Pregnant Rhesus Research Articles

Fetal CD34+ Cells in the Maternal Circulation and Long-Term Microchimerism in Rhesus Monkeys (Macaca mulatta)

Studies in humans have shown that during pregnancy fetal cells can enter the maternal circulation and persist for many years. While we have previously reported the presence of cell-free fetal DNA during pregnancy in rhesus monkeys, it is unknown whether cells circulate and persist long term in maternal tissues. In this study, we asked whether fetal CD34 cells can be found in the maternal circulation and if male fetal cells persist in maternal tissues postdelivery. The presence of the Y chromosome in maternal blood and tissues was assessed using real-time PCR assays for the sex determining region Y (SRY) and testes specific protein Y (TSPY) genes. Analysis was done on CD34 and CD34 cells isolated from maternal blood collected at select time points during gestation from gravid animals with male or female fetuses, and tissues were analyzed from nongravid animals that had previously delivered male offspring. All animals with male fetuses tested positive for the Y chromosome in CD34 cells (0-30 cells/50,000 genome equivalents). Y sequences were also found in one or more maternal tissues collected up to 3-years postdelivery (thyroid, heart, spleen, liver, pituitary, adrenals, skin, inguinal lymph nodes). These studies suggest transfer of fetal CD34 cells during pregnancy and persistent fetal microchimerism in the rhesus model. Thus, rhesus monkeys can be used to further our understanding of fetal:maternal microchimerism and the role of fetal cells in maternal health and disease.

Effects of prenatal nicotine exposure on primate brain development and attempted amelioration with supplemental choline or vitamin C: neurotransmitter receptors, cell signaling and cell development biomarkers in fetal brain regions of rhesus monkeys.

Studies in developing rodents indicate that nicotine is a neuroteratogen that disrupts brain development by stimulating nicotinic acetylcholine receptors (nAChRs) that control neural cell replication and differentiation. We administered nicotine to pregnant Rhesus monkeys from gestational day 30 through 160 by continuous infusion, achieving maternal plasma levels comparable to those in smokers (30 ng/ml). Fetal brain regions and peripheral tissues were examined for nAChR subtypes, other neurotransmitter receptors, and indices of cell signaling and cell damage. Nicotine evoked nAChR upregulation, but with distinct regional disparities indicative of selective stimulatory responses. Similarly, indices of cell loss (reduced DNA), cell size and neuritic outgrowth (protein/DNA and membrane/total protein ratios) were distinct for each region and did not necessarily follow the rank order of nAChR upregulation, suggesting the involvement of additional mechanisms such as oxidative stress. We then attempted to offset the adverse effects of nicotine with standard dietary supplements known to interact with nicotine. By itself, choline elicited nicotine-like actions commensurate with its promotion of cholinergic neurotransmission. When given in combination with nicotine, choline protected some regions from damage but worsened nicotine's effects in other regions. Similarly, Vitamin C supplementation had mixed effects, increasing nAChR responses while providing protection from cell damage in the caudate, the brain region most susceptible to oxidative stress. Our results indicate that nicotine elicits neurodevelopmental damage that is highly selective for different brain regions, and that dietary supplements ordinarily thought to be neuroprotectant may actually worsen some of the adverse effects of nicotine on the fetal brain.

Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers.

Intra-amniotic infection (IAI) is commonly associated with preterm birth and adverse neonatal sequelae. Early diagnosis of IAI, however, has been hindered by insensitive or nonspecific tests. To identify unique protein signatures in rhesus monkeys with experimental IAI, a proteomics-based analysis of amniotic fluid was used to develop diagnostic biomarkers for subclinical IAI in amniotic fluid and blood of women with preterm labor. Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry, gel electrophoresis, and tandem mass spectrometry were used to characterize amniotic fluid peptides in 19 chronically instrumented pregnant rhesus monkeys before and after experimental IAI. Candidate biomarkers were determined by liquid chromatography-tandem mass spectrometry. Polyclonal antibodies were generated from synthetic peptides for validation of biomarkers of IAI. Amniotic fluid peptide profiles identified in experimental IAI were subsequently tested in a cohort of 33 women admitted to Seattle, Wash, hospitals between June 25, 1991, and June 30, 1997, with preterm delivery at 35 weeks or earlier associated with subclinical IAI (n = 11), preterm delivery at 35 weeks or earlier without IAI (n = 11), and preterm contractions with subsequent term delivery at later than 35 weeks (n = 11). Identification of peptide biomarkers for occult IAI. Protein expression profiles in amniotic fluid showed unique signatures of overexpression of polypeptides in the 3- to 5-kDa and 10- to 12-kDa molecular weight ranges in all animals after infection and in no animal prior to infection. In women, the 10- to 12-kDa signature was identified in all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection (P<.001). Peptide fragment analysis of the diagnostic peak in amniotic fluid identified calgranulin B and a unique fragment of insulinlike growth factor binding protein 1, which were also expressed in maternal serum. Mapping of other amniotic fluid proteins differentially expressed in IAI identified several immunoregulators not previously described in amniotic fluid. This proteomics-based characterization of the differential expression of amniotic fluid proteins in IAI identified a distinct proteomic profile in an experimental primate chorioamnionitis model that detected subclinical IAI in a human cohort with preterm labor. These diagnostic protein expression signatures, complemented by immunodetection of specific biomarkers in amniotic fluid and in maternal serum, might have application in the early detection of IAI.

