JOURNAL OF INTERFERON RESEARCH 13:303-309 (1993)Mary Ann Lieber!, Inc., Publishers

Recombinant Interferon-T Regulates Secretion ofTwo Bovine Endometrial Proteins

BO R. RUEDA,1-2 KEITH A. NAIVAR,1 ERIC M. GEORGE,1 KATHY J. AUSTIN,1 HATTIE FRANCIS,3and THOMAS R. HANSEN1

ABSTRACT

Conceptus-derived interferon-T (IFN-t) has been implicated in the process of maternal recognition of preg-nancy in the bovine. This type IIFN interacts with a uterine receptor complex to elicit secondary maternalresponses, one of which is secretion of uterine proteins. We investigated the effects of pregnancy and recombi-nant (r) bovine (bo) IFN-t (107 antiviral units/mg) on secreted proteins by cultured bovine endometrialexplants. Explants were cultured for 24 h with [3H]Ieucine and dialyzed medium analyzed by one- andtwo-dimensional SDS polyacrylamide gel electrophoresis (PAGE) and fluorography. In one-dimensionalPAGE experiments, endometrium representing early pregnancy (days 16-21) and the estrous cycle (days16-19) was cultured in the presence of 5 nM rboIFN-T and showed an increase (two- to five-fold) in secretion of12- and 28-kD proteins. Further examination of these proteins by using two-dimensional PAGE indicated thatthe 12-kD protein was basic (pi > 7.5), whereas the 28-kD protein was acidic (pi ~ 5.0). Isoelectric focusing inthe acidic range revealed that the 28-kD protein was composed of several isoelectric variants (pi 4.5-5.5).Although the functions of these secretory proteins are currently unknown, they serve as useful markers for IFNaction and may act as secondary signals to protect the early developing conceptus.

INTRODUCTION

Interferons (iFNs) ARE CYTOKINES that have complex bio-logical effects besides protecting cells from viral infec-

tion."-2' Type I IFNs were originally classified as a, ß, or co

based on cell of origin and sérologie characteristics. This clas-sification has recently been expanded to encompass the majorsecretory proteins of preimplantation bovine and ovine concep-tuses. These conceptus proteins were originally named tropho-blastin or trophoblast protein-1. They were later identified as

IFN-t3 because these and other type I IFNs differed in aminoacid sequence, number of genes, binding to receptors, organiza-tion of promoters on genes, and distribution among species/3-7'

Bovine (bo) IFN-t is secreted by the conceptus from days16-24 of pregnancy.'8-9' During this period, bofFN-T attenu-ates the frequency and magnitude of prostaglandin F2ct release,which would otherwise cause luteolysis of the corpus luteum,terminate the pregnancy, and initiate a new estrous cycle/9-10'Receptors for IFN-t in ovine endometrium differ in complexityand binding affinity when compared with receptors for IFN-ct.(6> Thus, an avenue exists through which IFN-t might com-

municate with the maternal uterine endometrium to regulatesynthesis of secretory products.

After the cDNA encoding boIFN-T was isolated, it was pos-sible to study gene structure'5' and to produce and characterizenonglycosylated rboIFN-T."" Purity of rboIFN-T was con-

'Department of Animal Science, University of Wyoming, Laramie, WY 82071; 2present addrerss: Department of Physiology, College ofMedicine, University of Arizona, Tucson, AZ 85724; department of Animal Science, University of Missouri, Columbia, MO 65211.

303

304 RUEDA ET AL.

firmed by one-dimensional polyacrylamide gel electrophoresis(PAGE) analysis and by the presence of a single protein bandfollowing silver staining and Western blotting. Direct aminoacid microsequencing of rboIFN-T indicated that sequence was

identical to native boIFN-T. Recombinant boIFN-T binds bo-vine endometrial receptors (R.M. Roberts, University of Mis-souri, personal communication) and is more potent than nativeboIFN-T in inhibiting synthesis of prostaglandins by culturedbovine endometrial cells.'12'

