Quantification and Cellular Localization of Ovine Placental Lactogen Messenger Ribonucleic Acid Expression during Mid- and Late Gestation* STEVEN M. KAPPESt, WESLEY RUSSELL V. ANTHONY Department

of

Animal

C. WARRENS,

Sciences, University

of

Missouri,

SCOTT L. PRATT, Columbia,

Missouri

RONGTI

LIANG,

AND

65211

ABSTRACT Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supportive role in fetal growth. This study examined the concentration and cellular location of oPL mRNA at five stages of pregnancy (days 60,90, 105, 120, and 135) in 21 cross-bred ewes, and results were compared to maternal and fetal serum oPL concentrations, cotyledonary DNA and actin mRNA concentrations, and total fetal weight. The concentration of oPL mRNA in fetal cotyledonary tissue increased (P 5 0.05) from day 60 (15.4 pg/pg total cellular RNA) to day 120 (73.7 pg/pg total cellular RNA) of gestation and then plateaued, whereas no significant changes occurred in the concentration of actin mRNA over the gestational ages examined. The concentration of DNA in cotyledonary tissue (micrograms per mg wet tissue) increased (P 5 0.05) from days 60 through 120 and remained constant through day 135, such that when oPL mRNA was expressed on a picogram per rg DNA basis, no stage of gestation effect (P 2 0.10) was observed. The maternal serum oPL concentration increased (P s 0.05) from day 60 (7.1 rig/ml) to day 105 (417.7 rig/ml), followed by a large but nonsignificant (P 2 0.10) increase in maternal serum oPL occurring on day

135 (902.0 rig/ml). Fetal serum oPL concentrations increased (P 5 0.05) from day 60 (11.0 rig/ml) to day 90 (29.0 rig/ml) and then remained relatively constant. Maternal serum oPL (r = 0.68; P 5 0.01) and cotyledonary oPL mRNA levels (r = 0.61; P 5 0.05) were correlated with total fetal weight when adjusted for fetal number and gestational age, and together accounted for 80.6% (r* value) of the variation found in total fetal weight. The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (P 5 0.10). Examination of placentome cross-sections by immunocytochemistry and in situ hybridization at the five gestational ages indicated that the chorionic binucleate cell was the sole source of oPL. These data provide evidence that, like maternal serum concentrations of oPL, oPL mRNA expression by chorionic binucleate cells increases until late gestation, whereas fetal serum concentrations of oPL plateau during midgestation. The secretion of oPL into maternal and fetal circulations appears to be regulated separately, and secretion into the fetal circulation may result from a population of binucleate cells that do not migrate into the maternal-fetal interface of the placentome. However, transcriptional control of the oPL gene within binucleate cells has yet to be defined. (Endocrinology 131: 2829-2838, 1992)

0

tions by days 120-135 (10-13). Although a general pattern of oPL release into the fetal and maternal circulations emerges from these studies, considerable variation exists among the various sets of data. Some of the variation associated with maternal serum levels has been accounted for by the nutritional status (14, 15), litter size (12, 13, 16, 17), and breed (16) of the ewe, making it difficult to comparematernal serum 0PL levels acrossexperiments. Discrepanciesexist as to whether the chorionic epithelial primary or binucleate cells produce oPL. In a seriesof experiments using polyclonal antibodies prepared against oPL, some investigators identified chorionic primary cells as the site of oPL production (9, 18), while others determined it to be a product of chorionic binucleate cells (18-20). Wooding (19) localized oPL to the secretory granules of binucleate cells by electron microscopy, whereas Chan et al. (21) localized oPL to the chorionic primary cells using a monoclonal antibody prepared against 95% or more pure oPL. The identification of the chorionic cell type(s) that expressesoPL mRNA has not been reported. Migration of chorionic binucleate cells and fusion with the syncytial interface of the placentome (22-25) have been implicated as the route by which oPL is delivered to the maternal circulation, whereas the route of oPL delivery to the fetal circulation has not been defined.

VINE placental lactogen (oPL) is a secretory protein of placental origin that has both lactogenic and somatotropic activities in vitro (l-4). Structurally, 0PL is more similar to PRL than it is to GH (5,6), but an accumulation of evidence (7) suggeststhat oPL may function in altering maternal and fetal growth and metabolism during pregnancy. oPL is first detected in day 16 trophoblastic tissue (8, 9) and is found in the allantoic fluid (10) by day 18 (1 l), and in maternal and fetal circulations after 40 days gestation (11). The fetal serum oPL concentration appearsto reach its maximum by 80-120 days gestation (10-13). Maternal serum concentrations are lower than fetal concentrations until 70-90 days gestation, at which time they increaserapidly to maximum concentraReceived June 19, 1992. Address requests for reprints to: Dr. R. V. Anthony, Department of Animal Sciences, 164 Animal Sciences Center, University of Missouri, Columbia, Missouri 65211. * This work was supported in part by USDA-CSRS Grant 88-372403904 and Cooperative Agreement 58-5438-9-001 between the USDAARS U.S. Meat Animal Research Center and the University of Missouri. This manuscript is Journal Series 11,662 of the Missouri Agricultural Experiment Station. t Present address: U.S. Meat Animal Research Center, Box 166, Clay Center, Nebraska 68933. $ Present address: Monsanto Co., 800 North Lindberg Boulevard, St. Louis, Missouri 63197.