Determination of genes involved in the early process of embryonic implantation in rhesus monkey (Macaca mulatta) by suppression subtractive hybridization.

Embryonic implantation is a temporally and spatially restricted process that involves a precise cross talk between the embryo and the receptive maternal endometrium. Underlying the complex changes in the uterus during implantation is the alteration in gene expression pattern, which is not fully understood for the primates. In the present study, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the implantation site of the pregnant rhesus monkey, and a subtractive cDNA library was constructed. Furthermore, with dot blot analysis, reverse Northern blot analysis, and semiquantitative reverse transcription-polymerase chain reaction, 76 of 376 clones randomly selected from the library were proven to be differentially expressed in the implantation site. With DNA sequencing and BLAST analysis against the GenBank/EMBL database, it was demonstrated that the cDNA fragments carried by 73 clones shared high homology with 31 human genes. Among them, 15 positive clones represented the S100A10 gene and 10 positive ones corresponded with the secreted frizzled-related protein 4 gene. The other two clones shared homology with one human EST. There was one clone homologous to a human DNA sequence, which indicated that it might be a novel gene. To our knowledge, this is the first report to determine genes involved in the early implantation stage in the rhesus monkey with high throughput technology.

Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile Rhesus monkeys

Background Early life stress, including during fetal development, has been hypothesized to predispose individuals to several illnesses and psychiatric disorders later in adulthood. Methods To determine whether prenatal stress alters neural, hormonal, and behavioral processes in nonhuman primates, pregnant rhesus monkeys were acutely stressed on a daily basis for 25% of their 24-week gestation with an acoustical startle protocol. At 2 to 3 years of age, hippocampal volume, neurogenesis in the dentate gyrus, and cortisol levels were evaluated in the offspring generated from stressed and control pregnancies. Results Prenatal stress, both early and late in pregnancy, resulted in a reduced hippocampal volume and an inhibition of neurogenesis in the dentate gyrus. These changes were associated with increased pituitary–adrenal activity, as reflected by higher cortisol levels after a dexamethasone suppression test, and also with behavioral profiles indicative of greater emotionality. Conclusions These findings indicate that the prenatal environment can alter behavior, dysregulate neuroendocrine systems, and affect the hippocampal structure of primates in a persistent manner.

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy.

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.

Low-dose antiprogestin treatment prevents pregnancy in rhesus monkeys and is reversible after 1 year of treatment.

Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5-6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment. This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females). Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights. The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.

Dexamethasone or interleukin-10 blocks interleukin-1β-induced uterine contractions in pregnant rhesus monkeys

