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Endocrinology, doi:10.1210/en.2008-0084
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Endocrinology Vol. 149, No. 10 5012-5023
Copyright © 2008 by The Endocrine Society

Molecular and Morphological Changes in Placenta and Embryo Development Associated with the Inhibition of Polyamine Synthesis during Midpregnancy in Mice

Carlos López-García, Andrés Joaquín López-Contreras, Asunción Cremades, Maria Teresa Castells, Faustino Marín, Fernanda Schreiber and Rafael Peñafiel

Departments of Biochemistry and Molecular Biology B and Immunology (C.L.-G., A.J.L.-C., R.P.), Pharmacology (A.C.), Cell Biology (M.T.C.), and Human Anatomy and Psychcobiology (F.M.), Faculty of Medicine, University of Murcia, 30100 Murcia, Spain; and Team 15 (FS), Microbial Pathogenesis, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom

Address all correspondence and requests for reprints to: Dr. Rafael Peñafiel, Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain. E-mail: rapegar{at}um.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polyamines play an essential role in murine development, as demonstrated by both gene ablation in ornithine decarboxylase (ODC)-deficient embryos and pharmacological treatments of pregnant mice. However, the molecular and cellular mechanisms by which ODC inhibition affects embryonic development during critical periods of pregnancy are mostly unknown. Our present results demonstrate that the contragestational effect of {alpha}-difluoromethylornithine (DFMO), a suicide inhibitor of ODC, when given at d 7–9 of pregnancy, is associated with embryo growth arrest and marked alterations in the development of yolk sac and placenta. Blood island formation as well as the transcript levels of embryonary globins {alpha}-like x chain and β-like y-chain was markedly decreased in the yolk sac. At the placental level, abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer and a deficient development of the labyrinthine zone were evident. Real-time RT-PCR analysis showed that transcript levels of the steroidogenic genes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase VI, and 17{alpha}-hydroxylase were markedly decreased by DFMO treatment in the developing placenta at d 9 and 10 of pregnancy. Plasma values of progesterone and androstenedione were also decreased by DFMO treatment. Transcriptomic analysis also detected changes in the expression of several genes involved in placentation and the differentiation of trophoblastic lineages. In conclusion, our results indicate that ODC inhibition at d 8 of pregnancy is related to alterations in yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYAMINES (PUTRESCINE, SPERMIDINE, and spermine) are aliphatic cations synthesized and stored by most eukaryotic cells, which are essential for many cellular functions. It is clear that they play an important role in the regulation of cell proliferation and differentiation (1, 2), and they also appear to have a dual role in promoting cell survival or cell death (3, 4). The polyamine content of mammalian cells is tightly controlled by biosynthesis, degradation, uptake, and excretion (3). For each individual organ system, these processes are finely regulated by many different agents, including hormones and growth factors as well as the polyamines themselves (2, 3). So it is not surprising that alterations of polyamine homeostasis may impair normal cell function, which may have pathological consequences. In fact, different human diseases, including cancer, are related to abnormal polyamine metabolism (5, 6, 7). The molecular basis by which polyamines affect cellular functions is currently unknown. However, a large body of data indicates that polyamines can interact electrostatically with nucleic acids and proteins, affecting the packaging and replication of DNA (1, 2), gene transcription (8), and protein synthesis (9) as well as the functioning of different ion channels (10).

Ornithine decarboxylase (ODC), the enzyme that catalyzes the transformation of L-ornithine into putrescine, is a key regulatory member in the polyamine biosynthetic pathway. ODC is highly inducible by many trophic stimuli that increase ODC gene transcription or the translation efficiency of its mRNA (11). Moreover, this enzyme has a short half-life (about 15 min) and is negatively regulated by polyamines through the induction of regulatory proteins termed antizymes, which inhibit ODC activity and stimulate its degradation by the 26S proteasome (12, 13). Marked increases in ODC activity have been found in cultured cells stimulated to proliferate by mitogenic agents and in many different types of solid tumors (5). Current evidence supports the concept that ODC overexpression is required in the carcinogenic process (5, 14, 15). In reproductive tissues, ODC and polyamines have been related to critical events affecting spermatogenesis (16, 17, 18) or progesterone secretion by the corpus luteum (19, 20). In fact, transgenic mice overexpressing ODC in the testes have reduced fertility (21), whereas transgenic activation of polyamine catabolism causes female infertility (22).

ODC and polyamines are important for embryonic development as evidenced by many experimental results. Early pharmacological studies, using {alpha}-difluoromethylornithine (DFMO) or other ODC inhibitors, clearly indicated that increased polyamine synthesis during critical periods was essential for embryo development, both in vertebrate and invertebrate animals (23, 24). In fact, DFMO exerted contragestational effects when administered during a critical period after implantation to pregnant mice, rats, rabbits, or hamsters (25, 26, 27). These effects of DFMO were prevented by the concomitant administration of putrescine. More recent studies using lower eukaryotes and nematodes with targeted ablation of ODC gene indicated that deletion of ODC is lethal unless polyamines were exogenously administered (28, 29, 30). Homozygous deficiency of ODC or S-adenosylmethionine decarboxylase, another enzyme participating in the polyamine biosynthetic pathway, obtained by gene targeting, is incompatible with embryogenesis (31, 32). Whereas it was reported that the loss of ODC negatively affected the survival of the pluripotent cells of the inner cell mass (31), little is known about the cellular and molecular changes associated to pregnancy arrest produced by treating mice with DFMO.