Once boIFN-T was synthesized in ample amounts as a recom-

binant, bioactive protein, it became possible to study its effectson protein sythesis by the endometrium. The present study was

designed to determine the effects of rboIFN-T on synthesis ofproteins by cultured bovine endometrium during the estrous

cycle and early pregnancy.Implantation is noninvasive in cattle and begins as early as

days 18-20 of pregnancy,'13-15' a period during which boIFN-Tis produced in maximal amounts. In cattle, the trophoblast andendometrium fuse to form placentomes. The placentome iscomposed of maternal caruncles and fetal cotyledons, whichallow localized nutrient and hormonal exchange.'13-15' Thus,caruncular and intracaruncular tissues from day 21 pregnantcows also were examined for the capacity to secrete proteins inresponse to rboIFN-T.

MATERIALS AND METHODS

Materials: L-[3,4,5-3H]leucine (155 Ci/mmole) and fluoro-graphic enhancer (EN3HANCE) were from New England Nu-clear (Boston, MA). Culture medium, amino acids, and sup-plements were from Sigma (St. Louis, MO). Ampholines fortwo-dimensional PAGE were from Pharmacia LKB Biotech-nology (Piscataway, NJ). Lutalyse (PGF2a) was from Upjohn(Kalamazoo, MI). Recombinant doIFN-t was provided by Dr.R. M. Roberts (University of Missouri, Columbia). All otherchemicals were reagent grade or better.

Animals: Thirty multiparous cows (Angus-crossbred, 5-7years old) were administered PGF2ot to synchronize estrous

cycles and monitored twice daily for estrous activity. Thoserandomly assigned to the pregnant group were inseminatedartificially 12 h following standing estrus. Reproductive tractswere collected from cows on days 16, 17, 18, and 19 of theestrous cycle (numbers = 3, 3, 3 and 1, respectively) and days16, 17, 18, 19, 20, and 21 of pregnancy (numbers = 2, 4, 1, 3,4, and 6, respectively). Uterine homs were flushed with Dul-becco's phosphate buffered saline (PBS) solution. Pregnancywas verified by the presence of a conceptus.

Expiant Culture: Endometrium was removed by standardprocedures, minced into small pieces, rinsed twice with Dul-becco's PBS flushing media and once with Eagle's minimalessential medium (MEM) lacking serum and limiting in leucine(one-tenth normal MEM concentration of leucine). In additionto whole expiants, endometrium representing day 20-21 ofpregnancy also was subdivided into caruncular and intercarun-cular tissue and cultured. Tissue (500 mg) was placed in culturedishes (60 x 15 mm) with 5 ml of leucine-deficient medium

and 100 uCi of L-[3,4,5,3H]leucine for incorporation intonewly synthesized proteins.

The Kd for IFN-t receptors ranges from 10"10 to 10- ' ' M.'6'Thus, rboIFN-T was added to cultures at 0, 0.5, and 5 r\Mlevels. Cultures were performed as previously de-scribed.'8-16-17' Medium from each culture was collected, cen-

trifuged (2,500 x g, 20 min), treated with the proteaseinhibitor phenylmethylsulfonyl fluoride (0.1 mAi final concen-

tration), dialyzed (MT cutoff 6,000) against distilled water(0.1% wt/vol sodium azide) and stored at —20°C. Nondiffus-ible, and presumably macromolecular, components in the cul-ture medium were analyzed for incorporation of radiolabel intoprotein by liquid scintillation and spectrophotometric absor-bance (A2g0). The specific activity of radiolabeled proteins av-

eraged 1,172 ± 149 cpm/u,g protein and was not affected bystage of estrous cycle, pregnancy, or rboIFN-T treatment.

One-Dimensional SDS-PAGE: For one-dimensional PAGEanalysis, 10,000 cpm from each culture was lyophilized, recon-

stituted in Laemmli buffer,'18' boiled for 3 min, and loaded andseparated on reducing 15% polyacrylamide gels. Gels were

stained with Coomassie Blue R-250, impregnated with a fluoro-graphic enhancer, dried, placed on Kodak XRP film, and ex-

posed for 2 weeks at—

80°C. For ease of presentation andanalysis of one-dimensional PAGE results, days of pregnancyand the estrous cycle were grouped as follows: day 17 (16-17),day 19 (18-19), and day 21 (20-21).