Objective: The purpose of this study was to determine whether treatment with the immune modulators dexamethasone or interleukin-10 prevents interleukin-1β-induced uterine contractions in a nonhuman primate model. Study Design: Thirteen chronically instrumented rhesus monkeys at 135 ± 1 days of gestation (term, 167 days) received one of three interventions: (1) intra-amniotic interleukin-1β (10 μg) infusion with maternal dexamethasone (1 mg/kg) intravenously every 6 hours for 1 day before interleukin-1β and for 2 days thereafter (n = 4), (2) intra-amniotic interleukin-1β infusion with maternal interleukin-10 (25 μg/kg) given intravenously and 100 μg interleukin-10 given intra-amniotically before the interleukin-1β and continued every 8 hours for 3 days (n = 5), and (3) intra-amniotic interleukin-1β administered alone (n = 5). Uterine activity was monitored continuously and quantified as the hourly contraction area (millimeters of mercury times seconds per hour) in all groups until delivery. Amniotic fluid was sampled for leukocyte counts and assayed for prostaglandins E2 and F2α, cytokines interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-α, interleukin-10, and interleukin-1 receptor antagonist by specific assays. Maternal and fetal blood were assayed for cortisol, dehydroepiandrosterone sulfate, and estradiol. Results: Interleukin-1β infusion in the absence of immune modulators resulted in an increase in uterine activity and amniotic fluid proinflammatory cytokines, prostaglandins, and leukocytes. Dexamethasone and interleukin-10 treatment significantly reduced interleukin-1β-induced uterine contractility (P < .05) and amniotic fluid prostaglandins (P < .05) but not interleukin-8 or interleukin-1 receptor antagonist. Amniotic fluid interleukin-6 and maternal and fetal cortisol, dehydroepiandrosterone sulfate, and estradiol concentrations were reduced by dexamethasone (P < .05), whereas tumor necrosis factor-α levels and leukocyte counts were attenuated by interleukin-10 treatment (P < .05). An inverse relationship was noted between amniotic fluid interleukin-10 concentrations and interleukin-1β-induced uterine activity (r = −0.74, P < .05). Conclusion: Dexamethasone and interleukin-10 exert similar inhibitory effects on interleukin-1β-induced uterine activity, which appears to be mediated by a decrease in prostaglandin production. Reduced estrogen biosynthesis or suppression of tumor necrosis factor-α and leukocyte migration may contribute to the tocolytic actions of dexamethasone and interleukin-10, respectively. Dexamethasone and interleukin-10 are likely to be useful adjuncts in the treatment of preterm labor that is associated with inflammation or infection.Am J Obstet Gynecol 2003;188:252-63.

Identification and specific expression of matrix metalloproteinase-26 in rhesus monkey endometrium during early pregnancy.

Matrix metalloproteinases (MMPs) and their tissue inhibitors may play important roles in tissue remodelling processes of the uterus. This study identified MMP-26 (endometase/matrilysin-2) in the endometrium of pregnant rhesus monkeys (Macaca mulatta) and monitored the spatial and temporal expression of the transcript and protein in the uteri on days 12, 18 and 26 of pregnancy. The partial monkey MMP-26 gene sequence of 289 nucleotides was 98% identical to that of its human homologue and its protein fragment contained a PHCGVPDGSD sequence in the prodomain identical to that in human MMP-26. RT-PCR analysis demonstrated that the average level of MMP-26 mRNA in the endometrium was high on day 12 of pregnancy, but significantly decreased on days 18 and 26 (P < 0.05). In-situ hybridization confirmed that MMP-26 mRNA is specifically localized in the endometrial compartments, with intense signals in the glandular epithelium on day 12 and in the walls of spiral arterioles adjacent to the implantation site on day 26. The hybridization signal for MMP-26 mRNA in the glandular epithelium decreased dramatically on day 18 and was undetectable on day 26. No MMP-26 mRNA transcripts were detected in the placental villi on days 18 and 26. Immunohistochemistry showed that the expression pattern of MMP-26 protein was similar to that of its mRNA. The restricted expression pattern of MMP-26 in the monkey uterus implies that this new MMP is involved in the highly regulated tissue remodelling processes of the glandular epithelium and spiral arteries during early pregnancy.

The effects of photoperiod on the switching of myometrial contractility patterns of pregnant baboons: relationship to surgery and parturition.

Pregnant baboons were studied to determine the precise time of the switch from myometrial contractures to contractions in relation to photoperiod after laparotomy and at parturition. We compared the patterns recorded in baboons to those we have previously reported in pregnant rhesus monkeys to determine fundamental primate characteristics. Seven pregnant baboons (126-160 days' gestation) were instrumented with femoral arterial and venous catheters and electrodes for myometrial electromyogram. All animals were subjected to a 14-hour light:10-hour dark photoperiod. Myometrial activity was monitored using a computer-based data acquisition system. Onset time for all switches was noted and standardized against time of lights off. Animals were studied at three stages of pregnancy (stage 1, first 10 days after laparotomy; stage 2, more than 10 days after laparotomy and more than 10 days before cesarean; and stage 3, 10 days before cesarean section or vaginal delivery). All baboons demonstrated myometrial switches for a variable number of days preceding parturition. Onset of darkness was 0 hours. Average time of stage 1 switch onset was 2.17 +/- 0.60 hours and was not different from stage 3 switch onset, which was -1.00 +/- 0.27 hours. Myometrial contractile patterns showed clear photoperiodicity in the switch from contractures to contractions in late pregnancy in the baboon. The relationship of the switch from contractures to contractions was not altered by surgical laparotomy. There was a significant difference in the time of switch in relation to photoperiod between pregnant rhesus monkeys and baboons. However, the fact that a significant photoperiod exists in both species indicates a fundamental similarity in the switch from contractures to contractions in primate pregnancy.