2829

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EXPRESSION

2830

The availability of oPL mRNA-specific cDNAs (5, 6) has allowed us to examine the ontogeny of oPL mRNA expression during mid- and late gestation, compare oPL mRNA expression to maternal and fetal serum oPL concentrations and fetal weight, and examine the cellular source of oPL. Materials

and Methods

Materials Nitrocellulose membranes were obtained from MS1 (Westboro, MA), and nylon membranes from Cuno, Inc. (Meridian, CT). The in vitro transcription kit, pBluescript SK(-), and restriction enzymes were acquired from Stratagene (La Jolla, CA), and the 3’.end-labeling kit and ProtoBlot immunoscreening system from Promega (Madison, WI). The multiprime labeling kit and [“Pldeoxy-CTP were purchased from Amersham (Arlington Heights, IL), and Na”‘1 and [3’S]deoxy-ATP from New England Nuclear (Boston, MA). Iodogen and diaminobenzidine tetrahydrochloride were obtained from Pierce Chemical Co. (Rockford, IL), and the XRP film, D19 developer, and NTB-2 emulsion from Eastman Kodak (Rochester, NY). Normal goat serum was purchased from Jackson Laboratories (West Grove, PA), and the ABC Elite immunocytochemistry kit from Vector Laboratories (Burlingame, CA). Freund’s adjuvant, hematoxylin and eosin stain, and proteinase-K were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Tissuepreparation Twenty-one mature cross-bred ewes were mated at behavioral estrus and hysterectomized by midventral laparotomy while under halothane anesthesia at various stages of gestation, with four (days 60, 90, 120, and 135) or five (day 105) ewes randomly allotted per group. Blood samples were obtained from the fetal umbilical artery and maternal uterine vein at the time of surgery and allowed to clot at 4 C, and serum was collected by centrifugation at 2800 x g for 10 min and stored at -80 C. Fetal number and individual fetal body weights were recorded at surgery. Six to 10 placentomes were immediately excised from the pregnant uterus, rinsed in ice-cold PBS buffer (10 rnM NaPOa and 0.13 M NaCl, pH 7.4), sectioned into 3- to 5-mm sections with a Stadie Riggs hand-held microtome (Thomas Scientific, Swedesboro, NJ), fixed in icecold 4% paraformaldhyde in PBS for 16-18 h, washed in PBS, dehydrated in a series of graded alcohols, embedded in paraffin, and stored at -20 C (26). The remainder of the cotyledonary tissue was manually separated from the caruncular tissue, rinsed in 4 C physiological saline (0.86% NaCI, wt/vol), frozen in liquid N:, and stored at -80 C until needed for RNA isolation. All procedures were approved by the University of Missouri Animal Care and Use Committee, under Protocol 1041.

RIA Purified oPL (MU-oPL-5) (5) was used to generate polyclonal antiserum (MU-(uoPL-S) in a male New Zealand White rabbit. The rabbit was individually housed and fed Purina rabbit chow (Ralston-Purina, St. Louis, MO) and water ad libitum. Preimmune serum was obtained via the ear vein before multisite SC injection of oPL (250 pg in 1 ml 0.1 M NH,HCO,) that was emulsified with 1 ml Freund’s complete adjuvant. Booster injections of 100 pg purified oPL emulsified with 0.45 ml Freund’s incomplete adjuvant were administered monthly for 6 months, Blood was collected via the ear vein 2 weeks after each booster beginning with the second booster and allowed to clot at 4 C, and serum was collected by centrifugation. Antibody titer was evaluated by an enzymelinked immunosorbant assay. Brieflv, 0.1, 1, 10, 100, and 1000 ng oPL were blotted on nitrocellulose and incubated in the presence of rabbit antisera (1:5,000 to 1:500,000 dilutions). Visible detection of the titer \vas accomplished with the ProtoBlot immunoscreening system. oPL concentrations in maternal and fetal sera were determined by a homologous RIA procedure similar to that described by Chan et al.(11).

OF oPL mRNA

EdI) * 1992 Yr1l131-XI, 6

Purified oPL was iodinated to a specific activity of 60-90 pCi/pg, using 1,3,4,6-tetrachloro-3,6-diphenylglycouril (Iodogen) (5, 27). Serum samples (10, 50, or 100 ~1) or oPL standards (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 5, and 10 rig/tube) were diluted to 200 ~1 in RIA buffer (0.05 M NaP04, 0.15 M NaCI, 1% BSA, and 0.01% NaN3, pH 7.4) and added to 200 ~1 [‘2iI]oPL (50,000 cpm), 200 ~1 oPL antiserum (1:150,000 dilution of MI--aoPL-S4) containing 2% normal rabbit serum, and 200 ~1 RIA buffer. Duplicate tubes for each sample were incubated at 4 C for 24 h before adding 200 ~1 sheep antirabbit immunoglobulin G antiserum (1:15 dilution). The l-ml assay samples were incubated for 24 hat 4 C, and antibody-bound [“iI]oPL was collected by centrifugation at 2,800 X g for 10 min. The supernate was aspirated off, and the pellet was counted in a y-counter. Maximum binding was determined to be 31.76%, and nonspecific binding was 2.3%. Assay specificity was demonstrated by a lack of cross-reactivity in the presence of 1 ng to 10 rg oGH and oPRL. Dilutions of day 109 pregnant ewe serum (1, 2, 5, 7.5, 10, 15, 20, 25, 50, 75, and 100 ~1) and spiked nonpregnant ewe serum (0.1, 0.25, 0.325, 0.5, 0.75, 1, 2.5, 5, 10, and 25 ng oPL) were used to validate the assay for sheep serum. The standard spiked serum and serum dilution curves were logitlog transformed and compared for parallelism by the F test; no differences were found (P 2 0.10). The intra- and interassay coefficients of variation were 5.08% and 6.29%, respectively, for a pool of day 109 pregnant ewe sera (93 f 3.15 rig/ml). The sensitivity of the assay was 100 pg/tube.