Validation of the densitometry (LKB densitometer) for sensi-tivity and range of detection was accomplished by separatingtwo-fold amounts of labeled endometrial secretory proteins(1,000-64,000 cpm) on 15% one-dimensional PAGE gels andsubsequent fluorography. Labeled proteins from three cows

were loaded on separate gels and the resulting fluorographsscanned in the 28-kD protein area (gels placed on film for 2weeks at —80°C). Optical density values (area) were analyzedfor each level of protein loaded on gels. Detection of signals on

fluorographs was linear (Y = 0.002X-

0.17; R2 = 0.99) from1,000 cpm to 64,000 cpm protein loaded and from 0-14 opticaldensity units (area) detected. All subsequent one-dimensionalPAGE were evaluated at 10,000 cpm per lane with a 2-weekexposure time.

Two-Dimensional SDS-PAGE: Two-dimensional PAGE was

conducted as described by Roberts et a/.'19' Briefly, 250,000cpm (labeled protein) equivalence was loaded on isoelectricfocusing gels and proteins separated by isoelectric point. Tofacilitate two-dimensional PAGE analysis, equivalent cpm was

pooled to constitute the 250,000 cpm loaded on gels represent-ing each day of the estrous cycle (4 days evaluated: days 16, 17,18, and 19), day of pregnancy (6 days evaluated: days 16, 17,18, 19, 20, and 21), and dose of rboIFN-T (0 and 5 nM).Second-dimension separation was done on 15% PAGE gels thatwere impregnated with a fluorographic enhancer, dried, andplaced on Kodak XRP film for 2 months at -80°C.

Statistical Analysis: Optical density values from fluoro-graphs following one-dimensional PAGE were combined intomeans and analyzed by factorial analysis of variance [maineffects: day of estrous cycle or pregnancy (day), pregnancy,and rboIFN-T]. Main effects that were shown to be significant(P < 0.10) by analysis of variance were evaluated further byr-test.'20'

IFN-t REGULATES UTERINE PROTEINS 305

RESULTS

One-dimensional SDS-PAGEA fluorograph representing secreted proteins following cul-

ture on day 17 of the estrous cycle or pregnancy in the absenceor presence of 5 nM rboIFN-T is illustrated in Fig. 1. Two bandsof protein were consistently enhanced following culture withrboIFN-T. The proteins were estimated to be about 12- and 28-kD in size. Since 12- and 28-kD proteins appeared on one-

dimensional PAGE gels following both reducing and nonreduc-ing conditions, the 12-kD protein did not appear to be a disul-fide-linked subunit of the 28-kD protein (data not shown). Aslight enhancement of both proteins was apparent in culturesfrom pregnant (Fig. 1, lane 3) when compared to nonpregnantcows (Fig. 1, lane 1) in the absence of rboIFN-T. However, thisdegree of enhancement was not consistent throughout all of thefluorographs evaluated.

Dose of rboIFN-t: The effect of dose of rboIFN-T was ana-

lyzed by using day-17 endometrium collected from pregnantcows. Signals for both the 12- and 28-kD proteins increased(P < 0.05) as the amount of rboIFN-T added to each culturewas increased. Since the 5 nM dose of rboIFN-T seemed to elicitthe greatest response, this dose was used in subsequent experi-ments.

12-kDa Protein: Only rboIFN-T (P < 0.001) and day(P < 0.10) of estrous cycle or pregnancy affected secretion ofthe 12-kD protein. The main effect, pregnancy, and interactionsin the factorial model were not significant (P > 0.10). Lowsignals for the 12-kD protein were evident in cultures fromendometrium representing the estrous cycle on days 17 and 19(Fig. 2A). When rboIFN-T was added to these cultures, synthe-sis of the 12-kD protein increased (P < 0.01) only in culturesrepresenting day 19. Low levels of the 12-kD protein were

evident in control cultures from pregnant animals on all daystested (Fig. 2C). A marked enhancement (P <0.01) in thesynthesis of the 12-kD protein was noted following culture withrboIFN-T regardless of stage of pregnancy tested. When com-

paring control (0 nM rboIFN-T) nonpregnant with control preg-nant cultures, no difference in the amount of the 12-kD proteinwas detected.