In the present study, we further examined the influence of DFMO treatment on the mouse embryo and associated structures by studying the changes produced by this compound, both at the histological and molecular levels. Our results indicate that the inhibition of ODC peak in the mouse conceptus arrests embryonic growth, alters hematopoiesis in the yolk sac, and markedly decreases the steroidogenic capacity of the developing placenta.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Animal procedures were carried out according to the institutional guidelines of the University of Murcia, in compliance with national (RD 1201/2005) and international laws and policies (European Union normative 86/009). Female Swiss CD1 mice bred in the Service of Laboratory Animals of the University of Murcia were used. Animals were fed with standard chow (UAR A03; Panlab, Barcelona, Spain) and water ad libitum and maintained at 22 C and 55% relative humidity under a controlled 12-h light, 12 h dark cycle (light on from 0700 h). Timed-pregnant mice were obtained by housing the females with males of proven fertility. The day when the vaginal plug was observed was designated as d 1. Pregnant animals at different days of pregnancy were killed by cervical dislocation. When required, blood extraction was obtained by cardiac puncture after ether anesthesia. The abdominal cavity was opened and the uteri were removed, weighed, and opened. Up to d 10, embryos were dissected out together with their yolk sacs. Beyond d 11, the number of implantation sites, viable fetuses, and resorptions were recorded. They were photographed under a stereomicroscope and processed for histological or biochemical analysis. For morphological studies, four control and four DFMO-treated mice were used for each gestational day studied. At least three conceptuses from each animal were excised and analyzed. In all cases, uteri, conceptuses, and embryos were photographed, and the differences observed between control and treated animals were recorded and measured. For biochemical analysis, the different tissues were separated mechanically from the gravid uterus and immediately processed.

To study the influence of polyamine deprivation on prenatal development, DFMO (obtained from Illex Products, San Antonio, TX), a potent and specific inhibitor of ODC, was administered to pregnant mice by sc injection, twice a day at 1000 and 1900 h at a dose of 500 mg/kg in saline solution (pH 7), during different periods of pregnancy. To asses the possible influence of prolactin decrease on the effects of DFMO on mice during d 7–9 of pregnancy, 20 U prolactin (Sigma Chemicals Co., St. Louis, MO) were administered sc to four pregnant mice simultaneously to DFMO administration, and animals were autopsied at d 18 of pregnancy.

Enzyme measurements
For determining ODC activity, pools of two to three conceptuses from each animal were homogenized with the aid of a Polytron homogenizer in buffer containing 25 mM Tris (pH 7.2), 2 mM dithiothreitol, 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, and 0.25 M sucrose. The extract was centrifuged at 20000 x g for 20 min, and ODC activity was assayed in the supernatant as described elsewhere (20) by measuring 14CO2 release from L-[1-14C] ornithine. Activity was expressed as nanomoles CO2 produced per hour and per gram of wet weight. For the measurement of ODC activity in embryos and deciduas at d 8 of pregnancy, the embryos and its associated membranes were dissected out from the deciduas, and these tissues were separately homogenized by using a glass-glass tissue grinder. The resulting extracts were processed as above, but in this case ODC activity was expressed as nanomoles CO2 produced per hour and per milligram of protein. Protein concentration was determined in the supernatant using Bradford reagent (Bio-Rad, Hercules, CA).

Western blot analysis
The conceptuses were homogenized in 50 mM Tris-HCl (pH 8), 1% Igepal, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and an additional mixture of protease inhibitors (13 µM bestatin, 1.4 µM E-64, 100 µM leupeptin, and 30 nM aprotinin). The homogenates were centrifuged at 12,000 x g for 20 min, and equal amounts of protein from the supernatants were mixed with Laemmli sample buffer, heated at 95 C for 5 min, and fractionated by electrophoresis in 10% polyacrylamide-sodium dodecyl sulfate gels. The resolved proteins were electroblotted to polyvinyl difluoride membranes, and the resulting blots were incubated with 5% nonfat dry milk in PBS [0.01 M PBS (pH 7.4)] for 1 h. After washing in PBS+0.1% Tween 20, the blots were incubated at room temperature for 1 h with anti-ODC rabbit polyclonal antibody at a dilution 1:500 (Euro-Diagnostica, Malmö, Sweden). The blots were washed in PBS+0.1% Tween 20 and incubated at room temperature for 1 h with a horseradish peroxidase-labeled goat secondary antibody (dilution 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by using ECL+ detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ) and commercial developing reagents and films (Amersham).

