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BOR - Papers in Press, published online ahead of print August 10, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044628
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BIOLOGY OF REPRODUCTION 73, 1191–1198 (2005)
DOI: 10.1095/biolreprod.105.044628
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Effects of Parathyroid Hormone Like Hormone (PTHLH) Antagonist, PTHLH7–34, on Fetoplacental Development and Growth During Midgestation in Rats1

C.S. Thota , L.C. Reed , and C. Yallampalli 2 

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555-1062

ABSTRACT

Parathyroid hormone-like hormone (PTHLH) secretion has been reported in human amnion, chorion, decidual cytotrophoblast, syncytiotrophoblast, endometrium, and myometrium; however, the functions of PTHLH during pregnancy, particularly during placenta formation and fetal development, are not well understood. We examined whether neutralization of PTHLH action using PTHLH antagonist, PTHLH7–34, in rats during early gestation affects fetal and placental growth. Rats received s.c. a daily dose of either 0, 4, 12, or 36 µg of PTHLH7–34 infused continuously through mini-osmotic pumps from Day 8 through Day 15 of pregnancy. Fetal weights measured on Day 15 were significantly decreased in rats treated with all the doses of PTHLH7–34 compared to controls, and decreases in placental weights were significant at the 12-µg dose. TUNEL assay demonstrated an increased number of apoptotic cells in placenta of treated rats, including rats treated with the 4-µg dose. Cleaved caspase 3 (CASP3), caspase 9 (CASP9) (P < 0.05) and poly-ADP-ribose polymerase (PARP1) (P < 0.01) expression was increased and BCL2 (P < 0.01) expression was decreased in rats treated with 4 µg PTHLH7–34 compared to that in control. Placental cytochrome c expression was increased (P < 0.01) in cytosolic and decreased (P < 0.01) in mitochondrial fraction in PTHLH7–34-treated rats. Caspase 8 expression was not affected by the treatment. Immunohistochemical analysis of platelet endothelial cell adhesion molecule (PECAM1) showed higher staining intensity in control than in treated rats. In conclusion, these results suggests that PTHLH plays a role in early pregnancy, and that antagonization of PTHLH action causes fetoplacental growth restriction through activation of mitochondrial pathway of apoptosis in the placenta and through decreased expression of PECAM1.

apoptosis, early development, placenta, pregnancy

INTRODUCTION

Parathyroid hormone-like hormone (PTHLH), initially discovered in patients with humoral hypercalcemia of malignancy, more recently has been reported to express in most normal adult and fetal tissues, including uterine myometrium and endometrium [1, 2], ovary [3], placenta [4, 5], and mammary gland [6]. In the uterus, PTHLH expression is demonstrated by in situ hybridization in the circular and longitudinal layers of smooth muscle cells during late gestation, whereas it is greatly reduced or absent in the nongravid uterus [7]. Circulating concentrations of PTHLH have also been reported to be elevated during pregnancy [8, 9]. Spontaneous and phenyl epinephrine-stimulated myometrial contractility have been reported to be inhibited by PTHLH [10, 11], suggesting a role for PTHLH in uterine relaxation during pregnancy.

PTHLH mRNA expression was reported in antimesometrial luminal epithelial cells on Day 1.5 postcoitum [12], and in embryos during late morula and blastocyst stages [13]. PTHLH was also localized in luminal and glandular epithelium on Days 3, 4, and 5 and in decidualized stomal cells by Days 6 and 7 of pregnancy [14], suggesting a role for PTHLH in early embryonic development and implantation. In the placenta, PTHLH is expressed in syncytial trophoblasts [15], and is reported to stimulate Ca2+ transport across the basal membrane [16], indicating a role for PTHLH in the nutrient exchange between the mother and fetus during pregnancy. Decreased PTHLH expression observed in the placenta of spontaneously hypertensive rats is associated with fetal growth restriction [17]. Together, these findings provide evidence that PTHLH may be involved in fetoplacental growth during pregnancy.

