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BOR - Papers in Press, published online ahead of print June 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032086
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BIOLOGY OF REPRODUCTION 71, 1475–1483 (2004)
DOI: 10.1095/biolreprod.104.032086
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Adrenomedullin Antagonist Treatment During Early Gestation in Rats Causes Fetoplacental Growth Restriction Through Apoptosis1

Josthna Penchalaneni3, Sunil J. Wimalawansa4, and Chandrasekhar Yallampalli2,3

Department of Obstetrics and Gynecology,3 University of Texas Medical Branch, Galveston, Texas 77555 Regional Bone Center,4 Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adrenomedullin (AM), a potent vasorelaxant peptide, has been shown to function as an angiogenic and growth factor. The present study investigated whether antagonism of endogenous AM in rats during early gestation results in diminished placental and fetal growth and whether this occurs through induction of apoptosis. Rats on Gestational Day 8 were implanted s.c. with osmotic minipumps delivering 125 and 250 µg rat–1 day–1 of AM22–52 and were killed on Gestational Day 15. In AM22–52-treated rats, both placental and fetal weights were dose-dependently inhibited, with 50% reduction in the group receiving 250 µg rat–1 day–1. In these animals, fetal resorption sites were also increased. Apoptosis was demonstrated in placenta and uterus by the TUNEL method. Apoptotic changes were more apparent in trophoblast cells in the labyrinth zone of placenta and uterine decidua of AM22–52-treated rats when compared with vehicle-control rats. Immunoreactivity to active caspase-3 protein was abundant in the placenta and uterus of the AM22–52-treated group. Western blot analysis demonstrated that in homogenates of both the placenta and uterus of AM22–52-treated rats, levels of active caspase-9 and -3 as well as of Poly ADP ribose polymerase were significantly increased, whereas levels of Bcl-2 protein decreased, compared with controls. However, no significant treatment-associated changes were observed in Bid, Fas, Fas ligand, p53, and caspase-8 and -10 proteins in either placenta or uterus. Bad protein was undetectable in either tissue. In mitochondrial fractions from both placenta and uterus, the levels of Bax increased with decreases in cytochrome c on AM22–52 treatment. Conversely, in the cytosol, Bax levels decreased with increases in cytochrome c, demonstrating translocation of Bax from cytosol to mitochondria and release of cytochrome c from mitochondria with AM22–52 treatment. In conclusion, these findings show that antagonism of AM in rats during early pregnancy caused fetoplacental growth restriction through the activation of mitochondrial apoptotic pathways.

apoptosis, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Implantation, placental growth, and development are crucial to the overall well-being of the fetus and are controlled by a range of signals, including steroids and growth factors. Adrenomedullin (AM), a potent vasodilator peptide, has been demonstrated previously to exert various actions on cellular growth, central nervous system, and endocrine system. Recently, AM has been implicated in a variety of reproductive functions, specifically during pregnancy. In the human, plasma AM levels were elevated during pregnancy and decreased at term [1–4]. This increase in AM levels occurred immediately after implantation [1], and this decrease occurred in both plasma [5, 6] and amniotic fluid [7] later in gestation. Furthermore, Moriyama et al. [8] reported that the expression of AM in cytotrophoblasts is most abundant in the first-trimester human placenta. These observations implicate the involvement of AM during early placentation and fetal growth in human pregnancy; however, it is unclear whether this involvement is critical.

In rodents, evidence is increasing for a role of AM in fetoplacental development. We recently observed significant decreases in placental and fetal growth and impaired vasculogenesis when pregnant rats were infused with the AM-antagonist AM22–52 from Day 14 of gestation [9]. These studies demonstrate the requirement of AM for fetal growth and placental function after the formation of placenta. The impact of AM22–52 infusion on early placental formation and fetal growth, however, were not addressed in that study. In the pregnant mouse, AM has been localized to the endometrial/decidual layer, and AM expression peaks around Day 9.5 of gestation [10]. In a recent report, Caron and Smithies [11] reported fetal death at midgestation (Day 13.5) in AM knockout mice, whereas fetuses in heterozygous mice were viable. The cause of fetal death appeared to be severe nonimmune fetal hydrops associated with cardiovascular defects. These reports suggest that fetal AM plays a critical role in early fetal cardiovascular development and fetal viability. Because, in heterozygous mice, the fetal viability and growth do not appear to be altered, the role of AM in implantation, placental formation, and early fetal growth remains unclear. We therefore hypothesized that inhibition of endogenous AM function during implantation and early placental development (Days 8–15 of gestation) would result in impaired placental formation and fetal growth restriction in rats.