Plasma steroid measurements
Plasma steroid levels were measured by RIA using commercial kits, purchased from Immunotech (Marseille, France) for progesterone determination and Biosource Europe (Nivelles, Belgium) for androstenedione. Results were calculated from the standard curve by interpolation. Samples were assayed in duplicates. The analytical sensitivities were 0.05 ng/ml for progesterone and 0.03 ng/ml for androstenedione.

Semiquantitative RT-PCR analysis
Total RNA was extracted using Gen Elute total RNA miniprep kit (Sigma). Fresh tissues were homogenized by using a Polytron and the total RNA yield was determined by measuring the absorbance at 260 and 280 nm. RNA was analyzed by RT-PCR as described previously (20). The primers used were purchased from Sigma-Genosys (Suffolk, UK) and are summarized in Tables 1Go and 2Go.


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  		TABLE 1. Primers used for semiquantitative RT-PCR analysis of genes related to polyamine metabolism

 

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  		TABLE 2. Primers used for semiquantitative RT-PCR analysis of steroidogenic and growth factor genes

 
Quantitative real-time RT-PCR
Real-time-PCR was carried out using special primers deduced from published cDNA sequences that amplified fragments about 100 bp. When possible, primers were chosen such that the resulting amplicons would cross an exon junction, thereby eliminating false-positive signals from genomic DNA contamination (Table 3Go). The PCR mix contained 2.25 µl of 100 µM primers, 12.5 µl of SYBR Green PCR master mix (Applied Biosystems, Warrington, UK), 1 µl of template cDNA, and RNase-free water to 25 µl. The reaction was performed in a 7500 Real Time machine (Applied Biosystems) under the following cycling conditions: one cycle at 95 C for 10 min and then 40 cycles at 95 C for 15 sec and 60 C for 1 min. Fluorescence data were collected from each cycle and were analyzed by means of 7500 SDS software (Applied Biosystems).


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  		TABLE 3. Primers used for real-time RT-PCR

 
Microarray analysis
Total RNA was extracted from three conceptuses of each control and each 7–9 d DFMO-treated animals, as previously described. The RNAs isolated from conceptuses of three 9-d-pregnant control mice or three DFMO-treated mice were pooled and used as control RNA and DFMO-treated RNA, respectively. An aliquot of total RNA from each sample was run in an Agilent Bioanalyzer RNA Nanochip (Agilent Technologies, Berkshire, UK), and its quality was confirmed by calculating the 28S to 18S ratio. The total RNA was labeled by means of the GeneChip one-cycle target labeling (Affymetrix Corp., Santa Clara, CA) according to the manufacturer’s protocol. Labeled and fragmented cRNA was added to the microarray (GeneChip Mouse Genome 430 2.0; Affymetrix) and allowed to hybridize at 45 C with constant rotation for 16 h. Washes were carried out in a Fluidics Station 400 (Affymetrix), prior labeling of hybridized cRNA by addition of streptavidin-phycoerythrin, a second layer of biotinylated antistreptavidin antibody, and a third layer of streptavidin-PE. The microarray was scanned in an Agilent scanner (Affymetrix) and the raw data analyzed by Affymetrix Microarray Suite 5.0.

Histological study
After tissue collection, samples were fixed in 10% formalin in PBS (pH 7.4) for 10 h. The fixed samples were paraffin embedded and cut in 3- to 10-µm sections prior hematoxylin-eosin staining or neutral red staining.

Statistics
Data are expressed as mean ± SE. The significance of the differences observed was assessed by ANOVA, followed by the post hoc Newman-Keuls multiple range test or {chi}2 test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ODC and other genes related to polyamine metabolism in conceptus and decidua at midgestation
Figure 1AGo shows the evolution of ODC activity in the conceptus (embryo and associated membranes plus decidua) from d 6 to d 10 of pregnancy. ODC activity, which was low at d 6 and 7 of pregnancy, abruptly increased at d 8, and decreased sharply on d 9 and 10. At d 8, ODC protein was detected by Western blot analysis, but it dramatically decreased at d 9 and 10. ODC protein levels correlated well with ODC activity (Fig. 1CGo). However, no correlation was found between ODC activity or protein and ODC mRNA. Whereas ODC transcript levels declined from d 8 to d 9, they increased at d 10 to reach twice of their value at d 8 (Fig. 1DGo). This apparent discrepancy may be related to the fact that in placenta and fetus, the regulation of ODC at translational and posttranslational levels seems to be different (33). This observation, and the fact that at d 10 the relative contribution of placental and embryonic tissues to the whole conceptus with respect to that of d 8 and 9 are different, may explain the mentioned inconsistency. On the other hand, to determine the contribution of the decidual tissue and of the embryo itself on the peak of ODC activity found at d 8 of pregnancy, the embryos and associated membranes were surgically dissected out from the deciduas, and ODC activity was measured in different pools of embryos and deciduas. ODC activity values in both gestational compartments were almost identical (Fig. 1BGo). The expression of other genes related to polyamine metabolism was analyzed by semiquantitative RT-PCR, in RNA samples obtained from conceptuses at d 8–10 of pregnancy. Apparently, similar levels of transcripts of the biosynthetic genes S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase and spermine synthase were found in the three days studied (Fig. 2Go). Spermidine/spermine-acetyl transferase, the enzyme participating in the polyamine retroconversion pathway, was also fairly expressed in this period, whereas weaker signals were found for polyamine oxidase and spermine oxidase, genes implicated in polyamine catabolism. The ODC antizymes (AZ)-1 and AZ2 were also expressed during these days, but AZ3 expression was not detected. Of the two antizyme inhibitors, only AZIN1 was mostly detected. The expression of the polyamine-modulated transcription factor-1 was also evident, mainly at d 9 of pregnancy.