28-kD Protein: Secretion of the 28-kD protein was affectedby rboIFN-T (P < 0.0001), day of estrous cycle or pregnancy(P < 0.001), day X rboIFN-T (P < 0.01), and pregnancy xrboIFN-T (P < 0.10), but were not affected by other main ef-fects or interactions in the model. The 28-kD protein was se-creted by control days 17 and 19 endometrium from nonpreg-nant cows, and levels were higher (P < 0.01) following culturewith rboIFN-T (Fig. 2B). In endometrium collected from preg-nant cows, rboIFN-T caused an increase (F < 0.001) in synthe-sis of the 28-kD protein on day 17 and tended (P < 0.08) toelicit an increase on days 19 and 21 (Fig. 2D). The capacity ofthe endometrium to respond to rboIFN-T declined with advanc-ing stages of pregnancy. When comparing expression of the28-kD protein between pregnant and nonpregnant cows, nodifference in amount synthesized was detected.

Caruncular and Intercaruncular Tissue: Significant main ef-fects and interactions in the model for the 12-kD protein were:

rboIFN-T (P< 0.001), endometrial type (P<0.10), andrboIFN-T x endometrial type (P < 0.10). Significant main ef-fects in the model for the 28-kD protein were: rboIFN-T(P < 0.001) and endometrial type (P < 0.05). Treatment withrboIFN-T increased synthesis of both 12- and 28-kD proteins byintercaruncular (P < 0.01) and caruncular (P < 0.001) tissue(Fig. 3). There was no difference between caruncular and inter-caruncular tissue in the amount of these proteins synthesized bycontrol cultures. However, caruncular tissue synthesized more

28-kD (P < 0.05) and tended to synthesize more 12-kD(P<0A) protein following rboIFN-T treatment than inter-caruncular tissue.

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FIG. 1. One-dimensional SDS-PAGE (15% gels) analysis ofradiolabeled uterine secretory proteins. Each lane was loadedwith an equivalent amount (10,000 cpm) of radiolabeled pro-tein. Fluorographs were exposed to XRP Kodak film for 2weeks. Lanes 1-4 (all represent days 16-17) are identified asfollows: lane 1, non-pregnant, control; lane 2, non-pregnant, 5nM rboIFN-T; lane 3, pregnant control; lane 4, pregnant, 5 nMrboIFN-T. The dark arrow identifies enhancement of a band of28-kD proteins when cultured with rboIFN-T. The light arrowidentifies enhancement of a band of 12-kD proteins when cul-tured with rboIFN-T.

Two-dimensional SDS-PAGE

Fluorographs in Fig. 4 represent two-dimensional PAGEanalysis of secreted and radiolabeled endometrial proteins. Incultures representing day 16 of pregnancy (Fig. 4A,B), therewas an increase in secretion of an acidic (pi ~ 5.0) 28-kDprotein (dark arrow) and a basic (pi s= 7.5) 12-kD protein (lightarrow) following treatment with rboIFN-T. In the absence ofrboIFN-T, the first significant signal for the 28-kD protein wason day 17 of pregnancy. This 28-kD signal became enhanced as

pregnancy progressed (Fig. 4E, Fig. 5A). The 12-kD proteinwas on all gels examined representing the estrous cycle or

pregnancy. In the presence of rboIFN-T, synthesis of the 28-kDprotein was enhanced on all gels representing days 16-19 of theestrous cycle and days 16-21 of pregnancy. Likewise, synthe-sis of the 12-kD protein was enhanced on all gels except day 19of the estrous cycle following treatment with rboIFN-T.

