Baboon Placenta Research Articles

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

Baboon placental heparin-binding epidermal growth factor-like growth factor.

Placental extravillous trophoblast remodeling of the uterine spiral arteries is important for promoting blood flow to the placenta and fetal development. Heparin-binding EGF-like growth factor (HB-EGF), an EGF family member, stimulates differentiation and invasive capacity of extravillous trophoblasts in vitro. Trophoblast expression and maternal levels of HB-EGF are reduced at term in women with preeclampsia, but it is uncertain whether HB-EGF is downregulated earlier when it may contribute to placental insufficiency. A nonhuman primate model has been established in which trophoblast remodeling of the uterine spiral arteries is suppressed by shifting the rise in estrogen from the second to the first trimester of baboon pregnancy. In the present study, we used this model to determine if placental HB-EGF is altered by prematurely elevating estrogen early in baboon gestation. Uterine spiral artery remodeling and placental expression of HB-EGF and other EGF family members were assessed on day 60 of gestation in baboons treated with estradiol (E2) daily between days 25 and 59 of gestation (term = 184 days). The percentages of spiral artery remodeling were 90, 84 and 70% lower (P < 0.01), respectively, for vessels of 26-50, 51-100 and >100 µm diameter in E2-treated compared with untreated baboons. HB-EGF protein quantified by immunocytochemical staining/image analysis was decreased three-fold (P < 0.01) in the placenta of E2-treated versus untreated baboons, while amphiregulin (AREG) and EGF expression was unaltered. Therefore, we propose that HB-EGF modulates the estrogen-sensitive remodeling of the uterine spiral arteries by the extravillous trophoblast in early baboon pregnancy.

Down-Regulation of Placental Transport of Amino Acids Precedes the Development of Intrauterine Growth Restriction in Maternal Nutrient Restricted Baboons

Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P < 0.05) in MNR fetuses compared to controls. In this primate model of IUGR, placental MVM system A amino acid transporter activity is decreased prior to the onset of reduction in the fetal growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced placental amino acid transport may be a cause rather than a consequence of IUGR due to inadequate maternal nutrition.

Reduced placental amino acid transport in response to maternal nutrient restriction in the baboon.

Intrauterine growth restriction increases the risk of perinatal complications and predisposes the infant to diabetes and cardiovascular disease in later life. Mechanisms by which maternal nutrient restriction (MNR) reduces fetal growth are poorly understood. We hypothesized that MNR decreases placental amino acid (AA) transporter activity, leading to reduced transplacental transfer of AAs. Pregnant baboons were fed either a control (ad libitum, n = 7), or MNR diet (70% of control diet, n = 7) from gestational day (GD) 30. At GD 165 (0.9 gestation), placentas (n = 7 in each group) were collected, and microvillous plasma membrane vesicles (MVM) isolated. MVM system A and system L AA transport was determined in vitro using radiolabeled substrates and rapid filtration techniques. In vivo transplacental AA transport was assessed by infusing nine (13)C- or (2)H-labeled essential AA as a bolus into the maternal circulation (n = 5 control, n = 4 MNR) at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each essential AA was calculated. Fetal and placental weights were significantly reduced in the MNR group compared with controls (P < 0.01). The activity of system A and system L was markedly reduced by 73 and 84%, respectively, in MVM isolated from baboon placentas at GD 165 following MNR (P < 0.01). In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly reduced for leucine, isoleucine, methionine, phenylalanine, threonine, and tryptophan in MNR baboons (P < 0.05). This is the first study to investigate placental AA transport in a nonhuman primate model of MNR. We demonstrate that the downregulation of system A and system L activity in syncytiotrophoblast MVM in MNR leads to decreased transplacental AA transport and, consequently, reduced circulating fetal AA concentrations, a potential mechanism linking maternal undernutrition to reduced fetal growth.

Parasite accumulation in placenta of non-immune baboons during Plasmodium knowlesi infection.

