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BOR - Papers in Press, published online ahead of print August 6, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018150
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BIOLOGY OF REPRODUCTION 69, 1945–1950 (2003)
DOI: 10.1095/biolreprod.103.018150
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

11ß-Hydroxysteroid Dehydrogenase and Glucocorticoid Receptor Messenger RNA Expression in Porcine Placentae: Effects of Stage of Gestation, Breed, and Uterine Environment1

H.G. Klemcke2,4, R. Sampath Kumar3,5, K. Yang5, J.L. Vallet4, and R.K. Christenson4

USDA-ARS,4 Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933 The Lawson Research Institute,5 St. Joseph's Hospital, Departments of Obstetrics and Gynecology and Physiology, University of Western Ontario, London, Ontario, Canada N6A 4V2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD-1), 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24–40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11ßHSD-1 and 11ßHSD-2 and a cRNA for GR{alpha} were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11ßHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11ßHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11ßHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11ßHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11ßHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11ßHSD-1, 11ßHSD-2, and GR mRNA as well as 11ßHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.

conceptus, cortisol, developmental biology, embryo, glucocorticoid receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple mechanisms exist for regulating cellular actions of glucocorticoids such as cortisol. One such mechanism is the enzyme 11ß-hydroxysteroid dehydrogenase (11ßHSD) that reversibly converts biologically active cortisol to inactive cortisone. Two predominant 11ßHSD isoforms exist: 11ßHSD type 1 (11ßHSD-1), which has both dehydrogenase and oxoreductase activities and uses NADP+ and NADPH, respectively, as coenzymes [1, 2] but usually acts as a reductase enzyme [3, 4], and 11ßHSD type 2 (11ßHSD-2), which has only dehydrogenase activity and uses NAD+ as a coenzyme [5]. Previous work revealed placental 11ßHSD activity in numerous species (e.g., [6–10]). We demonstrated with pigs that maternal cortisol was converted to cortisone as it traverses the porcine placenta, and indeed, maternal cortisol provided 50% of fetal cortisone during mid and late gestation [11]. We subsequently showed that this placental cortisol conversion to cortisone resulted from the presence of NAD+- and NADP+-dependent 11ßHSD dehydrogenase activities [12]. We also used placental tissue fragment cultures with endogenous levels of substrates and coenzymes to show the following at 75 days of gestation: 1) both 11ßHSD oxoreductase and dehydrogenase activities were present, 2) dehydrogenase activity was 5-fold greater than oxoreductase activity, and 3) significant positive linear associations were present between net dehydrogenase activity and fetal or placental size [13].

A second cellular mechanism that regulates biological effects of glucocorticoids is the glucocorticoid receptor (GR). In humans, the GR consists of two protein isoforms, GR{alpha} and GRß, with GR{alpha} being the biologically relevant isoform [14, 15]. The GRß isoform is unable to bind glucocorticoid hormones but may form heterodimers with GR{alpha}, thereby altering the ability of GR{alpha} to regulate transcription [14, 15]. Thus, although the presence of cortisol in porcine embryos as early as Day 24 of gestation [16] suggests that cortisol may influence early embryonic development, this is necessary—but not sufficient—evidence that cortisol is active. The GRs must also be present within embryonic and placental target tissues for cortisol to exert its effects.

Therefore, to provide further suggestive evidence for an action of glucocorticoids on both porcine placental development and function and embryonic development, the objectives of the present study were as follows: 1) to determine if placental 11ßHSD-1 and 11ßHSD-2 mRNA expression and dehydrogenase activity are present in porcine placentae during early gestation (Days 24, 30, and 40); 2) to determine if this 11ßHSD expression is influenced by breed and uterine environment; and 3) to determine if mRNA expression of GR is present in embryonic placentae at these gestational ages. Because glucocorticoids are known to influence placental and fetal size as well as fetal survival in other species (e.g., [17, 18]), it was considered to be of interest to determine if these cellular mechanisms regulating cortisol actions might be altered by breed or uterine environments associated with altered fetal size and survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, Experimental Design, and Tissue Collections

White crossbred (WC) gilts remained intact (INT; n = 17) or were unilaterally hysterectomized and ovariectomized (UHO; n = 18) at 160 days of age as previously described [19]. Chinese Meishan gilts (ME; n = 18), which are known for their increased litter size [20], remained intact. Gilts were randomly assigned to be sampled at Day 24, 30, or 40 of gestation and were bred as previously described for this study [21]. On the appropriate day of gestation, gilts were slaughtered in the U.S. Department of Agriculture Meat Animal Research Center abattoir. Reproductive tracts were removed immediately, and placental samples were obtained as rapidly as possible. Tissue was rapidly frozen in liquid nitrogen and stored frozen at -80°C until used for 11ßHSD and GR analyses. All procedures involving use of animals were reviewed and approved by the Institutional Animal Care and Use Committee.