Figure 1
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  		FIG. 1. ODC expression in mouse conceptus at midpregnancy. A, ODC activity in conceptus (embryo+decidua). Values are mean ± SE of three to five animals per day and three to four conceptuses for each animal. *, P < 0.01 vs. the other days. B, ODC activity in embryo/associated membranes and decidua at d 8 of pregnancy. Values are the mean ± SE of pools of three embryos or three deciduas from three pregnant mice. C, Western blot analysis of ODC protein at d 8–10 of pregnancy. *, P < 0.01 vs. d 9 and 10. D, RT-PCR analysis of ODC mRNA at d 8–10 of pregnancy. *, P < 0.05 vs. d 8 and 10.

 

Figure 2
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  		FIG. 2. Semiquantitative RT-PCR analysis of mRNAs of genes related to polyamine metabolism. RNA was extracted from pools of three to four conceptus plus placenta of 8, 9, and 10 d. Experiments were performed three times with reproducible results. SpdST, spermidine synthase; SpmST, spermine synthase; SSAT, spermidine/spermine acetyl transferase; PAO, polyamine oxidase; SMO, spermine oxidase; AZIN, antizyme inhibitor; PMF-1, polyamine-modulated factor 1.

 
Morphological and histological changes produced by DFMO treatment
Figure 3Go shows the effect of the treatment of pregnant mice with DFMO at different periods of pregnancy. As reported by others, the maximal contragestational effect was found when DFMO was given between d 7 and 9 of pregnancy. Lower contragestational effect was observed when DFMO was given in the periods 4–6 d or 9–18 d. In the other periods, the contragestational effect of DFMO was almost absent. Our results are in agreement with those reported by Fozard et al. (25, 26). These authors reasoned that the close correlation between inhibition of the peak rise in ODC activity and the arrest of embryonic development supports the idea that the contragestational effect of DFMO is directly related to the inhibition of embryonic ODC activity. However, because it is known that DFMO treatment may affect prolactin secretion in the rat (34), we tested whether a hypothetical decrease in prolactin secretion produced by DFMO treatment could be related to the contragestational effect. The concomitant administration of exogenous prolactin to DFMO-treated pregnant mice during d 7–9 did not rescue the embryo for the contragestational effect of DFMO (results not shown).


Figure 3
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  		FIG. 3. Contragestational effect of DFMO administered to pregnant mice at different periods of gestation. In treatments that started on d 9 or earlier, DFMO was given by sc injection (500 mg/kg, twice a day). From d 10 onward, DFMO was administered as a 1% solution in the drinking water. Pregnant mice were autopsied at d 18 of gestation. The histograms represent the percentage of viable and nonviable fetuses, and the values are mean ± SE of three to four animals per group. *, P < 0.001 vs. control.

 
As shown in Fig. 4Go, in mice treated with DFMO during the period of maximal contragestational effect, changes in the weight of the fetoplacental units were significantly different from controls from d 11 onward. Thus, the marked increase in the weight of the fetoplacental units observed in control pregnant mice did not take place in the treated animals, remaining almost constant (Fig. 4Go). Accordingly, whereas in the control animals the fetal and placental units were clearly recognized and increased in size with age, in treated mice the gross morphology of conceptus and placenta was clearly different from that of controls. In fact, at d 12 the sizes of fetuses (9.75 ± 0.36 mm, n = 12) and placentas (7.30 ± 0.30 mm, n = 12) in control animals were markedly higher than those of conceptuses obtained from treated mice (4.86 ± 0.35 mm, n = 12, P < 0.001). At d 14, fetal development was drastically altered. The size of treated conceptuses (3.36 ± 0.53 mm, n = 12) was markedly lower than that of fetuses or placentas from control animals (11.12 ± 0.47 and 8.44 ± 0.40 mm, respectively, n = 12, P < 0.001). Dissection of the conceptuses at d 9 post conception (pc) in control and treated pregnant mice showed that the treatment affected the growth and development of the embryos, whereas decidua did not show significant differences (data not shown). At d 11 pc, the examination of the gravid uterus revealed smaller size of conceptuses plus deciduas in treated mice than in controls (3.08 ± 0.51 mm vs. 7.41 ± 0.32, n = 12, P < 0.001) as well as the presence of blood clots and collapsed mesometrial vessels in deciduas in all treated conceptuses analyzed (12 of 12), whereas none of these changes were observed in the controls (0 of 12, P < 0.001, {chi}2). Representative samples are shown in Fig. 5Go, A–C. Dissection of the conceptuses revealed a marked underdevelopment of the embryos in the DFMO-treated group that was clearly reflected not only in the smaller size but also in the absence of vascular structures (Fig. 5DGo).