Apoptosis of the placental tissue is involved in reduced growth and development of the fetus in many species, including humans [18] and rats [19]. Apoptosis occurs through either the extrinsic or the intrinsic pathways [20]. The extrinsic pathway involves activation of FAS, FASL, FADD, caspase 3 (CASP3), caspase 8 (CASP8), and caspase 10 (CARD10) [20], whereas the intrinsic pathway may involve decreases in BCL2 and release of cytochrome c from mitochondria, which, along with apoptotic protease activating factor 1 (APAF1) and caspase 9 (CASP9) [21] activates CASP3 [20, 22]. Adrenomedullin antagonist (ADM22–52), administered during midgestation in rats, increased apoptotic cell death through mitochondrial pathway [19]. Monoclonal antibodies to PTHLH1–34 were reported to increase apoptosis in chondrosomal cells [23], PTHLH1–34 has been reported to reverse tumor necrosis factor-alpha/interferon-gamma (TNF/IFNG) and staurosporine-induced apoptosis in trophoblast cells [24]. Therefore, we hypothesize that antagonism of PTHLH leads to decreased placental development and fetal growth retardation through increased apoptotic cell death in placenta.

Adhesion of trophoblast cells to arterial endothelium and cohesion between subpopulations of cytotrophoblast cells are important for normal functioning of the placenta [25]. Platelet endothelial cell adhesion molecule 1 (PECAM1), secreted by trophoblasts and localized on endothelium of vessels in placenta [26], has been implicated in the adhesive interaction between trophoblasts and vascular endothelium [27] and cohesion between subpopulations of cytotrophoblast cells [25]. Further, PTHLH secreted by trophoblast cells was reported to regulate transport of nutrients across the placenta. Therefore, PECAM1 secreted by trophoblast cells may mediate PTHLH regulation of nutrient exchange across maternal and fetal compartments. We hypothesize that PTHLH plays a role in fetoplacental development, and inhibition of action of endogenous PTHLH by PTHLH7–34 infusion, would decrease fetoplacental development by increasing apoptotic cell death and decreasing the expression of platelet cell adhesion molecules.

MATERIALS AND METHODS

Animals

Prepubertal female rats between 20 and 23 days of age were purchased from Harlan Sprague Dawley and were housed in a climate-controlled room with a 12L:12D schedule in the animal care facility. Animals were fed standard rat chow with water to drink ad libitum. All the procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX). Rats were superovulated on Day 26 with 4 IU eCG (Sigma Aldrich) administered s.c., and animals were mated 48 h later. The presence of sperm in the vaginal smear the following morning was taken as evidence of Day 1 of pregnancy. Pregnant females were maintained in the animal care facility until they were infused with PTHLH7–34 or saline.

Treatment

On Day 8 of pregnancy, rats were randomly allocated into 4 groups of 4 animals each. Mini-osmotic pumps (Model 2001: Durect Corp.) were inserted s.c. into the dorsum of pregnant rats that had been deeply anesthetized by an intraperitoneal injection of ketamine (45 mg/kg bw; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg bw). The mini-pumps were filled with saline alone or with PTHLH antagonist, PTHLH7–34 (Bachem Bioscience Inc.) in saline to continuously deliver 0, 4, 12, and 36 µg/day to each rat. All rats were killed on Gestational Day 15 using a CO2 inhalation chamber. Placentas and fetuses were carefully dissected out, their weights were recorded, and they were fixed in Bouins fluid for immunohistochemical analysis or snap-frozen in liquid nitrogen and stored at –80°C for further analysis.

TUNEL Assay

Placental tissues embedded in paraffin were cut into 5-µm-thick sections using a microtome. For TUNEL, slides with paraffin sections were immersed in xylene in a coupling jar to remove paraffin and were rehydrated in graded ethanol solutions. Fragmentation of DNA was detected using FragEL DNA Fragmentation Detection Kit (Calbiochem) according to the manufacturer's instructions with some modifications. After the tissue sections were deparaffinized and rehydrated, protein was digested with 20 µg/ml of proteinase K for 20 min at room temperature. Endogenous peroxidase activity was quenched with 5% H2O2 in methanol. After rinsing slides with 1x TBS, 1x deoxynucleotidyl transferase (DNTT) equilibration buffer was applied directly to the specimen. Terminal DNTT enzyme-diluted 1:100 in DNTT labeling reaction mixture was applied and incubated at 37°C for 1 h in a humidified chamber. The reaction was then stopped by incubating specimens for 5 min with stop buffer supplied with the kit followed by 10 min incubation in blocking buffer. Specimens in blocking buffer were then incubated with conjugate buffer for 30 min in a humidified chamber followed by colorization with diaminobenzidine (DAB)/H2O2 and counterstaining with hematoxylin. Negative controls were incubated with DNTT labeling reaction mixture without the DNTT 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 PTHLH7–34 treated (n = 4) groups are presented as the mean ± SEM.