Assay Procedures

11ßHSD dehydrogenase activity Detailed assay procedures and validation for placental 11ßHSD dehydrogenase were previously published [12]. Enzymatic activity was measured in the presence of saturating substrate (2.5 µM unlabeled + 0.86 µCi labeled cortisol) and coenzyme (800 µM NAD+) concentrations. Filtered placental homogenates containing protein concentrations of 450 ± 26 µg (mean ± SEM) per 100 µl were prepared as described previously [12]. For the current studies, seven assays were conducted that each included one replicate from all treatment groups. The same nonexperimental sample served as an internal standard in each assay and provided an interassay coefficient of variation (CV) of 23.9%. All samples were assayed in triplicate (average CV of triplicate determinations = 5.23%), and specific conversion of [1,2,6,7-3H]cortisol (Amersham, Arlington Heights, IL) into labeled cortisone was calculated by subtracting nonspecific conversion in the presence of a heat-denatured placental preparation from total conversion. Each assay was used as a blocking factor in the statistical analysis of enzymatic data. One assay (block) was repeated once, and the average of the two assays for each sample was used in subsequent analyses.

Numerous attempts were made to validate procedures for measurement of 11ßHSD oxoreductase activity in homogenates prepared from frozen tissues. This assay made use of [3H]cortisone and the coenzyme NADPH. Many steps in the validation (linearity with time, 0–30 min; linearity with protein concentration, 230–950 µg protein; optimum pH, 6.0) and determination of saturating coenzyme (Km = 18.9 µM) concentrations were accomplished. However, repeatability among experimental assays proved to be exceptionally poor, casting doubt on the reliability of the data and, perhaps, reflecting problems that other investigators have experienced using frozen and stored or homogenized tissues [9, 22]. Hence, no data are presented for 11ßHSD oxoreductase activity.

RNA isolation and Northern blot analyses Total RNA was isolated from tissues using RNeasy Kits (Qiagen, Chatsworth, CA). Placental RNA was loaded onto denaturing MOPS (3-[N-Morpholino] propanesulfonic acid) 1.25% agarose/formaldehyde gels, and electrophoresis was conducted. Subsequently RNA was transferred to nylon membranes (Hybond-N; Amersham) via capillary blotting and was fixed to the membrane using ultraviolet (UV) cross-linking (UV Stratalinker 2400; Stratagene, La Jolla, CA). Porcine cDNA for 11ßHSD-1 was cloned from adult porcine liver, and cDNA for 11ßHSD-2 was cloned from adult kidney. Both cDNAs were obtained by a modified 3'-rapid amplification of cDNA ends and cloned into a pBluescript KS vector (Stratagene). Briefly, first-strand cDNAs were synthesized using an oligo-dT-adapter primer (designed in house; 5' GTC GAC GGT ACC GAT ATC T17 3') in a total volume of 20 µl. An aliquot (2 µl) was then subjected to a standard polymerase chain reaction (PCR; 94°C, 55 sec; 50°C, 55 sec; 72°C, 2 min; 30 cycles) by using the adapter primer and a gene-specific primer (5' GGG GGG TAC CCG GGT AGA AAG CTC TGT AGG 3') that corresponds to nucleotides 1–20 in the ovine 11ßHSD-1 cDNA [23] and the primer 5' CTG AAG CTG CTG CAG ATG GA 3' that corresponds to nucleotides 405–424 in the murine 11ßHSD-2 cDNA [24]. These cDNAs were subsequently sequenced, determined to be more than 70% homologous with other mammalian counterparts, and submitted to GenBank (11ßHSD-1, accession no. AF 414124, 1348 base pairs [bp]; 11ßHSD-2, accession number AF 414125, 1304 bp). Shorter cDNAs to be used for Northern blot analyses were generated from these larger cDNAs using, for 11ßHSD-1, the forward-primer 5' TTC TGG GGA TCT TCT TGG C 3' and the reverse-primer 5' AGT GGA TTC GCC ATT TTC C 3' and, 11ßHSD-2, the forward-primer 5' GCC AGC AGA CAT TAG CCG 3' and the reverse-primer 5' AAG TAC ATG AGC CCC AGG C 3'. These primer pairs generated a cDNA for 11ßHSD-1 that contained a 505-bp fragment that was 88–90% homologous with other species. Primer pairs for 11ßHSD-2 generated a cDNA that contained a 624-bp fragment and was 83–87% homologous with other species. These cDNAs for 11ßHSD-1 and 11ßHSD-2 had no sequence similarity with the other 11ßHSD isoform.

