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BOR - Papers in Press, published online ahead of print April 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013458
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BIOLOGY OF REPRODUCTION 69, 679–686 (2003)
DOI: 10.1095/biolreprod.102.013458
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Vasopressin Receptor Expression in the Placenta

Irene Koukoulas2, John Risvanis3, Rebecca Douglas-Denton2,5, Louise M. Burrell3, Karen M. Moritz2,5, and E. Marelyn Wintour1,4

Howard Florey Institute of Experimental Physiology and Medicine,2 The University of Melbourne, 3010 Victoria, Australia Department of Medicine,3 The University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, 3084 Victoria, Australia Departments of Physiology4 Anatomy and Cell Biology,5 Monash University, Clayton, 3800 Victoria, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The arginine vasopressin (AVP) type 1a receptor (V1a) is well known to mediate vasoconstriction. In pregnancy, blood flow in the placenta is crucial for sustaining normal growth and development of the fetus. This is the first AVP receptor study in the placenta and fetal membranes. The aim was to compare, quantitatively, the level of V1a gene expression with that of a known marker for vascularization, aquaporin 1 (AQP1). V1a and AQP1 gene expression did not correlate; placental V1a mRNA levels were significantly upregulated at 45 and 66 ± 1 compared with 27, 100 ± 4, and 140 days (term ~150 days). V1a mRNA levels were much lower in fetal membranes in which no significant difference across gestation was observed. In situ hybridization histochemistry localized V1a gene expression in the maternal component of the placenta similar to the receptor-binding studies using 125I-labeled [d(CH2)5, sarcosine7] vasopressin. No AVP gene expression was observed in the placenta and fetal membranes, which eliminates local AVP production. This increase in V1a expression at 45 and 66 ± 1 days of gestation correlates with the period of maximal placental growth in the sheep and suggests that AVP and V1a receptors may play a hitherto unrecognized role in placental growth, differentiation, and/or function, particularly in the deleterious effects of heat stress, early in pregnancy, on fetal growth.

placenta, pregnancy, vasopressin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Over the past decade, there has been very convincing epidemiological evidence that inappropriately low birth weight, for gestational age, is correlated with an increased risk of the development of cardiovascular and metabolic disease (hypertension, coronary heart disease, late-onset diabetes, dyslipidemia) in adult life [1, 2]. The factors that result in intrauterine growth retardation include inadequate diet (in total calories or specific components) and exposure of the fetus to excess natural or synthetic glucocorticoids [3–5]. Placental insufficiency is also a potential cause of fetal programming [6]. The placenta serves the purpose of delivering nutrients to the fetus and removing unwanted waste products and also produces hormones that influence both maternal physiology and fetal growth so that the optimal environment is provided for the maintenance of the pregnancy and the ultimate birth of an appropriately grown neonate. For the placenta to function, there are two main requirements: The placenta itself must grow appropriately, and there must be a matching and adequate perfusion by blood on the maternal and fetal sides [7]. In the sheep maximal placental growth occurs in the first half of pregnancy [8], although the bulk of fetal growth occurs in the last third. In previous studies we had established that aquaporin 1 (AQP1) was expressed only in the vasculature of the ovine placenta throughout gestation [9]. In that study we looked at expression at three stages: 66 days, 99 days, and 140 days. These tissues were investigated, initially, for the expression of what we hoped would be another vascular marker, the type 1a receptor (V1a) for the pituitary hormone arginine vasopressin (AVP). When we found an unusual pattern of distribution and in a new location (the maternal stroma of the placenta), further investigations were carried out to define the ontogeny and potential function of this V1a receptor.