Prenatal stress diminishes the cytokine response of leukocytes to endotoxin stimulation in juvenile rhesus monkeys.

This study investigated whether exposing the fetal primate to repeated episodes of maternal stress would have long-lasting effects on the endotoxin-induced cytokine response and corticosteroid sensitivity of peripheral blood cells in juvenile animals. Pregnant rhesus monkeys were acutely aroused on a daily basis for 6 wk using an acoustical startle protocol, either early or late in the 24-wk pregnancy. To quantify cytokine responses and corticosteroid sensitivity in their offspring at 2 yr of age, whole blood cultures were stimulated with lipopolysaccharide and incubated with dexamethasone (DEX). TNFalpha and IL-6 levels were determined in the culture supernatants. The blood samples were collected from undisturbed monkeys under baseline conditions, as well as in an aroused state induced by a 2 h social separation. Juvenile monkeys from stressed pregnancies had significantly lower cellular cytokine responses compared with the undisturbed controls. When DEX was added to the cell cultures, it systematically inhibited TNFalpha and IL-6 production, bringing the values for control animals down into the range of the prenatally stressed animals. Lipopolysaccharide-induced cytokine production was also markedly suppressed by the experience of acute stress, reducing cytokine responses of controls to the levels found for prenatally disturbed monkeys under baseline conditions. Therefore, this study has demonstrated that prenatal disturbance can induce a lasting change in cytokine biology, which persists well beyond the fetal and infant stage. Further, these effects may be due to elevated hypothalamic-pituitary-adrenal activity in the prenatally stressed animals, because both DEX and acute arousal made the cells from control monkeys appear more similar to those from disturbed pregnancies.

Identification of matrix metalloproteinase-9 in amniotic fluid and amniochorion in spontaneous labor and after experimental intrauterine infection or interleukin-1β infusion in pregnant rhesus monkeys

Objective: The purpose of this study was to examine the roles of intrauterine infection, inflammation, and spontaneous labor on the expression of matrix metalloproteinase-9 in fetal membranes and amniotic fluid of late pregnant rhesus monkeys. Study Design: Pregnant rhesus monkeys with timed gestations were chronically catheterized to allow serial sampling of amniotic fluid before and during experimentally induced intrauterine inflammation. Six animals received group B streptococci into the chorionic-decidual space, and 4 animals received intra-amniotic interleukin-1β infusions (10 μg). Three additional animals were serially sampled by amniocentesis through late pregnancy until spontaneous term labor. Amniotic fluid samples were examined by zymography for matrix metalloproteinase-9 and -2 and Western immunoblot for matrix metalloproteinase-9 and -2 and tissue inhibitors of metalloproteinase-1 and -2. Fetal membranes were obtained at cesarean delivery during labor (before rupture), formalin fixed, and embedded in paraffin for immunocytochemistry of matrix metalloproteinase-9 and in situ hybridization of matrix metalloproteinase-9 messenger RNA. Tissues from 2 additional animals were collected as age-matched nonlabor controls. Results: In amniotic fluid, the 92-kd latent matrix metalloproteinase-9 was detectable in late pregnancy and increased dramatically, followed by the appearance of the 83-kd active form before spontaneous term delivery. Amniotic fluid matrix metalloproteinase-2 levels (both latent and active forms) remained relatively constant throughout pregnancy and in labor. Both bacteria and interleukin-1β rapidly increased the signal of latent matrix metalloproteinase-9 without a consistent increase in the active form before the onset of labor. Chorionic-decidual inoculation of group B streptococci was followed by the expression of latent matrix metalloproteinase-9 before the appearance of group B streptococci in amniotic fluid or the onset of labor. Matrix metalloproteinase-2 increased to a new steady-state level or remained unchanged after group B streptococci inoculation or interleukin-1β infusion, respectively. Amniotic fluid tissue inhibitors of metalloproteinase-1 declined and tissue inhibitors of metalloproteinase-2 remained unchanged during early group B streptococci infection, after interleukin-1β infusion and on the day of spontaneous term labor. However, both tissue inhibitors of metalloproteinase-1 and -2 levels increased after preterm labor that was induced by group B streptococci. Immunocytochemistry localized matrix metalloproteinase-9 protein to amnion and chorion epithelial and mesenchymal cells and decidual stromal cells. Granular matrix metalloproteinase-9 staining was observed in the connective tissue layer of inflamed fetal membranes. In situ hybridization for messenger RNA confirmed the production of matrix metalloproteinase-9 by amnion and chorion. Conclusion: Bacterial- and interleukin-1β–induced preterm labor and spontaneous term labor are preceded and accompanied by progressive increases in amniotic fluid matrix metalloproteinase-9 (92 kd) in rhesus monkeys. Amniotic fluid matrix metalloproteinase-9 may serve as a clinical marker for the onset of both preterm and term labor. (Am J Obstet Gynecol 2002;186:128–38.)