For the porcine GR, total RNA was isolated from female pig liver. Reverse transcription was then conducted as previously described [25] using random nonamer primers (Amersham) to produce cDNA. To amplify the GR, specific cDNA primers were designed for regions of the human GR{alpha} 3'-untranslated region [14]. These primers (forward primer, 5' TTG GTG CTT CTA ACC TGA TGG 3'; reverse primer, 5' GAT GGG AAT GTG AAA ATG GG 3') produced a 491-bp cDNA that would be specific for GR{alpha} [14] if the porcine GR is comparable to the human GR. This sequence was found to be 89.6% homologous with human GR 9{alpha} (U80946 and X03225) and was submitted to GenBank (accession no. AY007222). Porcine cDNA for porcine ß-actin was generated, and cDNA were cloned and sequenced as previously described [25].

For Northern blot analyses, 32P-labelled cDNAs were prepared using PCR procedures, [32P]dCTP (Dupont New England Nuclear, Wilmington, DE), and the above-noted primers specific for 11ßHSD and beta-actin transcripts. For Northern blot analyses of GR, and for ß-actin in the same Northern blot membrane preparations, cRNAs were prepared, and procedures detailed in Ambion's MAXIscript in vitro transcription kit (Ambion, Austin, TX) and [{alpha}-32P] UTP (Amersham) were used for preparation of 32P-labelled cRNA probes.

Northern blot analyses using these labeled cDNAs and cRNAs were conducted essentially as previously described for erythropoietin mRNA [25]. To insure that variability among processing operations was evenly distributed across all treatments, RNA from each treatment was equally represented on each gel/membrane, and membranes were used as a blocking factor in statistical analyses. Densitometric measures were conducted with a ChemImager (Alpha Innotech Corp., San Leandro, CA), or with Epi Chemi Darkroom (UVP, Inc., Upland, CA) used in conjunction with the NIH Image program (National Insitutes of Health, Research Services Branch, Bethesda, MD. Website at http://rsb.info.nih.gov/nih_image/).

Statistical Analyses

Data for mRNA and enzymatic activity were analyzed using the Statistical Analysis System [26]. All data were examined for normality of distribution (PROC UNIVARIATE NORMAL) and homogeneity of variance (Fmax test) and were transformed to a log or square root function when necessary to meet assumptions of analysis of variance. Enzymatic activity (mean of replicate analyses for each sample) was then analyzed using PROC MIXED and two-way analysis of covariance (ANOCVA) with treatments and day of gestation as main factors and assay as a random factor. Activity of the internal standard in each assay served as the covariate. All data for mRNA bands of interest were also analyzed using PROC MIXED and two-way ANOCVA with treatments and day of gestation as main factors and gel as a random factor. Each mRNA of interest was adjusted using ANOCVA and expression of ß-actin in the same lane as a covariate to account for potential differences in lane loading and membrane transfer. Comparisons of individual treatment means were made by a priori orthogonal contrasts. The least-squares mean ± SEM of original data are presented. A probability level of P <= 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fetal and placental data for the present study have been reported elsewhere in detail [21] and will not be repeated here. A single band of 11ßHSD-1 mRNA was observed at approximately 1.9 kilobases (kb) (Fig. 1A). Under conditions of the present study, placental mRNA expression of 11ßHSD-1 was undetectable on Day 24 of gestation (Fig. 1A), with the exception of one Meishan fetus. Hence, data for Day 24 were not included in the statistical analyses (Fig. 1B). The 11ßHSD-1 mRNA expression was modestly present by Day 30 (Fig. 1). Between Days 30 and 40, a 53% increase (P < 0.01) was observed in the expression of placental 11ßHSD-1 mRNA in all pig models (Fig. 1). No differences (P = 0.45) were observed among the three treatments (pig models).



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  		FIG. 1. A) Northern blot analysis of placental 11ßHSD-1 mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 10 µg of total RNA. B) Results of densitometric analysis (ChemImager; Alpha Innotech Corp., San Leandro, CA) of steady-state levels of placental 11ßHSD-1 mRNA. Values for 11ßHSD-1 were adjusted by analysis of covariance to those of ß-actin for the same sample. Results represent the least-squares mean + SEM of the number of replicates indicated by numbers within bars. WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts

On the contrary, 11ßHSD-2 mRNA expression was already readily apparent on Day 24 (Fig. 2). In contrast to 11ßHSD-1, 11ßHSD-2 mRNA had four distinct bands (Fig. 2A). A decidedly major band (band A) was present at approximately 2.3 kb, with more minor bands occurring at approximately 1.8, 1.5, and 1.2 kb. Statistical evaluations were conducted on each individual band and on the sum of the densitometric measurements for all bands. Analysis for the major band is presented (Fig. 2B). For this major band, ANOCVA did not reveal any treatment effects (P = 0.51) or treatment x day interactions (P = 0.10), but it did demonstrate marked day effects (P = 0.01) (Fig. 2B). These day effects represent an increase in mRNA expression across all treatments on Day 40 compared with Day 30 (P = 0.005). A priori orthogonal contrasts indicate that on Day 30, 11ßHSD-2 mRNA expression is less in WC-UHO pigs than in WC-INT pigs (P = 0.03). When the sum of bands A through D is evaluated, the interpretation of results is the same (data not shown).