We were aware that one maternal stress that has been used extensively to produce fetal growth retardation in an animal model is heat stress. It has been known for some time, particularly in livestock production, that exposure to excessive heat lowers the reproductive performance and can result in undergrown neonates [10]. Heat stress is known to cause increased secretion, in the maternal compartment, of various hormones, including prolactin and AVP [11, 12]. AVP is a well-established vasoconstrictor, acting via the major vascular receptor type, V1a, to produce an effect [13]. The mechanism(s) by which heat stress affects fetal growth have been the subject of intense investigation, particularly in sheep. Initially it was supposed that decreased placental perfusion was the main mechanism [14, 15]. More recent data demonstrate that when heat stress is applied for a prolonged period, starting in early gestation, it is placental growth that suffers initially, before fetal growth is compromised, and there is evidence that it is the fetal component of the placenta that is most restricted in growth [16–18]. Because the fetal trophoblast is responsible for the bulk of the hormone production (e.g., progesterone, placental growth hormones), this leads also to altered placental function.

The effect of heat stress on placental blood flow has been reported to differ, depending on the stage of gestation at which it has been applied. Early in gestation, perfusion has been claimed to be decreased by 20–30% [5, 7, 8]. When heat was applied acutely in the last third of gestation, however, it was reported that neither maternal nor fetal perfusion was compromised [19].

It was hypothesized that the level of expression of the V1a receptor in the placenta may change during pregnancy, with lower levels being found in the latter stages. We therefore set out to quantify the mRNA of the V1a receptor in the ovine placenta and fetal membranes, with the sensitive and highly reproducible technique of real-time polymerase chain reaction (PCR), as used previously [9]. In situ hybridization histochemistry was used to determine the tissue distribution of the V1a mRNA and ligand binding to explore the protein expression. When the tissue distribution of V1a mRNA and protein was shown to be very strong in an unexpected site, the maternal stroma, preliminary experiments using a specific antagonist were performed to gain some insight into the potential function of the receptor in this site. The effect of the antagonist was assessed by both measurement of placental weight and analysis of the volumes and composition of fetal fluids (amniotic and allantoic) after antagonist treatment. At the time of treatment (40–50 days of gestation), the amniotic fluid is very similar to extracellular fluid of the fetus, and the allantoic fluid represents fetal urine [20].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Animals

In accordance with the NH&MRC of Australia guidelines, all animal experiments were approved by the Howard Florey Institute Ethics Committee. Pregnant Merino ewes of known gestational age were killed by an overdose of sodium pentobarbitone (100 mg/kg body weight, Lethabarb, Arnolds, Boronia, Australia). Allantois, amnion, chorion, and cotyledon tissue samples were collected from four fetuses from the following age groups: 65–67, 96–104, and 140 days of gestation (term is 145–150 days) for real-time PCR and in situ hybridization histochemistry studies. The placentae were weighed before individual placentomes were counted and sorted (types A–D), and only A types were used in this study [21]. Cotyledon tissue samples were also collected from four fetuses at 27 days of gestation and six fetuses at 45 days of gestation. Uterine wall sections between cotyledons were also collected from four pregnant ewes at 27 days of gestation. Additional cotyledon samples were also collected at 41 and 51 days of gestation for in situ hybridization histochemistry and 63 days of gestation for in vitro autoradiography. Fresh tissues were rinsed in physiological saline (0.9%) and cleaned with gauze to remove excess blood prior to fixing and freezing. After cleaning, tissues were immediately frozen in liquid nitrogen and stored at -80°C until further use or fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 4 h at room temperature and routinely processed. Four pregnant ewes were also given the V1 antagonist OPC-21268, orally at 30 mg/kg in a solution containing 5% Arabic gum twice daily. Maximal and effective responses at this dose has previously been reported during in vivo dose-response studies in the rat [22, 23]. Treatment was carried out for 10 days, commencing from 40 days of gestation and ending at 50 days of gestation. Control animals had cannula placed in a jugular vein under local anesthetic at 38 days of gestation. Between 40 and 50 days of gestation, 0.9% saline was infused at the rate of 0.19 ml/h. Treated and control pregnant ewes were killed at 50 days of gestation and their tissues collected as above. Volume and composition of amniotic and allantoic fluids were analyzed as reported previously [20]. These data were used to assess the effects, if any, of the maternally administered antagonist.