RNA isolationand analysis Total cellular RNA (tcRNA) was isolated from cotyledonary tissue, as previously described by this laboratory (5, 28). The concentration (micrograms per mg tissue) of cotyledonary DNA was determined in tissue samples homogenized in 0.1 M PBS (pH 7.4) by the fluorometric assay of Labarca and Paigen (29). For Northern blotting, tcRNA was denatured in the presence of 2.2 M formaldehyde and 50% (vol/vol) deionized formamide, subjected to electrophoresis in a 1.2% agarose gel containing 0.66 M formaldehyde and 1 X MOPS [20 rnM 3-(N-morpholino)propanesulfonic acid, 5 rnM Na,ChH,O,, and 1 rnM EDTA], and electrophoretically transferred to a nylon membrane using 0.5 x TBE buffer (0.45 M Tris-borate and 1 rnM EDTA). The nylon membrane was baked for 2-4 h at 80 C and prehybridized in 50% (vol/vol) deionized formamide, 0.1% (wt/vol) Ficoll 400, 0.1% (wt/vol) polyvinylpyrrolidone (PVI’), 0.1% (wt/vol) BSA, 5 X SSPE (0.75 M NaCI, 50 rnM NaH,P04, and 5 rnM EDTA, pH 7.4), 0.1% (wt/vol) sodium dodecyl sulfate (SDS), and 100 pg/ml denatured salmon sperm DNA for 4 h at 42 C. An 866-basepair oPL cDNA was isolated from its pBluescript SK(-) vector with EcoRI (clone oPL-24) (5), and full-length (2.1 kilobases) y-actin cDNA was isolated from its Okayama-Berg expression vector with BanlHI (30, 31) and random prime labeled with [3’P]deoxyCTP to a specific activity of l-5 x 10” cpm/Fg DNA. The labeled probes were hybridized to the nylon membranes in the presence of 50% deionized formamide, 0.04% (wt/vol) Ficoll 400, 0.04% (wt/vol) PVP, 0.04% (wt/vol) BSA, 5 X SSPE, 0.1% (wt/vol) SDS, and 100 pg/ml denatured salmon sperm DNA for 12-18 h at 42 C. The membranes were washed twice in 2 x SSC (0.3 M NaCl and 30 mM NazChHiO;) and 0.1% (wt/vol) SDS for 20 min at 25 C and once at 42 C for 20 min. Membranes were exposed to XRP film at -80 C for 24 h. oPL and actin mRNA levels were quantified with RNA dot blots, and the standard curves were produced by it1rho transcription of a 533basepair cDNA fragment of oPL-24 (5) and the 2.1.kilobase cDNA of y-actin (pHFrA-1) (30). Varying concentrations (10 pg to 250 ng) of the sense strand transcripts were used to generate a linear standard curve, and pig ovary tcRNA was used as a negative control for the oPL probe. Total cellular RNA was denatured in the presence of 50% (vol/vol) formamide, 6% (vol/vol) formaldehyde, and 20 mM Tris (pH 7.5) and loaded, in duplicate, on nylon membranes that were prewetted with 20 X SSC. The membranes were baked, prehybridized, hybridized with the oPL probe, and washed in the same manner as the Northern blot membranes. The dot blot membranes were initially hybridized with the oPL probe, stripped with 5 rnM Tris (pH 8.0), 0.2 rnM EDTA, and 0.5% (wt/vol) NalPZ07 at 65 C for 2 h, and verified to be clean of the oPL probe by exposing the membranes to XRP film for 3 days at 25 C. The membranes were then reprobed with the labeled y-actin cDNA. Quan-

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EXPRESSION tification of radioactivity per sample was performed with an Ambis radioanalytic imaging system (Ambis System, San Diego, CA).