Tissue Preparation and Subcellular Fractionation

Placentas collected from control and PTHLH7–34 treated rats were homogenized (PowerGen 1251; Fisher Scientific, Houston, TX) at 15,000 rpm for 10 sec three times in Tris EGTA buffer (50 mM Tris, 0.1 mM EGTA, 1 mM PMSF) containing 1.4 µl ß-mercaptoethanol and 1 tablet of protease inhibitor cocktail (Roche Applied Sciences) for every 10 ml of buffer. After centrifugation of the homogenate at 1000 x g 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 (10 mM Tris acetate, 0.5% nonidet P, 40 and 5 mM CaCl2; pH 8.0) buffer containing protease inhibitors. Protein concentration was determined using BCA kit (Pierce Biotechnology).

Western Blot Analysis

Equal amounts of protein (30 µg) were resolved on a 10% or 15% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane by electroblotting. Membranes blocked with 10% nonfat milk in 20 mM Tris saline buffer with 0.095% Tween 20 (TTBS) were incubated with cleaved CASP3 (Cat. No. 9961), cleaved CASP8 (Cat. No. SC-7890), cleaved CASP9 (Cat. No. 9506), poly (ADP-ribose) polymerase (PARP1; Cat. No. SC-1562), BCL2 (Cat # SC-492), cytochrome c (Cat. No. SC-13156), BCL2-associated x protein (BAX) (Cat. No. SC-426), and BCL2-antagonist/killer 1 (BAK1) (Cat. No. SC-832) antibodies, diluted 1:750; 1:250, 1:500, 1:250, 1:500, 1:1000, 1:500, and 1:500, respectively. Antibodies to CASP3 and CASP9 were obtained from Cell Signaling Technology, and all other antibodies were from Santa Cruz Biotechnology. These membranes were further incubated with either anti-rabbit, anti-goat or anti-mouse IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology), depending on the source of primary antibody. Blots were washed in 1x TTBS and signals were captured on a Hyperfilm ECL after incubating in enhanced chemiluminescent reagents for 1.0 min. The intensity of specific immunoreactive bands was quantified by a densitometric scanning program (Sigma Gel Software; Sigma). Membranes with lysate and cytosol proteins were probed with ß-tubulin (TUBB-RS; 1:500) antibody, and membranes with mitochondrial fractions were probed with heat shock protein (HSP)-60 (HSPD1; 1:2000) antibody. All replicates from a group were run in one gel, and proteins are expressed as a ratio of protein signal to respective TUBB-RS/HSPD1 signals.

Immunohistochemistry

Placental tissue sections were deparaffinized in xylene and rehydrated in graded ethanol solutions. Antigen retrieval was performed by incubating tissue sections for 10 min in boiling citrate buffer (10 mM) adjusted to pH 6.0 at 25°C. Endogenous peroxidase activity was blocked with 3% H2O2. After blocking with avidin and biotin solutions, sections were incubated with 1:200 diluted PECAM1 goat polyclonal antibody (Santa Cruz Biotechnology) for 18 h at 4°C. Following primary antibody incubations, sections were rinsed in wash buffer and incubated with biotinylated rabbit anti-goat IgG (Santa Cruz Biotechnology) and avidin-biotin peroxidase solution (Vectastain ABC Elite Kit; Vector Laboratories). Color was developed by incubating sections for 3 min in DAB (Vector Laboratories) with 0.003% H2O2 in PBS. Specimens were lightly counterstained with hematoxylin and examined by light microscopy. Negative controls were obtained using normal goat serum in place of primary antibody with the other steps unchanged.

Statistical Analysis

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

RESULTS

Effects of PTHLH7–34 on Fetoplacental Weights

In the present study, groups of rats were s.c. infused with PTHLH7–34 using mini-osmotic pumps to continuously deliver 0, 4, 12, or 36 µg of PTHLH7–34 per day between Day 8 and Day 15 of pregnancy to study the effects of PTHLH7–34 on fetoplacental growth. Fetuses and placentas collected on Day 15 showed a significant decrease in their weights in treated rats. Fetal weights decreased significantly (P < 0.05) with all the doses used. However, the decreases observed in fetal weights were not dose-dependent (Fig. 1). Placental weights decreased (P < 0.05) with the 12-µg dose but not with the 4- or 36-µg dose of PTHLH7–34 (Fig. 1). No significant differences were observed either in the number of fetuses or in fetal resorptions in the PTHLH antagonist-treated rats (data not shown). Pilot studies showed an increase in apoptotic markers in all the doses of PTHLH7–34 used. Further studies on apoptotic pathway were carried out using placenta samples from rats treated with 4 µg PTHLH7–34.


