (Sharp, 1989b; Bazer et al., 1998), but apparently does not involve conceptus-produced IFN (Sharp, 1989a; Baker et al., 1991). Northern blot analysis revealed an mRNA of approximately 2.5 kb that comigrated with the Mx mRNA present in ewe, cow, and gilt endometrial RNA. However, slot-blot analysis of steady-state levels of Mx mRNA revealed very low levels of Mx expression that were not different between cyclic and pregnant mares. Consistent with this observation, a carboxyl terminus oMx antiserum detected multiple faint bands of the approximate size for Mx in both cyclic and pregnant mares (approximately 60, 72, 75, and 82 kDa). There was no apparent difference in the level of protein expression between cyclic and pregnant mares. In situ hybridization analysis revealed that Mx was confined primarily to the luminal epithelium. Although these results confirm that Mx protein and mRNA are expressed in the endometrium of mares, they do not indicate that Mx is induced during this period of early pregnancy—results that are highly consistent with the absence of conceptus-secreted IFN in this species (Sharp, 1989a; Baker et al., 1991). However, unlike ovine, bovine, and porcine conceptuses, the horse conceptus does not elongate during pregnancy-recognition signaling. Gene expression in endometrium closely associated with the equine conceptus should be studied, particularly after d 16, when the conceptus becomes immobilized in one uterine horn (Ginther et al., 1983), to determine the effects on uterine Mx expression.
This study demonstrated that Mx protein is expressed in the uterus of cows, gilts, and mares and is strongly upregulated in pregnant cows, as in ewes. The Mx protein expression was apparently upregulated to a lesser extent during pregnancy in gilts, a species in which conceptuses secrete IFN, but which utilizes estrogen for pregnancy recognition. Two to four bands of Mx protein were identified in the endometrium of the gilt, cow, mare, and ewe. These observations indicate that there are multiple Mx genes expressed in the uterus, or that there are differences in post-transcriptional mRNA processing that result in splice variants, or that proteolytic cleavage products of Mx are being detected. In support of the former possibility, other species express two to three distinct Mx genes in response to viral infection (Horisberger and Gunst, 1991; Melen et al., 1996). However, whether multiple Mx genes are expressed in the uterus during early pregnancy in the species studied here remains to be determined.
Results from this experiment indicate that Mx may be regulated differently in gilts than in cows and ewes, possibly by a direct effect of the conceptus interacting with the uterine wall or because Mx expression increases in response to conceptus-secreted IFN- and/or - between d 12 and 18 (Lefevre et al., 1998a). In addition, early pregnancy apparently reduced Mx expression in the lumenal epithelium, while increasing it in the stroma in gilts, indicating a more complex regulation than that described for ewes and cows.
The Mx protein expression in the uterus of domestic ungulate species other than sheep has not previously been reported, and evidence that Mx expression is regulated by factors other than Type I interferon or viral infection has not previously been postulated. Although Mx is expressed in the uteruses of these domestic species, the mechanism of regulation and the function of Mx in the uterus during early pregnancy remain to be determined.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
2 Idaho Agric. Exp. Stn. manuscript No. 02A04.
3 Current address: College of Veterinary Medicine, Texas A&M University, College Station 77843.
4 Current address: National Research Initiative Competitive Grants Program, USDA, 1400 Independence Ave. SW, STOP 2241, Washington, DC 20250-2241.
Received for publication August 7, 2002. Accepted for publication January 23, 2003.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
by the uterine endometrium. Prostaglandins 14:397–400.[Medline]
). Biol. Reprod. 60(Suppl. 1):211.
This article has been cited by other articles:
|
|
 |
|
  C.A. Gifford, A.M. Assiri, M.C. Satterfield, T.E. Spencer, and T.L. Ott Receptor Transporter Protein 4 (RTP4) in Endometrium, Ovary, and Peripheral Blood Leukocytes of Pregnant and Cyclic Ewes Biol Reprod, September 1, 2008; 79(3): 518 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Joyce, R. C. Burghardt, R. D. Geisert, J. R. Burghardt, R. N. Hooper, J. W. Ross, M. D. Ashworth, and G. A. Johnson Pig Conceptuses Secrete Estrogen and Interferons to Differentially Regulate Uterine STAT1 in a Temporal and Cell Type-Specific Manner Endocrinology, September 1, 2007; 148(9): 4420 - 4431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Stevenson, J. C. Dalton, T. L. Ott, K. E. Racicot, and R. C. Chebel Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers J Anim Sci, September 1, 2007; 85(9): 2163 - 2172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Joyce, J. R. Burghardt, R. C. Burghardt, R. N. Hooper, L. A. Jaeger, T. E. Spencer, F. W. Bazer, and G. A. Johnson Pig Conceptuses Increase Uterine Interferon-Regulatory Factor 1 (IRF1), but Restrict Expression to Stroma Through Estrogen-Induced IRF2 in Luminal Epithelium Biol Reprod, August 1, 2007; 77(2): 292 - 302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, E. Antoniou, Z. Liu, L. B Hearne, and R M. Roberts A microarray analysis for genes regulated by interferon-{tau} in ovine luminal epithelial cells Reproduction, July 1, 2007; 134(1): 123 - 135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Gifford, K. Racicot, D. S. Clark, K. J. Austin, T. R. Hansen, M. C. Lucy, C. J. Davies, and T. L. Ott Regulation of Interferon-Stimulated Genes in Peripheral Blood Leukocytes in Pregnant and Bred, Nonpregnant Dairy Cows J Dairy Sci, January 1, 2007; 90(1): 274 - 280. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bauersachs, S. E Ulbrich, K. Gross, S. E M Schmidt, H. H D Meyer, H. Wenigerkind, M. Vermehren, F. Sinowatz, H. Blum, and E. Wolf Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity. Reproduction, August 1, 2006; 132(2): 319 - 331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Klein, S. Bauersachs, S. E. Ulbrich, R. Einspanier, H. H.D. Meyer, S. E.M. Schmidt, H.-D. Reichenbach, M. Vermehren, F. Sinowatz, H. Blum, et al. Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period Biol Reprod, February 1, 2006; 74(2): 253 - 264. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