In situ hybridization The in situ hybridization procedures were similar to those reported by Farin et al. (32). The paraffin-embedded tissues were sectioned (6 pm) and placed on acid-washed slides that had been coated with 0.1% (wt/vol) gelatin and 0.01% (wt/vol) CrKO& and baked at 42 C overnight. The 6-Frn sections represented the full length of the placental villi (from the base of the placentome to the periphery) and were deparaffinized with xylene and postfixed in freshly filtered 4% paraformaldehyde, which was made in PBS (pH 7.4). The sections were then treated with proteinase-K (1 rg/ml in 0.1 M Tris and 50 rnM EDTA) for 20 min at 37 C, rehydrated in a graded alcohol series, and acetylated with 0.1 M triethanolamine-HCl for 10 min and with 0.1 M triethanolamine-HCl and 0.5% (vol/vol) acetic anhydride for 10 min. Sections were washed in 2 X SSC and 0.01% (vol/vol) diethylpyrocarbonate-treated water and dehydrated in 70% and 95% alcohol. Hybridization was performed in 50% (vol/vol) deionized formamide, 10 rnM Tris (pH 7.5), 1 mM EDTA, 0.6 M NaCl, 50 mM DTT, 1.25 mg/ml yeast transfer RNA, 2 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate, 0.02% (wt/vol) WI’, 0.02% (wt/vol) Ficoll 400, and 50 rig/ml 35S-labeled oligonucleotide probe. A 41-base sequence (5’ GGT GAT GGA TGA AGT GTG GCA GTT GAT GAC CTT GGA TTC GG 3’) was selected from the oPL cDNA sequence (5), and antisense and sense oligonucleotides were synthesized and 3’-end labeled with [35S]deoxy-CTP to a specific activity of 5-6.5 X lo8 cpm/pg oligonucleotide. The sense strand probe or RNase-treated sections (200 fig/ml at 37 C for 60 min) were used as negative controls. Twenty microliters of hybridization solution (400,BOO cpm) were placed on each tissue section and covered with an acid-cleaned siliconized coverslip. The coverslip was sealed with rubber cement, and the tissues were hybridized for 16 h at 42 C in a 2 x SSC humidified chamber. The coverslip was removed by soaking the slides in 2 X SSC and removing the rubber cement. The slides were washed in 2 x SSC-50 mM DTT for 15 min at 25 C, posthybridization solution [50% deionized formamide, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.6 M NaCl, and 50 mM DTT] for 1 h at 42 C, 0.1 X SSC-50 rnt.4 DTT for 30 min at 42 C, and 0.1 X SSC-50 mM DTT for 30 min at 25 C. The slides were dehydrated in ethanol, dipped in 50% (vol/vol) autoradiography emulsion, stored at 4 C in a dark dry box for 7-10 days, then developed for 3 min, rinsed in distilled Hz0 for 1 min, fixed for 8 min, and rinsed in running Hz0 for 30 min. The slides were air dried and counterstained with 10% (vol/vol) hematoxylin, washed in distilled H,O, and fixed in 0.5% (vol/vol) NH,OH. The sectioned tissues were then rinsed in distilled Hz0 and 70% ethanol, stained in eosin (1:25 dilution in 80% alcohol and 0.1 M acetic acid), rinsed, dehydrated in 95% and 100% ethanol, and coverslipped. The tissue sections were evaluated and photographed with a Zeiss Axiophot microscope (New York, NY).

OF

oPL

mRNA

The day of gestation and fetal number were used as dependent variables in the model. The independent variables were maternal and fetal serum oPL levels, cotyledonary DNA concentrations, cotyledonary oPL and actin mRNA concentrations, and total fetal weight. The fetal and maternal oPL levels were log transformed to account for the relatedness of the mean and the variance. The means were evaluated for significant differences by calculating the least significant difference. Residual correlation coefficients were determined by Statistical Analysis Systems (34) after adjusting for the effects of stage of gestation and fetal number. Standard partial regression coefficients were calculated to evaluate the effects of maternal and fetal serum oPL levels and oPL mRNA on total fetal weight.

Results

Maternal and fetal serum oPL concentrations, cotyledonary oPL and actin mRNA concentrations, and fetal weight were measured at five gestational stages(days 60, 90, 105, 120, and 135). The specificitiesof the random prime labeled oPL and y-actin cDNAs were assessedon Northern blots of all of the ovine cotyledonary total cellular RNA (tcRNA) samplesobtained at the five gestational agesand of porcine ovarian tcRNA (negative control for oPL cDNA). The oPL cDNA probe hybridized to a single classof mRNA (Fig. l),

A

123456 *

28S-

18S-

6

Immunocytochemistry Immunocytochemistry was completed using an immunoperoxidase procedure (ABC Elite kit) similar to that reported by Hsu et al. (33). Briefly, the sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase was quenched in four 30min incubations of 3% HZ02, and nonspecific binding was blocked by incubating the sections in 2% normal goat serum for 1 h. The sections were then incubated for 18 h with rabbit anti-oPL antiserum (1:25,000 MU-aoPL-S4), rinsed with PBS (pH 7.4), and incubated with biotinylated goat antirabbit immunoglobulin G antiserum and avidin-peroxidase conjugate for 1 h each, and diaminobenzidine tetrahydrochloride was used as the color substrate at 1 mg/ml for 2-5 min. The sections were then counterstained and photographed. The primary antibody was replaced by preimmune rabbit serum (MU-noPL-SP) or normal goat serum as the negative control.

Statistical Data General

analysis

were analyzed Linear Model

by least square analysis procedures of Statistical

of variance, using the Analysis Systems (34).

28S-

18S-

FIG. 1. Representative Northern blot analysis of ovine cotyledonary tcRNA with y-actin (A) or oPL (B) cDNA, demonstrating specificity of hybridization. Lanes l-5 contain 8 fig cotyledonary tcRNA from ewes at 1) 60, 2) 90, 3) 105, 4) 120, or 5) 135 days gestation. Lane 6 contains 8 pg porcine ovarian tcRNA as a negative control for the oPL cDNA probe. The positions of the ribosomal RNA bands are indicated.