Figure 4
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  		FIG. 4. Influence of DFMO administered to pregnant mice from d 7 to d 9 in the weight of conceptus. DFMO was administered by sc injection (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Control and treated pregnant mice were autopsied from d 8 to 15. Each point is the mean of the conceptuses obtained from three to four animals per group. SE is not shown because the limits of variability fall within the area of the symbol. *, P < 0.01 vs. control.

 

Figure 5
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  		FIG. 5. Influence of DFMO treatment on the morphology of gravid uterus, embryo, and conceptus analyzed at d 11 of pregnancy. DFMO was administered to four animals by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Four control animals received 0.9% saline solutions with the same schedule as treated mice. Pregnant mice were killed at d 11 of pregnancy. The gravid uteri were removed, studied macroscopically, and photographed. The uteri were opened and the conceptuses excised. At least three embryos from each animal were dissected out together with their yolk sacs. They were photographed under a stereomicroscope. A and B, Representative conceptuses obtained from the uteri of control and treated animals, respectively; d, decidua; mt, mesometrium. Asterisks indicate blood clots. C, Conceptus plus placenta extracted from the uterine wall. Left row, control; right row, DFMO treated. ys, Yolk sac; p, placenta. D, Control (left) and treated (right) embryos with their yolk sacs. h, Embryo head; t, embryo tail; ys, yolk sac; bv, blood vessels. Bars (A–C), 2 mm; (D), 1 mm. Note the different size from control to treated decidua as well as the presence of blood clots (asterisks) and collapsed mesometrial vessels in treated decidua (A–C). The underdevelopment of treated embryos is also visible (D).

 
Histological analysis of six fetoplacental units per group at d 11 pc revealed marked changes between control and DFMO-treated animals. Labyrinthine zone and spongiotrophoblasts seen in the placenta of control animals were absent in all treated mice analyzed (Fig. 6Go, A and B). Poor development of the yolk sac and impaired fusion of allantois and chorion membranes were also evident in all treated conceptuses (Fig. 6Go, C and D). Interestingly, blood islands present in the yolk sac of control embryos (Fig. 6EGo) were dramatically decreased in that of the treated group as shown in Fig. 6FGo (n = 6, P < 0.001, {chi}2). At d 14 pc, fetal and placental structures were still markedly underdeveloped in all DFMO-treated animals. The differentiation of embryonic structures appeared to be arrested, the formation of the fetal placenta was almost absent, and in which the labyrinth and the junction zone were missing (data not shown). Giant trophoblasts were present but spongiotrophoblasts could not be seen. The yolk sac also showed an altered structure lacking the typical convoluted form of the endodermal layer (data not shown).


Figure 6
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  		FIG. 6. Influence of DFMO treatment in the histology of placenta and extraembryonary membranes. DFMO was administered by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Pregnant mice were killed at d 11. Representative histological sections of control (A) and DFMO-treated (B) placenta. Db, Decidua basalis; lz, labyrinthine zone; st, spongiotrophoblasts; gt, giant trophoblasts. Bar, 100 µm. Note the absence of the labyrinth and spongiotrophoblasts in the treated animals. Yolk sac structure in control (C) and DFMO-treated mice (D) are shown. Bar, 100 µm. E and F, Higher magnification of yolk sac, allantois, and chorion showing the presence of blood islands in control (E) but not DFMO-treated animals (F). m, Mesodermal component of the yolk sac; en, endodermal component of the yolk sac; b, blood cells; e, embryo. Bar, 100 µm.

 
Hormonal and molecular changes associated to DFMO treatment
It is well known that adequate levels of steroid hormones are required for the proper development of pregnancy. To know whether DFMO treatment may affect the steroidogenic systems in the pregnant mice, we studied plasma levels of progesterone and androstenedione along the gestational period from d 7 to d 20. Figure 7AGo shows that there was a transient increase in progesterone on d 9 and 10, and from d 11 progesterone levels steadily increased to reach high values on d 15–18 and decreasing abruptly before of labor. In the treated mice, the rise in progesterone on d 9 and 10 was not produced, and the levels remained markedly low with respect to control values. Plasma androstenedione concentration showed a marked increase on d 10, declining thereafter. In DFMO-treated animals the androstenedione peak was dramatically reduced (Fig. 7BGo).


Figure 7
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  		FIG. 7. Progesterone (A) and androstenedione (B) levels in the plasma of control and DFMO-treated pregnant mice. DFMO was administered by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Results are expressed as the mean ± SE of two to six animals per group. When not shown, SE lies within symbols. *, P < 0.05 vs. control.

