Objective
Our objective was to determine signaling molecules and apoptosis rate in the term placenta of a baboon model of maternal nutrient reduction (MNR).
Study Design
Female baboons were fed ad libitum for controls (n = 7) or 70% of control baboon diet (MNR; n = 6) from 30-165 days of gestation with necropsy at 165 days of gestation. Placental tissues were collected and fixed for immunohistochemistry or snap frozen to measure extracellular signal-regulated kinases, protein kinase B, JUN NH 2 -terminal kinase, X-linked inhibitor of apoptosis protein, and caspase 3. Placental villous apoptosis was determined by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling and cytokeratin 18 cleavage.
Results
Compared with the control placentas, MNR placentas demonstrated reduced placental weight ( P < .02), decreased phospho (p)-ERK ( P < .04), increased placental villous apoptosis ( P < .001), increased villous cytokeratin 18 cleavage, increased X-linked inhibitor of apoptosis protein ( P < .007), and increased active caspase 3 ( P < .02).
Conclusion
We conclude that placental apoptosis is increased in this baboon model of MNR at term and that the increase in X-linked inhibitor of apoptosis protein may be a protective mechanism against this apoptosis.
Placental development is important for fetal growth and development. Abnormal placentation is associated with the development of complicated pregnancies in humans. Poor maternal nutrition is a common problem in the developing world, which can have many adverse effects on the fetus, including intrauterine growth restriction (IUGR). A higher degree of syncytiotrophoblast apoptosis is found in placentas from pregnancies that are complicated by fetal growth restriction and preeclampsia. Apoptosis, an active process of cellular destruction, serves an essential remodeling function in multicellular organisms and is a component of normal development and differentiation in most tissues, including the placenta. Abnormal regulation of apoptosis in different tissues has been implicated in the onset and progression of a broad range of diseases.
The X-linked inhibitor of apoptosis protein (XIAP) is the most potent member of a group of inhibitor of apoptosis proteins that regulate cell death. Inhibitor of apoptosis proteins possess a defining structural motif, called Baculovirus Inhibitor of Apoptosis Repeat (BIR), which is responsible for the inhibition of caspases 3, 7, and 9. Caspases are a family of cysteine proteases that play a central role in initiating and executing the apoptosis cascade. In trophoblast cells, caspases are known to cleave the cytokeratin 18 protein, which produces a neopeptide that is used as a marker of apoptosis. Controlling the activity of caspases is essential for the appropriate execution of cell death and the regulation of cell survival. XIAP is present in trophoblast throughout placental development, but expression is decreased significantly near delivery when apoptosis is maximal. A decrease in XIAP protein is associated with the presence of increased apoptosis during IUGR in pregnant sheep. This suggests a role for XIAP in the regulation of trophoblast apoptosis in normal and abnormal processes in pregnancy.
Signal transduction cascades serve to elicit a cellular response that is initiated at the cell surface. These cascades contain multiple components that involve a series of phosphorylation steps. The extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein kinase B (AKT) pathways are found in many cell types and play various roles. These proteins mediate biologic responses like cellular growth, proliferation, and cell survival. The c-Jun amino terminal kinase (JNK) is another signaling molecule that is activated to diverse stimuli that include DNA damage, ultraviolet radiation, heat shock, and others. There is a reduction of these signaling proteins in other models of IUGR.
Although there are many studies on the effects of controlled reductions in maternal nutrient intake on apoptosis in rodents and sheep, data are lacking in nonhuman primates. In the present study, we evaluated placental apoptosis and changes in key signaling molecules in the placentas of control pregnant baboons that were fed ad libitum and mothers that were exposed to nutrient reduction (MNR) and fed 70% of the feed consumed by the controls. Previous studies on the placentas showed that MNR decreased placental size near-term when villous volume and surface area, capillary surface area, and the villous isomorphic coefficient were all decreased and intervillous space hydraulic diameter was increased.
The pregnant baboon presents several advantages for the study of effects of maternal nutrition on placental and fetal growth, compared with other nonhuman primates, including fetal size and similarities in placentation and placental structure with those of human pregnancy. We hypothesized that MNR would increase placental villous apoptosis and decrease abundance of XIAP at the end of gestation. Because this model is associated with a decrease in placental size, we further hypothesized an increase in cytokeratin 18 cleavage and active caspase 3 in placentas from MNR mothers. We also sought to determine changes in cell signaling proteins that are associated with cell proliferation (ERK), cell survival (AKT), and cell stress (JNK) in the placenta of controls and MNR animals.