Chronic Maternal Nicotine Exposure Alters Neuronal Systems in the Arcuate Nucleus That Regulate Feeding Behavior in the Newborn Rhesus Macaque

It is well known that maternal smoking during pregnancy can lead to low birth weight and low body fat in human newborns. The purpose of this study was to determine whether chronic maternal nicotine treatment alters levels of known regulators of energy balance in the newborn offspring. Pregnant rhesus monkeys were treated with nicotine tartrate (1.5 mg/kg·d) starting on d 26 of pregnancy and maintained through d 160 of gestation. Nicotine exposure had no significant effect on absolute birth weights of the neonatal monkeys, although there was a 10% reduction in birth weights with nicotine exposure when they were normalized to maternal weight. Postnatal d 1 plasma leptin levels were significantly reduced by about 50% in the nicotine treatment group compared with saline controls, suggesting that the infant monkeys exposed to nicotine may also have lower body fat levels. In situ hybridization studies demonstrated that chronic nicotine exposure resulted in a significant decrease in arcuate NPY mRNA expression in the neonatal monkeys. In addition, there was a 2-fold increase in POMC mRNA in the arcuate nucleus in the nicotine-exposed group. These data suggest that nicotine exposure during pregnancy may increase energy expenditure in the developing fetus through actions on hypothalamic systems, resulting in lower birth weights and body fat levels.

Chronic maternal nicotine exposure alters neuronal systems in the arcuate nucleus that regulate feeding behavior in the newborn rhesus macaque.

It is well known that maternal smoking during pregnancy can lead to low birth weight and low body fat in human newborns. The purpose of this study was to determine whether chronic maternal nicotine treatment alters levels of known regulators of energy balance in the newborn offspring. Pregnant rhesus monkeys were treated with nicotine tartrate (1.5 mg/kg x d) starting on d 26 of pregnancy and maintained through d 160 of gestation. Nicotine exposure had no significant effect on absolute birth weights of the neonatal monkeys, although there was a 10% reduction in birth weights with nicotine exposure when they were normalized to maternal weight. Postnatal d 1 plasma leptin levels were significantly reduced by about 50% in the nicotine treatment group compared with saline controls, suggesting that the infant monkeys exposed to nicotine may also have lower body fat levels. In situ hybridization studies demonstrated that chronic nicotine exposure resulted in a significant decrease in arcuate NPY mRNA expression in the neonatal monkeys. In addition, there was a 2-fold increase in POMC mRNA in the arcuate nucleus in the nicotine-exposed group. These data suggest that nicotine exposure during pregnancy may increase energy expenditure in the developing fetus through actions on hypothalamic systems, resulting in lower birth weights and body fat levels.

ANDi – the first transgenic monkey

The first transgenic primate, created by researchers at the Oregon Health Sciences University, was born in October of last year [Chan, A.W.S. et al. (2001) Science 291, 309–312]. The rhesus monkey, named ANDi (for ‘inserted DNA in a reverse-transcribed direction’), was designed to produce GFP fluorescence. The animal shows the presence of the transgene in genetic analyses but does not show GFP fluorescence. A stillborn twin expresses GFP in all tissues examined. The reason for the miscarriage of the twin is probably the rare and high-risk nature of twin pregnancy in rhesus monkeys. However, it remains formally possible that this was due to the inserted gene – possibly direct toxicity of the GFP protein. The work is aimed at creating better animal models of human diseases such as Alzheimer's and Parkinson's disease. D.S.

