Rhesus Monkey Placenta Research Articles

The pattern of expression of insulin-like growth factor (IGF), IGF-I receptor and IGF binding protein (IGFBP) mRNAs in the rhesus monkey placenta suggests a paracrine mode of IGF-IGFBP interaction in placental development

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) act as paracrine factors at or close to the sites of biosynthesis, i.e. cellular sites of expression of specific mRNAs. To determine the developmental pattern of expression of IGF-I, IGF-II, IGF-I R and IGFBP 1–6 mRNAs in the rhesus monkey placenta, in situ hybridization histochemistry was performed in placentae and fetal membranes from 65 days until term (165 ± 5 days). IGF-I mRNA was not detectable in any of the specimens examined. IGF-11 mRNA was localized abundantly in the placenta (syncytiotrophoblasts of the chorionic villi and the anchoring villi, and the extravillous cytotrophoblasts), and in the fetal membranes (chorion and amnion). IGF-I R mRNA was expressed predominantly in the decidua. All IGFBP mRNAs (IGFBP-1 to −6) were expressed in the maternal decidua in variable abundance. Only some IGFBP mRNAs, notably IGFBP-3 mRNA, was expressed in the fetal tissues, such as the chorionic mesoderm, some extravillous cytotrophoblasts and in the amnion and chorion. Gestational age did not alter the localization or relative abundance of all of the mRNAs studied. These findings suggest a role for IGF-11 in the regulation of nutrient transport or placental hormone synthesis and/or secretion in the syncytiotrophoblasts, and a role for IGF-1I and IGFBPs in the cell to cell communication and interaction at the feto-maternal interface.

MHC class I expression in the rhesus monkey placenta

MHC class I expression in the rhesus monkey placenta

Re-association of human and ovine placental eluate immunoglobulins with acidified trophoblast vesicles

The aim of this study was to characterize the expression of the rhesus HLA-E ortholog Mamu-E, particularly at the maternal–fetal interface. Mamu-E expression was confirmed by locus-specific RT-PCR in the placenta as well as in peripheral blood mononuclear cells (PBMC) and other organs. We evaluated the utility of antibodies recognizing HLA-E (MEM-E/06 against native HLA-E, MEM-E/02 against denatured HLA-E) to detect Mamu-E by flow cytometry/immunofluorescence, Western blot, and immunohistochemistry (IHC). Western blot analysis of cells and selected transfectants confirmed the recognition of Mamu-E but not Mamu-AG by antibodies MEM-E/06 and HC10 but not MEM-E/02. Immunohistochemical staining of frozen sections of rhesus placenta with the MEM-E/06 antibody demonstrated expression in most populations of rhesus monkey trophoblast cells, including villous cytotrophoblasts (strong positive staining), apical membrane of syncytiotrophoblasts (light to moderate staining) and extravillous cytotrophoblasts (moderate to strong staining, especially endovascular trophoblasts in early pregnancy). Expression was not trophoblast cell-specific, especially at term, when endothelial cells in both the chorionic plate and placental villi showed strong staining for Mamu-E. Staining of rhesus extravillous trophoblast cells suggested the co-expression of Mamu-E and Mamu-AG (the rhesus HLA-G homolog) on these cells. MEM-E/06 was shown also to react with differentiating rhesus placental syncytiotrophoblasts in primary culture, detecting intracellular and weak surface expression of Mamu-E. We conclude that the gestation-dependent co-expression of Mamu-E with Mamu-AG in villous and extravillous trophoblast cells suggests important and perhaps complementary but distinct roles of these two non-classical MHC class I loci in pregnancy at the maternal–fetal interface. In addition, the MEM-E/06 antibody will be useful for the detection of Mamu-E at the maternal–fetal interface in the rhesus monkey.

Keratinocyte growth factor and its receptor in the rhesus macaque placenta during the course of gestation

Keratinocyte growth factor (KGF) is synthesized and secreted exclusively by mesenchymal cells, and acts through its receptor (KGFR) to stimulate epithelial proliferation. In vivo, KGF and KGFR comprise a mesenchymal-epithelial cell paracrine system that can mediate epithelial cell mitosis. In preliminary work, we noted that KGF was expressed in the rhesus monkey placenta, and we now report on the expression of placental KGF and KGFR mRNAs during the course of gestation in this species. In-situ hybridization revealed that during early gestation, KGF mRNA was strongly expressed in placental mesenchymal cells. These cells, which were also immunoreactive for vimentin, were mainly located on the periphery of the mesenchymal cores of both anchoring and floating villi. KGFR mRNA was expressed in the adjacent trophoblastic epithelium, which was immunoreactive for cytokeratin. In-situ hybridization revealed that KGF mRNA expression was very high in the youngest placentae (34-days gestation) and decreased gradually to minimal levels by late gestation (157 days). Northern blot analysis indicated also that the KGF MRNA signal was strongest in early gestation samples and weakest by late gestation. Analysis for KGFR mRNA by a reverse transcriptase-polymerase chain reaction technique showed that KGFR mRNA expression could be detected at all stages. However, in-situ hybridization indicated that KGFR mRNA expression was highest in early gestation placentae and least in the oldest placentae. Autoradiographs of frozen sections of placenta that had been incubated with [125I]KGF to detect receptor binding showed that grain density over the trophoblast was highest in the youngest and least in the oldest placentae. PCNA and Ki-67 expression followed this same temporal trend. We conclude that the KGF/KGFR system may be important in proliferation of the placental trophoblast during early- to mid-pregnancy in rhesus monkeys.