BackgroundPlacental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease.MethodsTen pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by Plasmodium knowlesi blood-stage parasites through the femoral vein on 160th day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn’s Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman’s rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant.ResultsLevels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score (rs = 0.7650, P <0.05) and inflammatory score (rs = 0.8590, P <0.05). Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons.ConclusionThis study has demonstrated accumulation of parasitized red blood cells and infiltration of inflammatory cells in the placental intravillous space (IVS) of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy.

Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.

Metabolism of 17α-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons

Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon ( Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16′ and M17′) that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

Regulation of placental villous angiopoietin‐1 and ‐2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.

The kinetics for glyburide metabolism by human and baboon livers and placentas

Glyburide is under investigation for treatment of gestational diabetes. The previously identified metabolites of glyburide were 4-trans (M1) and 3-cis (M2) hydroxycyclohexyl glyburide. Recently, we identified four additional metabolites formed by human and baboon liver and placental microsomes. Baboon is a primate that can be used when investigations in human are impossible. The aim of this work was to determine the apparent Km of glyburide and the rates for formation of each metabolite (Vmax) by microsomal preparations from human and baboon placentas and livers.

Identification of glyburide metabolites formed by hepatic and placental microsomes of humans and baboons

Glyburide (glibenclamide) is a second-generation sulfonylurea used for treatment of type-2 and gestational diabetes mellitus. To date, two glyburide metabolites have been identified in maternal urine: namely, 4- trans-hydroxycyclohexyl glyburide and 3- cis-hydroxycyclohexyl glyburide. The use of glyburide to treat gestational diabetes prompted us to investigate its metabolism by the placenta. The metabolism of glyburide by microsomal preparations from human and baboon placenta was compared with metabolism by their livers. The metabolites formed by the microsomes of the four tissues were identified by high-performance liquid chromatography–mass spectrometry using retention times, ion current (extracted at m/ z 510), and selected-ion monitoring. The data obtained revealed the formation of six distinct hydroxylated derivatives of glyburide by each of the four microsomal preparations. However, the amounts of the six metabolites formed by the placentas were a fraction of that formed by the livers. Moreover, the relative quantities of each metabolite formed differed between species as well as between the two tissues. Also, the structure of the unidentified metabolites was determined by comparison with synthesized standards. These metabolites were identified as the 4- cis-hydroxycyclohexyl glyburide, 3- trans-hydroxycyclohexyl glyburide, and 2- trans-hydroxycyclohexyl glyburide. Therefore, one glyburide metabolite remains to be identified, but the data we obtained allowed us to suggest its structure.

Developmental Regulation of the Sodium/Hydrogen Ion Exchangers and Their Regulatory Factors in Baboon Placental Syncytiotrophoblast

Although Na+/H+ exchange is important to maintenance of pH and volume and thus placental-fetal homeostasis, regulation of the Na+/H+ exchange system is incompletely understood. We previously showed that Na+/H+ exchanger (NHE)1 and -3 and their regulatory factors NHERF1 and -2 were expressed in human and nonhuman primate placenta. Our laboratories have also shown that estrogen regulates key aspects of primate placental function and development including the 11beta-hydroxysteroid dehydrogenase enzymes controlling cortisol metabolism. Therefore, it is possible that localization and/or expression of components of the syncytiotrophoblast NHE system are also estrogen dependent. As a first step in testing this possibility, the current study compared the immunocytochemical localization and level of NHE1, NHE3, NHERF1, and NHERF2 in baboon placentas obtained at mid- (d 100) and late gestation (d 165; term = d 184). NHE3 and NHERF2 were abundantly expressed at midgestation and localized to the cytoplasm and juxtanuclear compartment but were not detected in the microvillus membrane. By late gestation, NHE3 and NHERF2 expression were markedly reduced in the juxtanuclear compartment but not the cytoplasm. NHERF2 was also abundantly expressed in fetal vascular endothelium in which levels, as assessed by immunoblot exhibited a 3-fold developmental increase. In contrast, levels of NHE1 and NHERF1, which were abundantly expressed in and localized almost exclusively to syncytiotrophoblast microvillus membrane, were similar at mid- and late gestation. We conclude that the subcellular distribution and levels of key components of the Na+/H+ system in the baboon syncytiotrophoblast are developmentally regulated.