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  		FIG. 2. A) Northern blot analysis of placental 11ßHSD-2 mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 10 µg of total RNA. B) Results of densitometric analysis (ChemImager; Alpha Innotech Corp., San Leandro, CA) of steady-state levels of placental 11ßHSD-2 mRNA for only the major band in lane A. Values for 11ßHSD-2 were adjusted by analysis of covariance to those of ß-actin for the same sample. Results represent the least-squares mean + SEM of the number of replicates indicated by numbers within bars. Statistics were conducted on log10-transformed data. A priori orthogonal contrasts indicate that on Day 30, 11ßHSD-2 mRNA expression is less in WC-UHO pigs than in WC-INT pigs (P = 0.03, a vs. b). WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts

Placental 11ßHSD dehydrogenase activity displayed marked, day-dependent increases in activity between Days 30 and 40 of gestation (P = < 0.01) (Fig. 3). On Day 24 (using an a priori orthogonal contrast), 11ßHSD dehydrogenase activity in ME placentae was greater than in WC-INT placentae on the same days (P < 0.037). Although 11ßHSD-2 mRNA and dehydrogenase activity increased in parallel between Days 30 and 40, ANOCVA procedures using 11ßHSD-2 mRNA expression as a covariate did not detect a significant linear relationship between dehydrogenase activity and mRNA expression as measured by the most prominent band or by the sum of all bands (P > 0.47).



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  		FIG. 3. Effects of breed, uterine environment, and gestational age on placental NAD+-dependent 11ßHSD dehydrogenase activity under optimal in vitro conditions of pH (9.0), coenzyme concentration (800 µM), substrate concentration (2.5 µM plus 0.86 µCi [3H]cortisol), time (15 min), and temperature (37°C). Each bar represents the mean + SEM of the number of pigs in parentheses. Statistics were conducted on square-root-transformed data. A priori orthogonal contrasts show that this enzymatic activity was greater in Meishan placenta compared with WC-INT placenta on Day 24 (P < 0.037, a vs. b). WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts

Expression of mRNA for GR was detected in placentae at each stage of gestation (Fig. 4). Labeled cRNA hybridized with a mRNA of approximately 8.8 kb. An increase (P = 0.013) was observed in GR expression with age that was independent of treatment (P = 0.83) (Fig. 4B) and that represented a 37% increase (P < 0.01) between Days 30 and 40. Placental ß-actin expression was not affected by treatment (P > 0.22).



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  		FIG. 4. A) Northern blot analysis of placental GR mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 30 µg of total RNA. B) Results of densitometric analysis (Epi Chemi Darkroom; UVP, Inc., Upland, CA) of steady-state levels of placental GR mRNA expression. Values for GR were adjusted by analysis of covariance to those of ß-actin for the same sample. Results represent the least-squares mean + SEM of the number of replicates indicated by numbers within bars. Statistics were conducted on log10-transformed data. WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the findings of primary importance are as follows: 1) 11ßHSD-2 mRNA expression and enzymatic activity are present within porcine placentae as early as Day 24 of gestation; 2) placental 11ßHSD-1 mRNA expression was not detected on Day 24 of gestation; 3) both 11ßHSD-1 and 11ßHSD-2 mRNA expression as well as 11ßHSD dehydrogenase activity increased between Days 30 and 40 of gestation; 4) placental mRNA expression of GR was present as early as Day 24 of gestation, and this expression increased with gestation; 5) 11ßHSD-2 mRNA expression on Day 30 of gestation decreased in embryos of WC-UHO gilts relative to WC-INT gilts; and 6) on Day 24 of gestation, placental 11ßHSD dehydrogenase activity was greater in Meishan compared with WC-INT gilts.

For all species in which detailed studies have been conducted, 11ßHSD-2 functions primarily as a dehydrogenase (converts active cortisol to inactive cortisone) [27], and 11ßHSD-1, although capable of oxidative and reductive activity, functions primarily as a reductase (converts cortisone to cortisol) [27]. In the present study, we cannot say with certainty that dehydrogenase activity represents only 11ßHSD-2 function. However, the presence of 11ßHSD-2 mRNA expression and assumed (dehydrogenase) biological activity at Day 24, along with the absence of 11ßHSD-1 mRNA expression, suggest an importance for lower active glucocorticoid concentrations in both placentae and embryos. This importance is further suggested by the decline in placental 11ßHSD-2 mRNA expression on Day 30 of gestation in embryos of WC-UHO gilts relative to WC-INT gilts. It was previously shown that decreased embryonic survival occurred in the crowded uterine environment on Day 40 compared with Day 30, although placental weights were not compromised [21]. However, more recent results demonstrated 38% decreases (P = 0.01) in placental weight on Day 35 in WC-UHO gilts [25].

