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Biology of Reproduction 66, 555-561 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Placental Growth Hormone and Lactogen Production by Perifused Ovine Placental Explants: Regulation by Growth Hormone-Releasing Hormone and Glucose

M.C. Lacroix1,,a, P. Bolifrauda, D. Durieuxa, A. Pauloina, M. Vidaudb, and G. Kanna

a Unité de Biologie Cellulaire et Moléculaire, I.N.R.A. 78352 Jouy en Josas, France b Laboratoire de Génétique Moléculaire, Faculté de Pharmacie, 75006 Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 ± 0.2 and 353.6 ± 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.

growth hormone, placenta, pregnancy, syncytiotrophoblast, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In sheep, as in other mammals, fetal growth is conditioned by a normal placental development. Factors underlying placental growth, particularly in early pregnancy, are poorly understood. Insulin-like growth factors (IGFs) and their binding proteins seem to be involved in placental development [1, 2]. Growth factors belonging to the growth hormone (GH)/prolactin (PRL) family may also be involved. In sheep, production of a placental lactogen (oPL) by binucleated trophoblast cells occurs from Day 16 until the end of pregnancy [3, 4]. This hormone can act as a growth-promoting agent as it binds GH/PRL receptors [5–7] and may influence metabolic activities during pregnancy [8–12]. A second hormone of the GH/PRL family produced by the ovine trophoblast and syncitium is placental growth hormone (oPGH) [13, 14]. This hormone is produced between Days 30 and 60 of pregnancy, a key phase when the placental growth rate is very high, and levels peak near Day 55 [14, 15]. The placenta is a potential target for oPGH, as GH receptors are expressed in the trophectoderm, syncitium, fetal mesoderm, and maternal uterine stroma [14]. Thus, oPGH may influence placental growth in the first half of pregnancy.

The regulation of oPGH production is not understood, but could involve hypothalamic-like type factors, including placental growth hormone-releasing hormone (GHRH) [16]. The influence of the metabolic environment on oPGH production remains to be investigated. Modulation of maternal nutrition in early pregnancy influences fetal-placental development in sheep [17, 18]. Wallace et al. [19] developed a model of singleton pregnancies in peripubertal adolescent sheep, which resulted in a population of pregnant animals that were highly sensitive to nutritional status. In this model, high nutrient intake throughout early and mid pregnancy was characterized by higher insulin and glucose levels in the maternal circulation and a decrease in placental and fetal weights [19]. Nutritional imbalance probably affected production of some placental hormones, and this stimulated our interest in the effects of high glucose levels on oPGH and oPL production. The aim of this study was to investigate the regulation of oPGH and oPL production by GHRH and glucose in a perifusion system of ovine placental explants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Pregnant ewes of the Préalpes du Sud breed were used. All experiments were conducted in accordance with French guidelines on the care and use of experimental animals. The pregnant ewes were fed according to French nutritional recommendations covering 100% physiologic needs at 45 days of gestation [20]. Placentae were collected on Day 45 of pregnancy, which corresponds to the period of maximal placental GH production [13, 14]. After removal of the fetus, cotyledons (the fetal side of each placentome) were manually separated from the caruncle (the maternal side of each placentome) and minced into explants of about 1 mm3. For each experiment, 500 mg of explant tissue was immediately frozen in liquid nitrogen at time zero (T0), and stored at -20°C until protein or RNA extraction. Remaining explants were divided into aliquots and immediately perifused.