Real-Time PCR

Total RNA was extracted from frozen tissue by the acid guanidinium thiocyanate-phenol-chloroform extraction method, DNAase treated, and cDNA synthesized as described previously [9]. For the relative quantitation of gene expression, real-time PCR was performed using an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). Primers and TaqMan probes for real-time PCR were designed using Primer Express version 1.0 (Applied Biosystems). The nucleotide sequences for the AQP1, AVP, V1a, and V2 primers and probes are listed in Table 1, and their positions relative to GenBank/EMBL data entries are also provided. Ribosomal 18S (18S) TaqMan probe and primers were supplied by Applied Biosystems in a reagent kit.


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  		TABLE 1. TaqMan probe and primer sequences and their positions relative to the EMBL/GenBank databases and their final concentrations.*

For each TaqMan probe, 6-carboxy-fluorescein (FAM) was attached to the 5' end for AQP1, V1a and V2, and VIC for 18S. For all probes, 6-carboxytetramethylrhodamine (TAMRA) was attached to the 3' end. No TaqMan probe was used in the AVP assay. For the relative quantitation of gene expression, a multiplex comparative CT method was employed. Separate AQP1, V1a, and V2 reactions were set up in which 18S was detected in all of these reactions, although the primers for 18S were limited. In pilot experiments, multiplex versus nonmultiplex CT values were compared, and for all genes studied, the CT values were identical, suggesting that multiplex reactions did not affect CT values. A validation experiment was also performed to test whether the comparative CT method could be used for the relative quantitation of gene expression. Here approximately equal efficiencies of AQP1, V1a, and V2 amplifications together with 18S were tested using different template concentrations, and in all sets of multiplex reactions, approximately equal PCR efficiencies were obtained. A TaqMan assay was not developed for the study of AVP expression because a SYBR Green assay was used instead. Nonmultiplex PCR reactions were carried out in 25-µl volumes consisting of 1 x SYBR Green Master Mix [Applied Biosystems], 900 nM of forward and 300 nM reverse of primers for AVP. The cDNA (5 ng) and no reverse transcriptase preparations were amplified using the identical PCR conditions used for the above TaqMan assays.

Real-Time PCR Calculations

Comparative CT calculations for the expression of AQP1, V1a, and V2 were all relative to a chosen calibrator. In the first ontogeny study, gene expression was studied in the fetal membranes at 66 ± 1, 100 ± 4, and 140 days of gestation; the amnion at 66 ± 1 days of gestation was the chosen calibrator because this tissue had the lowest value. It also allowed the results of the previous study on AQP1 expression [9] to be compared.

The second study, which focused on the placenta at 27, 45, 66 ± 1, 100 ± 4, and 140 days of gestation, included the 140-day placenta as the calibrator. Each AQP1, V1a, and V2 study was performed separately. A multiplex reaction was performed in each assay, in which both a test gene (AQP1, V1a, or V2) and the endogenous control gene 18S were amplified together. To achieve quantitative values, 18S CT values were first subtracted from a test CT value for each well to give a {Delta}CT value. {Delta}{Delta}CT values were achieved by subtracting the average calibrator {Delta}CT value from the {Delta}CT value in every other tissue at each age group. The expression of AQP1, V1a, and V2 relative to the calibrator at each gestational age was evaluated using the expression 2-{Delta}{Delta}CT. For the SYBR Green (Applied Biosystems) assays, CT values were tabulated after each run, and the presence (CT <40) or absence (CT = 40) of AVP expression was subsequently determined.

Riboprobe Preparation

A 6.1-kb XbaI-HindIII genomic fragment was subcloned and used to generate smaller fragments for further subcloning experiments and subsequent riboprobe synthesis [24].

After the recombinant plasmid was linearized, both antisense and sense (negative control) riboprobes were prepared by in vitro transcription using a riboprobe kit (Promega, Madison, WI) in which [{alpha}-35S]uridine 5-triphosphate (100 Ci mmol-1) was incorporated (Bresatec, Thebarton, Australia). The riboprobes were hydrolyzed, precipitated, and resuspended in 10 mM dithiothreitol prior to hybridization histochemistry.

In Situ Hybridization Histochemistry

This was carried out as described previously [9] on paraffin sections (4 µm).

