0013-7227/92/1305-2557$03.00/O Endocrinology Copyright 0 1992 by The Endocrine Society Vol. 130, No. 5 Printed in U.S.A. Steroid Regulation of the Synthesis and Secretion of Retinol-Binding Protein by the Uterus of the Pig* WILLIAM E. TROUT, JEFFREY A. HALL?, MELODY L. STALLINGS-MANN, JULIE M. GALVIN, RUSSELL V. ANTHONY, AND R. MICHAEL ROBERTS Departments of Animal Sciences and Biochemistry, University of Missouri, Columbia, Missouri 65212 ABSTRACT. The endometrium of the pig secretes retinolbinding protein (RBP) under the influence of progesterone (Pl). The objective of this study was to determine how conceptusderived estrogen might modulate this production of RBP around days 11-13 of pregnancy when conceptuses elongate from spheres to long thread-like forms. Concentrations of retinol and RBP were low (35 f 7 rig/ml) in uterine flushings obtained on days lo-12 of the estrous cycle or from pregnant gilts in which conceptuses had not elongated. Concentrations of retinol and RBP increased 7- to S-fold (P c 0.01) in flushings where filamentous conceptuses were present. Size exclusion and ion exchange chromatography demonstrated that virtually all retinol assaved in uterine flushings was associated with RBP.

Northern blotanalysis with a cDNA representing uterine RBP revealed a single endometrial mRNA 1.1 kilobases in length. Expression of RBP mRNA in uterine endometrium was measured in ovariectomized prepubertal gilts after the administration of steroids according tithe following regimens: I, corn oil (days O-16; n = 10): II. estradiol benzoate (EB; davs 13-14: n = 11); III, EB (days i-2; n = 12); IV, EB ‘(days 112) plus P, (days 3-16; n = 12); and V, EB (days l-2) plus P, (days 3-16) plus EB (days 13- 14; n = 12). EB (200 rg) and P, (100 mg) were administered twice daily. Treatment IV was designed to simulate the estrous cycle, and treatment V simulated early pregnancy. All gilts were hysterectomized on day 16, and total uterine mRNA (3 pg) was analyzed by Northern blotting. No RBP mRNA was detected in groups I, II, or III. In group IV, 5 of 12 gilts had detectable RBP mRNA, as measured by densitometric scanning (OD = 0.35 + 0.14). RNA isolated from all gilts in group V (12 of 12) gave a strong hybridization signal (OD = 1.58 + 0.22) for RBP. Finally, RBP mRNA was examined in the uterine endometrium of mature gilts on day 13 of the estrous cycle (n = 4), day 13 of pseudopregnancy (2.5 mg EB given on days 11-12; n = 4), or day 13 of pregnancy after conceptuses had elongated (n = 4). RBP mRNA was present in all groups, but was enhanced approximately 12-fold (P < 0.01) in pregnant and pseudopregnant gilts compared to that in control gilts.

These data strongly suggest that while P, may be sufficient to allow expression of uterine RBP, estrogen secreted by the conceptus can have a major influence on the amount and timing of RBP production. This combined effect of estrogen and progesterone may be critical during the periimplantation phase of pig conceptus development, a time when embryonic losses are high. (Endocrinology 130: 2557-2564, 1992) T HE SECOND week of embryonic development in the pig appears to represent a critical period for embryonic survival (1, 2). It is around this time that the spherical hatched blastocysts begin to elongate to longer thread-like forms (3-6), to synthesize estrogens (7-ll), and to ensure that the pregnancy is recognized by the mother (12). The estrogen produced is regarded as an essential factor in maternal recognition of pregnancy in swine and is probably responsible for extending the lifespan of the corpus luteum in the mother (8, 13, 14), although by mechanisms still not understood. This es- Received October 11, 1991. Address all correspondence and requests for reprints to: Dr. R. M. Roberts, 158 Animal Science Research Center, University of Missouri, Columbia, Missouri 65211. *This work was supported by USDA Grant 89-37240-4586 (to R.M.R.), Grant 88-37240-3904 (to R.V.A.), and USDA Postdoctoral Fellowship 89-37240-4772 (to J.A.H.). This is publication 11,522 of the University of Missouri Agricultural Experiment Station. t Present address: Baxter Healthcare Corp., Infectious Disease Research and Development, 1851 Delaware Parkway, Miami, Florida 33125. trogen also causes the relatively sudden release of uterine secretory proteins (often called histotroph) from secretory vacuoles in the surface and glandular epithelial cells of the endometrium into the uterine lumen, where the secretory products then bathe and presumably provide nutrient support to the rapidly growing conceptuses (5, 15).