Human endogenous retroviruses in health and disease: a symbiotic perspective

In the early 1970s, scientists scanning baboon placentas with the electronmicroscope were startled to discover endogenous retroviruses budding from the syncytiotrophoblast. Similar viruses were subsequently detected in the placentas of healthy cats, mice, guineapigs and humans.1 Retroviruses were originally discovered when Peyton Rous noticed the effects of the avian sarcoma virus in 1910, but their infectious and pathogenic potential for humans only became clear with the discoveries of the sexually transmitted human T-cell leukaemia viruses, HTLV-I and HTLV-II, and even more so with the arrival of the human immunodeficiency viruses, notably HIV-1, which ushered in the lethal pandemic now underway. The genomes of retroviruses are based on RNA. To insert themselves into the DNA-based genome of humans they possess a unique enzyme, reverse transcriptase (RT), that copies the viral RNA template to its complementary DNA, which is then integrated into the chromosomes. Retroviruses have two very different life strategies. Exogenous retroviruses, like HIV, reproduce within the somatic tissues of infected individuals and spread in the human population through sexual intercourse and contaminated blood products as well as from mother to child by passage of virus transplacentally or during parturition, or via breast milk. Although it is likely that the HTLV and HIV viruses first infected humans through cross-species spread from animal hosts, their natural host is now the human species. Endogenous retroviruses (ERVs) have invaded the germ cell lines of every species of vertebrate. Here they replicate in Mendelian fashion, as an integral part of the sexual reproduction of the host, to inhabit the genome of all future generations. This is known as germline transmission. It has important evolutionary, physiological and pathological implications. Genome sequencing reveals that 8% of the human genome consists of human endogenous retroviruses, or HERVs, and, if we extend this to HERV fragments and derivatives, the retroviral legacy amounts to roughly half our DNA.2,3 Although many HERVs have been degraded into fragments, they are still readily identifiable from the presence of any of three pathognomonic genes, gag, pol and env, and their flanking long terminal repeats or LTRs (see Figure 1). These genes, which they share with HTLV and HIV, are subdivided into regions with different functions. For example, the env gene codes for the surface protein of the virus as well as regions responsible for inducing human cell fusion and immunosuppression. Tens of thousands of HERVs have entirely lost their genes, leaving a legacy of vast numbers of solitary LTRs that still retain a variety of genetic potentials. Human endogenous retroviruses are believed to be the legacy of ancient germ cell infections by exogenous retroviruses, dating from 60 million years ago to the present. Pandemic plagues are a brutal manifestation of an evolutionary mechanism resulting in changes to the species gene pool I have labelled 'plague culling'. Assuming that the exogenous forebears of our human endogenous retroviruses once behaved as highly contagious infections, following transmission pathways akin to that of HIV-1, their potential for pandemic spread through the original African-based population of primates and subsequently hominids implies a protracted series of invasions with repeated large-scale attrition. It is salutary to realize that we modern humans are descended from the survivors of this terrible evolutionary legacy. The most recently integrated human endogenous retrovirus yet discovered is HERV-K113, found on chromosome 19 in just 29% of people of mainly African, Asian and Polynesian extraction. It could only have been incorporated into the human genome after the last great migration from Africa, certainly less than 200 000 years ago and possibly much more recently.4 Like the virus designated HERV-H/RGH-2, which is associated with multiple sclerosis, it may retain some degree of horizontal transmissibility.5 However, the great majority of HERVs have lost the capacity for horizontal transmission and have entered into a long-standing union with the rest of the human genome. Figure 1 Geonomic structure of a human endogenous retrovirus

The effects of betamethasone (BM) on endothelial nitric oxide synthase (eNOS) expression in adult baboon femoral arterial endothelial cells