Figure 1
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  		FIG. 1. Effect of PTHLH antagonist, PTHLH7–34, on fetoplacental weights during pregnancy. Rats were continuously infused from Day 8 to Day 15 of pregnancy with 0 (control), 4, 12, or 36 µg/day of PTHLH7–34, and fetuses and placentas were collected on Day 15. Bars represent the mean ± SEM of weight of fetus (A) and placenta (B) from 4–8 rats. Groups with asterisks are significantly (*P < 0.05 and **P < 0.01) different from controls

Apoptotic Changes in Placental Tissues with PTHLH7–34 Infusion in Pregnant Rats

We examined the possible involvement of apoptosis in the decrease of fetoplacental weights in rats treated with PTHLH7–34. Apoptosis is demonstrated within the placental tissue by the TUNEL method (Fig. 2). TUNEL-positive reaction was detectable in trophoblast cells in placenta. The number of cells with TUNEL-positive staining increased (P < 0.01) in PTHLH7–34 treated animals compared with the number in untreated control rats. In placenta, the number of TUNEL-positive cells per section was 13.5 ± 1.93 in PTHLH7–34-treated compared to 3.0 ± 0.7 in control animals. Specificity of staining was demonstrated by the absence of staining when DNTT enzyme was eliminated.


Figure 2
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  		FIG. 2. TUNEL staining in placenta on Day 15 of gestation in rats treated continuously with 0 or 4 µg/day of PTHLH7–34, a PTHLH antagonist, from Day 8 to Day 15 of gestation. Brown staining of nuclei indicates a positive reaction; this was absent when DNTT enzyme was omitted (E and F). TUNEL positive cells (arrows) were more abundant in the labyrinth zone (C and D) of placenta of PTHLH7–34 treated rats compared to that of control rats (A and B). Original magnification A, C, and E x100; B, D, and F x200

Effects of PTHLH7–34 on Expression of the BCL2 Family of Proteins in Placenta

To study the pathways involved in PTHLH7–34-induced apoptosis in placenta, we examined the changes in antiapoptotic and proapoptotic proteins of the BCL2 family in both PTHLH7–34-treated and untreated control rat placentas. A significant (P < 0.01) decrease in the expression of the antiapoptotic BCL2 was observed in the PTHLH7–34 treated group when compared with untreated controls (Fig. 3). BAK1, a proapoptotic protein, increased significantly (P < 0.05) in rats treated with PTHLH7–34 compared to the control. However, no significant changes were observed in BAX with PTHLH7–34 treatment (Fig. 3). BH3 interacting domain death agonist (BID) was undetectable in both the control and treated group (data not shown).


Figure 3
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  		FIG. 3. Expression of BCL2 family of proteins in placenta from rats on Day 15 of gestation following treatment with 4 µg/day of PTHLH7–34, a PTHLH antagonist, from Day 8 to Day 15. A) Western blot analysis of placental homogenates for BCL2, BAK1, TUBB-RS and placental cytosol for BAX in PTHLH7–34 treated and control rats. B) Summary of densitometric analysis normalized to TUBB-RS. Data are presented as the mean ± SEM from four replicate animals in each group. Groups with asterisks are significantly (*P < 0.05 and **P < 0.01) different from controls

Release of Cytochrome c into Cytosol

Intrinsic or mitochondrial pathway of apoptosis is said to be initiated with the release of cytochrome c from mitochondria into cytosol. We measured the levels of cytochrome c in both cytosol and mitochondrial fractions of control and PTHLH 7–34-treated rat placentas to assess the involvement of intrinsic pathway in the apoptotic process. Western blot analysis using a specific antibody to cytochrome c demonstrated a significant (P < 0.01) increase in cytosol and a significant (P < 0.01) decrease in mitochondrial fractions of the placenta in PTHLH7–34-treated compared to control rats (Fig. 4).