Perifusion System

One placenta was used for each perifusion experiment. Cotyledon explants (800 mg) were packed into 8 sterile perifusion chambers made from 2-ml disposable plastic syringes (the outlet being fitted with a 20-µm nylon filter mesh; Pharmacia, Orsay, France) and positioned vertically in a 37°C water bath. Chambers were perifused using an 8-channel peristaltic pump (Gilson minipulse, Villiers le Bel, France) for 8 h. The perifusion medium was Dulbecco modified Eagle medium (DMEM0; without glucose, phenol red, sodium pyruvate, sodium bicarbonate; Sigma, Saint Quentin Fallavier, France) supplemented with 0.5% BSA (Sigma), 4 mM L-glutamine, 3.7 g/L sodium bicarbonate, 1 mM sodium pyruvate, 2 mM insulin, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 400 mU trypsin inhibitor (aprotinin; Sanofi Synthelabo, Gentilly, France). The medium was constantly gassed with 95% O2 and 5% CO2. In some experiments the medium was further supplemented with 5.5 mM, 12 mM, or 25 mM glucose or 25 mM mannose as the control for changing osmolarity. The rate of perifusion was 600 µl/h. Media were collected in a refrigerated chamber (4°C) every hour and immediately stored at -20°C until RIA for oPGH and oPL. At the end of the perifusion period, explants in each chamber were collected and frozen in liquid nitrogen after eliminating the remaining perifusion medium by a short centrifugation step (4000 x g for 10 min). The oGH concentration in tissues was evaluated in the supernatant after homogenization of 400 mg of explants in 10 mM phosphate buffer, 0.15 M NaCl, 0.1% gelatin pH 7.2, and centrifugation at 17 000 x g for 30 min. RNA was extracted from the remaining 400 mg of explant tissue as previously described [13].

Treatments

Basal oPL and oPGH production by perifused placental explants Four independent perifusion experiments (4 placentae) were performed as described above. For each perifusion, the 8 chambers were perifused with DMEM0 supplemented with 5.5 mM glucose.

Glucose Three independent experiments (3 placentae) were performed to investigate the effect of the glucose concentration on oPGH production. In each experiment, 2 chambers were perifused simultaneously with DMEM0 (controls), 3 with DMEM0 supplemented with 12 mM glucose, and 3 with DMEM0 supplemented with 25 mM glucose. All perifusion chambers and tubing were initially filled with DMEM0 medium. At the beginning of the perifusion period (T0) the tubing to each chamber was connected to flasks containing either DMEM0 (control) or glucose-supplemented DMEM0 (treated). Preliminary results (data not shown) indicated that in these experimental conditions, it took 56 min for the medium to reach the perifusion chambers. Therefore, effluents collected during the first hour of perifusion (washout period) were considered as control preexperimental effluents. The effect of 25 mM glucose on oPGH production was studied more extensively in 11 independent experiments (11 placentae) using the same conditions (4 chambers perifused with DMEM0 and 4 with DMEM0/25 mM glucose). In 4 experiments, 25 mM glucose was replaced by 22 mM mannitol to control for the effects of osmotic pressure (mean osmotic pressure of 25 mM glucose or 22 mM mannitol = 310 ± 2 mOsm/kg).

Growth hormone-releasing hormone Regulation of placental GH production by GHRH was investigated in 4 independent perifusion experiments (4 placentae) as described above. In each experiment, 4 chambers were perifused with 5.5 mM glucose-DMEM0 and 4 with 5.5 mM glucose-DMEM0 supplemented with 100 nM human GHRH (hGHRH 1–29 NH2; n = 4) kindly supplied by Sanofi Synthelabo Recherche (Monpellier, France). The biological activity of hGHRH was first tested in vivo in sheep, as previously described [21]. The GHRH reached the perifusion chamber after 1 h of perifusion. After 5 h of perifusion, the same dose of GHRH was added to the medium of the four treatment chambers, as GHRH is rapidly degraded at 37°C [16, 22].

Metabolic labeling and evaluation of newly synthesized proteins by polyacrylamide gel electrophoresis Three independent perifusions (3 placentae) were used to monitor changes in protein synthesis during the 8-h perifusion period. Perifusion was stopped in one chamber each hour, and 200 mg of cotyledon explant tissue was collected. The tissue was pulse-labeled for 10 min with 1.85 Mbq (50 µCi) of [35S]methionine/cysteine (Tran35S-label; S.A.: 44 TBq/mmol) in 400 µl of Hanks medium (Life Technologies, France) and then chased for 60 min by adding 3.6 ml of Hanks medium. These incubation steps were carried out at 37°C in an atmosphere of 95% O2 and 5% CO2. Tissue fragments were collected after a short centrifugation step (12 000 x g, 1 min), then weighed and sonicated in 1 ml of 0.4% SDS in 12 mM Tris pH 6.8 buffer. The resulting extracts were centrifuged (12 000 x g for 15 min in an Eppendorf centrifuge), the supernatants were saved, and the total protein concentration was evaluated [23]. Proteins in the supernatant were subjected to denaturing electrophoresis in SDS slab gels (10% or 15% acrylamide; SDS-PAGE) according to the method of Laemmli [24]. Autoradiography was performed by exposing dried gels to X-Omat AR film (Kodak, Rochester, NY) at -70°C. The total amount of newly synthesized proteins was evaluated using a PhosphorImager SI with Image Quant version software (Molecular Dynamics, Sunnyvale, CA).