 
The possible influence of DFMO on the expression of steroidogenic genes was studied by semiquantitative RT-PCR. In the ovary of pregnant mice, no significant changes in the levels of mRNA of the steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (CytP450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) I, or aromatase were found between control and treated animals. On the contrary, DFMO treatment was associated with significant decreases in the levels of expression of different steroidogenic genes in the fetoplacental unit. These changes were clearly observed at d 10 and involved StAR, 3β-HSD VI, an isoenzyme of the enzyme expressed in the ovary, and 17{alpha} steroid hydroxylase (17{alpha}H), the enzyme required for androgen synthesis. Cyt P450scc, 20{alpha} hydroxysteroid dehydrogenase, enzyme participating in progesterone catabolism, and the transcription factor activator protein-2{gamma}, which regulates placental steroidogenesis, were not affected by the treatment. To confirm the decrease in the expression of the genes mentioned above, mRNA levels were more precisely analyzed by real-time RT-PCR. Figure 8Go shows that DFMO treatment elicited significant drops (P < 0.01) in the levels of 3β-HSD VI (80%), 17{alpha}H (79%) and StAR (54%) at d 9, which were even higher at d 10 (87, 89, and 88%, respectively).


Figure 8
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  		FIG. 8. Influence of DFMO treatment in the expression of steroidogenic genes in mouse conceptus and placenta at d 9 and 10 of pregnancy. DFMO was administered by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Mice were killed at d 9 (A) and 10 (B) of pregnancy, and RNA was isolated and analyzed by real-time RT-PCR. Values are the mean ± SE of three animals per group. *, P < 0.01 vs. control. Expression values of the steroidogenic genes are normalized with respect to those of β-actin.

 
Because the histological analysis of the conceptuses from animals treated with DFMO suggested a possible alteration in embryo hematopoiesis, the expression of embryonary hemoglobins in control and DFMO-treated embryo were studied by real-time RT-PCR. A substantial fall in the levels of {alpha}-like x chain (Hba-x) and β-like y-chain (Hbb-y) of the embryonic hemoglobin, higher than 50%, was observed (Fig. 9Go).


Figure 9
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  		FIG. 9. Influence of DFMO treatment in the expression of embryonary globin genes in mouse conceptus and placenta at d 9 of pregnancy. DFMO was administered by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Mice were killed at d 9 of pregnancy, and RNA was isolated and analyzed by real-time RT-PCR. Results are the mean ± SE of three animals per group. *, P < 0.01 vs. control. Expression values of the globin genes are normalized with respect to β-actin.

 
Many genes and factors have been related to embryo and placental development. Among them, IGF-I and IGF-II and its receptor (IGFR) have been implicated in the regulation of fetoplacental growth through gestation (35). Transcription factors, heart and neural crest derivatives transcription factor (Hand)-1 and mammalian achaete-scute complex homolog-like 2 are essential for placental development in mice (36, 37). Placental growth factor (PGF), proliferin (PLF), and adrenomedulin (ADM) are implicated in the growth and vascularization of the decidua and in embryo development (38, 39). Figure 10Go shows that the expression of none of these genes appeared to be altered by DFMO treatment in the days studied. Interestingly, the expression of antizyme inhibitor-2, protein recently characterized in our laboratory (40), was increased by DFMO treatment. However, mRNA levels of the other genes related to polyamine metabolism expressed in the conceptus (Fig. 2Go), including ODC, were not significantly affected by DFMO treatment, although ODC activity was nondetectable in the conceptuses of DFMO-treated mice (results not shown).


Figure 10
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  		FIG. 10. Gene expression of different transcription and growth factors related to embryo development in mouse conceptus. DFMO was administered by sc injection d 7–9 of pregnancy (500 mg/kg, twice a day). Control animals received 0.9% saline solutions with the same schedule as treated mice. Mice were killed at d 8–10 of pregnancy, and RNA was isolated and analyzed by semiquantitative RT-PCR. Results are the mean ± SE of repeated experiments using RNA from separate pools of three animals per group. Relative intensity values were calculated with respect to β-actin. *, P < 0.05 vs. control. PLF, proliferin; mPL-1, placental lactogen 1 (chorionic somatomammotropin hormone precursor); GCM1, glial cells missing homolog transcription factor 1.

 
To find more genes that could be implicated in the contragestational effect of DFMO, the microarray technology was used to analyze changes in transcripts between RNA obtained from conceptus and placenta of control mice and RNA obtained from DFMO-treated mice. A preliminary transcriptomic analysis revealed differences in 1965 transcripts if a 1.5 factor was considered. The transcripts of embryonary hemoglobins were among the most decreased transcripts by DFMO. This decrease is consistent with the results found in the real-time RT-PCR analysis shown in Fig. 9Go. On the other hand, the analysis also showed decreases of about 50% in the expression of several genes (Table 4Go) corresponding to factors and markers of the trophoblastic lineage, such as Tpbpa, a spongiotrophoblast cell marker (41); the labyrinth and spongiotrophoblast-expressed factors placental specific protein 1 (Plac1), reproductive homeobox 6 (Psx1), Cbp/p300-interacting transactivator (Cited1) (42, 43, 44); {alpha} 4-integrin, implicated in the chorioallantoic fusion (45); and eomesodermin, a protein involved in the differentiation of trophoblastic stem cells (46).