Figure 4
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  		FIG. 4. Effect of PTHLH7–34 treatment on cytochrome c content in mitochondrial and cytosolic fractions of placenta. Rats were treated with PTHLH7–34, a PTHLH antagonist, continuously from Day 8 to Day 15 of gestation and cytochrome c measured in cytosolic and mitochondrial fractions of placenta on day 15. A) Western blot analysis of cytochrome c from both the cytosol and mitochondrial fractions in PTHLH7–34 treated and control rats. B) Summary of densitometric analysis normalized to TUBB-RS for cytosolic and HSPD1 for mitochondrial fractions. Data are presented as the mean ± SEM from four replicate animals in each group. Groups with asterisks are significantly (**P < 0.01) different from controls

Activation of Caspases in Rats Treated with PTHLH7–34

Caspases are considered to be executioners of cell death. Therefore, we examined the expression of caspases and the downstream target, PARP1, in the placenta of rats treated with PTHLH7–34. Cleaved CASP3, executioner of cell death, showed a significant (P < 0.05) increase in rats treated with PTHLH7–34 compared to its expression in control rats (Fig. 5). Increases (P < 0.01 ) observed in PARP1 in PTHLH7–34-treated rats further confirmed the CASP3 involvement in this process (Fig. 5). Cytochrome c activates CASP9, one of the major initiator caspases. In the present study, PTHLH7–34 treatment caused a significant (P < 0.05 ) increase in cleaved CASP9 levels compared to the control group (Fig. 5).


Figure 5
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  		FIG. 5. Expression of CASP3, CASP9, and PARP1 proteins in placenta of rats. Rats were treated continuously with PTHLH7–34, a PTHLH antagonist, from Day 8 to Day 15 of gestation, and apoptotic markers were measured in the Day 15 placental homogenates. A) Western blot analysis of cleaved CASP3, CASP9, PARP1, and TUBB-RS in PTHLH7–34-treated and control rats. B) Summary of densitometric analysis normalized to TUBB-RS. Data are presented as the mean ± SEM from four replicate animals in each group. Groups with asterisks are significantly (*P < 0.05 and **P < 0.01) different from controls

Involvement of CASP8 and CARD10

CASP8 and CARD10 are activated downstream of the FAS/FASL pathways via what is called the extrinsic pathway. We examined the involvement of this pathway of apoptosis in placenta of rats treated with PTHLH7–34. Western analysis of CASP8 in placental homogenate from rats treated with PTHLH7–34 did not show any significant difference compared to the levels observed in control samples (Fig. 6). CARD10 was below the detection limit in both control and treated samples (data not shown).


Figure 6
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  		FIG. 6. Expression of CASP8 in placenta of pregnant rats. Rats were treated continuously with PTHLH7–34, a PTHLH antagonist, from Day 8 to Day 15 of gestation and CASP8 was measured in placenta on Day 15. A) Western blot analysis of placental homogenates for caspase-8 and TUBB-RS in PTHLH7–34 treated and untreated control rats. B) Summary of densitometric analysis normalized to TUBB-RS. Data are presented as the mean ± SEM from four replicate animals in each group

Expression of Platelet Endothelial Cell Adhesion Molecule

Immunohistochemical staining of PECAM1 was conducted to assess the cellular integrity and function in placental sections obtained from rats treated with PTHLH7–34. PECAM1 expression was abundant all along the endothelial lining in the labyrinth zone in the control placenta. However, the expression of PECAM1 is substantially lower in the placenta obtained from rats treated with PTHLH7–34 (Fig. 7). Specificity of the staining was confirmed by the absence of staining when primary antibody was omitted in the reaction.


Figure 7
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  		FIG. 7. Immunohistochemical staining of platelet endothelial cell adhesion molecule (PECAM1) in sections of placenta. Rats were treated with 0 or 4 µg/day of PTHLH7–34 between Days 8 and 15 of pregnancy. Placentas were collected on Day 15 and processed for immunohistochemical staining. PECAM1 immunoreactivity was seen in endothelial cell lining of fetal vessels. Brown staining indicates a positive reaction; this was absent when primary antibody was omitted (G). Staining was observed in the labyrinth zone in the control (A) and treated rats (D). Control placenta shows intensive brown staining (arrows) on endothelial cells (B and C) whereas staining is lower in the PTHLH7–34 treated placenta (E and F). Tissues were counterstained with hematoxylin. Original magnification A and D x40; B, E, and G x200; C and F x600