The role of estrogen in this regard has generally been held to be on the secretory, rather than the synthetic, limb of the process because the response is rapid (15), and estrogen alone has no direct inductive effect on the synthesis of uterine secretory proteins (16, 17). Several progesterone (P1)-induced proteins that are synthesized by the endometrium have been identified in the uterine histotroph. These proteins include uteroferrin, which transports iron to the fetus (17, la), a family of serine protease inhibitors of the Kunitz class (19), a glycoprotein that belongs to the serpin superfamily of proteins,
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whose function during pregnancy remains obscure (20, 21), and, most relevant to this paper, a group of retinol-binding proteins (RBP) that presumably sup- 2557 Downloaded from endo.endojournals.org on January 23, 2006 2558 STEROID REGULATION OF RBP Endo. 1992 Vol130. No 5 ply vitamin A to the conceptus (22, 23). Classically, RBP is described as a product of the liver, which regulates circulating concentrations of retinol and RBP over a wide range of dietary vitamin intakes (see Refs. 24-26 for reviews).

During pregnancy, the uterus presumably garners retinol from the circulation for delivery to the conceptuses, and the liver must compensate by releasing retinol complexed with RBP. At least four different isoelectric forms of these RBP were identified in uterine histotroph by ion exchange chromatography (23). We have recently determined that all four isoforms of uterine RBP appear to be closely related, if not identical, to serum RBP by sequencing the NHs-termini of the proteins after electroblotting onto polyvinylidene difluoride membranes (Stallings-Mann, M., W. E. Trout, and R. M. Roberts, unpublished results). The basis of the variability in pI remains unclear. In this paper, we have investigated how estrogen and P4 control the production of uterine RBP and, hence, the amount of vitamin A available to the conceptuses. Specifically, we indicate that conceptus-derived estrogen plays a critical role in causing increased synthesis and secretion of uterine RBP at the time of trophoblast elongation.

Materials and Methods Guanidine thiocyanate, cesium chloride, and formamide were obtained from the Fisher Chemical Co. (Fairlawn, NJ). Herring sperm DNA and the random prime labeling kit were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). [cu-32P]Deoxy-ATP (3000 Ci/mmol) and [11,12-3H]retinol (47.8 Ci/mmol) were purchased from ICN Biomedical, Inc. (Costa Mesa, CA) and DuPont-New England Nuclear Research Products (Boston, MA), respectively. Sephadex G-50 and the Superose 12 fast protein liquid chromatography (FPLC) and diethylaminoethyl (DEAE) Ultropac-HPLC columns were products of Pharmacia/LKB (Piscataway, NJ). Exp la: quantitation of vitamin A in uterine flushings Uterine luminal proteins were obtained by surgically flushing (27) 1 or both uterine horns of 25 gilts with 30 ml each of physiological saline, Dulbecco’s PBS, or Hanks’ Balanced Salt Solution on day 6 (n = 4) of pregnancy, days lo-13 of pregnancy (n = 15), or days 11-12 (n = 6) of the estrous cycle. Concentrations of vitamin A in uterine flushings were determined in duplicate 5-ml samples by absorbance at 325 nm, after extraction with ethanol and hexane, according to the method of Johnston and Chew (28). Exp lb: Superose-12 chromatography of uterine flushings To characterize RBP in uterine flushings, 4 ml flush obtained from nonpregnant pigs on days 11-12 of the estrous cycle (n = 4) and from pregnant pigs on days 11-13 of pregnancy (n = 4) were dried in a Speed-Vat (Savant Instruments, Inc., Farmingdale, NY) and resuspended in 0.5 ml distilled water. For each gilt, an aliquot (0.25 ml) of concentrated uterine flushing was applied to a Superose-12 FPLC column and eluted with 0.02 M Tris-HCl (pH 8.2) and 1.5 M NaCl at a flow rate of 0.5 ml/min. Protein in the eluant was monitored by absorbance at 280 nm. Vitamin A was monitored by relative fluorescence with a Hoefer DNA Fluorometer (model TKO-100, Hoefer Instruments, San Francisco, CA; excitation wavelength, 365; emission wavelength, 460 nm). The elution positions of vitamin A and uterine RBP from Superose-12 were determined in flushings obtained from two gilts on day 12 of pregnancy after incubation with 5 PCi [3H]retinol for 4 h before concentration by Speed-Vat and chromatography on Superose-12 FPLC.