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EXPRESSION

2832

approximately 1000 basepairs in length, whereas the y-actin cDNA probe hybridized to a single class of mRNA, approximately 2100 basepairs in length. The fetal serum oPL concentration (Fig. 2) increased (P 5 0.05) from days 60-90 and then remained relatively constant through day 135 (P 2 0.10). Concentrations of oPL in maternal serum increased (P 5 0.05) from days 60-105, with no significant (P 2 0.10) changes thereafter. The maternal serum oPL concentration was higher than the fetal concentration at all stages of gestation except day 60 (7 ‘us. 11 ng/ ml, respectively). Mean maternal (multiple, 232.4 rig/ml; single, 59.9 rig/ml; P = 0.002) and fetal (multiple, 25.65 ng/ ml; single, 19.9 rig/ml; P = 0.09) serum 0PL concentrations were higher in ewes carrying multiple fetuses. Total fetal weight (Fig. 2) increased steadily (P 5 0.05) from days 60-135 of gestation. The oPL mRNA concentration (picograms per pg tcRNA) increased from days 60-120 '-‘ 1500 E

OF oPL mRNA

(P 5 0.05) and then stabilized (Fig. 2), whereas the actin mRNA concentration (picograms per pg tcRNA) was relatively constant from days 60-135 (P L 0.10) of gestation (Fig. 3). When the oPL mRNA concentration was expressedon a picogram per pg actin mRNA basis,the mRNA concentration ratio increased (P I 0.05) from days 60-105 and remained relatively constant (P 2 0.10) through day 135 (Fig. 3). The concentration (micrograms per mg wet tissue) of DNA in cotyledonary tissue increased (P I 0.05) from days 60-120 (Fig. 3) and remained constant through day 135. Due to the similar concentration profiles for oPL mRNA (picogramsper pg tcRNA) and cotyledonary DNA (micrograms per mg wet tissue),when oPL mRNA was expressedon a picograms per pg DNA basis, no stage of gestation effect (P > 0.10) was observed (Fig. 3). Correlation coefficients were determined for the relationships among oPL mRNA concentrations, maternal and fetal _ E

1.2

F1250

k .3

1.0

2

s t

0.8

f

maiernal

0PL

\

1000

0

f fY K < z k + 2

750

: 2 E E 2 0

500 250

Endo. 1992 Vol 131. No 6

0

m

mRNA/ACTIN

200

mRNA

180

2

160

g

140 120

2 ? 1

0.6

100

.z

0.4

80 60 40

s : z =

20

a

ACTIN

mRNA

0.2 0.0

100

0

20

-2

Z CY

oPL

hr

80

p

2

oPL

mRNA/DNA

DNA

concentration

15

El

t

2 q a z E 2 0

10

5

0

0

60

90

105

120

135

60

DAY OF GESTATION FIG. 2. Concentrations of fetal and maternal serum oPL and oPL mRNA concentrations and total fetal weight in day 60-135 gestating ewes. Top, Serum oPL concentrations were determined by a homologous RIA. Bottom, oPL mRNA concentrations were determined by blotting 4 or 8 pg tcRNA on a nylon membrane and hybridization with ‘“P-labeled y-actin or oPL cDNA. Radioactivity per sample was compared to known amounts of sense strand transcript. Total fetal weights were determined at surgery. Values represent the mean f SEM, and means with different superscripts differ (P 5 0.05) among gestational ages within a given variable. n = 4 ewes for days 60,90, 120, and 135; n = 5 ewes for day 105.

90

105

120

135

DAY OF GESTATION 3. Cotyledonary concentrations of actin mRNA, DNA, and oPL mRNA, expressed on a per actin mRNA or tissue DNA basis in day 60-135 gestating ewes. Top, Actin mRNA concentration and oPL mRNA/actin mRNA. Bottom, Cotyledonary tissue DNA concentration and oPL mRNA/tissue DNA. Concentrations of mRNA were determined by blotting 4 or 8 rg tcRNA on a nylon membrane and hybridizing with “P-labeled y-actin or oPL cDNA. Radioactivity per sample was compared to known amounts of sense strand transcript. Values represent the mean f SEM, and means with diferent superscripts differ (P 5 0.05) among gestational ages within a given variable. n = 4 ewes for days 60,90, 120, and 135; n = 5 ewes for day 105. FIG.

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EXPRESSION serum oPL concentrations, cotyledonary DNA concentrations, and total fetal weight, while adjusting for fetal number and gestational age. The maternal oPL serum concentration (r = 0.684; P = 0.0035) and cotyledonary oPL mRNA concentration (r = 0.607; P = 0.0126) were correlated with total fetal weight, and together accounted for 80.6% (r’ value) of the variation in total fetal weight. Additionally, the DNA concentration in cotyledonary tissue was correlated (P 5 0.05) with the cotyledonary oPL mRNA concentration (r = 0.570) and total fetal weight (r = 0.509). The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (P 2 0.10). Cellular localization of oPL expression was examined at all five gestational ages by immunocytochemistry and in situ hybridization, oPL was immunolocalized specifically to the chorionic binucleate cells at all five gestational ages, with no specific immunostaining observed in chorionic epithelial primary cells. Figure 4 (A and B) presents the positive immuno-