The cause of decreased 11ßHSD-2 mRNA is unknown. Of the mRNA measured (ß-actin, GR, 11ßHSD-1, and 11ßHSD-2), only 11ßHSD-2 mRNA expression differed between WC-UHO and WC-INT gilts. This suggests a specificity of response in terms of at least these limited numbers of mRNAs. Hence, lower 11ßHSD-2 at Day 30 may be associated with an increased number of compromised embryos that will die by Day 40. Whether altered placental 11ßHSD expression is one cause of subsequent mortality, merely symptomatic of embryonic morbidity, or unrelated to mortality and morbidity, however, remains to be determined. The 11ßHSD dehydrogenase activity was not affected by the crowded uterine environment on Day 30, suggesting different sensitivities of mRNA and active protein to uterine crowding and associated biological alterations. Depending on the half-life of the 11ßHSD-2 protein, reduced mRNA expression would be expected to precede a subsequently lowered activity. An alternative hypothesis would be that 11ßHSD-1 protein is partially contributing to measured dehydrogenase activity in addition to its reductase activity. However, placental 11ßHSD-1 dehydrogenase activity is NADP+-dependent [10, 12, 28], and in the present assays, NAD+ was used as a coenzyme. Furthermore, although intraplacental concentrations of cortisol substrate are not known, plasma cortisol concentrations on Days 16–18 of pregnancy vary from 10 to 38 ng/ml [29]. Embryonic cortisol concentrations on Day 25 are quite low (~1.5 ng/mg DNA, or <10 ng cortisol/g tissue [17]). Such cortisol concentrations are incompatible with the previously measured Km of placental NADP+-dependent dehydrogenase activity (849 nM [12]). It is unlikely, therefore, that placental 11ßHSD-1 provides a substantial contribution to either measured or actual dehydrogenase activity at this stage.

It is also of interest to note the increased placental 11ßHSD dehydrogenase activity in Meishan versus WC-INT gilts. This is additional evidence for differences in aspects of glucocorticoid concentrations or metabolism in Meishan pigs during gestation [30] that could influence fetal development and survival. This finding is also contrary to what one might anticipate if glucocorticoids were the primary factor regulating fetal size; that is, with higher placental 11ßHSD-2, one would expect lower fetal cortisol and larger fetal weights [27, 31]. This might be especially true at Day 24, when cortisol impinging on the embryo is predominantly of maternal origin [16, 32]. However, many factors regulate fetal size (e.g., [33, 34]). Additionally, our interpretation of the potential effects of glucocorticoids on fetal size and the regulation of these effects by placental 11ßHSD and GR are confounded in the present study by an absence of information on embryonic glucocorticoid regulatory mechanisms.

The present study demonstrates the presence in porcine placentae of multiple transcripts of 11ßHSD-2 mRNA expression. The most prominent band occurred at approximately 2.3 kb. In mouse kidney and colon, multiple 11ßHSD-2 mRNA transcripts were evident at approximately 5 and 2.0 kb [24]. In term human placentae, 11ßHSD-2 mRNA was present at 1.9 and 4.0 kb [35]. Hence, precedents exist for multiple 11ßHSD-2 transcripts. However, in other studies, only single transcripts of 11ßHSD-2 mRNA were observed [36–38]. The biological relevance of these various mRNA species remains to be determined. For example, which bands are translated into active protein is not known, although it might be presumed that the major band (2.3 kb) represents the mRNA that contributes most to active protein. Mechanisms contributing to the various bands may represent alternative cleavage and polyadenylation patterns, alternative splicing of transcripts from a single gene, or transcription from more than one gene. The specific mechanism was not determined, but to date, evidence exists for only one gene encoding 11ßHSD-2 in human tissues [39].

Expression of 11ßHSD-1 mRNA was almost completely absent in Day 24 placentae with the amount of total RNA (10 µg) used. Such absence on Day 24 again suggests an additional mechanism by the developing placenta to minimize exposure of the placenta and embryo to biologically active glucocorticoids, because 11ßHSD-1 may serve to amplify glucocorticoid action at the cellular level [40].

Day 24 of gestation—relative to term in pigs (114 days; 21% or 0.21 of gestation completed)—represents the earliest measures of placental 11ßHSD mRNA expression in any species. Consequently, it is difficult to compare the relative onset of mRNA expression of these enzymes in pigs with those of other species. However, in human placenta (8–12 wk, or ~0.21–0.32 of gestation), there was a 4% conversion of cortisone to cortisol (reductase activity), but a 79% conversion of cortisol to cortisone (dehydrogenase activity [41]). In baboon placentae at early (0.33) gestation, mRNA and protein expression for both isozymes were present, and both increased with advancing gestation [38]. However, to our knowledge, earlier ages have not been measured. Therefore, the currently reported pattern of initiation of 11ßHSD mRNA expression is most nearly similar to that of enzymatic activities in humans at a comparable age (relative to term). Although, as noted above, data for oxoreductase activity were inconsistent, our measures do indicate the existence of this activity and, thereby, substantiate that translation of this 11ßHSD-1 mRNA into biologically active protein does occur.