In addition, flushings (4 ml) obtained on day 12 of pregnancy (n = 2) were concentrated by Speed-Vat, and each sample was resuspended in 1 ml distilled water. Vitamin A was then removed from the samples by hexane extraction, and 250 ~1 of the aqueous fraction were applied to the Superose-12 FPLC column. The relative fluorescence of the eluting fractions was measured and corrected for dilution during the extraction procedure. DEAE-HPLC chromatography of uterine RBP Superose-12 FPLC fractions that contained uterine RBP were obtained from uterine flushings of pigs on day 12 of pregnancy (n = 2). The two fractions were pooled, concentrated by centrifugation in a Centricon 10 ultrafiltration unit (Amicon Division, W.R. Grace & Co., Danvers, MA), and washed with 4-5 vol distilled water. This material was then incubated with 1 &i [3H]retinol for 4 h before application on a DEAE-Ultropac HPLC ion exchange column (Pharmacia-LKB). The DEAE column was equilibrated in 0.01 M Tris-HCl, pH 8.2, and eluted with a 60-min linear gradient to 0.25 M NaCl at a flow rate of 1 ml/min. Exp 2: induction of uterine RBP mRNA in prepubertal pigs by gonadal steroids Fifty-seven prepubertal gilts were ovariectomized at 120-130 days of age. Two weeks after ovariectomy, gilts were randomly assigned to treatment, as described in Fig. 1. The dose of estradiol (400 Kg/gilt .day) was chosen to be approximately twice the minimum amount required to induce estrus in ovariectomized pigs (29). All gilts were hysterectomized on day 16 of treatment. Total cellular RNA (3 pg) was examined by Northern blot analysis and hybridized with cDNA probes for QROUP n II co 1 10 II co m l1 III co 12 I" g#J&&&;, $&&&&* …:yx$;, _ 1 12 v 12 1-a 13-14 16 DAY OF TREATMENT t HY8TERECTOMY FIG. 1. Protocols of steroid replacement therapy administered to ovariectomized gilts in Exp 2. Injections of corn oil, EB (200 fig), and P4 (100 mg) were given twice daily at 0600 and 1800 h. n, Number of gilts per treatment group.

Downloaded from endo.endojournals.org on January 23, 2006 STEROID REGULATION OF RBP 2559 RBP (30) and porcine uterine serpin [uteroferrin-associated basic glycoprotein (UABP)] (21). Statistical analysis and densitometry Exp 3: induction of uterine RBP mRNA by estrogen and pregnancy Twelve mature cyclic gilts were randomly assigned to be hysterectomized 1) on day 13 of the estrous cycle (n = 4), 2) on day 13 of the estrous cycle after being injected once daily with 2.5 mg estradiol benzoate (EB) on days 11 and 12 to induce a pseudopregnant state (n = 4) (31), or 3) on day 13 of pregnancy (n = 4). Forty micrograms of total cellular RNA were examined by Northern blot analysis and hybridized with cDNA probes to RBP, UABP, and y-actin (32). The hybridization signal obtained for actin served as an index of RNA loading. Isolation of RNA from uterine endometrium and Northern analysis Uterine tissues collected for all experiments were snapfrozen in liquid nitrogen at the time of hysterectomy and stored at -80 C until processed for isolation of RNA. In Exp 2 and 3, total cellular RNA was isolated from 3-8 g uterine tissue by homogenization in 25 ml buffer containing 4 M guanidine thiocyanate, 0.5% N-lauryl sarcosine, 0.1 M P-mercaptoethanol, 0.1% Antifoam-A, and 0.02 M sodium citrate (pH 7.0). This homogenate was centrifuged at 1,000 x g for 20 min to pellet debris. The supernatant fraction was applied to a lo-ml cushion of 5.7 M cesium chloride-O.1 M EDTA (pH 7.0) and subjected to ultracentrifugation for 20-24 h at 25,000 rpm in a Beckman SW28 rotor (Palo Alto, CA). The pelleted RNA was extracted twice with phenol-chloroform (1:l) and once with chloroformisoamylalcohol (24:1), and concentrated by ethanol precipitation. Concentrations of vitamin A determined in uterine flushings obtained in Exp 1 were analyzed by one-way analysis of variance by means of the General Linear Models (GLM) procedure of the Statistical Analysis System (SAS User’s Guide: Statistics, SAS Institute, Cary, NC). Fluorescence profiles generated after Superose-12 chromatography of uterine flushings from pregnant and nonpregnant gilts were analyzed by split plot analysis of variance with the GLM procedure of SAS. The intensity of autoradiographic signals obtained from Northern blots was determined by densitometry with a Bio- Rad model 620 video densitometer and the accompanying onedimensional analysis and integration software (Bio-Rad, Richmond, CA). Signal intensity is expressed as the area of the spot, i.e. height in optical density units, and width in millimeters.