To further characterize the 28-kD protein, isoelectric focus-ing was conducted within the acidic range to accomodate pro-teins with pi values of 4-6. Figure 5 represents synthesis of the28-kD protein by endometrium representing day 21 of preg-

306 RUEDA ET AL.

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FIG. 2. Quantitative densitometric analysis of one-dimensional SDS-PAGE (15% gels) fluorographs. Equivalent radiolabeledprotein (10,000 cpm) was loaded on one-dimensional PAGE gels, and resulting fluorographs (exposure time 2 weeks) werescanned in 12- and 28-kD-regions. Radiolabeled proteins (12-kD, A and C; 28-kD, B and D) were analyzed following culture ofendometrium representing the estrous cycle (A,B) and pregnancy (C,D). Values represent the mean ± SE. Means within day differ(P < 0.05) when depicted with an asterisk.

nancy in the absence (A) or presence (B) of 5 nM rboIFN-T.Expression of the 28-kD protein was determined by two-dimen-sional PAGE and fluorography appeared to be highest on day 21of pregnancy following culture with rboIFN-T and to consist ofseveral isoelectric variants (Fig. 5B). Synthesis of the 28-kDprotein was apparent on all two-dimensional PAGE gels (sixgels) representing days 16-21 of pregnancy and on only one offour gels (i.e., day 19) representing days 16-19 of the estrous

cycle.

DISCUSSION

We have identified two secretory proteins (~ 12-kD, basicand ~28-kD, acidic) that are synthesized by the endometriumof pregnant cows in response to rboIFN-T. Although functionsof these proteins are unknown, we have demonstrated that theyserve as useful markers for IFN-t action on bovine endo-metrium. The 28-kD protein may be specific to pregnancy butbecause of the small two-dimensional PAGE sample size, this

IFN-t REGULATES UTERINE PROTEINS 307

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UTERINE PROTEINS (Mr X 10"3)FIG. 3. Synthesis of 12- and 28-kD proteins in caruncularand intercaruncular tissues from endometrium representing day21 of pregnancy. Radiolabeled proteins (10,000 cpm) wereloaded on 15% one-dimensional SDS-PAGE gels and resultingfluorographs (exposed for 2 weeks) scanned with an LKB den-sitometer in 12- and 28-kD regions. Values represent means ±SE of 10 separate determinations (i.e., endometrial culturesfrom 10 different pregnant cows on day 21). Means within Mrclass differ (P < 0.05) when designated with an asterisk.

could not be tested. There also is a differential response torboIFN-T in caruncular and intercaruncular tissues.

In the cow, the conceptus is relatively free-floating in theuterine lumen until about day 18 of pregnancy.'13-15' From days18 to 20 of pregnancy, cytoplasmic protrusions from the con-

ceptus engage intercaruncular tissues to keep the conceptus inclose contact with histotrophic secretions.'21' Caruncular tis-sues are infiltrated by chorionic villi to form the placentome.Caruncles contain uterine glands, and by virtue of their anatom-ical relationship to chorionic tissue, contribute significantly tomaternal-fetal exchange of nutrients and hormones. In thisstudy, caruncular tissue had a higher capacity to synthesize bothuterine proteins in response to rboIFN-T than intercarunculartissue from day-21 pregnant cows. The responsiveness ofcaruncular tissue to rboIFN-T probably reflects the secretorynature of this tissue and communication between the conceptusand maternal systems. It also may be due to a greater number ofhigh-affinity receptors for boIFN-T in this tissue.

The 28-kD protein is secreted by endometrium as early as day17 of pregnancy and is further enhanced following exogenoustreatment with rboIFN-T. Although one-dimensional PAGE re-

vealed significant signals for the 28-kD protein by days 16-17of pregnancy and the estrous cycle, this analysis represents bothbasic and acidic polypeptides within the same one-dimensionalPAGE band of proteins. Presence of basic radiolabeled proteinsin the 28-kD band of proteins confounds interpretation and mayexplain why analysis by using one-dimensional PAGE did not

differ between pregnant and nonpregnant tissues. The decline inresponsiveness of endometrium to rboIFN-T as pregnancy pro-gressed also may have been due to confounding effects ofexpression of basic radiolabeled proteins. It also could be due to

down-regulation/desensitization of the boIFN-T receptor/signaltransduction system caused by large amounts of boIFN-T fromthe growing conceptus'8-10' and addition of 5 nM rboIFN-T invitro.