Cloning of four growth hormone/chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta.

The close homology of the chorionic somatomammotropin (CS) genes with pituitary GH is unique to primates. Southern blots of rhesus genomic DNA probed with a human CS cDNA demonstrated that the rhesus gene family consists of at least five EcoRI fragments between 2.3-9.5 kilobases. Rhesus pituitary and placental cDNA libraries were screened for hCS-hybridizing clones. A pituitary GH cDNA was isolated that was 95% and 96% identical to human GH at the mRNA and protein levels, respectively. Thirteen placental clones representing four different cDNAs were identified and sequenced. The relationships of the rhesus placental clones among themselves were similar to those among human placental mRNAs. Two cDNAs (mCS1 and mCS2) differed by two bases and coded for identical proteins. A third cDNA (mCS3) was 94% homologous to mCS1/2 in mRNA sequence as well as in deduced amino acid sequence. A fourth cDNA (mGH-V) was 84-86% homologous to the other placental cDNAs at the mRNA and protein levels, similar to the homology between human placental GH-V and CS mRNAs. To determine the relative expression of placental mRNAs, we developed a quantitative reverse transcription-polymerase chain reaction assay using diagnostic NlaIV restriction enzyme sites in cDNAs amplified from the placental mRNAs. All four mRNAs were expressed in the placenta throughout most of pregnancy, with the mCS1 and mGH-V mRNAs generally expressed at 2- to 10-fold higher levels than mCS2 and mCS3. Additionally, mCS3 and mGHV were expressed at relatively higher levels during the second and third trimesters than during the first trimester.

Cloning of four growth hormone/chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta

Cloning of four growth hormone/chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta

Isolation and morphologic differentiation in vitro of villous cytotrophoblast cells from rhesus monkey placenta

Trophoblast is the major functional cell type of the placenta. The purpose of this study was to devise a means to isolate trophoblast cells from the monkey placenta and to examine their capacity to differentiate in vitro. Methods originally devised for the isolation of human cytotrophoblast cells produced poor yields and a low degree of purity when applied to the near-term rhesus monkey placenta. However, a procedure has been developed which allows the isolation of a cell population consisting of more than 95% cytotrophoblast based on intermediate filament immunocytochemistry. The cells sedimented between densities of 1.040 and 1.053 g/ml on continuous Percoll density gradient centrifugation. When maintained in culture the cells adhered and formed aggregates of mononuclear cells by 24 h. By 5 d in culture, immunofluorescent staining using antidesmoplakin and antinuclear antibodies revealed that most colonies consisted of large multinucleated masses similar to syncytiotrophoblast. These results demonstrate trophoblast cells from monkey placental villi can be isolated with a high degree of purity and undergo morphologic differentiation in vitro. This preparation should enable investigators to study many functional characteristics of these cells throughout gestation.

Species-specific placental corticotropin releasing hormone messenger RNA and peptide expression

The human placenta is a major site of synthesis of the hypothalamic neuropeptide corticotropin releasing hormone (CRH). We have examined placentae of several other species for expression of the CRH gene in this tissue. CRH mRNA was detected in human, gorilla, and rhesus monkey placentae, but not in rat, guinea pig and lemur placentae. A nonhuman primate may therefore be a suitable experimental model for studying placental CRH gene expression in vivo.