Early pregnancy in rodents is characterized by a progressive interaction between the embryo and the maternal compartment. Rodent uterine epithelium around the embryo undergoes apoptosis in response to the presence of the blastocyst [12–14]. The blastocyst signals that induce the apoptotic cascade, as well as the genes that regulate this local event, are still unknown. Recent reports suggest that AM is a regulator of cell growth and differentiation [4] and that it plays a potent protective role against apoptosis [3] and in maintaining cellular integrity. Moreover, AM has been shown to inhibit hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells [15]. Thus, optimal AM function may be required to prevent apoptosis and maintain normal implantation, placental development, and fetal growth. We therefore further hypothesized that the fetal and placental growth restriction in AM-antagonized rats might occur through enhanced apoptosis in the uteroplacental tissues during early development. At the molecular level, apoptosis is highly regulated and is mainly orchestrated by activation of the aspartate-specific cysteine protease (caspase) cascade. Two reported pathways lead to the activation of caspases [16–18]. The first is the mitochondrial (receptor-independent) pathway, and the second involves interaction of death receptor and its ligand. Both proapoptotic (Bak, Bid, Bad, and Bax) and antiapoptotic Bcl-2 (Bcl-2 and BclXL) family members regulate the mitochondrial pathway [19] by modulating cytochrome c release from mitochondria into cytosol. The cytosolic cytochrome c then induces oligomerization of Apaf-1 [20], resulting in the activation of procaspase-9, which then activates procaspase-3. The second is the extrinsic pathway through the induction of Fas/ Fas ligand (FasL), causing activation of procaspase-8 and -10 or of p53, which then can directly activate procaspase-3. Activation of procaspase-8 can also cleave proapoptotic protein, Bid, which subsequently induces cytochrome c release from mitochondria [21, 22]. In the present study, the AM-antagonist AM22–52 was continuously infused during Days 8–15 of gestation, and on Day 15, we assessed fetoplacental growth and examined changes in a variety of caspases, Bcl-2 family proteins, and extrinsic pathway mediators of apoptosis.


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

Adult male and female nonpregnant rats (body weight, 200–300 g) were purchased from Harlan Sprague Dawley (Houston, TX). All animals were housed in a climate-controlled room with a 12L:12D photoperiod and were fed standard rat chow with water to drink ad libitum. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX). Female rats were mated with male proven breeders, and the pregnant females (presence of sperm in the vaginal flush was recorded as Day 1) were maintained in the animal care facility until they were infused with AM22–52 or saline on Day 8 of gestation. Three to four pregnant rats were used in each experimental group.

Treatments

The mean (± SEM) body weight of the rats on Day 8 of gestation (study initiation) was 224 ± 4.8 g. On Day 8 of gestation, osmotic minipumps (model 2ML1; 10 µl/h; Alza, Palo Alto, CA) were inserted s.c. into the dorsum of pregnant rats while the animals were under anesthesia. Anesthesia consisted of a combination of ketamine (45 mg/kg; Fort Dodge Laboratories, Fort Dodge, IN) and xylazine (5 mg/kg; Burns Veterinary Supply, New York, NY). The minipumps were filled with saline alone or with saline containing different concentrations of AM22–52. These concentrations were chosen to deliver AM22–52 at 125 and 250 µg rat–1 day–1. Based on the pumping rate and duration of infusion, we prepared the drug concentration in the pumps to provide the specified daily dose of the drug. One of the authors (S.J.W.) synthesized human AM antagonist (22–52) by use of solid-phase t-butoxycarbonyl chemistry that was purified and characterized by mass spectrometry, amino acid analysis, and sequencing [9]. The dose of AM22–52 was similar to that used in previous studies [9]. All rats were killed on Gestational Day 15 using a CO2 inhalation chamber. The uteri were removed, and the total number of fetoplacental units and resorption sites were counted. A resorption site is identified by the presence of an implantation site with a complete absence of fetal tissue. Placentas and fetuses were carefully dissected out, and weights were recorded. In addition, placentas and uterine tissues were collected for immunohistochemistry and Western blot analysis. Tissues from animals receiving AM22–52 at 250 µg rat–1 day–1 and from controls were either fixed in Bouin fluid for immunohistochemistry or frozen in liquid nitrogen and stored at –80°C until further analysis of various proteins.

In Situ Detection of DNA Nicking

Tissue samples were embedded in paraffin, and sections (thickness, 5 µm) were cut with a microtome and placed on coated slides. Paraffin was removed from the tissue sections with xylene, and the sections were rehydrated in graded ethanol solutions. Fragmentation of DNA was detected by the TUNEL method using an ApopTag Red In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions with slight modifications. After the tissue sections were deparaffinized, protein was digested with 20 µg/ml of proteinase K for 15 min at room temperature. Endogenous peroxidase activity was quenched with 3% H2O2 in PBS. After washing with PBS, an equilibration buffer was applied directly to the specimen. Terminal deoxynucleotidyl transferase (TdT) enzyme and deoxyuridine triphosphate (dUTP) digoxigenin were added and incubated at 37°C for 1 h in a humidified chamber. The reaction was then stopped using a stop/wash buffer supplied with the kit, and the slides were incubated with an antidigoxigenin-peroxidase solution for 30 min at room temperature, colorized with diaminobenzidine (DAB)/ H2O2, and counterstained with methyl green. Negative controls were processed with labeled dUTP in the absence of TdT enzyme. To assess the extent of apoptosis, the number of TUNEL-stained nuclei were counted by two investigators who were blinded with regard to the treatments in four randomly selected microscopic fields (at 200x magnification) per section. Data obtained from two sections per animal were then averaged. Values for the control (n = 4) and AM22–52-treated (n = 3) groups are presented as the mean ± SEM.