In Exp 2, only the five gilts in group 4 that had a detectable hybridization signal for RBP were used to calculate the mean signal intensity presented in Table 1. The differences in intensity of signal for RBP in the two groups were examined by t test. Signal intensities obtained from Northern blots probed with RBP, UABP, or y-actin in Exp 3 were examined with a randomized complete block design. The analysis was performed by the GLM procedure of SAS. Results Concentrations of vitamin A and RBP in uterine flushings of pregnant and nonpregnant gilts For Northern blot analysis, total cellular RNA was separated by formaldehyde-agaroseg el electrophoresisa nd transferred to nylon filters (Zetabind, Cuno, Inc., Meriden, CT) according to the method of Sambrook et al. (33). The filters were prehybridized overnight at 42 C with a buffer containing 50% (vol/vol) formamide, 5 x SSC (0.15 M NaCl and 0.015 M Na3 citrate, pH 7.0), 1 x Denhardt’s solution, 0.1% sodium dodecyl sulfate (SDS; wt/vol), and 0.5 mg/ml sonicated herring sperm DNA. The filters were then hybridized at 42 C for 16 h in the above buffer containing the appropriate P,-labeled cDNA probe. After hybridization, the filters were washed at 42 C with three or four changes of buffer containing 0.1 x SSC and 0.1% (wt/vol) SDS. Filters were then used to expose x-ray film (XAR, Eastman Kodak, Rochester, NY) for 24-72 h at -80 C in the presence of intensifying screens. Amounts of retinol were low in uterine flushings obtained on days 11-12 of the estrous cycle and in flushings obtained from pregnant pigs whose conceptuses had not yet elongated (Fig. 2). However, concentrations of retinol in uterine flushings were increased 7- to &fold in flushings obtained on days 11-13 of pregnancy, provided that the majority of conceptuses present had reached the fiiamentous stage of development. Flushings from gilts containing only spherical conceptuses had no more retino1 than those recovered from nonpregnant gilts.

Gel filtration and ion exchange chromatography were employed to measure the quantity of RBP in uterine flushings from both pregnant gilts that had provided filamentous conceptuses as well as nonpregnant gilts. Superose-12 size-exclusion FPLC of samples (n = 4) Labeling of cDNA probes TABLE 1. Detection of RBP mRNA by Northern hybridization of total cellular RNA (3 wg) isolated from uterine endometrium of ovariectomized prepubertal pigs after steroid replacement therapy cDNA probes (-250 ng) corresponding to porcine RBP, Group Gilts with UABP, and human y-actin were radiolabeled by random prim- signal ing with [a-32P]dATP to specific activities of approximately 1 I, co x 10’ cpm/pg. Labeled probes were separated from free [a-“‘PI o/10 II, EB (13-14) o/11 dATP by centrifugation at 1000 x g through 0.8 ml Sephadex III, EB (l-2) o/12 G-50 (33). The RBP probe employed corresponded to the 3’- IV, EB + P, 5112 terminal 618-basepairso f the 937-basepairp orcine RBP cDNA V, EB + P, + EB 12112 (30). Probes for UABP and y-actin were the full-length cDNA, ’ Mean + SE, spot area (OD X mm). 1.4 (21) and 2.1 (32) kilobases, respectively. b.c Means within a column differ (P < 0.01). Signal intensity” 0.35 + 0.14* 1.58 + 0.22’ Downloaded from endo.endojournals.org on January 23, 2006 2560 STEROID REGULATION OF RBP Endo. 1992 Voll30. No 5 ;; 0 G ;; FIG. 2. Concentrations (mean + SE) of vitamin A (retinal) in uterine flushings obtained from gilts on days 11-12 of the estrous cycle (NP), day 6 of pregnancy, and days lo-13 of pregnancy (P). Flushings obtained on days lo-13 of pregnancy were subclassified depending upon whether all of the embryos in the litter were spherical (P-S) or whether some embryos had reached the filamentous stage (P-F) of development. equivalent to 2 ml of 30-ml uterine flushings obtained from pregnant gilts revealed two late eluting peaks of fluorescence (Fig. 3A, 0) that appeared after 32 and 36 min, respectively. Extraction of the uterine flushings (n = 2) with hexane to remove retinol before Superose-12 FPLC resulted in the disappearance of the peak of fluorescence eluting at 32 min, but did not remove the fluorescent peak eluting at 36 min (Fig. 3B). Hence, only the peak at 32 min was considered likely to contain retinol associated with uterine RBP. Superose-12 FPLC of identical samples (n = 2) of uterine flushings after they had been incubated with [3H]retinol revealed only one major peak of radioactivity that eluted at 32 min (Fig. 3A, 0).