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  		TABLE 4. Differential expression of genes related with placental development in conceptus from control and DFMO-treated mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Early studies about the implication of polyamines in prenatal development underlined the importance of ODC in mouse pregnancy. The pharmacological treatment of pregnant mice with DFMO indicated that there is a critical period for the contragestational effect of DFMO, which correlates with the peak of ODC activity in the developing embryo, seen at d 8 of pregnancy (25, 26). On the other hand, the elimination of ODC or SAMDC functions by gene targeting resulted in death of the embryo before gastrulation, demonstrating that polyamines play an essential role in murine development (31, 32). Whereas it was shown that in mice harboring a disrupted ODC gene, the abortive development of ODC-deficient embryos was related to altered survival of the pluripotent cells of the inner cell mass because of high rates of apoptosis (31), the cellular and molecular mechanisms by which ODC inhibition produced by DFMO treatment affects embryonic development are mostly unknown.

Our present results about the effect of DFMO administration on mouse pregnancy corroborate the findings of Fozard et al. (25, 26) on both the existence of a critical period around d 8 of pregnancy for the maximum contragestational action of DFMO and the lack of effect of this drug when administered before d 7 of pregnancy. The apparent discrepancy between the death of the mutant embryos, between d 3.5 and 6.5 of pregnancy, and the lack of contragestational effect of DFMO when administered during this period, could be related to a limited accessibility of the administered DFMO into the mouse embryo in the first third of gestation.

Our findings indicate that DFMO treatment at d 8 of pregnancy arrests both embryo growth and the proper development of extraembryonic structures, such as the yolk sac and placenta, which are critical for survival of developing embryos. The effect of DFMO on embryo growth and development could be related to the inhibition of embryonary ODC, which peaked at d 8 with an activity level similar to that found in extraembryonary structures of conceptus (Fig. 1BGo). However, the embryo arrest could also be due to indirect effects produced as a consequence of the changes observed in yolk sac and placenta.

The yolk sac is a highly vascularized structure that allows gases and nutrients exchanges to the embryo without directly exposing it to maternal blood and immune system. After implantation, the yolk sac is the first and single link between maternal and fetal compartments. Alterations of this primitive placentation could result in embryonic death during early postimplantation period due to abnormalities of the vascular network (47). In our study, the histological analysis of the yolk sac showed important alterations in the development of this structure. The formation of the yolk sac depends on massive proliferation and differentiation of multiple cell types (47, 48). In fact, many genes have revealed to be important for yolk sac development, with Hand 1 having a major role in such process (49). In our study, the expression of this gene was not modified by DFMO treatment, suggesting that the implication of polyamines in yolk sac development is not exerted through this transcription factor (at least at the transcriptional level). The alterations encountered in the yolk sac also affected its associated blood islands. It is well known that early embryonic hematopoiesis takes places in the blood islands of the yolk sac from d 7.5 until d 11 of pregnancy, i.e. when the embryonic liver assumes hematopoiesis (50, 51). Therefore, a deficient hematopoiesis in the yolk sac at this stage of pregnancy could result in embryonic hypoxia and subsequent death. Interestingly, we found a marked drop in the expression of embryonic globins that could be related either to a diminished transcription of Hba-x and Hbb-y genes or to the deficit in blood islands formation. This effect of DFMO on globins expression could be related to the high levels of ODC found at d 8 of pregnancy in the extraembryonic mesodermal components of the yolk sac, which will subsequently form blood islands (52). Because early hematopoiesis in the yolk sac is subject to a complex regulation, the influence of DFMO in the expression of transcription factors involved in globin biosynthesis (53, 54) or other factors that regulate the extraembryonic hematopoiesis from mesodermal progenitors (48) cannot be excluded. Nonetheless, the transcriptomic analysis did not show alterations in the expression of these factors. On the other hand, the possibility that DFMO may affect yolk sac vasculogenesis or angiogenesis is in agreement with reports that indicated that ODC overexpression promotes neovascularization in nude mice (55, 56).

Our results also show that DFMO treatment markedly affected the development of a functional placenta. Whereas in the control mice, the histological analysis at different days of pregnancy revealed a normal formation of the choriallantoic placenta, in the DFMO-treated mice different alterations in placental structures were evident. The study showed abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer, and a deficient development of the labyrinthine zone, alterations that appear to be incompatible with normal embryo development. However, the trophoblastic giant cell zone isolating the fetoplacental unit from the mother was not significantly affected by the treatment. In agreement with this result, the mRNA levels of PLF, PGF, and ADM, genes that are expressed in trophoblast giant cells and related to the regulation of maternal blood flow (38), were not affected by the treatment. Although we did not find changes in the transcripts of Gcm1, Hand 1, and mammalian achaete-scute complex homolog-like, important transcription factors involved in labyrinth and spongiotrophoblast development (39, 57), alterations in multiple factors implicated in placentation cannot be excluded. Interestingly, the transcriptomic analysis showed changes in the expression of a subset of factors involved in placentation and in the differentiation of several trophoblastic lineages.