Immunohistochemistry

After deparaffinization of tissue sections, antigen retrieval was performed by heating in 10 mM citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 min. After blocking with 5% normal rabbit serum, sections were incubated with the primary antibody for caspase-3 (polyclonal antibody at 1:100 dilution; Cell Signaling Technology, Beverly, MA) at 4°C overnight. They were rinsed in PBS and incubated with biotinylated goat anti-rabbit immunoglobulin (Ig) G (Vector Laboratories, Burlingame, CA), followed by avidin-biotin-peroxidase solution (Vectastain ABC Elite Kit; Vector Laboratories). Next, DAB with 0.003% H2O2 in PBS was added to each slide. The tissues were lightly counterstained with hematoxylin and examined by light microscopy. Negative controls were obtained using normal rabbit serum in place of primary antibody, with all other steps unchanged.

Tissue Preparation and Subcellular Fractionation

We homogenized 100 mg of placenta or full-thickness uterus in 500 µl of Tris buffer (50 mM Tris, 0.1 mM EGTA, 100 mM PMSF, 1.4 µl of ß-mercaptoethanol, and one mini-tablet of protease-inhibitor cocktail [Roche Applied Sciences, Mannheim, Germany] per 10 ml) with a homogenizer (PowerGen 125l; Fisher Scientific, Houston, TX) at 15 000 rpm for 10 sec. After centrifugation of the homogenate at 3500 rpm for 10 min at 4°C, the supernatant fraction was aliquoted and stored at –80°C. For subcellular fractionation and to obtain mitochondria, the supernatant was centrifuged at 10 000 x g for 20 min. The mitochondrial pellet was washed three times in homogenizing buffer and then solubilized in TNC buffer (10 mM Tris-acetate [pH 8.0], 0.5% Nonidet p-40, and 5 mM CaCl2) containing protease inhibitors. Protein concentration was determined by BCA kit (Pierce Biotechnology, Rockford, IL).

Western Blot Analysis

Equal amounts of protein (30 µg) were separated by various appropriate concentrations of SDS-PAGE: 8% for endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively); 12% for Bcl-2, Bax, Bak, Bad, caspase-8, caspase-9, caspase-10, p53, Fas, and FasL; and 15% for cytochrome c and caspase-3. Then, the SDS-PAGE-separated proteins were equilibrated in transfer buffer (25 mM Tris [pH 8.3], 190 mM glycine, 0.05% SDS, and 20% methanol) and electrotransferred to nitrocellulose membranes. Blocking of membranes was performed with TTBS buffer (20 mM Tris [pH7.4], 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk for 1 h and then washed with TTBS buffer. For the detection of proteins on nitrocellulose membrane, antibodies to eNOS (catalog no. 610296), iNOS (catalog no. 610328; BD Transduction Laboratories, Lexington, KY), cytochrome c (catalog no. SC-13156), Poly ADP ribose polymerase (PARP; catalog no. SC-1562), Bcl-2 (catalog no. SC-492), BclXL (catalog no. SC-1041), Bax (catalog no. SC-426), Bak (catalog no. SC-832), Bid (catalog no. SC-6291), Fas (catalog no. SC-716), FasL (catalog no. SC-834), p53 (catalog no. SC-100), caspase-8 (catalog no. SC-7890; Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (catalog no. 9961), caspase-9 (catalog no. 9506), and caspase-10 (catalog no. 9752; Cell Signaling Technology, Beverly, MA) were diluted 1:500, 1:500, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1: 1000, 1:1000, 1:1000, 1:250, 1:500, 1:500, and 1:1000, respectively. Polyclonal ß-tubulin antibody (catalog no. SC-9104) and monoclonal heat shock protein 60 (HSP60) antibodies (catalog no. SC-13115; Santa Cruz Biotechnology) were used at a dilution of 1:1000. Blots were exposed to horseradish peroxidase-conjugated anti-rabbit IgG (caspase-3, caspase-8, caspase-9, caspase-10, Fas, FasL, Bak, Bcl-2, and BclXL), anti-goat IgG (Bid and PARP), or anti-mouse IgG (cytochrome c, p53, Bax, Bad, and HSP60) secondary antibodies (diluted 2000- to 5000-fold) for 1 h. The anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. The blots were rinsed, and the enhanced chemiluminescence reagent (ECL Kit; Amersham Life Science, Piscataway, NJ) was added and incubated for 1 min and then exposed to Hyperfilm ECL. The intensity of specific immunoreactive bands was quantified by a densitometric scanning program (SigmaGel Software; Sigma, St. Louis, MO). All replicates from each group were run in one gel, and the proteins are expressed as a ratio of protein signal to the ß-tubulin signal.