In Vitro Autoradiography

The selective V1a antagonist [1-(ß-mercapto-ß, ß-cyclopentamethylene propionic acid), 7-sarcosine] vasopressin ([d(CH2)5, sarcosine7]) (Auspep, Melbourne, Australia) was radioiodinated with 125I (specific activity: 2394 Ci/mmol) and purified as previously described [25, 26]. Frozen sections (15 µm) were prehybridized for 15 min with a chilled buffer consisting of 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 0.1% BSA. Sections were then placed into a chilled hybridization buffer consisting of 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 100 U/ml Aprotinin (Sigma, St. Louis, MO), 0.5 mg/ml Bacitracin (Sigma), 0.1% BSA, and either 1.3 x 106/10 ml of 125I-labeled [d(CH2)5, sarcosine7] vasopressin for total binding or, for nonspecific binding, a mixture of cold 2 x 106 M vasopressin and 2 x 106 M [d(CH2)5, sarcosine7] vasopressin. Sections were then incubated for 8 h at 4°C followed by five 30-sec washes, in which four washes were in a chilled wash buffer consisting of 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 0.1% BSA, and the remaining wash was performed in chilled double-distilled water. The sections were finally air dried before autoradiographic exposure to Agfa-Scopix CR3B film (Nunawading, VlC, Australia) for 10 days.

Image Acquisition

For in situ hybridization histochemistry, light-field and dark-field images were captured on a Microphot microscope (Nikon, FSE Pty Ltd, Melbourne, Australia) linked to a Sony 930P video camera (Sony, North Ryde, Sydney, Australia). Digitized images were subsequently processed using microcomputer imaging device software (MCID) (Imaging Research Inc., St. Catherine's, Canada). For in vitro autoradiography, 125I binding intensities on the x-ray films were analyzed using the MCID coupled to a Pericom computer system (Pericon Nu Horizons, Melville, NY).

Sample Analysis

Sodium, potassium, chloride, total carbon dioxide, urea, creatinine, glucose, fructose, lactate, and total protein concentrations were measured using a Synchron CX5 clinical system (Beckman, Fullerton, CA) as described previously [27]. Both allantoic and amniotic fluids were analyzed for osmolality by freezing point depression using an Advanced osmometer (Advanced Instruments, Norwood, MA).

Statistical Analyses

Data from the real-time PCR studies were measured by one-way ANOVAs with all pairwise multiple comparison procedures (Tukey test). All other data were measured by a Student t-test (placental and fetal weights and fluid composition and volumes). All data are reported as mean ± SEM unless otherwise stated. The level of significance for all tests was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Quantitation of AQP, V1a, and V2 Gene Expression

An initial screen of samples of cotyledon, amnion, allantois, and chorion from three ages (66, 99, 140 days) showed a very high expression of V1a mRNA at 66 days only in the placental tissue (data not shown). This was completely different from the data with AQP1. The study then concentrated on the expression in the cotyledons.

To establish whether V1a receptor gene expression was also high prior to the 66 ± 1 day cotyledon, further studies were performed at 27 and 45 days of gestation, and the 140 day cotyledon was used as the calibrator. Intercotyledonary uterus at 27 days of gestation was also analyzed for V1a receptor gene expression. In Figure 1, V1a receptor expression was observed to be significantly upregulated at both 45 (10.5 ± 2.6) and 66 ± 1 (11.9 ± 2.9) days of gestation but not at 27 (0.45 ± 0.1), 100 ± 4 (2.5 ± 1.4), and 140 (1.3 ± 0.2) days of gestation. Levels of V1a receptor mRNA in the 27-day intercotyledonary portion of the uterus (0.57 ± 0.15) was similar to the 27-day cotyledon, whereas V1a receptor mRNA content in the adult kidney (13.3 ± 1.1) was analogous to the 45- and 65-day cotyledons.