DISCUSSION

The present study demonstrated that PTHLH plays a role in fetal growth and placental development during midgestation. Although infusion of PTHLH7–34, an antagonist to PTHLH, did not abolish placental formation, it reduced early placental development and restricted fetal growth. The weight of the placenta and fetus decreased significantly on Day 15 in rats infused continuously with PTHLH antagonist from Day 8 through Day 15 of pregnancy. However, no significant changes were observed in the number of resorption sites. TUNEL assay conducted to see the possible involvement of apoptosis during the placental growth in rats treated with PTHLH7–34 showed an increase in apoptotic cells in the treated rat placentas. Apoptotic markers CASP9, CASP3, and PARP1 were increased in treated placental tissues, confirming the increased apoptotic activity. Further studies to identify the initiators of apoptotic process revealed an increase in cytochrome c in cytosol with a corresponding decrease in mitochondrial fractions. The levels of BCL2, an antiapoptotic marker, in placentas were decreased in rats treated with PTHLH antagonist. However, the levels of proapoptotic protein, BAK1, were elevated in antagonist-treated rat placenta, whereas BAX levels were unchanged compared to controls. Western analysis did not show any differences in the placental CASP8 expression between the control and treated groups. CARD10 and BID levels were below the detection limits. Immunohistochemical analysis of the placental sections revealed decreases in PECAM1 in treated animals, suggesting a decreased endothelial cell function.

In this study, we examined the role of PTHLH in fetoplacental development during midgestation by neutralizing the actions of endogenous peptide using continuous infusion of PTHLH7–34, an antagonist to PTHLH. Pregnant rats treated with 4, 12, and 36 µg of PTHLH antagonist between Days 8 and 15 of pregnancy in the present study showed a significant decrease in the fetoplacental weights. Fetal weights were lower with all the doses used, whereas decreases in placental weights compared to controls occurred with 12-µg but not with 4- or 36-µg doses of PTHLH7–34. In the present study, even though the placental weights were unchanged with 4- and 36-µg doses, decreases were observed in the fetal weights at these doses, suggesting an altered function of placenta in rats with all the doses used. Pups from PTHLH knockout mice die immediately after delivery [28] because of abnormalities in various fetal organs [29, 30], suggesting involvement of PTHLH in the fetoplacental development. In PTHLH knockout mice, chondrocytes differentiate early; as a result, the extracellular matrix is mineralized prematurely and undergoes early apoptosis [31]. By contrast, in mice that overexpress PTHLH, chondrocyte maturation is severely delayed and apoptosis is suspended [32], suggesting that PTHLH is essential for normal fetal growth. PTHLH was also reported to stimulate Ca2+ release from human syncytiotrophoblast basal membranes via a common receptor [16]. Therefore, neutralization of PTHLH action in this study might have led to altered placental function, resulting in reduced fetal growth.

The reduction in the placental and fetal weight in rats treated with PTHLH7–34 in the present study could be related to apoptotic cell death in placenta. Our data on the apoptotic changes further confirmed the changes in placenta of rats treated with 4 µg, at which the levels of caspases are increased in treated rats compared to controls. TUNEL staining indicated increased frequency of apoptotic cell death in placentas of rats treated with PTHLH7–34. Apoptotic changes were observed in trophoblast cells in the junctional and labyrinth layers of the placenta, with occasional apoptosis of giant trophoblast cells, and these were abundant in treated compared to control animals. PTHLH1–34 was reported to evoke a dose-dependent rescue of both TNF/IFNGRL and staurosporine-induced apoptosis in trophoblast cells [24] suggesting its involvement in placental growth. Thus, the increased apoptotic effects of PTHLH7–34 in the labyrinth zone of the placenta, an important region for fetomaternal exchange, could lead to observed reduction in placental function as well as in fetal growth.