We have previously demonstrated that intrauterine cortisol concentrations increase dramatically between Days 10 and 13 of pregnancy in gilts [42]. We have also demonstrated the presence of low cortisol concentrations in porcine embryos at Day 25 of gestation, with an 8-fold increase occurring by Day 35 [16]. Hence, at these early gestational stages in pigs, cortisol is present in the environment and in the embryos themselves, and it has the potential for influencing development. The currently reported early appearance of GR mRNA expression—with the caveat that it is translated into active protein—further suggests the involvement of glucocorticoids in early porcine placental development and/or function and indirectly in embryonic development. It has been previously suggested for other species that the presence of placental 11ßHSD not only reflects a regulation of transplacental passage of glucocorticoids but also a local regulation of placental glucocorticoid actions [43, 44]. Although only minimal indirect evidence exists in pigs [45], abundant direct and indirect evidence indicates a variety of effects of glucocorticoids on placental function in other species (e.g., [46–48]). Therefore, the presence of GR mRNA in the early porcine placenta is consistent with data in other species for a function of cortisol in porcine placentae.

The present study was not designed to evaluate the placental cellular location of either 11ßHSD or GR mRNA expression, as has been done for humans [38, 49, 50], baboons [49], and rats [51]. In contrast to hemochorial placentae of rat and humans, the porcine placenta is epitheliochorial in nature. Such a placenta consists of an outer layer of chorionic epithelial cells (trophectoderm) that contact the uterine endometrial epithelium, a somatic mesodermal layer, a splanchnic mesodermal layer, and finally, an inner thin layer of entodermal cells [52]. Hence, our preparations contained all these layers combined, and only by subsequent use of immunohistochemical and in situ hybridization techniques will it be possible to discern the cellular relationships of these enzymatic and receptor regulators of glucocorticoid actions in the porcine placenta. Nonetheless, as has been expressed for other species [44] and tissues [40], the colocalization of 11ßHSD-1, 11ßHSD-2, and GR in the porcine placenta suggests an intricate regulation of glucocorticoid action in this tissue at these early developmental stages.

In summary, these studies demonstrate the presence of 11ßHSD-1, 11ßHSD-2, and GR mRNA expression as well as 11ßHSD dehydrogenase activity in porcine placentae. Their presence suggests a role for glucocorticoids in porcine placental and embryonic development and function at these early stages of gestation.


   ACKNOWLEDGMENTS
 
We express our appreciation to Frank Reno, David Sypherd, and Terri Alberts for technical assistance. We also express our apologies to those investigators whose publications have contributed significantly to this area of research but could not be cited because of space constraints.


   FOOTNOTES
 
1 Mention of trade names is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. Back

2 Correspondence: H.G. Klemcke, U.S. Army Institute of Surgical Research, 3400 Rawley E. Chambers Ave., Fort Sam Houston, TX 78234. harold.klemcke{at}cen.amedd.army.mil Back

3 Current address: CuraGen Corporation, 16 Commercial Street, Branford, CT 06405 Back

Received: 18 April 2003.

First decision: 13 May 2003.