Anion exchange chromatography of this peak gave the radioactivity and fluorescence profiles shown in Fig. 3C. The radioactivity eluting first had clearly failed to bind to the column and was suspected to be free [3H]retinol. However, fluorescence and radioactivity were noted at 34, 39, and 44 min. The first of these three late eluting peaks was asymmetrical and likely, therefore, to contain more than one component. This fluorescence-radioactivity profile was essentially identical to that observed by Clawitter et al. (23) in their -1A MINUTES – 1B 0 10 20 3.0 4‘0 5’0 MINUTES 0 10 20 30 40 50 MINUTES FIG. 3. Superose-12 FPLC size exclusion chromatography of uterine flushings obtained from a representative gilt on day 12 of pregnancy. Filamentous conceptuses were recovered in the flushings. 0, Relative fluorscence. 0, Counts per min associated with eluted fractions after incubation of uterine flushings with [3H]retinol for 4 h. The abscissa represents the time of elution in minutes. B, Superose-12 FPLC of uterine flushings obtained from a representative gilt on day 12 of pregnancy. 0, Fluorescence profile presented in A. 0, Fluorescence profile obtained after the uterine flushings had been extracted with hexane before chromatography. C, DEAE ion exchange chromatography of the putative RBP peak eluting from Superose-12 at 32 min (A, 0). 0 and 0, Counts per min and relative fluorescence of the eluted fractions, respectively. analysis of uterine RBP in which at least four different isoelectric variants of uterine RBP were detected. These results confirm that the peak eluting at 32 min during Superose-12 chromatography did indeed represent the uterine RBP. Superose-12 profiles of samples equivalent to 2 ml uterine flushings obtained from either gilts with filamentous embryos (n = 4) or nonpregnant gilts on day 12 of the estrous cycle (n = 4) are shown in Fig. 4. The relative Downloaded from endo.endojournals.org on January 23, 2006 STEROID REGULATION OF RBP 2561 1500 , 1 – PREGNANT – NON-PREGNANT 25 30 35 40 45 MINUTES FIG. 4. Relative fluorescence profiles (mean + SE) obtained after Superose- 12 FPLC of uterine flushings obtained on day 12 of pregnancy (n = 4; 0) or on day 12 of the estrous cycle (n = 4; 0). Filamentous embryos were flushed from the uteri of all pregnant gilts. The abscissa represents the time of elution in minutes. fluorescence was greater (P < 0.001) in the peak eluting at 32 min for the samples from pregnant gilts than for samples from nonpregnant gilts.

Indeed, little or no RBP appeared to be present, based upon fluorescence, in the nonpregnant group. Assuming an approximate 1:l correspondence between the amount of retinol present and uterine RBP, it can be calculated from the data presented in Fig. 2 that between 1.5-2 mg RBP were present in the flushings from the gilts with elongated conceptuses. Induction of RBP mRNA by gonadal steroids RBP mRNA was not detectable on Northern blots of endometrial RNA obtained from ovariectomized prepubertal gilts that received injections of corn oil for 15 days (group 1; Table 1). Treatment of ovariectomized prepubertal gilts with injections of estradiol alone on either days l-2 or days 13-14 (groups 2 and 3; Fig. 1) failed to increase uterine concentrations of RBP mRNA to detectable levels (Table 1). Simulation of an estrous cycle by treatment of the gilts with estradiol on days l-2 followed by P, on days 3-16 (group 4) produced detectable levels of RBP mRNA in 5 of 12 gilts. Gilts in group 5 were given steroids to mimic the estrous cycle and additional injections of estradiol on days 13 and 14 in an attempt to simulate estrogen production by the conceptuses during early pregnancy (5). The intensity of the hybridization signal for gilts in group 5 was increased approximately 4-fold (P < 0.01) over that obtained for the 5 gilts with detectable signal in group 4 (Table 1). RNA from all gilts in group 5 contained detectable amounts of RBP mRNA (Fig. 5). Although the signal intensity was much weaker, expression of the mRNA for the UABP identified by Malathy et al. (21) appeared to be similar to that for the RBP mRNA across treatment groups. For example, while only 3 of 12 gilts in group 4 had a detectable signal for UABP mRNA, all 12 gilts in group 5 expressed the mRNA (data not shown). Effect of estrogen on uterine RBP mRNA expression Northern blots of total cellular RNA obtained from the uterine endometrium of mature gilts on day 13 of the estrous cycle gave clear signals for RBP and UABP mRNA (Fig. 6) when 40 /lg RNA were blotted compared to only 3 pg in Exp 2. Injection of gilts with EB on days 11 and 12 increased concentrations of RBP and UABP mRNA approximately 12-fold (P < 0.01) and lo-fold (P < 0.05), respectively, when tissue was examined on day 13.