Statistical Analysis

Placental and fetal weights are expressed as the mean ± SEM and were analyzed for differences using one-way ANOVA followed by the Bonferroni t-test. The Student t-test was used to analyze differences in protein signals in Western blots. Values were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of AM22–52 on Fetoplacental Weights

In the present study, we evaluated the role of AM in the regulation of fetoplacental growth during pregnancy. An antagonist of AM, AM22–52, was continuously infused through osmotic minipumps beginning on Gestational Day 8 in rats. These animals received either 125 or 250 µg rat–1 day–1 of AM22–52 or vehicle only and were killed on Day 15 of gestation to assess placental and fetal weights. Table 1 shows that both placental and fetal weights in rats receiving two doses of AM antagonist were significantly lower than in controls (P < 0.05). These reductions in fetal and placental weights were more substantial with AM22–52 at 250 µg rat–1 day–1 compared with 125 µg rat–1 day–1. The proportion of implantation sites that were resorbed in animals receiving AM22–52 were higher than in controls. Although the litter sizes were similar in all groups, the percentage of resorption sites were 22.7% and 23% in 125 and 250 µg rat–1 day–1 of AM22–52-treated animals, respectively, compared with 5% in controls (Table 1). Because the effects of AM22–52 on both the fetus and the placenta were substantial at a dose of 250 µg rat–1 day–1, we used this dose in all our subsequent studies.


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  		TABLE 1. Placental weight, pup weight, and mortality data for rats killed on Day 15 of gestation.a

Apoptotic Changes in Uteroplacental Tissues on AM22–52 Infusion in Pregnant Rats

Because fetal growth as well as placental weights were substantially lower in AM22–52-treated rats, we examined the possibility of the involvement of apoptotic changes in the uterine and placental tissues in the observed reduction in placental and fetal growth. Apoptosis is demonstrated within the placental and uterine tissue by the TUNEL method, and the TUNEL-positive reaction was detectable in trophoblast cells in placenta. The majority of apoptotic cells were trophoblasts and were found primarily in the labyrinth zone in placenta. The number of cells with TUNEL-positive staining in the labyrinth zone increased in the AM22–52-treated animals compared with the number in untreated control rats (Fig. 1). In placenta, the number of TUNEL-positive cells per microscopic field at 200x magnification was 64 ± 8 in AM22–52-treated animals compared to 3 ± 1 in control animals. Similarly, TUNEL-positive cells in the decidual compartment of the uterus were also more abundant in AM22–52-treated rats compared to untreated controls (Fig. 1). The number of TUNEL-positive cells per microscopic field at 200x magnification was 33 ± 6 in AM22–52-treated animals compared to 2 ± 1 in control rats. Specificity of staining was demonstrated by the absence of staining when TdT was eliminated from the staining protocol.



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  		FIG. 1. TUNEL staining in uterus (A–D) and placenta (E–H) of AM22–52-treated and control rats on Day 15 of pregnancy. Immunofluorescent staining indicates a positive reaction, and the TUNEL-positive cells are abundantly seen in the uterus (A and B) and placenta (E and F) of AM22–52-treated rats compared with untreated control (D and H, respectively) rats. Increased TUNEL-positive staining was seen in the deciduas of the uterus (A and B) and in syncytiotrophoblast cells in the labyrinth layer of placenta (E and F). No positive staining was detected in sections when TdT was omitted in the reaction mixture (C and G). Only photomicrographs of uterine deciduas and placental labyrinth are presented, because no staining occurred in the uterine myometrium or placental basal layer. Magnification, x200 (A, C–E, G, and H) and x400 (B and F)

Immunohistochemical staining for the presence of active caspase-3 protein in the uterus and placental tissues of AM22–52-treated animals is presented in Figure 2. The active caspase-3 protein appeared to be abundant in the uterine decidual cells of the AM22–52-treated rats compared with untreated controls. Similarly, active caspase-3 protein staining was undetectable in untreated controls, whereas staining was abundant in AM22–52-treated rats. Specificity of the staining was confirmed by the absence of staining when primary antibody was omitted in the reaction.