Accepted: 6 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Agarwal AK, Monder C, Eckstein B, White PC. Cloning and expression of rat cDNA encoding corticosteroid 11ß-dehydrogenase. J Biol Chem 1989 264:18939-18943[Abstract/Free Full Text]
  2. Agarwal AK, Tusie-Luna MT, Monder C, White PC. Expression of 11ß-hydroxysteroid dehydrogenase using recombinant vaccinia virus. Mol Endocrinol 1990 4:1827-1832[Abstract/Free Full Text]
  3. Lakshmi V, Monder C. Purification and characterization of the corticosteroid 11ß-dehydrogenase component of the rat liver 11ß-hydroxysteroid dehydrogenase complex. Endocrinology 1988 123:2390-2398[Abstract/Free Full Text]
  4. Jamieson PM, Chapman KE, Edwards CR, Seckl JR. 11ß-Hydroxysteroid dehydrogenase is an exclusive 11ß-reductase in primary cultures of rat hepatocytes: effect of physicochemical and hormonal manipulations. Endocrinology 1995 136:4754-4761[Abstract]
  5. Li KX, Obeyesekere VR, Krozowski ZS, Ferrari P. Oxoreductase and dehydrogenase activities of the human and rat 11ß-hydroxysteroid dehydrogenase type 2 enzyme. Endocrinology 1997 138:2948-2952[Abstract/Free Full Text]
  6. Osinski PA. Steroid 11ß-dehydrogenase in human placenta. Nature 1960 187:777[CrossRef][Medline]
  7. Bernal AL, Flint AP, Anderson AB, Turnbull AC. 11ß-Hydroxyteroid dehydrogenase activity (E.C. 1.1.1.146) in human placenta and decidua. J Steroid Biochem 1980 13:1081-1087[CrossRef][Medline]
  8. Baggia S, Albrecht ED, Pepe GJ. Regulation of 11ß-hydroxysteroid dehydrogenase activity in the baboon placenta by estrogen. Endocrinology 1990 126:2742-2748[Abstract/Free Full Text]
  9. Burton PJ, Waddell BJ. 11ß-Hydroxysteroid dehydrogenase in the rat placenta: developmental changes and the effects of altered glucocorticoid exposure. J Endocrinol 1994 143:505-513[Abstract/Free Full Text]
  10. Yang K. Co-expression of two distinct isoforms of 11ß-hydroxysteroid dehydrogenase in the ovine placenta. J Steroid Biochem Mol Biol 1995 52:337-343[CrossRef][Medline]
  11. Klemcke HG. Placental metabolism of cortisol at mid and late gestation in swine. Biol Reprod 1995 53:1293-1301[Abstract]
  12. Klemcke HG, Christenson RK. Porcine placental 11ß-hydroxysteroid dehydrogenase activity. Biol Reprod 1996 55:217-223[Abstract]
  13. Klemcke HG. Dehydrogenase and oxoreductase activities of porcine placental 11ß-hydroxysteroid dehydrogenase. Life Sci 2000 66:1045-1052[CrossRef][Medline]
  14. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM. Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 1985 318:635-641[CrossRef][Medline]
  15. Bamberger CM, Schulte HM, Chrousos GP. Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr Rev 1996 17:245-261[Abstract/Free Full Text]
  16. Klemcke HG, McGuire WJ, Christenson RK. Cortisol concentrations in early porcine embryos. Life Sci 1999 64:1307-1312[CrossRef][Medline]
  17. Blackburn WR, Kaplan HS, McKay DG. Morphological changes in the developing rat placenta following prednisolone administration. Am J Obstet Gynecol 1965 92:234-246[Medline]
  18. Roberts LG, Hendrickx AG. Hydrocortisone-induced embryotoxicity and embryonic drug disposition in H-2 congenic mice. J Craniofac Gen Dev Biol 1987 7:341-356[Medline]
  19. Christenson RK, Leymaster KA, Young LD. Justification of unilateral hysterectomy-ovariectomy as a model to evaluate uterine capacity in swine. J Anim Sci 1987 65:738-744
  20. Christenson RK, Vallet JL, Leymaster KA, Young LD. Uterine function in Meishan pigs. J Reprod Fertil Suppl 1993 48:279-289[Medline]
  21. Pearson PL, Klemcke HG, Christenson RK, Vallet JL. Uterine environment and breed effects on erythropoiesis and liver protein secretion in late embryonic and early fetal swine. Biol Reprod 1998 58:911-918[Abstract/Free Full Text]
  22. Lopez Bernal A, Anderson AB, Turnbull AC. Cortisol: cortisone interconversion by human decidua in relation to parturition: effect of tissue manipulation on 11ß-hydroxysteroid dehydrogenase activity. J Endocrinol 1982 93:141-149[Abstract/Free Full Text]
  23. Yang K, Smith CL, Dales D, Hammond GL, Challis JR. Cloning of an ovine 11ß-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid: tissue and temporal distribution of its messenger ribonucleic acid during fetal and neonatal development. Endocrinology 1992 131:2120-2126[Abstract/Free Full Text]
  24. Cole TJ. Cloning of the mouse 11ß-hydroxysteroid dehydrogenase type 2 gene: tissue specific expression and localization in distal convoluted tubules and collecting ducts of the kidney. Endocrinology 1995 136:4693-4696[Abstract]
  25. Klemcke HG, Vallet JL, Christenson RK, Pearson PL. Erythropoietin mRNA expression in pig embryos. Anim Reprod Sci 2001 66:93-108[CrossRef][Medline]
  26. SAS Online DOC, Version 8. Cary, NC: Statistical Analysis System, Inc.; 1999
  27. Stewart PM, Krozowski ZS. 11ß-Hydroxysteroid dehydrogenase. Vitam Horm 1999 57:249-324[Medline]