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  		FIG. 1. V1a and V2 receptor gene expression in the developing placenta (open bars) at 27, 45, 66 ± 1, 100 ± 4, and 140 days of gestation as well as at 27 days of gestation in the intercotyledonary uterus (filled bar). Gene expression in the adult kidney is also presented (striped bar). Fold expression is relative to the 140-day placenta. Values are expressed as the mean ± SEM (n = 4, except for the 45-day placenta where n = 6 and adult kidney where n = 3). *Statistically significant difference (P < 0.05)

V2 receptor gene expression was also analyzed throughout gestation in the placenta. At 27 (2.7 ± 0.56), 45 (1.6 ± 0.89), 66 ± 1 (0.7 ± 0.33), 100 ± 4 (1.2 ± 0.36), and 140 (1.0 ± 0.43) days of gestation, little expression was observed. The intercotyledonary portion of the uterus was similar also, whereby little V2 mRNA was identified (2.5 ± 1.1). The adult kidney, which expresses large amounts of V2 receptor, was used as positive control.

To detect any local AVP gene expression, an ovine SYBR Green real-time PCR assay was developed. All of the above cotyledon samples (45–140 days of gestation) as well as the 27-day uterus samples were screened for AVP gene expression. Adult kidney was used as a negative control, whereas adult hypothalamus was used as a positive control. A significant amount of AVP gene expression was detected in the adult hypothalamus (CT values at approximately 24), whereas the remainder of the samples contained no AVP gene expression (CT values at 40).

Localization of V1a Receptor Gene Expression

V1a receptor gene expression was most abundant in the 45 and 66 ± 1 day cotyledon in which it was localized specifically in the maternal tissue surrounding the fetal villi (Fig. 2). In Figure 2E, conspicuous expression was confined to the maternal part of the cotyledon, adjacent to the invading fetal villi. V1a receptor mRNA was predominant in stromal cells and cells that appeared undifferentiated (Fig. 2, E, F). Trophoblast cells of the fetal villi were void of V1a receptor mRNA. In large arteries of the uterus, between cotyledons, the V1a signal was seen, predominantly in the tunica adventitia (Fig. 3).



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  		FIG. 2. Dark-field photomicrographs (A, C, and E) and bright-field photomicrographs (B, D, and F) of tissue hybridized with the V1a receptor riboprobe labeled with 35S. cotyledon samples at 30 (A and B), 45 (C and D), and 65 (E and F) days of gestation are presented; hybridization is clearly represented by the white labeling in the dark-field photomicrographs (A, C, and E). An invading fetal villus is clearly seen in photomicrographs (C and D) at 45 days of gestation as well as 65 days of gestation at the bottom of photomicrographs (E and F). A significant amount of labeling is observed in the 65 cotyledon in the maternal stromal cells (E). Inserts in photomicrographs (A, C, and E) represent the corresponding sense slide. Magnification, x100. BV, Blood vessels; FV, fetal villus; MS, maternal stromal cells



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  		FIG. 3. Dark-field photomicrographs (A and C) and bright-field photomicrographs (B and D) of the 27-day intercotyledonary uterus with 35S-labeled riboprobe for the V1a receptor. Blood vessels (arrow) within the uterus are clearly labeled with the V1a receptor riboprobe (A and B, x40) (nonspecific binding to blood is also apparent). On closer examination, only the tunica adventitia (composed of collagen and elastin) displayed white labeling in the dark-field photomicrographs and not the tunica media (smooth muscle layer) and tunica intima (endothelial layer) (C and D, x400). LA, Large arterioles; TA, tunica adventitia

AVP-Binding Sites in the Sheep Cotyledon

In the 63-day cotyledon, AVP-binding sites were primarily localized in maternal stromal cells closest to the capsule (Fig. 4). However, the protein was expressed at a lower level than the mRNA.



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  		FIG. 4. Computer-generated images of 125I-labeled [d(CH2)5, sarcosine7]vasopressin binding in the sheep 63-day cotyledon. Red represents high level of binding, whereas yellow represents intermediate binding. Green represents undetectable levels of binding. A) Total binding. B) Nonspecific binding

Effects of Antagonist Treatment

Allantoic fluid OPC-21268 treatment caused a significant increase in phosphate concentration, compared with controls (Table 2). A significant increase in fluid volume was also observed in the OPC-21268 group, compared with the saline controls (Table 2).