The concentrations of RBP and UABP mRNA observed in the uterine endometrium of day 13 pregnant gilts were essentially identical to those in the gilts treated with estradiol. There were no significant differences in actin mRNA concentrations among any of the treatment groups. Discussion Geisert et al. (5) noted that a pair of proteins, subsequently identified as RBP (23), were present in uterine flushings around day 12 of pregnancy, but were absent at earlier times. It was of interest, therefore, to measure the retinol content of uterine flushings around this period and to determine whether this value was influenced by the stage of development of the conceptuses. Our results clearly show that as conceptuses began to elongate from spheres to long thread-like forms on or about day 12 of pregnancy, there was a marked and relatively sudden increase in the amount of retinol that could be flushed from the uterine lumen. In gilts that lacked elongated conceptuses on day 12, no such increase in retinol relative to that in nonpregnant controls was noted. This retinol was associated with a group of at least three or four low mol wt RBP identifiable by the same high performance anion exchange procedures used by Clawitter et al. (23) to demonstrate RBP in uterine flushings of pseudopregnant gilts. Geisert et al. (15) also noted that the appearance of the proteins we now know to be RBP could be mimicked in nonpregnant, but intact, gilts by a single injection of estradiol on day 11 of the estrous cycle. Similarly, Geisert et al. (34) and Morgan et al. (35) have noted that these same polypeptides of uterine flushings could be induced prematurely by the administration of estradiol on days 9 and 10 of pregnancy, even though this treatment resulted in the subsequent death of the embryos and termination of pregnancy. The above data along with the observation that conceptuses begin to synthesize estrogen as they elongate (3, 5, 8, 9, ll), suggested to us that conceptus-derived estrogen might be a critical factor controlling the availability of retinol in the uterine histotroph. However, induction of uterine RBP mRNA is also clearly dependent upon provision of Pq, since injections of estradiol valerate alone were unable to increase steady Downloaded from endo.endojournals.org on January 23, 2006 2562 FIG. 5. Northern blot of uterine endometrial RNA (3 fig) isolated from ovariectomized prepubertal gilts on day 16 after various regimens of steroid replacement therapy. RNA from pigs in group II (n = 5), group IV (n = 5), and group V (n = 5) was probed with a porcine RBP cDNA probe. On this blot, only gilts in group 5 that had received a steroid replacement regimen designed to mimic early pregnancy gave a detectable hybridization signal. The positions of the 28s and 18s ribosomal RNA bands are indicated.

STEROID REGULATION OF RBP GROUP II IV Endo. 1992 Voll30. No 5 10 cl RBP 8 q UABP ACTIN DlB-NP DlB-PP D13-P A B C FIG. 6. Quantitation by videodensitometry of autoradiographic signals obtained from Northern blots of uterine endometrial RNA after hybridization with RBP, UABP, or y-actin cDNA probes. RNA was isolated on day 13 of the estrous cycle (A), pseudopregnancy (B), or pregnancy (C). Data represent the mean rt SE signal obtained for four pigs in each group. state RBP mRNA to detectable levels in ovariectomized gilts. These data are in agreement with those of Adams et al. (22), who demonstrated in ovariectomized mature gilts that secretion of uterine RBP occurred in response to P4 or a combination of P, plus estrogen, but not in response to estrogen alone. Others have noted RBP production at a variety of extrahepatic sites (36, 37), including the visceral yolk sac (38), and there have been reports of estrogen induction of RBP synthesis in both rat kidney (39) and the liver of amphibians (40), although no estrogen response elements have so far been reported for the genes. Estrogen, injected on a background of P, in Exp 2 (to ovariectomized prepubertal gilts on days 13-14 of Pq treatment) or Exp 3 (to mature nonpregnant gilts on days 11 and 12 of the estrous cycle) markedly enhanced the expression of RBP mRNA. It seems likely that such injections successfully mimicked the local production of estrogen by elongating conceptuses, since the observed results in the two situations were identical, namely an increase in RBP and retinol in the uterine lumen and an elevation of RBP mRNA in the tissue. Thus, as conceptuses elongate, they may directly control the quantity of vitamin A available to them. Curiously, at about this time conceptuses themselves also begin to synthesize and secrete RBP (29, 41). Whether this apparent “extra” production of RBP serves to buffer the embryo from the potentially damaging action of the bioactive derivative of retinol, retinoic acid, or whether it somehow assists in retinol uptake and distribution is unclear. Whatever the explanation, this attention to retinol does emphasize the likely importance of the vitamin at this stage of pregnancy. Brief and Chew (42) demonstrated that injections of supplemental vitamin A and/or P-carotene increased litter size and decreased embryonic mortality in pigs. Recently, Coffey and Britt (43) also reported that injections of P-carotene increased litter size when given to multiparous sows approximately 6 days before breeding. Together with the present data, these results suggest that the beneficial effects of supplemental vitamin A might Downloaded from endo.endojournals.org on January 23, 2006 STEROID REGULATION OF RBP 2563 be manifested within the first weeks of pregnancy, when the rate of embryonic mortality is highest (1, 2).