Ovine GH and oPL radioimmunoassays Concentrations of oPGH in tissue extracts and perifusion media were determined using an oGH RIA kit (Antiserum AFP-C0123080 and oGH I-4; National Hormone and Pituitary Program, Bethesda, MD) kindly supplied by the National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], Bethesda, MD) [13]. Ovine pituitary hormones and oPL did not cross-react in the RIA at concentrations of up to 2 µg/tube. The inhibition curves of serial dilutions of culture medium and placental extract were parallel to the standard curve (data not shown). Intraassay and interassay coefficients of variation were 5.8% and 11%, respectively. The detection limit of the assay was 0.62 ng GH/ml. The oPL concentrations in perifusion medium were evaluated using a previously described RIA [13] with a detection limit of 2.5 ng oPL/ml. No cross-reactivity with oGH (NIDDK oGH I-4; 1.7 IU/mg) or oPRL (NIH PS7; 24.8 IU/mg) at concentrations of up to 1 µg/tube was detected.

RNA isolation Total RNA was extracted from cotyledon explants collected after various perifusion periods using a modified guanidium-thiocyanate-phenol-chloroform procedure as previously described [13].

Real-time quantitative reverse transcription-polymerase chain reaction Real-time quantitative polymerase chain reaction (PCR) analyses of oPGH and oPL were performed on cotyledon total RNA using an ABI PRISM 7700 Sequence Detection System instrument and software (Perkin Elmer, Orsay, France). The theoretical bases of the method have been described elsewhere [14, 25, 26]. Real-time PCR was performed using specific primers as follows: oPGH, forward (5'-CCCAGGTTGCCTTCTGCTTC-3') and reverse (5'-GCGAAGCAGCTCCAAGCTG-3') primers [14]; oPL forward (5'-AGCAACAACGGTGGCTAACT-3') and reverse (5'-GCCATACTGTTCATCAAATCTGTT-3') primers. In addition, the levels of transcripts for the constitutive housekeeping gene product, 36B4, coding for acidic ribosomal phosphoprotein (PO; [27]) or the constitutive housekeeping gene coding for the TBP (TATA box binding protein, a component of the DNA-binding protein complex TFIID [28]) were measured in each sample to control for sample-to-sample differences in RNA loading. In each case, 1 µg of total RNA was reverse transcribed for 30 min at 42°C with 1.5 mM random hexamers (Amersham-Biosciences, Orsay, France), 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl buffer pH 8.3, 500 mM each dNTP, 10 mM dithiothreitol, 10 units of RNasin ribonuclease inhibitor (Promega, Charbonnière, France), and 50 units of Moloney virus reverse transcriptase (Superscript II; Life Technologies, Gergy-Pontoise, France) in a total volume of 20 µl. Amplification reactions were run in a reaction volume of 50 µl using the SYBR Green DNA PCR Core Reagent Kit (Perkin Elmer, Foster City, CA). One microliter of the reverse transcription reaction mix was used for real-time PCR, which consisted of one-step denaturation at 95°C for 10 min followed by 35 cycles of amplification (denaturation at 95°C for 15 sec and annealing at 65°C for 1 min) in the presence of 200 nM specific forward and reverse primers; 5 mM MgCl2; 50 mM KCl; 10 mM Tris buffer pH 8.3; 200 mM each dATP, dGTP, and dCTP; 400 mM dUTP, and 1.25 units of AmpliTaq Gold (Perkin Elmer). Each sample was analyzed in duplicate and levels of oPGH and oPL mRNAs were expressed as a ratio of PO or TBP mRNA values.