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  		TABLE 2. Allantoic (1) and amniotic (2) fluid volume and composition after 10 days of OPC-21268 (O) or saline (S) administration.{dagger}

Amniotic fluid OPC-21268 treatment caused a significant increase in sodium, creatinine, calcium, phosphate, magnesium, fructose, lactate, and total protein concentrations, compared with controls (Table 2). It also caused a significant decrease in potassium concentrations, compared with controls (Table 2). No significant difference was observed between the OPC-21268 and saline group fluid volumes (Table 2).

Placental and fetal weights After administration of either OPC-21268 or saline for 10 days from 40 to 50 days of gestation, no significant difference was observed in placental weight between the OPC-21268 (93.5 ± 8.6 g) and saline (90.8 ± 4.9 g) groups at 50 days of gestation. There was also no significant difference between the OPC-21268 (17.9 ± 0.75 g) and saline (16.1 ± 0.42 g) groups in fetal weight at 50 days of gestation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Novel Localization and Timing of V1a Receptor Expression in the Ovine Placenta

When the human V1a receptor was first cloned and various tissues screened for gene expression, a low level was detected by Northern blot analysis in the human placenta [28]. This study is the only detailed report of placental expression of this gene. Because this is the major receptor to which AVP binds, it was expected that it would also be in placental vessels [13, 29]; however, the changes in V1a receptor mRNA did not parallel those of another vascular marker, the AQP1 gene [9]. Moreover, in the early blood vessels (at 27 days) when mRNA for the V1a receptor was detected, it was stronger in the adventitia than in the media. This may be due to the fact that in newly forming blood vessels, myofibroblasts of the adventitia are the precursors of the smooth muscle of the media, as has been proposed in some models of remodeling after balloon stretch [30]. V2 receptor mRNA also did not significantly change throughout gestation. In addition, when hybridization histochemistry was performed to delineate the cell site of V1a receptor expression, it was found, unexpectedly, in the maternal stroma. The greatest magnitude of expression occurred in the first half of pregnancy, at 45 and 66 ± 1 days when maximal growth of the placenta is known to take place [8, 31–33]. In fact, it is generally accepted that adequate placental growth must occur in the first half of pregnancy in the sheep to support normal fetal growth during the second half of pregnancy when maximal fetal body growth occurs [34]. To be able to speculate about the significance of the new findings and try and understand why peak placental expression of the V1a receptor might occur in the first half of pregnancy, it is necessary to understand how the ovine placenta develops. It is also necessary to understand what other potential growth factors/hormones might be involved during the rapid phase of placental growth.

The ovine placenta consists of a number (70–100) of individual placentomes, each formed when the fetal trophoblast interdigitates with the maternal uterine lining at specific sites called caruncles [35]. Attachment begins at about Day 16–18; even before then the conceptus starts communicating with the uterus. Pregnancy recognition in the sheep involves production by the trophoblast of interferon-{tau}, which acts on the uterus to decrease estrogen receptor alpha and oxytocin receptor gene expression, thus inhibiting pulsatile prostaglandin 2-alpha production, which would cause luteolysis in the ovary of a nonpregnant ewe [36, 37]. Significant interdigitation occurs between Days 25 and 30, accompanied by a marked increase in the expression of c-fos, a transcription factor controlling growth and differentiation [38]. During the time of maximal placental growth (28–80 days), a number of genes show peak placental expression, and these include aromatase (necessary for estrogen biosynthesis), estrogen receptor, oxytocin receptor, insulin-like growth factor II (IGF-II) and IGF binding proteins 2, 3, 4, and 5 as well as the V1a receptor, as demonstrated in the current study [39, 40]. The expression characteristics of all these genes are not the same, however.