Glucocorticoids have significant effects on endothelium mediated vascular function throughout life. The baboon model has been used extensively to study cellular responses to glucocorticoids at several stages of the lifespan. Endothelial nitric oxide synthase (eNOS) is a major regulator of endothelium dependent arterial vasodilation. We have previously demonstrated that synthetic glucocorticoids down regulate eNOS in the baboon placenta. We have now conducted studies to determine whether glucocorticoids would alter eNOS expression in adult systemic vascular endothelial cells in this important animal model. We explored this potential mechanism in endothelial cells from femoral arteries of adult baboons at necropsy and cultured to the fourth passage. Endothelial cells were treated with 10–100 nM betamethasone for 24 h at 37 °C. Vascular endothelial growth factor (VEGF) was used as a positive control and medium as negative controls. The role of glucocorticoid receptor mediation in betamethasone-induced eNOS changes was investigated with the glucocorticoid receptor antagonist mifepristone. RNA (real-time quantitative RT-PCR) and protein (ELISA) were extracted and measured for eNOS. Expression and subcellular distribution of glucocorticoid receptor were detected with fluorescence labeled antibody microscopy. eNOS mRNA and protein in baboon endothelial cells were downregulated 25% by betamethasone treatment. This effect was attenuated by pre-incubation with mifepristone ( P < 0.01). VEGF upregulated eNOS transcription and translation ( P < 0.001), medium did not alter eNOS expression. We observed that mifepristone and VEGF increased glucocorticoid receptor cytoplasmic accumulation by fluorescence microscopy. We conclude that betamethasone can downregulate eNOS in cultured baboon endothelial cells via the glucocorticoid receptor pathway.

Expression of placental low-density lipoprotein receptor-related protein and scavenger receptors AI/AII transcripts in the baboon

Objective: This study was undertaken to assess potential secondary lipoprotein-dependent mechanisms, scavenger receptor AI and AII (SR-AI and SR-AII), and low-density lipoprotein (LDL) receptor-related protein (LRP) in the baboon (Papio sp) placenta. Study Design: Baboon placental villous tissue and enriched fractions of syncytiotrophoblast cells were collected at early (~day 60, n = 3), mid (~day 100, n = 4), and late (~day 160, n = 4) pregnancy (term = ~184 days). The abundance of messenger RNA (mRNA) transcripts for SR-AI, SR-AII, and LRP were determined relative to constitutively expressed, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: No quantitative differences (P >.05) in SR-AI, SR-AII, or LRP mRNAs were noted in respect to stage of gestation. However, SR-AII/GAPDH mRNA ratios in syncytiotrophoblast cells (0.38 ± 0.09, n = 11) were significantly (P <.05) higher than in villous tissue (0.15 ± 0.04, n = 11), whereas LRP/GAPDH mRNA ratios were higher in villous tissue (0.43 ± 0.10, n = 11) than in syncytiotrophoblast cells (0.18 ± 0.02, n = 11). Conclusion: SR-AII mRNA in baboon placenta appears to be expressed, in greatest abundance, in the endocrinologically active syncytiotrophoblast. (Am J Obstet Gynecol 2003;188:1211-3.)

Gestation-related and betamethasone-induced changes in 11β-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta