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  		FIG. 2. Immunohistochemical staining for active caspase-3 protein in sections of the uterus (A–C) and placenta (D–F) from the AM22–52-treated and control rats on Day 15 of pregnancy. Active caspase-3 positive immunoreactivity was abundantly seen in labyrinth layer of placenta and deciduas of the uterus in the AM22–52-treated group (A and D) compared to untreated control (C and F) rats. Brown staining indicates a positive reaction, and this was absent when primary antibody was omitted (B and E). Tissues were counterstained with hematoxylin. Only photomicrographs of uterine deciduas and placental labyrinth are presented, because no staining occurred in the uterine myometrium or placental basal layers. Magnification, x200

Uteroplacental NOS Expression in AM22–52-Infused Rats

Because the effects of AM have been suggested to be mediated by nitric oxide [23], we examined the changes in protein levels for NOS enzymes, eNOS and iNOS, in uterine and placental tissues of AM22–52-treated and untreated control animals. A specific band at 140 kDa corresponding to the size of the eNOS protein was detectable in both the placenta and uterus from AM22–52-treated and untreated rats (Fig. 3). However, no significant differences in the amount of eNOS protein was found among the groups in either the placenta or the uterus. Also, iNOS protein was undetectable in the placenta of both AM22–52-treated and control rats. The iNOS protein was detectable in the uterus, but no significant changes were associated with AM22–52 treatment.



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  		FIG. 3. Expression of NOS enzymes in the uterus and placenta on Day 15 of pregnancy in rats. A) Western blot analysis of NOS enzymes, eNOS and iNOS, and ß-tubulin in AM22–52-treated and untreated control rats. Note that iNOS protein bands were not detectable in either tissue on Day 15 of gestation. B) Summary of densitometric values normalized to ß-tubulin. Data are presented as the mean ± SEM for at least three to four replicate animals

Effects of AM22–52 on Expression of the Bcl-2 Family of Proteins in Uteroplacental Tissues

The Bcl-2 family of proteins has been demonstrated to play an important role in the regulation of apoptosis in a variety of cells [24]. To study the pathways involved in AM22–52-induced apoptosis, we examined the changes in proteins of proapoptotic and antiapoptotic Bcl-2 family members in both uterus and placenta from AM22–52-treated and untreated control rats. Figure 4 shows the changes associated with AM22–52 treatment in Bcl-2, BclXL, Bak, and Bid proteins in both the uterus and placenta. Significant decreases in expression of the antiapoptotic Bcl-2, but not BclXL, protein in the uterus and placenta were observed in the AM22–52-treated group when compared with untreated controls. However, no differences were found in the expression of proapoptotic proteins, Bak and Bid, among treated and untreated groups in either of the tissues.



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  		FIG. 4. Expression of Bcl-2 family proteins in the uterus and placenta of Day 15 pregnant rats. A) Western blot analysis of Bcl-2 family proteins and ß-tubulin in AM22–52-treated and untreated control rats. B) Summary of densitometric analysis normalized to ß-tubulin. Data are presented as they mean ± SEM for three to four replicate animals in each group. *P < 0.05 vs. control

Additionally, the changes associated with AM22–52 treatment were also assessed in proapoptotic proteins, Bax and Bad. Because translocation of these protein levels from cytosolic to mitochondrial compartments appears to trigger cell death, we measured the levels of these proapoptotic proteins in mitochondrial and cytosolic fractions. Figure 5 shows that in the uterus and placenta, Bax levels decreased in cytosol and increased in mitochondria in AM22–52-treated compared to control rats. However, Bad protein was undetectable in both the cytosol and mitochondria of the uterus and placenta (data not shown).



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  		FIG. 5. A) Western blot analysis for cytochrome c and Bax of mitochondrial and cytosolic fractions of the uterus and placenta from control and AM22–52-treated rats on Day 15 of pregnancy. HSP60 and ß-tubulin proteins in mitochondrial and cytosolic fractions, respectively, were also immunoblotted. B) Summary of densitometric analysis normalized to HSP60 in the mitochondria and ß-tubulin in cytosol presented as histograms. Bars represent the mean ± SEM for three to four replicate animals in each group. *P < 0.05 vs. control

Changes in Mitochondrial Cytochrome c Associated with AM22–52 Treatment

Cytochrome c release from mitochondria is a critical component in the apoptotic process [25–27]. It occurs upstream of caspase activation and is inhibited by Bcl-2 or BclXL overexpression. We measured cytochrome c content in mitochondrial and cytosolic fractions of placental and uterine tissues from control and AM22–52-treated rats. As shown in Figure 5, Western blot analysis revealed that cytochrome c levels in mitochondria of both the uterus and placenta were significantly (P < 0.05) lower in AM22–52-treated than in untreated rats, indicating an increase in the release of cytochrome c from mitochondria. Conversely, cytochrome c levels in cytosol were elevated in both the uterus and placenta in AM22–52-treated rats compared to controls.