Materials and Methods
Animal care
All procedures were approved by the Southwest Foundation for Biomedical Research Institutional Animal Care and Use Committee and conducted in American Association for Accreditation of Laboratory Animal Care–approved facilities. Animal care was carried out as described by Schlabritz-Loutsevitch et al Briefly, the subjects of this study were 13 female baboons aged 8-15 years that were maintained in group housing and bred as previously described in detail. Baboons were observed twice a day for well-being and 3 times a week for turgescence (sex skin swelling) and signs of vaginal bleeding to enable timing of pregnancy. Pregnancy was dated initially by observing the changes in the swelling of the sex skin and confirmed at 30 days of gestation by ultrasonography. Animals (Papio sp) were fed Purina Monkey Diet 5038 (Purina, St. Louis, MO). Diet was closely monitored in 7 animals that were fed ad libitum (control group) and their food intake was calculated weekly on a per kilogram basis. From confirmation of pregnancy, the 6 pregnant baboons in the MNR group received 70% of the average daily amount of feed eaten (on a weight-adjusted basis) by control animals at the same gestational age. Cesarean section deliveries were performed at 165.9 ± 1.1 days of gestation in control and at 162.1 ± 4.6 days of gestation in MNR animals. Placental tissues were obtained from a central area of a cotyledon, fixed in formalin (10% buffered formalin), and embedded in paraffin for immunohistochemistry or snap frozen in liquid nitrogen for protein studies.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
Placental sections were used for these experiments. TUNEL protocol was followed, as suggested by the manufacturer (Chemicon, Inc, Temecula, CA). Briefly, slides were dewaxed and postfixed with a solution of ethanol:acetic acid (2:1) for 5 minutes. The equilibration buffer was added to the slide for 10 seconds, followed by incubation with the terminal deoxynucleotidyl transferase enzyme for 1 hour at 37°C. After this, the anti-digoxigenin conjugate was incubated on the slide for 30 minutes and 4′,6-diamidino-2-phenylindole,dihydrochloride was used for nuclear staining. Slides were viewed with fluorescein excitation and emission filters. Analysis was performed in 2 placental slides per animal (3 controls; 3 MNR, selected randomly), and 20-30 fields were counted per slide with a mean count that was generated per slide for analysis purposes. The percent apoptosis was calculated in the placental slides (12 slides total) as the number of TUNEL-positive cells divided by the total number of cells in 20-30 fields × 100.
Western blot analysis
Western blot was performed as previously described by Arroyo et al. Briefly, frozen placental tissues were homogenized, and protein tissue lysates (50 μg) were separated on 4-12% Bis-Tris gel sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated with an antibody against mouse XIAP (at a dilution of 1:200; Transduction Laboratories, Lexington, KY) or antibodies against rabbit active (cleaved) caspase 3, caspase 3, phospho-AKT, total AKT, phospho-ERK or total ERK, and phospho-JNK or total JNK (all at 1:500; Cell Signaling Technology, Danvers, MA). A secondary anti-mouse or anti-rabbit immunoglobulin horseradish peroxidase antibody (dilution 1:10,000; Upstate Cell Signaling Solutions, Lake Placid, NY) was incubated for 1 hour at room temperature. The membranes were incubated with enhanced chemiluminescense substrate (Amersham, Princeton, NJ) for 5 minutes, and the emission of light was detected with x-ray film. To determine loading consistencies, each membrane was stripped of antibodies and reprobed with an antibody against mouse beta-actin at a dilution of 1:4000 (MP Biomedicals, Solon, OH). The presence of these proteins was confirmed and quantified.
M30 cytodeath immunohistochemistry
Immunohistochemistry was performed on paraffin-embedded placental sections. Slides were dewaxed with 100% xylene. Slides were washed in phosphate-buffered saline solution (PBS), and sections were blocked for 1 hour with 10% normal goat serum/PBS. Slides were then incubated for 1 hour with a mouse monoclonal primary antibody against M30 cytodeath (the neopeptide produced from cytokeratin 18 cleavage; dilution 1:200; Roche Diagnostics Corporation, Indianapolis, IN) or a mouse immunoglobulin G1 (dilution of 1:500) for negative control. Sections were washed in 1 × PBS. Sections were then incubated for 45 minutes with a biotin-labeled anti-mouse secondary antibody. Slides were washed in 1 × PBS, incubated in streptavidin-biotin-horseradish peroxidase solution, and developed with diaminobenzidine Vectastain ABC DAB kit (Vector Laboratories, Inc, Burlingame, CA). Diaminobenzidine (brown) was used to stain for the M30 positive cells in serial placental sections. Hematoxylin was used for nuclear counterstaining. Slides were mounted with Permount mounting media (Fisher Scientific, Pittsburgh, PA).
Statistical analysis
Comparisons were made between control and MNR groups with a rank sum test (Mann-Whitney U test) for the following elements: fetal and placental weights and XIAP, caspase 3, ERK, and AKT Western blot analysis. For TUNEL assay, a comparison was made of mean number of apoptotic cell counts per slide between both groups (n = 5); slides for each group were checked for normality and Student t test was performed. Data are shown as mean ± SE; a probability value of < .05 was considered significant for the statistical comparisons.
Results
MNR baboons showed a significant decrease in placental weight (177 g ± 8 g vs 145 g ± 7 g; P < .02) and a mean fetal weight decrease of 7% (744 g ± 32 g vs 669 g ± 33 g; P = .1) at 165 days of gestation ( Figure 1 ). In our study, there were no pregnancy miscarriages or fetal deaths, fetal growth restriction, or fetal abnormalities.