Objective: We determined developmental and labor-related changes in 11β-hydroxysteroid (HSD) 1 and 2 expression in baboon placentas during the final third of gestation and labor. We examined whether maternal glucocorticoid administration alters placental 11β-HSD 2 expression. Study Design: Maternal and fetal plasma cortisol concentrations were measured in five animals. Types 1 and 2 11β-HSD messenger RNA (mRNA) and protein in placentas obtained at 121 to 185 days' gestation (dGA, term approximately 185 dGA, n = 16), during labor between 141 and 193 dGA (n = 8), and after maternal administration of four doses of 87.5 μg/kg betamethasone (n = 5) at 12-hour intervals at 121 to 135 dGA were analyzed by Northern and Western blot. Results: Cortisol levels were higher in maternal plasma than fetal (4-fold, P < .mob031). Placental 11β-HSD 2 mRNA and protein decreased after 0.9 gestation (P < .001). 11β-HSD 1 mRNA remained unchanged. There was no effect of labor on placental 11β-HSD 1 and 2 mRNA and protein levels. Maternal betamethasone administration dramatically increased (P < .05) 11β-HSD 2 mRNA as well as protein without effect on 11β-HSD 1 mRNA and protein expression. Conclusions: The late-gestation baboon maternal plasma cortisol concentration is four times the fetal plasma concentration. Decreased placental 11β-HSD 2 may enhance maternal cortisol passage to the fetus at the end of gestation, thereby contributing to cortisol-mediated changes within the placenta and cortisol in fetal plasma at this stage of fetal development. The positive effect of betamethasone on placental 11β-HSD 2 induction further suggests an ability of the placenta to regulate glucocorticoid transfer in the presence of elevated maternal glucocorticoid. (Am J Obstet Gynecol 2003;188:13-21.)

Baboon placentas express soluble and membrane-bound Paan-AG proteins encoded by alternatively spliced transcripts of the class Ib major histocompatibility complex gene, Paan-AG.

The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta contains messages encoded by a novel MHC gene, Paan-AG, which is evolutionarily related to the HLA-A locus, but shares unique characteristics with HLA-G. In this study, we show that the Paan-AG message is alternatively spliced to generate at least seven transcripts. One of these transcripts retains intron 4 and encodes a soluble glycoprotein with three external domains and a unique 21-amino-acid sequence at the carboxyl terminus, similar to soluble HLA-G1. This glycoprotein was detected in first trimester placental villous cytotrophoblast and syncytiotrophoblast, and in extravillous cytotrophoblast cells in the basal plate in term placenta. Four of the transcripts ( Paan-AG1, Paan-AG2, Paan-AG3, Paan-AG4) encode membrane-bound class Ib MHC glycoprotein isoforms. Paan-AG1 protein expression was similar to that of sPaan-AG, while Paan-AG2 protein was not detected in these tissues. The other two transcripts ( Paan-AGx and Paan-AGxi) contain a truncated exon 3 and multiple stop codons. Paan-AG1 and Paan-AGx transcripts were detected in a number of non-placental tissues, but these transcripts contained multiple stop codons. Because of the structural similarities and common features of organ-specific expression and splicing of the message, studies on Paan-AG may be of value in dissecting the functions of the class Ib proteins in human pregnancy.

Developmental maturation of primate placental trophoblast: placental cytosolic and secretory phospholipase A 2 expression after estrogen suppression of baboons

We have demonstrated that the baboon placenta expressed the mRNAs and proteins for secretory and cytosolic phospholipase A 2 (PLA 2) enzymes and that cPLA 2 expression increased with advancing gestation in association with the increase in placental estrogen production. To determine whether estrogen regulates placental PLA 2 expression, as it does other aspects of syncytiotrophoblast functional differentiation, we compared sPLA 2 and cPLA 2 mRNA levels in placentas obtained on day 165 of gestation (term = day 184) from baboons that were untreated or treated during the second half of gestation with the aromatase inhibitor CGS 20267 or CGS 20267 and estradiol. Maternal saphenous and uterine vein estradiol levels were reduced ( P < 0.05) by approximately 95% by treatment with CGS 20267 and restored by concomitant administration of CGS 20267 and estrogen. However, sPLA 2 and cPLA 2 mRNA levels expressed as a ratio of β-actin were similar in whole villous placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus estrogen. PLA 2 expression in an enriched fraction of nontrophoblast cells of the baboon placenta was also not altered by CGS 20267 treatment. Collectively these findings indicate that placental cPLA 2 and sPLA 2 expression is not estrogen-dependent. Because estrogen has been shown to regulate other aspects of placental steroidogenesis, we suggest that the regulatory role of estrogen on syncytiotrophoblast functional maturation is specific.