Involvement of Caspase Activation in AM22–52-Induced Apoptosis

Activation of caspases is a central mechanism of apoptosis, and caspases are considered to be the "executioners" of cell death [28]. We examined whether the caspase family of proteases is activated in the apoptotic process induced by AM22–52. Because caspase-3 is a central effector of apoptosis, we examined the proteolytic processing of caspase-3 and its downstream target, PARP protein, by Western blot analysis. Consistent with cytochrome c efflux from mitochondria, the proteolytically cleaved, active caspase-3 fragments were more abundant in AM22–52-treated animals compared with untreated controls. In addition, we investigated the involvement of caspase-9, because mitochondrial dysfunction results in cytochrome c release with subsequent activation of caspase-9, one of the major initiator caspases. Active caspase-9 protein levels in both uterus and placenta in AM22–52-treated animals were significantly (P < 0.05) higher compared to those in untreated animals (Fig. 6). The levels of cleaved-fragment PARP in the uterus and placenta were increased with AM22–52 treatment, providing further evidence for activation of mitochondria-related apoptotic pathway (Fig. 6) in AM22–52-induced apoptosis in uteroplacental tissues.



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  		FIG. 6. Expression of active caspase-9, caspase-3, and cleaved PARP in the uterus and placenta of pregnant rats. A) Western blot analysis of cleaved caspase-9, caspase-3, PARP, and ß-tubulin in AM22–52-treated and untreated control rats. B) Summary of densitometric analysis normalized to ß-tubulin. Data are presented as the mean ± SEM for three to four replicate animals in each group. *P < 0.05 vs. control

Fas, FasL, and p53-Mediated Extrinsic Pathway Signaling Does Not Contribute to AM22–52-Induced Apoptosis

Several recent studies have suggested that Fas/FasL signaling contributes to cell death during development and/or in response to apoptotic stimuli [29]. To study the contribution of Fas/FasL pathways and p53 in AM22–52-induced apoptosis, we examined Fas, FasL, and p53 protein levels in uteroplacental tissues of AM22–52-treated and untreated rats. The data shown in Figure 7 revealed no significant differences in Fas and FasL protein levels in both uterus and placenta associated with AM22–52 treatment. No significant differences were also observed in p53 levels in both these tissues among treated and untreated groups (Fig. 7). Because it has been reported that caspase-8 and -10 are rapidly activated after Fas engagement, we examined caspase-8 and -10 in both groups. Figure 7 shows the isoforms of caspase-8 and -10 in both the uterus and placenta; however, no significant changes with treatment were detectable. Several isoforms of caspase-10 have been previously reported [30]. Figure 7 indicates that both the 28- and 66-kDa bands were expressed in the placenta and that the 28-kDa protein appeared to be the primary form expressed in the uterus.



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  		FIG. 7. Expression of Fas, FasL, p53, caspase-10, and caspase-8 in the uterus and placenta of Day 15 pregnant rats. A) Western blot analysis for Fas, FasL, p53, caspase-8, and ß-tubulin in AM22–52-treated and untreated control rats. B) Western blot analysis for caspase-10 and ß-tubulin in AM22–52-treated and untreated control rats. C) Summary of densitometric analysis normalized to ß-tubulin. Data are presented as the mean ± SEM for three to four replicate animals in each group. Note that both the 28- and 66-kDa isoforms of caspase-10 are present in placenta, whereas only the 28-kDa isoform is detectable in the uterus


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previously, we reported significant decreases in fetal and placental growth in pregnant rats when AM22–52 was continuously infused from Day 14 of gestation, several days after implantation and placental formation [9]. Increases in fetal resorptions were also observed in that study [9]. In the present study, we examined two questions: Does infusion of AM22–52 from Day 8 of gestation abolish placental formation or inhibit early placental development and cause impairment of fetal development, and are apoptotic pathways involved in the pathophysiology associated with fetal growth retardation in AM22–52-treated rats? Although AM22–52 infusion did not completely abolish placental formation, it severely reduced early placental development and restricted fetal growth. Resorption of implantation sites was also significantly greater in AM22–52-treated rats compared with control rats. In addition, we identified enhanced apoptotic changes, including mitochondrial-related apoptotic pathway mediators, in both placenta and uterine decidua of AM22–52-treated rats. These data implicate an important role for endogenous AM in the regulation of early placental and fetal development in rats through its protection against apoptosis.

In a previous study, we demonstrated a role for endogenous AM in normal placental function and fetal growth, as evidenced from impairment of placental and fetal growth when AM22–52 was infused from Day 14 of gestation [9]. The present study was designed specifically to address the role of endogenous AM in early fetoplacental development by continuously infusing AM22–52 during Days 8–15 of gestation. Our results provide strong evidence that endogenous AM is, indeed, essential for early placental development and, therefore, fetal growth. The adverse effects of AM22–52 on both fetal and placental development were apparent with the group given 125 µg rat–1 day–1, the lowest dose used in the present study. Although these effects were statistically significant, the reductions in both fetal and placental weights were only 15%. On the other hand, AM22–52 at 250 µg rat–1 day–1 caused a more than 50% decrease in both the placental and fetal weights. Effects of AM22–52 at these doses on early placental development and fetal growth are consistent with those in our previous report on dose-related adverse effects of AM22–52 infused during midgestation in rats [9].