  28. Lakshmi V, Nath N, Muneyyrci-Delale O. Characterization of 11ß-hydroxysteroid dehydrogenase of human placenta: evidence for the existence of two species of 11ß-hydroxysteroid dehydrogenase. J Steroid Biochem Mol Biol 1993 45:391-397[CrossRef][Medline]
  29. Klemcke HG, Pearson PL, Vallet JL, Christenson RK. Plasma cortisol and aldosterone during early porcine pregnancy. J Anim Sci 1998 76:suppl 1241
  30. Klemcke HG, Christenson RK. Porcine fetal and maternal adrenocorticotropic hormone and corticosteroid concentrations during gestation and their relation to fetal size. Biol Reprod 1997 57:99-106[Abstract]
  31. Benediktsson R, Lindsay RS, Nobile J, Seckl JR, Edwards CRW. Glucocorticoid exposure in utero: new model for adult hypertension. Lancet 1993 341:339-341[CrossRef][Medline]
  32. Whitehead RH. The histogenesis of the adrenal in the pig. Am J Anat 1903 2:349-360[CrossRef]
  33. Wolpert L, Beddington R, Brokes J, Jessell T, Lawrence P, Meyerowitz E. Principles of Development. New York: Oxford University Press; 1998:417–441
  34. Evain-Brion D. Hormonal regulation of fetal growth. Horm Res 1994 42:207-214[Medline]
  35. Sun K, Yang K, Challis JR. Differential expression of 11ß-hydroxysteroid dehydrogenase types 1 and 2 in human placenta and fetal membranes. J Clin Endocrinol Metab 1997 82:300-305[Abstract/Free Full Text]
  36. Langlois DA, Matthews SG, Yu M, Yang K. Differential expression of 11ß-hydroxysteroid dehydrogenase 1 and 2 in the developing ovine fetal liver and kidney. J Endocrinol 1995 147:405-411[Abstract/Free Full Text]
  37. Campbell LE, Yu M, Yang K. Ovine 11ß-hydroxysteroid dehydrogenase type 2 gene predicts a protein distinct from that deduced by the cloned kidney cDNA at the C-terminus. Mol Cell Endocrinol 1996 119:113-118[CrossRef][Medline]
  38. Pepe GJ, Babischkin JS, Burch MG, Leavitt MG, Albrecht ED. Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11ß-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta. Endocrinology 1996 137:5678-5684[Abstract]
  39. Agarwal AK, Rogerson FM, Mune T, White PC. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11ß-hydroxysteroid dehydrogenase. Genomics 1995 29:195-199[CrossRef][Medline]
  40. Seckl JR, Walker BR. Minireview: 11ß-hydroxysteroid dehydrogenase type 1—a tissue-specific amplifier of glucocorticoid action. Endocrinology 2001 142:1371-1376[Abstract/Free Full Text]
  41. Giannopoulos G, Jackson K, Tulchinsky D. Glucocorticoid metabolism in human placenta, decidua, myometrium and fetal membranes. J Steroid Biochem 1982 17:371-374[CrossRef][Medline]
  42. Klemcke HG, Kattesh HG, Vallet JL, Roberts MP, McGuire WJ, Christenson RK. Intrauterine cortisol, aldosterone, and corticosteroid binding globulin-like activity during early porcine pregnancy and the estrous cycle. Biol Reprod 1998 58:240-247[Abstract/Free Full Text]
  43. Burton PJ, Smith RE, Krozowski ZS, Waddell BJ. Zonal distribution of 11ß-hydroxysteroid dehydrogenase types 1 and 2 messenger ribonucleic acid expression in the rat placenta and decidua during late pregnancy. Biol Reprod 1996 55:1023-1028[Abstract]
  44. Burton PJ, Waddell BJ. Dual function of 11ß-hydroxysteroid dehydrogenase in placenta: modulating placental glucocorticoid passage and local steroid action. Biol Reprod 1999 60:234-240[Abstract/Free Full Text]
  45. Gibb W, Randall GC. Prostaglandin production by porcine allantochorion in vitro: effect of cortisol infusion in vivo. Can J Physiol Pharmacol 1992 70:990-995[Medline]
  46. Steele PA, Flint AP, Turnbull AC. Activity of steroid C-17,20 lyase in the ovine placenta: effect of exposure to foetal glucocorticoid. J Endocrinol 1976 69:239-246[Abstract/Free Full Text]
  47. Ringler GE, Kallen CB, Strauss JF III. Regulation of human trophoblast function by glucocorticoids: dexamethasone promotes increased secretion of chorionic gonadotropin. Endocrinology 1989 124:1625-1631[Abstract/Free Full Text]
  48. Jones SA, Challis JR. Steroid, corticotrophin-releasing hormone, ACTH and prostaglandin interactions in the amnion and placenta of early pregnancy in man. J Endocrinol 1990 125:153-159[Abstract/Free Full Text]
  49. Pepe GJ, Burch MG, Albrecht ED. Expression of the 11ß-hydroxysteroid dehydrogenase types 1 and 2 proteins in human and baboon placental syncytiotrophoblast. Placenta 1999 20:575-582[CrossRef][Medline]
  50. Driver PM, Kilby MD, Bujalska I, Walker EA, Hewison M, Stewart PM. Expression of 11ß-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies. Mol Hum Reprod 2001 7:357-363[Abstract/Free Full Text]
  51. Waddell BJ, Benediktsson R, Brown RW, Seckl JR. Tissue-specific messenger ribonucleic acid expression of 11ß-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action. Endocrinology 1998 139:1517-1523[Abstract/Free Full Text]
  52. Patten BM. Embryology of the Pig, 3rd ed. New York: McGraw Hill; 1948



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