Phenotypic and functional characterization of rhesus monkey decidual lymphocytes: rhesus decidual large granular lymphocytes express CD56 and have cytolytic activity

In this study, we carried out a phenotypic and functional characterization of lymphocytes isolated from the uterine endometrium of the pregnant rhesus monkey. A majority (80%) of these cells were CD56 bright+, CD3− had typical large granular lymphocyte/uterine natural killer (NK) cell morphology and contained numerous cytoplasmic granules. Flow cytometric evaluation showed that rhesus decidual CD56 bright+ cells shared other phenotypic features of human uterine NK cells, including low levels of CD45RA and CD62L expression. A majority of the rhesus uterine CD56 bright+ cells expressed low levels of CD 16 but were CD2−. In contrast, most rhesus CD16+ peripheral blood cells were CD56−. In addition to the primary population of CD56 bright+ cells, a minor subset of smaller and less granular CD56 intermediate+ decidual lymphocytes was identified, the majority of which were CD16−, CD2 +. Decidual CD56+ cells did not express monocyte/macrophage markers, including CD14, CD64 and CD68. Decidual lymphocytes effectively lysed K562, Raji and particularly 721.221 targets in cytotoxicity assays. Together, these results suggest that as in human pregnancy, rhesus decidual CD56 bright+ cells represent a distinct lymphocyte subset that belongs to the NK cell lineage.

Corticotropin-releasing hormone-binding protein in primates.

In humans, placental corticotropin-releasing hormone (CRH) production has been linked to the determination of gestational length, and a late gestational fall in CRH-binding protein (CRH-BP) has been linked to the onset of parturition. Expression of placental CRH mRNA is limited to primates, and only in man has a circulating CRH-BP been described. As the fall in CRH-BP in late gestation has been associated with parturition in humans, we sought to determine whether a CRH-BP circulated in the plasma of other primates. It is unclear whether maternal plasma CRH concentrations are elevated in New World monkeys and prosimians. We have therefore performed CRH plasma measurements in the blood of pregnant marmosets, in several species of lemur, and in pregnant and fetal rhesus monkeys as a positive control. Using gel chromatography, CRH-BP was detected in the human, gorilla, chimpanzee, orangutan, gibbon, macaque, squirrel monkey, and marmoset, but was absent in the mandrill, spider monkey, and lemur. CRH was detected in the plasma of pregnant marmosets and rhesus monkeys. CRH was also detected in the fetal rhesus monkey, but at lower concentrations than in maternal plasma. CRH immunoreactivity was not detectable in the plasma of pregnant lemurs or in extracts of lemur placenta. In conclusion, a circulating binding protein for CRH exists in all species of apes but occurs variably among New World and Old World monkeys and is absent in lemurs. The variable occurrence of the CRH-BP does not support a role for this protein in the mechanism of parturition in primates. Maternal CRH is elevated in the pregnant marmoset and rhesus, and may play a role in the pregnancy of New and Old World monkeys.

Synergistic effects of DL111-IT combined with mifepristone on termination of early pregnancy in rhesus monkeys

The objectives of this study were to determine the synergistic effects of DL111-IT in combination with mifepristone (RU486) on termination of early pregnancy in rhesus monkeys. Pregnancy was confirmed by tactile sensation of pregnant uterus via anus with finger and ultrasound examination. Pregnancy termination was obtained with vaginal bleeding and abortion materials including fetuses and placentae after treatment. With multiple doses of DL111-IT or RU486 given alone between d24 and d50 of gestation, pregnancy arrests were obtained in 40% (2/5) of monkeys treated with DL111-IT intramuscularly (im) (25 mg · kg −1 · d −1 × 3 days), in 20% (1/5) of monkeys treated with 9 mg · kg −1 · d −1 × 2 days, and 4.5 mg · kg −1 on day 3 with RU486 intragastrically (ig). DL111-IT (25 mg · kg −1 on day 1, im) in combination with RU486 (the same treatment as above) resulted in 100% (10/10) termination of pregnancy and uterine bleeding lasted 6.6 ± 1.3 days. RU486 (as above treatment) in combination with misoprostal (Miso, 109 μg · kg −1 on day 3, ig) showed 71.4% (5/7) termination of pregnancy, and uterine bleeding lasted 12.9 ± 9.6 days. The synergistic effect of DL111-IT plus RU486 enhances termination of early pregnancy and significantly shortens the bleeding time than RU486 plus Miso does in rhesus monkeys.