The present study suggests an important role for AM in early placental function and fetal growth, but our data are somewhat at variance with those of Caron and Smithies [11]. In their report, lack of fetal AM was lethal to 100% of fetuses. This occurred on day 13.5 in mice, whereas only 23% of the fetuses of the AM22–52-treated rats died in the present study. These discrepancies may result from differences in the species or models used (knockout vs. antagonist infusion from Day 8), incomplete inhibition of AM activity, or inability of AM22–52 to cross the placenta to inhibit fetal AM.

Data from the studies with AM knockout mice and AM22–52 infusion to pregnant rats provide strong evidence for a role of AM in early placental development and fetal growth; however, such a role for AM in the human remains to be demonstrated. Nevertheless, several reports provide strong support for such a role. Plasma AM levels were reported to be elevated during pregnancy in both humans and rats [2, 5, 31], and this occurred immediately after implantation and decreased later in gestation [1, 5, 6]. The AM is highly expressed in trophoblast cells and decidua during early pregnancy, at 6–10 wk of gestation [1, 32]. The expression of AM in trophoblasts is maximal in the first-trimester placenta [8], and immunoreactive AM at the fetomaternal interface as well as AM immunopositivity in both trophoblasts and decidual cells were lower in women with spontaneous abortion compared with controls [32, 33].

Because the action of AM appears to involve activation of the nitric oxide pathway in a variety of cells, including endothelial cells [23], we investigated whether AM22–52 treatment inhibits the expression of eNOS and iNOS enzymes in the placenta and uterus. Our results indicate that changes in eNOS do not appear to be involved in the AM22–52-induced fetoplacental growth restriction. In both tissues, iNOS protein levels were very low; therefore, it was difficult to interpret the data regarding the changes in iNOS because of treatment. The effects of AM22–52 treatment on the NOS activity cannot be excluded, because we did not address these questions in the present study. Regardless of whether the NO pathway is involved, the AM22–52 infusion induced apoptotic changes in uteroplacental tissues and caused fetal growth restriction. In addition, it has been suggested that AM is a placental growth factor [34] and that it might be involved in the regulation of trophoblast invasion during implantation and/or in embryonic development, including growth and tissue differentiation [11].

In the present study, we hypothesized that the reductions of placental and fetal development in AM22–52-infused rats may involve apoptotic cell death in uteroplacental tissues. Both TUNEL staining and immunolocalization of the executioner caspase, caspase-3, indicated abundant apoptotic changes in both the placenta and uterus. The TUNEL staining was predominant in trophoblast cells in the labyrinth layer of placenta, and caspase-3 staining was abundant in decidual cells of the uterus. It is possible that intense TUNEL staining also may reflect increased necrosis in the labyrinth zone, and the results of our previous studies suggested that AM22–52 infusion increases necrosis in the labyrinth zone of the placenta in pregnant rats on Day 18 of gestation [9]. Additionally, these changes in the labyrinth may be related to hypoxia induced by the possible reductions in maternal blood supply when AM22–52 is infused. Thus, the increased apoptotic effects of AM22–52 in the labyrinth zone of the placenta, an important region for fetomaternal exchange, could lead to the observed reduction in placental weights as well as fetal growth. To our knowledge, the present study is the first in vivo examination demonstrating that the inhibition of endogenous AM action leads to induction of apoptotic changes in the placenta that result in fetal growth restriction.

In the present study, we measured changes in mediators of both intrinsic and extrinsic pathways for apoptosis on AM22–52 infusion. In both placental and uterine decidua, the mitochondrial pathway appears to be activated in these animals. This conclusion is supported by several observations. These include decreased antiapoptotic Bcl-2, increased levels of activated caspase-9 and -3, reduced mitochondrial cytochrome c, and elevated cytosolic cytochrome c in both the placenta and uterus of AM22–52-treated rats. The increased cytochrome c release from mitochondria to cytosol then presumably binds to Apaf-1 [20], enabling recruitment of procaspase-9 with its subsequent processing to initiator caspase-9 and, finally, activation of caspase-3. In the present study, cytochrome c levels were decreased in mitochondria, with increases in the cytosol in AM22–52-treated animals indicating enhanced release into the cytosol (Fig. 6), which is in agreement with previous reports that cytochrome c is required for caspase activation [17, 19, 35]. Several previous studies have suggested that overexpression of Bcl-2 and BclXL prevents mitochondrial cytochrome c release and, therefore, the cascade of caspase activation [19]. Because AM has been shown to upregulate Bcl-2 in Ishikawa cells [15], and because Bcl-2 is a known protector against many adverse stimuli, including hypoxia, we suggest that inhibition of AM action could have caused decreases in Bcl-2 in AM22–52-treated rats. The Bcl-2 is thought to block apoptosis by retaining cytochrome c in the mitochondria, thereby inhibiting caspase-3 activation and subsequent downstream apoptotic events [36]. Additionally, Bcl-2 can act at the level of inhibitor caspases, as was recently proposed for stress-induced apoptosis [37]. In the present study, the lowered Bcl-2 expression in placental and uterine tissues would allow cytochrome c release from mitochondria to initiate the cascade of caspase activation and cell death.