The massive increase in uterine concentrations of retinol and RBP elicited by conceptus estrogen suggests that pig conceptuses may require very high concentrations of vitamin A at the time of transition from spherical to filamentous forms, and as discussed above, RBP may ensure the appropriate presentation of the vitamin to the conceptuses, thereby avoiding potentially toxic and teratogenic effects (44, 45). The data of Pope et al. (46), who found that supplemental injections of estradiol on days 12-13 of pregnancy could also increase embryonic survival, are particularly interesting in view of the likely involvement of this hormone in provision of retinol to the conceptus. We speculate that supplemental injections of estradiol at this critical period of pregnancy may further enhance retinol availability and, by mechanisms presently unclear, increase the number of embryos likely to survive. Acknowledgments The authors wish to express their appreciation to Drs. N. Mathialagan and S. Xie, Mr. K. Kramer, and Ms. H. Francis for surgical and technical assistance. We also thank Mr. A. Rieke for the handling and preparation of the animals, and Ms. G. Foristal for typing this manuscript. 1. 6. 7. 8. 9. 10. 11. 12. References Polge C 1982 Embryo transplantation and preservation. In: Cole DJA, Foxcroft GR (eds) Control of Pig Reproduction. Butterworth, London, pp 277-291 Pope WF, First NL 1985 Factors affecting the survival of pig embryos. Theriogenology 23:91-105 Perry JS, Rowlands IW 1962 Early pregnancy in the pig. J Reprod Fertil 4:175-188 Anderson LL 1978 Growth, protein content and distribution of early embryos. Anat Ret 190:143-154 Geisert RD, Renegar RH, Thatcher WW, Roberts RM, Bazer FW 1982 Establishment of pregnancy in the pig. I. Interrelationships between preimplantation development of the pig blastocyst and uterine endometrial secretions. Biol Reprod 27:925-939 Geisert RD, Brookbank JW, Roberts RM, Bazer FW 1982 Establishment of pregnancy in the pig. II. Cellular remodeling of the porcine blastocvst during elongation on day 12 of pregnancy. Biol keprod 27:941-955 – - Perrv JS. Hean RB. Amoroso EC 1973 Steroid hormone nroduction by pig blastocysts. Nature 245:45-47 Flint APF, Burton RD, Gadsby JE, Saunders PTK, Heap RB 1979 Blastocyst oestrogen synthesis and the maternal recognition of pregnancy. In: Whelan J (ed) Maternal Recognition of Pregnancy.

Ciba Foundation Symposium on Nutrition, New Series 64, Excerpta Medica, Amsterdam, pp 209-288 Gadsby JE, Heap RB, Burton RD 1980 Oestrogen synthesis by blastocyst and early embryonic tissue of various species. J Reprod Fertil60:409-417 Heap RB, Flint APF, Gadsby JE, Rice C 1979 Hormones, the early embryo and the uterine environment. J Reprod Fertil55:267-275 Heap RB, Flint APF, Hartmann PE, Gadsby JE, Staples LD, Ackland N, Hamon M 1981 Oestrogen production in early pregnancy. J Endocrinol89:77P-94P Dhindsa DS, Dziuk PJ 1968 Influence of varying the proportion of uterus occupied by embryos on maintenance of pregnancy in the 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. pig. J Anim Sci 27:668-672 Gardner ML, First NL, Casida LE 1963 Effect of exogenous estrogens on corpus luteum maintenance in gilts. J Anim Sci 22:132- 134 Bazer FW, Thatcher WW 1977 Theory of maternal recognition of pregnancy in swine based on estrogen controlled endocrine versus exocrine secretion of prostaglandin Fsa by the uterine endometrium. Prostaglandins 14:397-400 Geisert RD, Thatcher WW, Roberts RM, Bazer FW 1982 Establishment of pregnancy in the pig. III. Endometrial secretory response to estradiol valerate administered on day 11 of the estrous cycle. Biol Reprod 27:957-965 Roberts RM, Bazer FW 1980 The properties, hormonal control and synthesis of uteroferrin, the purple protein of the pig uterus. In: Beato M (ed) Steroid-Induced Proteins. Elsevier/North-Holland, Amersterdam, pp 133-149 Roberts RM, Bazer FW 1988 The functions of uterine secretions. J Reprod Fertil82:875-892 Roberts RM, Raub TJ, Bazer FW 1986 The role of uteroferrin in transplacental iron transport in the pig. Fed Proc 45:2513-2518 Fazleabas AT, Bazer FW, Roberts RM 1982 Purification and properties of progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus. J Biol Chem 257:6886-6897 Murray MK, Malathy PV, Bazer FW, Roberts RM 1989 Structural relationship, biosynthesis and immunocytochemical localization of uteroferrin-associated basic glycoproteins.