Timing of Prenatal Androgen Exposure: Anatomical and Endocrine Effects on Juvenile Male and Female Rhesus Monkeys

Prenatal androgen shapes genital differentiation. In humans, genital anatomy determines sex of rearing and subsequent behavioral development. Rhesus monkey genital anatomy and neuroendocrine function are sexually differentiated, and behavioral development occurs in a complex social environment. We investigated prenatal hormonal influences on sexual differentiation by suppressing or increasing androgens in male and female rhesus monkeys. Pregnant multiparous female rhesus monkeys received 35–40 days of testosterone enanthate (TE) treatment, androgen antagonist (flutamide, FL) treatment, or vehicle starting on gestation day (GD) 35 or 40 (early) or GD 110 or 115 (late). Exogenous androgen increased neonatal LH secretion in females when given early and altered female genital differentiation when administered either early or late. TE treatment, early or late in gestation, had no measurable effects on male genital differentiation or neuroendocrine function. Early FL treatment, however, radically altered male genital differentiation, producing in two cases males with a urethral opening separate from the glans. In females, early FL treatment produced detectable alterations in genitalia consistent with a reduced exposure to prenatal androgen, suggesting that female rhesus monkeys are naturally exposed prenatally to meaningful levels of T. Late FL treatment reduced male penis size and increased neonatal T secretion, but had no effect in females. This is the first study to block endogenous prenatal testosterone in rhesus monkeys, thereby altering sexual differentiation. These findings illustrate the complexity of prenatal influences on anatomical and neuroendocrine development. The relationship between the anatomical changes reported here and sex differences in behavior is currently under investigation.

Indomethacin blocks interleukin 1β–induced myometrial contractions in pregnant rhesus monkeys*1

Objective: We sought to determine whether blockade of prostaglandin synthesis with indomethacin prevents interleukin 1β–induced increases in uterine contractions in a nonhuman primate model. Study Design: Maternal and fetal vascular catheters, intra-amniotic fluid pressure catheters, and fetal electrocardiographic and myometrial electromyographic electrodes were implanted in 11 rhesus monkeys at 124 ± 2 days’ gestation (term, 167 days). After postsurgical stabilization (136 ± 2 days) indomethacin 50 mg was administered orally twice daily for 5 days (n = 6). On day 3 human recombinant interleukin 1β 10 μg was infused into the amniotic cavity over 2 hours. Five days after the last indomethacin dose the study was repeated without indomethacin treatment. Uterine activity was continuously monitored and quantified as the hourly contraction area (millimeters of mercury · seconds per hour) in the experimental group and a control group (n = 5) that received interleukin 1β alone. At timed intervals amniotic fluid was sampled for leukocyte counts and assayed for prostaglandin E2 and F2α, the inflammatory cytokines interleukin 1β, interleukin 6, interleukin 8, tumor necrosis factor α, and interleukin 1 receptor antagonist by specific assays. Results: Uterine activity was increased severalfold from baseline after interleukin 1β infusion alone and in the absence of indomethacin treatment (P <.05). There was no increase in uterine contractility when interleukin 1β was infused concurrently with indomethacin treatment. Concentrations of amniotic fluid leukocytes and cytokines increased significantly after interleukin 1β infusion in both the presence and absence of indomethacin. Amniotic fluid prostaglandins E2 and F2α were suppressed during indomethacin treatment but rose significantly after interleukin 1β infusion in the absence of indomethacin. Except for higher interleukin 6, cytokine levels were unaltered by indomethacin. Conclusions: After interleukin 1β infusion, indomethacin blocked the development of uterine activity. Amniotic fluid prostaglandins were suppressed by indomethacin treatment, but cytokines and leukocytes were not. These results suggest that prostaglandins or possibly other indomethacin-suppressible compounds stimulate uterine activity after interleukin 1β infusion in late-gestation rhesus monkeys or that indomethacin has direct tocolytic effects. (Am J Obstet Gynecol 2000;183:173-80.)