The fetal trophoblast contains two cell types (~80% uninucleate, ~20% binucleate) [41], and the binucleate cells are the site of synthesis of steroid (estrogen and progesterone) and protein (ovine placental lactogen) hormones [35, 42]. Binucleate cells are migratory, and throughout pregnancy they can fuse with maternal uterine epithelial cells to form a fetomaternal hybrid tissue called the syncytium [35]. Aromatase is expressed in the fetal trophoblast binucleate cell, IGF-II in the mesoderm just adjacent to the fetal trophoblast, and the IGF-binding proteins, and the estrogen, oxytocin, and V1a receptors are expressed in the maternal stroma. Thus, there is a good morphological basis for the hormones from the fetal trophoblast exerting biological effects on the development of the maternal component of the placenta. Less is known of how factors made in the maternal stroma may influence the development and function of the fetal trophoblast. There is experimental evidence of decreased maternal progesterone concentrations (and decreased placental growth) in the second half of pregnancy when heat stress was applied to the mother in the first half of pregnancy [12, 14]. In addition, overfeeding of adolescent ewes in the first half of pregnancy can also result in later decreased placental growth and function [43]. In these situations, it is suggested that the early insult to the mother altered the trajectory of growth and development of the fetal component of the placenta, which only becomes obvious in the second half of pregnancy.

Source of Ligand for V1a Receptor

No AVP mRNA was detected in the placenta, and it is unlikely that the V1a receptor "sees" AVP from a local source. It could potentially be accessed by AVP produced by the fetal hypothalamic-pituitary because by 42 days of gestation, AVP immunoreactivity is very strong in the fetal brain, particularly in the external layer of the median eminence [4]. AVP levels in the maternal plasma are low throughout pregnancy [45]; however, heat stress is known to cause hyperventilation in sheep and increase maternal plasma AVP concentrations [11]. This could then act on the V1a receptor in the maternal component of the placenta and alter function in some, as-yet-unidentified way to compromise the fetal trophoblast development.

Explanation of Altered Fetal Fluids with V1a Antagonist

There was no obvious effect of V1a antagonism on placental weight. The only effects seen were on the composition of amniotic fluid and volume of allantoic fluid. At this stage (50 days of gestation), amniotic fluid is close to, but not identical with, fetal extracellular fluid, and allantoic fluid represents fetal urine, predominantly from the developing metanephros [20]. The increase in allantoic fluid is reminiscent of the changes recorded when the progesterone/estrogen ratio was altered in ewes in early pregnancy [46]. It is purely hypothetical, but one could speculate that the OPC-21268 may have interfered with progesterone production (by an agonist action on the V1a receptor) or interfered with the action of progesterone on the progesterone receptor. Interactions between oxytocin/AVP receptors and progesterone have been reported [47, 48]; however, in neither case was there evidence of the peptide hormone altering the effectiveness of the steroid receptor. It is unlikely that the effect of the antagonist was an indirect one, such as lowering placental perfusion because V1a antagonism has been shown to be without cardiovascular consequences in normal sheep [49], and AVP itself does not cross the ovine placenta [50]. Further experiments are required to test these hypotheses. It would be interesting to follow the plasma progesterone levels and placental growth in late pregnancy of sheep treated with AVP or the antagonist, between 40 and 70 days of gestation.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In summary, an unexpectedly high expression of both mRNA and protein for the V1a receptor was found in the maternal stroma of the ovine placenta during the maximal growth phase. It is suggested that this receptor would be activated by elevated plasma AVP concentrations in heat-stressed sheep and may be involved in the mechanism(s) by which heat stress in the first half of pregnancy leads to decreased placental growth and function later in pregnancy.


   ACKNOWLEDGMENTS
 
The authors would like to thank Jo Culican for administering the OPC-21268 compound to sheep at the Howard Florey Institute farm. The purchase of the ABI PRISM 7700 sequence detection system was possible with funding from the following foundations: the Clive and Vera Ramaciotti Foundation, Harold and Cora Brennen Benevolent Trust, Phillip Bushell Foundation, and Sylvia and Charles Viertel Foundation.


   FOOTNOTES
 
1 Correspondence: Marelyn Wintour, Department of Physiology, Monash University, Clayton, 3800 Victoria, Australia. FAX: 61 3 9905 2547; mwc{at}med.monash.edu.au Back

Received: 27 November 2002.

First decision: 23 December 2002.

Accepted: 9 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

  1. Barker DJ. The malnourished baby and infant. Br Med Bull 2001 60:69-88[Abstract/Free Full Text]
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