Pregnancy-Specific Glycoprotein (PSG) in Baboon (Papio hamadryas): Family Size, Domain Structure, and Prediction of a Functional Region in Primate PSGs1

Pregnancy-specific glycoprotein (PSG) constitutes a major component of serum of pregnant women and appears to be essential for a successful pregnancy. Its function is, however, still unknown. Because of the evolutionary divergence between human and rodent PSG, functional studies may require a primate animal model. We have characterized PSG transcripts in a baboon placenta cDNA library and analyzed baboon genomic DNA. The main PSG isoform had the domain structure N-A1-B2-C similar to the human type IIa isoform. The type I isoform (N-A1-A2-B2-C) was also expressed. Fifteen similar PSG genes were identified of which at least nine were simultaneously expressed in third trimester baboon placenta. Thus, the baboon PSG family was as complex as that of humans. Recombinant baboon PSG (isoform IIa) had a molecular weight of 38 kDa and reacted with antibodies against human PSG. Comparative analysis of 43 N-domain amino acid sequences of PSG from four species and nine primate carcinoembryonic antigen subgroup N domain sequences identified a number of residues in the GFCC'C" ss-sheet and FG loop that are probable candidates for PSG binding to its putative ligand.

Regulation of pathways determining cholesterol availability in the baboon placenta with advancing gestation.

Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

Developmental maturation of baboon placental trophoblast: expression of messenger ribonucleic acid and protein levels of cytosolic and secretory phospholipases A2.

Although the human placenta at term exhibits high levels of phospholipase A2 (PLA2) enzyme activity, our understanding of the ontogenesis, regulation, and specific roles of placental phospholipases A2 remains relatively incomplete. Using the baboon as the experimental model, the present study determined whether the levels of the messenger ribonucleic acid (mRNA) for the cytosolic (cPLA2) and/or type IIa, nonpancreatic secretory (sPLA2) enzymes are developmentally regulated and modulated by glucocorticoid treatment. Total RNA was extracted from whole villous placenta obtained on days 60 (early; n = 3), 100 (mid; n = 3), and 165 (late; n = 4) of gestation (term = day 184) from untreated baboons and on day 100 (n = 4) after maternal administration on days 60-99 of betamethasone (3 mg/day). The complementary DNA to cPLA2 recognized a single 2.9-kb mRNA transcript in both baboon and human placenta. Mean (+/-SE) levels of cPLA2 mRNA, expressed, in arbitrary units as a ratio to that of beta-actin, were similar at early (0.19 +/- 0.05) and midgestation (0.34 +/- 0.17) and increased (P < 0.005) 10-fold (2.53 +/- 0.53) by late gestation. Levels of cPLA2 protein were also greater (P < 0.05) on day 165 (2.6 +/- 0.3 arbitrary units) than on day 60 (0.6 +/- 0.2). Like that in the human, the baboon placenta contained very high levels of a single 0.9-kb mRNA transcript for sPLA2. In contrast to that of cPLA2, normalized levels of sPLA2 mRNA were similar at all three time points and were associated with high levels of sPLA2 protein throughout gestation. Treatment with betamethasone increased (P < 0.02) cPLA2 mRNA levels on day 100 by over 4-fold, but had no effect on sPLA2 mRNA levels. Additional studies indicated that the mRNAs for sPLA2 and cPLA2 were detected in an enriched fraction of nontrophoblast cells isolated by collagenase dispersion and Percoll density centrifugation. The mRNA for cPLA2 was also expressed in cytotrophoblast and syncytiotrophoblast cell fractions. Collectively, these findings indicate that the baboon placenta expresses mRNA and protein for both the cytosolic and secretory PLA2 enzymes, and that expression of cPLA2 is developmentally regulated and modulated by glucocorticoids. We previously demonstrated an estrogen-dependent developmental increase in placental expression of specific components of the progesterone steroidogenic pathway during the second half of baboon pregnancy. The current findings indicate that the developmental increase in placental function also includes expression of at least one specific PLA2 enzyme controlling arachidonic acid mobilization and eicosanoid synthesis.