Statistical Analysis

Data are expressed as means ± SEM. Differences between treatment groups were analyzed using ANOVA followed by the Scheffé F test. Statistical analyses were performed using Statview software (Abacus Concepts, Inc., Berkeley, CA). Probabilities of less than 5% were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ovine PGH and oPL Production by Perifused Placental Explants

Placental explants (800 mg; 12 ± 2 mg total protein) perifused with DMEM0 supplemented with 5.5 mM glucose, released oPGH and oPL into the medium for 8 h (Fig. 1A). The mean hourly rates of oPGH and oPL production were 2.45 ± 0.2 and 353.6 ± 13.6 ng/g/h, respectively (n = 4). In 3 other experiments (Fig. 1, B and C), perifusion was stopped in 1 chamber each hour and explants (200-mg aliquots) were collected for metabolic labeling and quantification of newly synthesized proteins by SDS-PAGE and autoradiography. After 1 h of perifusion, protein synthesis was equivalent to 54% ± 10% of that measured at T0. This capacity then decreased and remained at a mean of 16.4% ± 1.6% of the T0 value for the next 7 h (Fig. 1C). The levels of mRNAs encoding for oPGH and oPL were evaluated in the remaining 600 mg of explants by means of real-time reverse transcription-PCR and were expressed as the percentage of T0 values. Expression of oPGH mRNA was similar at 1 h and at T0, but was decreased at 2 h and then remained essentially unchanged (50% ± 3% of T0 values) for the next 5 h (Fig. 1B). At 8 h of perifusion, oPGH mRNA expression further decreased to 29% ± 3%, whereas oPL mRNA expression remained at 77% ± 3% of the T0 value until 7 h of perifusion. Then, like oPGH mRNA, the oPL mRNA level further decreased (51% ± 19% at 8 h; Fig. 1B).



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  		FIG. 1. Ovine PGH and oPL production by Day 45 placental explants in vitro. A) Explants (800 mg) were perifused with DMEM0 supplemented with 5.5 mM glucose for 8 h. Concentrations of oPGH and oPL in the perifusion medium were determined by RIA. Values represent the mean ± SEM of 4 independent experiments. In each experiment, mean hourly hormone production was evaluated in the medium of 8 perifusion chambers. In further experiments (n = 3, same conditions), perifusion was stopped each hour in 1 chamber and explants were collected and divided into 2 aliquots of 200 mg. B) Levels of expression of oPGH and oPL transcripts were evaluated in 1 aliquot by real-time quantitative reverse transcription-PCR. Values are expressed as a ratio of the PO housekeeping gene and compared with values from explants at T0 (100%). C) Capacity for de novo protein synthesis was evaluated in the second aliquot by SDS-PAGE analysis after metabolic labeling. Values represent the mean ± SEM of 3 independent experiments

Regulation of oGH and oPL Release by GHRH and Glucose

GHRH Perifusion of explants with medium continuously supplemented with GHRH had no effect on either oPGH or oPL production (Fig. 2; n = 4).



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  		FIG. 2. Absence of regulation of oPGH and oPL production by GHRH. Explants (800 mg) were perifused during 8 h with DMEM0 5.5 mM glucose, supplemented with 0 or 100 nM GHRH. GHRH was introduced into the perifusion chamber after 1 h of perifusion. Given the rapid GHRH degradation, the same amount was also added to the perifusion medium after 5 h. Values represent the mean ± SEM of 4 independent experiments. In each experiment, mean hourly hormone production was evaluated in the medium of 4 control and 4 treated perifusion chambers