The present study reveals an interesting effect of DFMO on plasma levels of androstenedione and progesterone in the pregnant mice. In the treated mice, there was a marked decrease in the androstenedione peak that occurs at midpregnancy, whereas the progesterone increase, observed in the second half of pregnancy, was completely abolished. This fall in steroid hormones may be critical for the maintenance of gestation because it is known that these hormones play an indispensable role in pregnancy (58). In rodents, unlike in humans, the ovarian steroidogenesis is required during the whole pregnancy (59, 60, 61), whereas the murine placenta maintains a limited steroidogenesis, first in the dedidual tissue and then in the trophoblastic giant cells (62, 63, 64). This effect of DFMO on plasma steroid hormones appears to be primarily related to the diminished expression of several steroidogenic proteins, such as StAR, 3β-HSD VI, and 17{alpha}H, observed in the conceptus, rather than to a direct effect of the drug in the expression of the steroidogenic genes in the ovary. Further supporting this possibility is the observation, stated above, that DFMO may affect the differentiation of placental trophoblasts, and especially the steroidogenic capacity of trophoblast giant cells (63). In this regard, it is noteworthy to mention that in previous studies we demonstrated that in other steroidogenic tissues, such as the corpus luteum and the adrenal glands, the rise in ODC activity elicited by different peptidic hormones is required for the acquisition of the steroidogenic secretory capacity (20, 65). The decrease in androstenedione synthesis elicited by DFMO in the mouse placenta may affect progesterone secretion by the ovary because placental steroidogenesis at midpregnancy appears to be critical for the correct development of ovarian corpus luteum, which is required for sex hormone production during the second half of pregnancy (61, 66, 67). In turn, it was shown that the addition of androstenedione to cultured luteal cells increased progesterone release by a nongenomic mechanism (68).

The results presented here, together with those found in ODC-null embryos (31), clearly indicate that there are two different periods in mouse pregnancy in which ODC is critical for embryo survival. Whereas in the latter case, it was suggested that the death of the ODC-deficient embryos was not related to an extraembryonic defect, in the DFMO-treated animals, the contragestational effect of this drug appears to be related to defects in both embryonic and extraembryonic compartments. Evidently the inhibition of embryonic ODC may arrest embryo growth because polyamine synthesis is required for cell proliferation (2, 3). Nevertheless, the embryo’s death may be also the consequence of an inappropriate supply of gases and nutrients because of the defects produced by DFMO in the formation of yolk sac and placental structures. Collectively the available evidence suggests that the embryo lethality produced by DFMO may be a combination of simultaneous defects. This pleiotropic effect associated with polyamine depletion is not surprising if one takes into account the multiple roles played by polyamines in different cellular processes and particularly in cell growth and differentiation. From our present data, it is difficult to establish cause-and-effects relationship. Indeed, it cannot be discerned whether the effects produced by putrescine depletion on steroidogenesis and hematopoiesis are independent or are mutually related. The fact that embryos with ablation of steroidogenic genes died at midpregnancy with defects in vascularization (69) may suggest that in our case the effects on vasculogenesis could be secondary to altered steroidogenesis.

In conclusion, our results indicate that the contragestational effect of DFMO is related to alterations in yolk sac formation and trophoblast differentiation, including vasculogenesis and steroidogenesis. The detailed molecular mechanisms by which polyamine depletion affects the expression of different genes in the conceptus warrants further investigations.


   Footnotes
 
This work was supported by Grants 00466/PI/04 from the Seneca Foundation (Autonomous Community of Murcia) and BFU2005-09378-C02 from the Spanish Ministry of Education: ''and FEDER funds'' C.L.-G. and A.J.L.-C. are recipients of fellowships from the Seneca Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2008

Abbreviations: ADM, Adrenomedulin; AZ, antizyme; CytP450scc, cytochrome P450 cholesterol side-chain cleavage enzyme; DFMO, {alpha}-difluoromethylornithine; 17{alpha}H, 17{alpha} steroid hydroxylase; Hand, heart and neural crest derivatives transcription factor; Hba-x, {alpha}-like x chain; Hbb-y, β-like y-chain; 3β-HSD, 3β-hydroxysteroid dehydrogenase; IGFR, IGF receptor; ODC, ornithine decarboxylase; pc, post conception; PGF, placental growth factor; PLF, proliferin; SAMDC, S-adenosylmethionine decarboxylase; StAR, steroidogenic acute regulatory protein.

Received January 17, 2008.

Accepted for publication June 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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