OF oPL mRNA

2833

staining (Fig. 4A) and the preimmune serum control (Fig. 48) observed in day 105 tissue, demonstrating specific localization within chorionic binucleate cells. The distribution of oPL-positive cells within the chorionic villi is presented in Fig. 4 (C and D). The mRNA encoding oPL was also localized exclusively in the chorionic binucleate cells, as determined by in situ hybridization. Figure 5 presents representative light- and darkfield photomicrographs of in situ hybridizations of day 60 (A and B) and day 135 (E and F) tissue with the antisense oligonucleotide and day 135 (C and D) with the sense oligonucleotide (negative control). Specific oPL mRNA hybridization within chorionic binucleate cells was observed at all five gestational ages, and hybridization of oPL mRNA was observed in all cells that could be identified as binucleate at these gestational ages. The number of chorionic cells (binucleate cells) expressing oPL mRNA appeared to increase with increasing gestational age, as evidenced by comparison of the darkfield photomicrographs of oPL mRNA

FIG. 4. Cellular localization of oPL by immunocytochemistry in day 105 ovine placentomes. Micrographs represent sections that were incubated in the presence of oPL antiserum (A and C) or preimmune serum (B and D). Arrows indicate binucleate cells of the chorionic epithelium (A and B). Notice the adjacent primary (uninucleated) chorionic epithelium cells that are not immunostained in micrograph A (A and B, ~400 magnification; bar = 2.5 Fm). C and D illustrate the frequency and location of the binucleate cells within the placentome (X100 magnification; bar = 10 pm). F, Fetal chorionic connective tissue; E, chorionic epithelium; M, maternal tissue.

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2834

EXPRESSION

‘-

OF oPL mRNA

Endo. Voll31.

1992 No 6

a b/ es.i E

FIG. 5. Cellular localization of oPL mRNA by in situ hybridization in day 60 and 135 ovine placentomes. Brightfield (A) and darkfield (B) micrographs of a placentome from a day 60 pregnant ewe hybridized with [%]antisense oPL oligonucleotide. Brightfield (C) and darkfield (D) micrographs of a day 135 placentome hybridized with [%i]sense oPL oligonucleotide as a negative control are shown. Brightfield (E) and darkfield (F) micrographs of a day 135 placentome hybridized with [%]antisense oPL oligonucleotide are shown. Arrows indicate binucleate cells of the chorionic epithelium. Note the lack of silver grains in adjacent primary cells of the chorionic epithelium. X400 magnification; bars = 2.5 pm.

in situ hybridizations of day 90 (Fig. 6; A, antisense;B, sense

oligonucleotides) and day 120 (Fig. 6; C, antisense;D, sense oligonucleotides) tissues. Light microscopy of hematoxylinand eosin-stainedplacentomal sectionsindicated that on day 60 a large percentage of the cotyledon was chorionic connective tissue, and primary branching was observed in the chorionic villi (Fig. 7A). By day 90 (Fig. 7B), an increasewas seen in secondary branching of the chorionic villi, which increased the surface area of the chorionic epithelium that was apposedto maternal tissues.On day 120 (Fig. 7C), more extensive secondary branching had occurred, while the proportion of the placentome that was occupied by chorionic connective tissue had been reduced and replaced by chorionic epithelium and maternal tissues. The reduction in chorionic connective tissue and replacement by chorionic

epithelium could account for the apparent gestational age increase in the number of oPL mRNA-positive cells (Fig. 6, A and C). Discussion The quantity and location of oPL mRNA expressionhave been examined during mid- and late gestation and have been related to the concentrations of oPL in maternal and fetal circulations and to total fetal weight. The cotyledonary oPL mRNA concentration appears to be more closely related to the maternal serum oPL profile than to the fetal serum oPL profile, since both maternal serum oPL and oPL mRNA concentrations increased until late gestation, whereas fetal

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EXPRESSION OF oPL mRNA

2835

FIG. 6. Low power magnification (x100) of cellular localization of oPL mRNA by in situ hybridization in day 90 and 120 ovine placentomes. Darkfield micrographs of a placentome from day 90 (A and B) and 120 (C and D) pregnant ewes hybridized with [%]antisense (A and C) or sense (B and D) oPL oligonucleotides. Note the localization of silver grains to specific cells (binucleate) with the antisense oligonucleotide. Bars = 10 pm.

serum oPL concentrations reached maximum values during midgestation. It should be kept in mind that fetal blood volume increases with increasing gestational age, which could effectively dilute out any gestational increasesin oPL synthesis and secretion into the fetal circulation. Recent data (35) on the entry rate of oPL into fetal circulation indicate that the entry rate increasesapproximately 2-fold from mid (day 75)- to late (day 115) gestation. These data suggestthat there may well be a strong relationship between oPL mRNA concentrations and the entry rate of oPL into the fetal circulation. The increasein oPL mRNA levels as gestation progresses could be explained by an increase in oPL mRNA levels per cell or an increase in the percentage of oPL-producing cells in the placentome. Resultsfrom this study provide evidence that the binucleate cells of the chorionic epithelium are the sole site of oPL production during mid- and late gestation. It has been reported that the percentage of chorionic epithelial cells that are binucleate cells remains constant until the last week of gestation, when a decline is seen (36). There was no gestational age effect on oPL mRNA expressionon a per cell basis(i.e. picogramsper pg DNA), nor was there an observable difference in oPL mRNA concentrations per cell from days 60-135 in our in situ hybridizations. However, there was a change in the morphology of the placentome as gestation progressed.This suggeststhat the increase in oPL mRNA concentrations seen as gestation progressesis due to an increase in total chorionic epithelial cells, including a proportional increase (constant percentage) in binucleate cells, at the expense of chorionic connective tissue. The lack of gestational age responsefor actin mRNA may result from the fact that total cotyledonary mass does not appear to increasebeyond midgestation (8, 37), such that tissue mass