Studies were conducted to measure the changes in the markers of both intrinsic and extrinsic pathways of apoptosis in placenta of rats treated with PTHLH7–34. Proapoptotic markers CASP3, CASP9, and PARP1 were increased in the placenta of rats treated with PTHLH7–34. Cytochrome c levels were increased in cytosol and decreased in mitochondrial fraction. The changes observed in cytochrome c in cytosol and mitochondria in the present study were supported by others [20, 22, 33, 34], confirming that release of cytochrome c from mitochondria initiates the apoptotic process. The cytochrome c in cytosol binds to APAF1, enabling recruitment of pro-CASP9 with its subsequent processing to initiator CASP9. In the present study, PTHLH7–34 treatment decreased BCL2 expression and increased BAK1 expression, whereas BAX expression was unaltered. BID was undetectable in the placentas of both control and treated rats. Members of the BCL2 family of proteins are central regulators of the apoptotic pathway. Certain family members (BCL2) act as potent suppressors of the apoptotic process, whereas other members (BAX and BAK1) have an opposing function and promote cell death. The ratio of antiapoptotic to proapoptotic molecules of BCL2 determines the response to a death signal [21]. BCL2 inhibits apoptosis, whereas an increase in BAX or BAK1 initiates apoptosis [35]. An increase in the ratio of BCL2 to BAX or BAK1 may inhibit apoptosis, whereas an increase in the ratio of BAX or BAK1 to BCL2 can lead to increased apoptosis. TNF decreases expression of BCL2 in deciduae in failing first trimester pregnancy [36], whereas monoclonal antibody to PTHLH1–34 was reported to induce apoptosis via an imbalance of BCL2/BAX ratio in chondrosarcoma cells [23]. Decreases observed in BCL2 to BAK1 ratio in placenta of rats treated with PTHLH7–34 in the present study indicate a possible involvement of intrinsic pathway in triggering apoptosis. Decrease in BCL2 causes release of APAF1, which, along with cytochrome c and pro-CASP9, forms apoptosome [37]. The apoptosome then recruits pro-CASP3, which is cleaved and activated [22]. Cytochrome c binds to APAF1 and drives cell death through activation of CASP3. Active CASP3 cleaves PARP1 [20, 38], setting off the cascade of events leading to cell death. In placenta of rats treated with PTHLH7–34, CASP3, CASP9, and PARP1 levels are elevated. These results suggest that PTHLH7–34 treatment induced increases in mitochondrial pathway-related caspase cascade and subsequent apoptosis in placenta.

CASP8 and CARD10 are major initiator caspases in death receptor-mediated apoptosis [20, 39, 40]. These are reported downstream of the FAS-dependent extrinsic pathway of apoptosis [20, 39, 40]. In the present study, we examined the changes in CASP8 and CARD10 proteins in placenta of rats treated with PTHLH7–34 to assess the involvement of death receptor-mediated pathway. CASP8 expression was seen in both control and PTHLH7–34 treated rat placentas. However, no significant differences were observed between treated and control placentas. CARD10 was undetectable in the placenta samples obtained from both control and PTHLH7–34-treated rats, suggesting that the extrinsic pathway is not involved in the apoptotic process. In the present study, PTHLH7–34 infusion appears to increase apoptosis by decreasing the expression of antiapoptotic marker BCL2 expression and increasing the proapoptotic marker BAK1 with changes in cytochrome c, active CASP3, CASP9 and cleaved PARP1.

Immunohistochemical analysis of PECAM1 was performed on placental sections of both control and PTHLH7–34-treated rats to assess the cellular integrity. Abundant PECAM1 expression was observed in fetal endothelial cells of control rats; these results are consistent with the PECAM1 localization to fetal capillary endothelium of human placenta [27]. Placental sections from rats treated with PTHLH7–34 showed little or no expression of PECAM1 compared to controls. PECAM1 reportedly increases adhesive interaction between trophoblast and maternal vascular endothelium [27] and also between some subpopulations of cytotrophoblast cells [25]. Therefore, the decreased expression of PECAM1 observed in rats treated with PTHLH7–34 in this study may reduce the interactions between trophoblast and endothelial cells, and may therefore alter placental integrity and transport of nutrients.

In summary, we have demonstrated that the administration of PTHLH antagonist, PTHLH7–34, during midgestation causes fetal growth restriction in rats. We also demonstrated an increase in apoptotic process, particularly through the mitochondrial pathway in the placenta of PTHLH-antagonist-treated rats. In addition, decreases in the expression of PECAM1 in placenta of treated rats may mediate PTHLH-antagonist-induced fetal growth restriction. These studies suggest that PTHLH may play an important role in early placental function and fetal growth during midgestation in rats.

ACKNOWLEDGMENTS

We thank Mrs. Cheryl Welch for administrative support.

FOOTNOTES

1 Supported by NIH grants HL-58144, HL-72650, and HD-40828. Back

2 Correspondence: C. Yallampalli, Dept. of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., MRB, 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu Back

Received: 24 June 2005.

First decision: 7 July 2005.

Accepted: 8 August 2005.

REFERENCES

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