Glucose In 3 preliminary experiments, placental explants were perifused with DMEM0 supplemented with various glucose concentrations (0, 12, or 25 mM). The presence of additional glucose in the medium decreased oPGH production in a concentration-dependent manner; this effect was most significant during the last 4 h of perifusion (P <= 0.05 for 25 mM glucose; Fig. 3A). Mean concentrations of oPL in the same medium were not affected by glucose treatment (Fig. 3B). Intrachamber and interchamber variations in oPGH release were significant; therefore, subsequent perifusion experiments (n = 11) were designed to assess the negative effect of 25 mM glucose on placental oPGH production. Perifusion of placental explants with DMEM0 supplemented with 25 mM glucose decreased oPGH production from 4 h of perifusion until the end of the perifusion period (Fig. 4A; P <= 0.03). However, oPL production by the same explants was not affected by glucose treatment (Fig. 4B). In 5 of 11 experiments, total protein and oPGH concentrations in explants were evaluated at T0 and at the end of the perifusion period (8 h). In control and glucose-treated explants, protein concentrations at the end of perifusion were comparable, at about one-half of T0 values (T0, 12.7 ± 2.5; 8-h control explants, 6.98 ± 0.35; 8-h glucose explants, 6.3 ± 0.43 mg/800 mg fresh tissue). The oPGH concentration per milligram of protein did not change significantly between T0 and 8 h of perifusion with either treated or control explants (T0, 6.75 ± 2.32; 8-h control explants, 9.32 ± 3.22; treated explants, 9.26 ± 2.8 ng/mg total protein). In the same 5 experiments, oPGH and oPL mRNA levels in control and treated explants after 8 h of perifusion were not affected by glucose treatment (Fig. 4, E and F). Perifusion of explants with mannose (22 mM) rather than glucose did not affect oPGH release (Fig. 4C) or the total protein or oPGH content of explants at the end of the perifusion period compared with values obtained when explants were perifused with DMEM0. Like glucose, mannitol had no effect on oPL release by placental explants (Fig. 4D).



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  		FIG. 3. Concentration-dependent effect of glucose on oPGH production. Explants (800 mg) were perifused for 8 h with DMEM0 supplemented with 0, 12, or 25 mM glucose. Ovine PGH and oPL levels in the perifusion medium were evaluated by RIA. Values represent the mean ± SEM of 3 independent experiments. In each experiment mean hourly hormone production was evaluated in the medium of 2 (0 mM), 3 (12 mM), and 3 (25 mM) perifusion chambers. Production of oPGH was decreased by 25 mM glucose (*P <= 0.05)



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  		FIG. 4. Glucose regulation of oPGH and oPL production. Explants (800 mg) were perifused for 8 h with DMEM0 supplemented with 0 or 25 mM glucose. Ovine PGH and oPL levels in the perifusion medium were evaluated by RIA. Values represent the mean ± SEM of 11 independent experiments. In each experiment, mean hourly hormone production was evaluated in the medium of 4 (0 mM) and 4 (25 mM) perifusion chambers. oPGH production was decreased by glucose after 4 h of perifusion (A; *P <= 0.03), whereas oPL production was not affected (B). Replacement of glucose with mannitol (22 mM, osmotic pressure control) did not modify oPGH (C) or oPL (D) production. In 5 of the above experiments, oPGH (E) and oPL (F) transcripts were evaluated after 8 h of perifusion (G-, DMEM0; G+, 25 mM glucose) by real-time quantitative reverse transcription-PCR. Levels are expressed as a ratio relative to the TBP housekeeping gene


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a perifusion system specifically designed to study the regulation of placental oPL and oPGH production by placental explants, in which oPGH is produced at low levels [14], the present results are the first to indicate the hourly rates of production of these placental hormones. Because the perifusion medium is renewed and collected continuously in this model, hormone production can be evaluated hourly, and the effects of effectors such as GHRH, which have rapid effects and are then rapidly degraded, can be measured [16, 29]. Furthermore, using this perifusion system, short-loop regulatory mechanisms are unlikely to affect the results, as accumulation of secretagogues and metabolic products in the medium bathing the cells is avoided. oPGH and oPL production were monitored for 8 h. Preliminary experiments showed that oPGH production fell between 10 and 24 h of perifusion, whereas oPL production was unchanged (data not shown). Rates of oPGH and oPL release into the perifusion medium were constant for 8 h. Hormone concentrations measured in the perifusion medium resulted from both de novo synthesis (about 20%) and release. Levels of mRNAs coding for oPGH and oPL in explants followed similar patterns of change. Both mRNAs decreased slightly during the first 2 h of perifusion, were constant for the next 5 h, and then decreased at 8 h. The rate of decrease was greater for oPGH mRNA (29% of the T0 value at 8 h) than for oPL mRNA (65% of the T0 value at 8 h). The amount of oPL released into the medium was about 200-fold higher than that of oPGH, in keeping with in vivo observations (mean oPGH and oPL concentrations in the placenta on Day 45 are 10 ng/g and 1 µg/g, respectively [13]). This perifusion system thus represents a useful in vitro model to study short-term regulation of oPGH production.