Several proapoptotic members, such as Bax, Bid, Bad, and Bak, have been reported to induce cytochrome c release from mitochondria [17, 19]. Our results show that Bad and phospho-Bad were undetectable (data not shown) and that both Bid and Bak levels were unaltered in both the placenta and uterus on AM22–52 infusion. However, we found that in both placenta and uterus, Bax levels are decreased in cytosol and increased in mitochondria in AM22–52-treated compared to control rats, providing evidence for its involvement in cytochrome c release from mitochondria. Translocation of Bax from cytosol to mitochondria has been shown to increase mitochondrial membrane pores and, thus, cytochrome c release [19] and subsequent activation of caspases. Therefore, translocation of Bax from cytosol to mitochondria could trigger cytochrome c release and initiate caspase activation and apoptosis when AM22–52 is infused. Furthermore, this process could have been enhanced by decreases in Bcl-2 protein in AM22–52-treated rats.

In both the uterus and placenta, we examined activation of caspase-9 and -3, as judged by cleavage of the substrate PARP (Fig. 6), because PARP cleavage has been attributed to caspase-3 activity [17, 38]. Figure 6 shows that the generation of apoptosis-specific PARP cleavage fragment (85 kDa) and the activated caspase-3 (17-kDa subunit) in same tissues. Both PARP and activated caspase-3 levels are elevated in both these tissues in AM22–52-treated compared to control rats. In addition, caspase-9 levels are also elevated in AM22–52-treated rats. These results suggest that AM22–52 treatment induced mitochondrial pathway-related caspase cascade and subsequent apoptosis in both tissues.

Several agents can induce upregulation of Fas/FasL expression, initiating the Fas-dependent extrinsic pathway of apoptosis [17, 18, 39]. In the present study, we found that AM22–52 did not alter the expression of Fas, FasL, and p53. Therefore, we suggest that AM22–52-mediated caspase-3 activation and apoptosis do not appear to involve p53 and the Fas/FasL-dependent mitochondrial pathway. In addition, we examined caspase-8 and -10, because they are major initiator caspases in death receptor-mediated apoptosis [17, 18, 39]. We did not detect any cleaved forms of caspase-8 and -10, but we did observe small isoforms of both caspases. Although the caspase activation cascade during the apoptosis induced by AM22–52 still needs to be fully understood, it appears to involve the Bax translocation, cytochrome c release, and caspase-3 activation.

Thus, AM22–52 infusion appears to have increased release of cytochrome c in a Fas/FasL-independent manner, with activation of downstream executioner caspase-3 and cleavage of specific substrates (i.e., PARP) leading to the process of apoptosis. These changes occurred with the downregulation of Bcl-2 and translocation of Bax from cytosol to mitochondria, indicating the involvement of mitochondrial pathway activation in AM22–52-mediated apoptosis. However, it is unclear how AM22–52 caused the translocation of Bax from cytosol to mitochondria. Regardless of the mechanisms of action, AM appears to protect against apoptosis in uteroplacental tissues in rats. This conclusion is supported by reports that AM, acting through its receptors, markedly reduces the rate of apoptosis in many cell types [3], including endothelial cells [40–42], zona glomerulosa cells [43, 44], mesangial cells [45], and several carcinoma cell lines [15, 46]. These reports concerning cells in vitro may indicate possible direct effects of AM in regulating apoptosis in uteroplacental tissues. However, the possibility that apoptosis in placenta may be secondary to hypoxia [47] induced by possible reduced blood flow in AM22–52-infused rats cannot be excluded. Several mechanisms have been described in the pathophysiology of pregnancy complications, such as intrauterine growth retardation, gestational hypertension, and preeclampsia. We suggest that one such mechanism may be increased apoptosis caused by decreases in the levels or effects of AM during pregnancy.


   ACKNOWLEDGMENTS
 
We thank Drs. P.R.R. Gangula and Madhir Chankar for helpful discussions and Uma Yallampalli for technical assistance.


   FOOTNOTES
 
1 Supported by the NIH through grants HL-058144, HL-072650, and HD-40828. Back

2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics & Gynecology, 301 University Blvd., MRB, Rm. 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu Back

Received: 13 May 2004.

First decision: 28 May 2004.

Accepted: 18 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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