(i.e. actin expression) does not increase coincidentally with cell number. The syncytiotrophoblastic tissuein the human, the site of human PL (hPL) production, alsoincreasesin size as gestation progressesand is thought to be responsible for the 5-fold increase in hPL mRNA concentrations from the first to the last trimester (38). This increase in syncytiotrophoblastic tissueis at the expenseof cytotrophoblastic tissue. Measurement of the binucleate cell-specific transcription rate of oPL mRNA at differing gestational agesis needed to verify our hypothesis that the gestational age-related increase in oPL mRNA expression results from increasedchorionic binucleate cell numbers rather than increased expression per cell. With larger litter sizes there is an increase in placental weight (8, 37, 39), and a simultaneous increasein total oPL mRNA and maternal serum oPL concentrations would be expected. The effect of fetal number on maternal oPL serum concentration is consistently seen acrossexperiments (8, 12, 13, 16, 17, 40) and has been correlated (r = 0.69-0.77) with placentomal weight across litter sizes (17, 41) and within singletons (42). When maternal serum oPL levels were expressedon a per g cotyledon basisand compared acrosslitter sizes(8, 37) or in ewes with restricted placental growth (42, 43), no significant changeswere observed, except by Schoknecht et al. (17). However, maternal serum oPL concentrations per g cotyledon have been reported to increase from days loo-130 (8), and the increase in mass of chorionic epithelium at the expense of the chorionic connective tissue would account for this observation, since total cotyledonary massonly increasesuntil days 70-85 (8, 37). Overall, these resultsimply that fetal growth and maternal serum oPL levels are tightly linked to placentomal mass. Further evidence for for such a relationship was seenwhen

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2836

EXPRESSION

OF oPL mRNA

Endo. Voll31.

1992 No 6

FIG. 7. Low power magnification (X25) of day 60,90, and 120 ovine placentomes. Hematoxylin- and eosin-stained placentomal sections from day 60 (a), 90 (B), and 120 (C) day pregnant ewes. F, Fetal chorionic connective tissue; E, chorionic epithelium; M, maternal connective tissue and epithelium. The chorionic connective tissue is very prominent in the day 60 and 90 placentomes, hut is barely visible in the day 120 placentome at this magnification (~25; bars = 40 Fm). The separation of the chorionic epithelium from the maternal epithelium and connective tissue is an artifact of fixation and sectioning. maternal serum oPL levels and oPL mRNA levels were correlated with total fetal weight. Together they accounted for 80.6% of the variation observed in total fetal weight. Taylor et al. (12) and Schoknecht et al. (17) reported similar correlation coefficients (r = 0.624 and 0.707, respectively) between maternal serum oPL levels and fetal weight. Schoknecht et al. (17) also reported that placental weight (r = 0.761; P 5 0.01) and fetal serum oPL levels (r = 0.699; P 5 0.05) were correlated to fetal weight in single fetuses and accounted for 81% of the variation in fetal weight. Unfortunately, total placentomal weights were not determined in our study, and insufficient numbers of ewes carrying singletons (one or two ewes per stage of gestation) prevented us from determining these correlations and coefficients of determination. Results from our immunocytochemistry and in situ hybridizations clearly indicate that the binucleate cells of the chorionic epithelium are the exclusive source of oPL during midand late gestation. These results agree with most studies on the immunolocalization of oPL where polyclonal antibodies were used (18-20). Since binucleate cells of the chorionic epithelium contain oPL mRNA, the current hypothesis of oPL delivery to the maternal circulation is still applicable. The binucleate cells migrate through the apical tight junction of the primary chorionic epithelium, cross the microvillar junction of the fetal-maternal interface, and fuse with the maternal syncytium (22, 36,44-46). The result of this migration and fusion is the displacement of uterine epithelial cells,

which die and are removed by phagocytosis by the primary chorionic epithelial cells (23, 47, 48). When the binucleate cells fuse with the syncytium, the oPL-containing granules are released into the syncytium by exocytosis (24). Since long branched processes of the syncytium extend toward the maternal capillaries, and the syncytium and maternal capillaries appear to lack definite basement membranes (49), it has been proposed that a function of binucleate cell migration and fusion may be the delivery of oPL to the maternal circulation (25). In conclusion, the correlations between fetal weight and maternal and fetal (17) serum oPL concentrations support the hypothesis that oPL is involved in supporting fetal growth. Since chorionic binucleate cells produce oPL mRNA, their migration appears to be the mode of transportation of oPL toward the maternal circulation. Little evidence exists for the mechanism of oPL release into the fetal circulation, but it does appear that it is controlled independently of the oPL released into the maternal circulation (17, 50, 51). The control of oPL mRNA transcription has yet to be examined, which could provide insight into the potential extrinsic control of oPL synthesis and secretion. Acknowledgments

Theauthorsaregratefulto the NIH-NIDDK National Hormone and Pituitary Program, University of Maryland School of Medicine (Baltimore, MD), for supplying the oGH and oPRL, to Dr. L. Kedes, University of Southern California, for supplying the y-actin cDNA, to Dr. L. Young

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EXPRESSION and Brad Freking graphic assistance, script.

for statistical assistance, to Penny Bures for photoand to Marsha Lewis for preparation of the manu-

OF oPL 21.