We have previously reported GHRH production by ovine placenta [16]. In the present study, we determined that GHRH did not modify placental oPGH production in vitro. GH production in other extrapituitary sites such as human placenta, mammary gland, ovary, thymus, lymphocytes, and fibroblasts [30–35] is also independent of traditional GH secretagogues, even if they are present in the same cells or tissues (reviewed in [36, 37]). Furthermore, administration of GHRH (sermorelin) to pregnant women elicits a small increase in pituitary GH, but does not modify placental GH production [38]. These reports indicate that GH production by extrapituitary tissues and by the anterior pituitary is differentially regulated. Furthermore, GHRH did not affect oPL production, in keeping with the results of an in vivo experiment indicating that treatment of pregnant rats with GHRH antiserum increases placental IGF levels but do not affect placental lactogen levels [39]. Results of in vitro experiments are conflicting. Kishi et al. [40] and Hochberg at al. [41] reported that GHRH stimulated placental lactogen production by rat and human trophoblasts, respectively, but Evain-Brion et al. [37] did not confirm their results; likewise, the present study does not support the concept of an autocrine loop of oPGH or oPL regulation by GHRH.

Nutrient supply to the pregnant uterus plays a key role in fetal development. Earlier studies focused on late pregnancy, but recent results indicate that early and midgestation are crucial periods of modulation of feto-placental growth due to changing maternal nutritional status. During these earlier stages it is placental rather than fetal development that is affected by nutritional status [42]. In adult sheep, undernutrition in early and midpregnancy stimulates placental growth which, later in gestation, enhances fetal growth and birth weight [43–45]. However, earlier studies by Robinson [46] and Quirke and Sheehan [18] indicated that high planes of nutrition during the same period caused a marked reduction in birth weight. These reports suggest that control of placental growth by nutrients is not a linear relationship between maternal and fetal compartments, but implies the importance of changes in the placental endocrinology. The adolescent sheep made pregnant through single embryo transfer seems to be a good model to investigate the effects of overfeeding in early pregnancy on feto-placental development. These animals still grow, and their anabolic drive required for tissue growth is maintained at the expense of the gravid uterus nutrient requirements. In this model, high nutritional status resulted in restricted placental growth and high maternal glucose levels [47]. This is why we investigated regulation of oPGH production by glucose in the present study. A decrease in oPGH production by placental explants was observed after 4 h of perifusion and was significant at glucose concentrations between 12 and 25 mM. Using a similar range of glucose concentrations, Weiss et al. [48] described the effect of hyperglycemia on trophoblastic cells lines in vitro. On the basis of their data and results obtained with rat blastocysts cultured in hyperglycemic medium [49] or recovered from diabetic rats [50], trophoblast cells appeared to have a low susceptibility to hyperglycemia. This could partially explain why relatively high glucose concentrations were necessary to obtain significant effects in the present study. Beyond 24 h of glucose treatment, depending on the cell line considered, Weiss et al. [48] observed a decrease in protein content and hyperosmolarity effects. We found no such effects after 8 h of perifusion, as the total protein content was comparable between control explants and explants treated with glucose or mannitol, at the end of the perifusion period. Furthermore, decreased oPGH production was not a result of osmotic pressure, as mannitol, perifused at a concentration yielding the same osmotic pressure as glucose, failed to modify oPGH release. The effect of glucose was specific to oPGH, as there was no effect on oPL production. Brinsmead et al. [51] also reported that hyperglycemia induced in fetal lambs and pregnant ewes did not affect fetal or maternal oPL concentrations. However, oPL concentrations were increased in the fetus when ewes were treated with glucose following a 3-day fast [52]. Glucose regulation of