J Biol Chem 264:4143- 4150 Malathy P-V, Imakawa K, Simmen RCM, Roberts RM 1990 Molecular cloning of the uteroferrin-associated protein, a major progesterone- induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy. Mol Endocrinol4:428- 440 Adams KL, Bazer FW, Roberts RM 1981 Progesterone-induced secretion of a retinol-binding protein in the pig uterus. J Reprod Fertil62:39-47 Clawitter J, Trout WE, Burke MG, Araghi S, Roberts RM 1990 A novel family of progesterone-induced, retinol-binding proteins from uterine secretions of the pig. J Biol Chem 265:3248-3255 Goodman DS 1984 Plasma retinol-binding protein. In: Sporn MB, Roberts AB, Goodman DS (eds) The Retinoids. Academic Press, Orlando, vol 2:177-208 Blaner WS 1989 Retinol binding protein: the serum transport protein for vitamin A. Endocr Rev 10:308-316 Blomhoff R, Green MH, Berg T, Norum KR 1990 Transport and storage of vitamin A. Science 250:399-404 Bazer FW, Roberts RM, Sharp DC 1978 Collection and analysis of female genital tract secretions. In: Danial JC (ed) Methods in Mammalian Reproduction. Academic Press, New York, pp 503- 527 Johnston LA, Chew BP 1984 Peripartum changes in plasma and milk vitamin A and /3- carotene among dairy cows with or without mastitis. J Dairy Sci 67:1832-1840 Day BN, Anderson LL, Hazel LN, Melampy RM 1959 Svnchronization of estrus and ovulation in swine. J .&rim Sci 18:969-919 Trout WE. McDonnell JJ. Kramer KK. Baumbach GA. Roberts RM 1991 The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc. Mol Endocrinol 5:1533-1540 Frank M, Bazer FW, Thatcher WW, Wilcox CJ 1977 A study of prostaglandin F, as the luteolysin in swine. III. Effects of estradiol valerate on prostaglandin F, progestins, estrone and estradiol concentrations in the utero-ovarian vein of non-pregnant gilts. Prostaglandins 14:1183-1196 32. Gunning P, Ponte P, Okayama H, Engel J, Blau H, Kedes L 1983 Isolation and characterization of full-length cDNA clones for human cy-, fl- and y-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol 3:787-795 33. Sambrook J, Fritsch EF, Maniatis T (eds) 1989 Molecular Cloning- A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor Downloaded from endo.endojournals.org on January 23, 2006 STEROID REGULATION OF RBP Endo – 1992 Voll30. No 5 but not in the liver. J Lipid Res 31:1483-1490 McKearin DM, Barton MC, Keder MJ, Shapiro DJ 1987 Estrogen induces transcription of the Xenopus laevis serum retinol-binding protein gene. J Biol Chem 262:4939-4942 Harney JP, Mirando MA, Smith LC, Bazer FW 1990 Retinolbinding protein: a major secretory product of the pig conceptus. Biol Reprod 42:523-532 Brief S, Chew BP 1985 Effects of vitamin A and &carotene on reproductive performance in gilts. J Anim Sci 60:998-1004 Coffev MT. Britt JH 1989 Effect of f3 carotene injection on renroductile performance of sows. J Anim Sci [Suppl1]67:251 (Abst’ract 615) Kochar DM 1967 Teratogenic activity of retinoic acid. Acta Path01 Microbial Stand 70:398-404 Lammer EJ, Chen DT, Hoar RM, Agnish ND, Benke PJ, Braun JT. Currv CJ. Fernhoff PM. Brix AW. Lott IT. Richard JM. Sun SC’ 1985 ketinoic acid embryopathy. N Engl J Med 313:837-841 Pope WF, Lawyer MS, Butler WR, Foote RH, First NL 1986 Doseresponse shift in the ability of gilts to remain pregnant following exogenous estradiol-178 exposure. J Anim Sci 63:1208-1210

34. 35. 36. 37. 38. 39. Geisert RD, Zavy MT, Wettemann RP, Biggers BG 1987 Length of pseudopregnancy and pattern of uterine protein release as influenced by time and duration of oestrogen administration in the pig. J Reprod Fertil79:163-172 Morgan GL, Geisert RD, Zavy MT, Fazleabas AT 1987 Development and survival of pig blastocysts after oestrogen administration on day 9 or days 9 and 10 of pregnancy. J Reprod Fertil 80:133- 141 Soprano DR, Soprano KJ, Goodman SD 1986 Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat. J Lipid Res 27:166-171 Makover A, Soprano PR, Wyatt MC, Goodman DS 1989 Localization of retinol-binding protein
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messenger RNA in the rat kidney and in perinephric fat tissue. J Lipid Res 30:171-180 Soprano DR, Soprano KJ, Goodman DS 1986 Retinol-binding protein and transthyretin mRNA levels in visceral yolk sac and liver during fetal development in the rat. Proc Nat1 Acad Sci USA 83:7330-7334 Whitman MM, Harnish DC, Soprano KJ, Soprano DR 1990 Retinol- binding protein mRNA is induced by estrogen in the kidney 40. 41. 42. 43. 44. 45. 46. Downloaded from endo.endojournals.org on January 23, 2006