1.

Handwerger

lactogen

using

monoclonal

Wooding FBP 1980 Electron cells in the sheep placenta 221357-365

23.

S, Maurer W, Barrett J, Hurley T, Fellows RE 1974

Evidence for homology between ovine and human placental lactogen. Endocr Res Commun 1:403-413 2. Martal J, Djiane J 1975 Purification of a lactogenic hormone in sheep placenta. Biochem Biophys Res Commun 65:770-778 3. Chan JSD, Robertson HA, Friesen HG 1976 The purification and characterization of ovine placental lactogen. Endocrinology 98:6576 4. Reddy S, Watkins WB 1978 Purification and some properties of ovine placental lactogen. J Endocrinol 78:59-69 5. Warren WC, Liang R, Krivi GG, Siegel NR, Anthony RV 1990 Purification and structural characterization of ovine placental lactogen. J Endocrinol 126:141-149 6. Colosi P, Thordarson G, Hellmiss R, Singh K, Forsyth IA, Gluckman P, Wood WI 1989 Cloning and expression of ovine placental lactogen. Mol Endocrinol 3:1462-1469 7. Handwerger S 1991 Clinical counterpoint: the physiology of placental lactogen in human pregnancy. Endocr Rev 12:329-336 8. Martal J, Djiane J 1977 The production of chorionic somatomammotrophin in sheep. J Reprod Fertil 49:285-289 9. Carnegie JA, Chan JSD, McCully ME, Robertson HA, Friesen HG 1982 The cellular localization of chorionic somatomammotrophin in ovine chorion. J Reprod Fertil 66:9-16 10. Handwerger S, Crenshaw C, Maurer WF, Barrett J, Hurley TW, Golander A, Fellows RE 1977 Studies of ovine placental lactogen secretion by homologous radioimmunoassay. J Endocrinol72:27-34 11. Chan JSD, Robertson HA, Friesen HG 1978 Maternal and fetal concentrations of ovine placental lactogen measured by radioimmunoassay. Endocrinology 102:1606-1613 12. Taylor MJ, Jenkin G, Robinson JS, Thorburn GD, Friesen H, Chan JSD 1980 Concentrations of placental lactogen in chronically catheterized ewes and fetuses in late pregnancy. J Endocrinol85:2734 13. Gluckman P, Kaplan SL, Rudolph AM, Grumbach MM 1979 Hormone ontogeny in the ovine fetus. II. ovine chorionic somatomammotropin in mid- and late gestation in the fetal and maternal circulation. Endocrinology 104:1828-1833 14. Brinsmead MW, Bancroft BJ, Thorburn GD, Waters MJ 1981 Fetal and maternal ovine placental lactogen during hyperglycaemia, hypoglycaemia and fasting. J Endocrinol 90:337-343 15. Gluckman I’D, Barry TN 1988 Relationships between plasma concentrations of placental lactogen, insulin-like growth factors, metabolites and lamb size in late gestation ewes subject to nutritional supplementation and in their lambs. Dom Anim Endocrinol 5:209217 16. Butler SR, Fullenkamp SM, Cappiello LA, Handwerger S 1978 The relationship between breed and litter size in sheep and maternal serum concentrations of placental lactogen, estradiol and progesterone. J Anim Sci 53:1077-1081 17. Schoknecht PA, Nobrega SN, Petterson JA, Ehrhardt RA, Slepetis R, Bell AW 1991 Relations between maternal and fetal plasma concentrations of placental lactogens and placenta and fetal weights in well-fed ewes. J Anim Sci 69:1059-1063 18. Martal J, Djiane J, Dubois Ml’ 1977 Immunofluorescent localization of ovine placental lactogen. Cell Tissue Res 184:427-433 19. Wooding FBP 1981 Localization of ovine placental lactogen in sheep placentomes by electron microscope immunocytochemistry. J Reprod Fertil 62:15-19 20. Watkins WB, Reddy S 1980 Ovine placental lactogen in the cotyledonary and intercotyledonary placenta of the ewe. J Reprod Fertil 58:411-414

Chan JSD, Nie Z-R, Pang SC 1990 Cellular placental 23:33-40

22.

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2838

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EXPRESSION

intrauterine death and fetectomy on ovine placental lactogen production. Res Vet Sci 35:22-24 Assheton R 1906 The morphology of the ungulate placenta, particularly the development of that organ in the sheep, and notes upon the placenta in the elephant and hyrax. Phil Trans R Sot Br 198:143225 Wimsatt WA 1951 Observations on the morphogenesis, cytochemistry, and significance of the binucleate giant cells of the placenta of ruminants. Am J Anat 89:233-282 Amoroso EC 1952 Placentation. In: Parkes AS (ed) Marshalls Physiology of Reproduction, ed 3. Longmans, London, pp 127-311 Wathes DC, Wooding FBP 1981 An electron microscope study of

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Quantification and cellular localization of ovine placental lactogen messenger ribonucleic acid expression during mid- and late gestation.

Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supporti...
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