oPL production seems to differ between the maternal and fetal compartments and is more acute in animals on a low plane of nutrition. A decrease in placental GH production induced by hyperglycemia, with no effect on placental lactogen production, was also reported in human trophoblast culture experiments [53, 54]. Thus, oPGH and oPL, although belonging to the same GH/PRL family, are differentially regulated. In human placenta, in vitro experiments suggest that GH can regulate placental lactogen production [55], but oPGH was not apparently involved in short-loop regulation of oPL production in the present study. However, potential regulation of oPL production by oPGH may need to be investigated over longer periods. Our results support those of Bauer et al. [56] showing that treatment of the ovine fetus in utero with recombinant bovine GH does not affect oPL concentrations in either the maternal or the fetal circulation. The modality of oPGH regulation by glucose remains to be elucidated but seems to be posttranscriptional in nature, as oPGH transcript levels were not modified by glucose treatment. The oPGH production of explants perifused with hyperglycemic medium was 1.8 ± 0.1 times less than that of explants perifused without glucose. Such an amplitude of GH modulation associated with biological effects has previously been reported in pregnant ewes treated with GH (0.1 mg/kg/day, resulting in a 2-fold increase in maternal GH levels) [12, 57, 58]. Consequently, decreased oPGH production could be involved in impaired placental growth. Future experiments should consider simultaneous modifications of IGF I and IGF II, and of their binding proteins in a hyperglycemic environment [59]. Wallace et al. [47] and Gadd et al. [60] showed that high levels of nutrition during early to midgestation in ewes affected the IGF system at the maternal, placental, and fetal levels. In the maternal circulation the IGF I concentration was increased and the GH level decreased [47]. Placental exposure to high IGF I levels could induce a down-regulation of oPGH production. Prolonged exposure could result in autocrine regulation of placental IGF I production or down-regulation of placental IGF I receptors, leading to placental growth restriction. Furthermore, given the presence of GH receptor in the endometrium and placenta during early to midgestation [14], oPGH could also affect the utero-placenta IGF environment, either directly or by regulation of its receptors.

This study demonstrates that oPGH production by the ovine trophoblast can be rapidly down-regulated in a high-glucose environment; this may modulate glucose partitioning between the fetus and placenta. Glucose transfer between the maternal and fetal compartments is regulated by 2 types of glucose transporter (GLUT1 and GLUT3) expressed at the trophectoderm surface [61]. GH has been implicated in GLUT regulation [62, 63]. GLUT-1 and GLUT-3 transcripts are first detected in the sheep placenta on Day 45 of pregnancy. During the period of oPGH production, between Days 45 and 60, GLUT-1 gene expression increases 3- to 4-fold [64]. The significance of oPGH regulation of GLUT gene expression remains to be investigated.

It is now well established that maternal nutrition during pregnancy can reprogram fetal growth and even induce adult diseases. The role of the placenta in these metabolic adaptations remains to be investigated. Rat and sheep models are now available to study fetal-placental growth reprogramming by dietary factors in early and midgestation. The diabetic ewe model is used to study disorders induced by gestational diabetes in pregnancy. Our present results establish that oPGH, which is considered important for early placental growth, is regulated by metabolic factors such as glucose. Thus, changes in oPGH levels should be taken into account in metabolic experiments targeting the first trimester of pregnancy in sheep.


   ACKNOWLEDGMENTS
 
The authors thank M. Olivi and I. Laurendeau for technical assistance.


   FOOTNOTES
 
First decision: 17 July 2001.

1 Correspondence: M.C. Lacroix, Unité INSERM 427, Faculté PARIS V, 4 avenue de l'Observatoire, 75270 Paris Cedex 6, France. FAX: 33 1 44 07 39 92; lacroix{at}pharmacie.univ-paris5.fr Back

Accepted: October 4, 2001.

Received: June 19, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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