Other investigators'22-24' have described regulation of 10- to15-kD uterine proteins by conceptus secretory proteins. The12-kD protein described in this study may be similar to an

acidic (pi 6.0) 12-kD protein in the ewe recently described byVallet et a/.'25' as ß2-microglobulin-like. However, the bovine12-kD protein identified by our group is much more basic(pi 2* 7.5) than the protein described by Vallet et a/.'25-26'Attempts to immunoprecipitate and Western blot the bovineprotein with antisera against human ß2-microglobulin havebeen unsuccessful (data not shown). Thus, it is not currentlyknown if the acidic 12-kD protein isolated from the ewe byVallet et a/.'25' is the same as the basic 12-kD protein describedin the cow by our group.

Also, in the ewe, an acidic, high MTprotein (~70-kD, pi 4) issecreted during early pregnancy and regulated by ovine IFN-tand rboIFN-T.'11-16-17-26' This protein was not consistentlypresent following addition of bovine conceptus secretory pro-teins to cultured endometrium in the cow,'23' or followingrboIFN-T treatment in the present experiment. This differentresponse to the IFN-t may involve a general IFN transducingpathway which is activated by type I IFNs. It may also involvespecies- or IFN-specific transducing pathways and transcriptionfactors.'27-28' We are currently studying endometrial responsesto other IFNs because binding to endometrial IFN receptorsappears to be a complex'6' and dynamic'29' process.

In summary, we have identified two novel uterine proteinsthat are regulated by rboIFN-T. To our knowledge, this is thefirst report of regulation of these endometrial proteins byrboIFN-T. The 12-kD protein is not specific to endometriumfrom pregnant cows. Based on the limited two-dimensionalPAGE analysis in this study, the 28-kD protein might be spe-cific to endometrium from pregnant cows and is produced byday 17 of pregnancy. However, a broader isoelectric focusingthat encompasses basic pis, and a larger two-dimensionalPAGE sample size may be required to resolve these questions.The 28-kD protein exists as several different isoforms (pi 4.5-5.5) that may reflect glycosylation and/or multiple gene prod-ucts. We conclude that these uterine proteins serve as usefulmarkers for IFN-t action. It also is probable that these uterineproteins deliver secondary signals that aid in the establishmentand protection of early bovine pregnancy.

ACKNOWLEDGMENTS

The authors thank Dave Moore, Donalee Martin, DeeHakert, Mel Riley, and Shawn Moore for assistance in collect-ing tissue. This work was supported in part by BiomédicalResearch Grant SO7RR07157-15, Faculty Grant in Aid from

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E «fe <3> FFIG. 4. Representative two-dimensional PAGE gels (15%) and resulting fluorographs of secreted and radiolabeled proteins fromcultured endometrial expiants. Fluorographs represent radiolabeled proteins following culture of endometrium from day-16pregnant (A,B), day-17 non-pregnant (C,D), and day-18 pregnant (E,F) cows either in the absence (A,C,E) or presence (B,D,F) of5 nM rboIFN-T. The dark arrow identifies the acidic 28-kD protein, whereas the light arrow identifies the basic 12-kD protein.Non-radiolabeled albumin is represented by the letter a. The isoelectric focusing gradient was generated in a broad range (pi 4-7.5)to illustrate both 12- and 28-kD proteins.

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BFIG. 5. Representative two-dimensional PAGE analysis of radiolabeled endometrial proteins in the acidic range. Endometriumrepresenting days 20-21 of pregnancy was cultured either in the absence (A) or presence (B) of 5 nM rboIFN-T. Non-radiolabeledalbumin is represented by the letter a. The dark arrow identifies the 28-kD protein. The isoelectric focusing gradient was generatedin the acidic range (pi 4-6) to illustrate 28-kD isoelectric variants.

IFN-T REGULATES UTERINE PROTEINS

the University of Wyoming and USDA grant 9102611 (to Dr.T.R. Hansen). This is Wyoming Agricultural Experiment Sta-tion Paper # JA 1680.

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Address Reprint Requests to:Dr. T.R. Hansen

Department of Animal Science112 Animal Science/Molecular Biology Building

P.O. Box 3684University of Wyoming

Laramie, WY 82071

Received 28 September 1992/Accepted 17 May 1993