Circadian hormonal interactions among the mother, fetus, and amniotic fluid

Circadian rhythms and hormonal interactions among the maternal, fetal, and amniotic fluid compartments were studied in long-term catheterized rhesus macaque monkeys between days 127 and 138 of gestation (term = 167 days). Blood samples were collected at 3-hour intervals for 48 hours and analyzed by radioimmunoassay for estrone, estradiol, cortisol, progesterone, dehydroepiandrosterone sulfate, and prolactin. Distinct circadian rhythms were present for cortisol and progesterone in the maternal circulation and for progesterone and dehydroepiandrosterone sulfate in the fetal circulation (p < 0.05). Although maternal and fetal estrogen levels were higher in am samples than in pm samples, a statistically significant circadian rhythm was not present (p > 0.10). Fetal levels of progesterone and dehydroepiandrosterone sulfate and maternal levels of progesterone were highest between 9:00 pm and 3:00 am and lowest between 9:00 am and 3:00 pm. Maternal levels of cortisol were highest between 6:00 am and 9:00 am and lowest between 6:00 pm and 12 midnight. The circadian patterns of maternal cortisol and progesterone were inversely related to each other (r = −0.68; p < 0.01). Amniotic fluid cortisol levels were highest between 9:00 am and 12 noon and lowest between 6:00 pm and 3:00 am (p < 0.10). With the possible exception of cortisol, amniotic fluid steroid hormones did not demonstrate distinct diurnal fluctuations, nor did they correlate with steroid changes in maternal or fetal blood. Because the rhesus placenta is permeable to glucocorticoids it is likely that transplacental passage of maternal cortisol influences the activity of the fetal pituitary and adrenal so that the circadian rhythm in the fetal axis is 180 degrees out of phase with that of the maternal axis. The circadian rhythms in fetal dehydroepiandrosterone sulfate and progesterone in late gestation parallel the biorhythm in uterine contraction frequency and amplitude, with peaks during periods of darkness between 9:00 pm and 3:00 am.

Patterns of 3β-hydroxysteroid [formula omitted] activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid dehydrogenase Δ 5−4isomerase activity (3Δ-HSDH) was examined in the rhesus monkey ( Macaca mulatta ) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [ 3H]pregnenolone to [ 3H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta. Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone in vivo . The physiological importance of this remains to be determined.

3β-Hydroxysteroid [formula omitted] activity in the rhesus monkey placenta and fetal adrenal, testis and ovary during late gestation

3β-Hydroxysteroid dehydrogenase Δ 5−4isomerase (3β-HSDH) was measured in the rhesus monkey ( Macaca mulatta ) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [ 3H]-pregnenolone to [ 3H]progesterone. The maximum enzyme velocity (V m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg −1protein) than in microsomes from the other tissues. Testicular V m was greater than either ovarian or placental V m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg −1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes. Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.

Preferential expression of endogenous type C viral antigen in rhesus placenta during ontogenesis

A radioimmunoassay for the major internal structural protein (p26) from Macaca arctoides type C virus (MAC-1) was used to score antigen expression in rhesus placenta and other fetal organs obtained at various times of gestation. Antigen expression was detected in 16 out of 16 placental specimens but not in fetal heart, lung, spleen, liver, kidney, intestines, skeletal muscle, testes, and endometrial scrapings of decidua from eight selected animals, nor in thymus from two animals. The levels of antigen detected in placenta ranged between 2 and 218 ng/mg protein with a correlation between lower antigen expression and term gestation or parity greater than 10. A 10-fold higher level of antigen expression was detected at the external surface of placenta near decidua than in the remainder of the placenta toward the amniotic surface. Thus, even within the placenta, there was a preferred site for endogenous retroviral antigen expression. Last, substantial interindividual variation exists in regard to the amount of placental p26 expression in preterm animals of low or intermediate parity.

Developmental changes in the fine structure of the chorion laeve (smooth chorion) of the rhesus monkey placenta.

Developmental changes in the fine structure of the chorion laeve (smooth chorion) of the rhesus monkey were studied at two time periods during gestation: 1) Early (19-60 days of gestation), before the chorionic epithelium fuses with the parietal decidua, and 2) near term, when the fused chorioamnion has also fused with the parietal decidua. Early in gestation the chorionic epithelium consisted of columnar and cuboidal cells one or two layers thick. The apical border of the cells had microvilli and coated pits, and adjacent cells were joined by tight junctions and desmosomes. The chorionic epithelial cells during this early period contained numerous large vesicles and vacuoles of varying electron-density. The apical cytoplasm contained various small coated vesicles and tubules. Taken together these observations were interpreted as indicating a possible role for these cells in endocytosis or phagocytosis of substances from the uterine lumen; i.e., a potential role in histiotrophic nutrition during this early period. Late in gestation the trophoblastic cells were more irregular in shape. The cells contained abundant granular endoplasmic reticulum (ER) and mitochondria, and a well-developed Golgi complex, suggesting the cells were actively synthetic late in gestation. The numerous cytoplasmic vacuoles characteristic of the trophoblastic cells of early gestation were absent near term. Glycogen deposits and lipid droplets were moderately well-developed near term. Most of the cells were joined by desmosomes but wide intercellular spaces, unobstructed by any cell junctions, were frequently observed. This observation provides at least one explanation for the increase in permeability of the chorion laeve later in gestation. Cells of the parietal decidua associated with the chorion laeve were also examined. These cells generally had a well-developed granular ER and Golgi apparatus, and numerous mitochondria. Limited numbers of membrane-bounded secretory bodies, similar to those in human decidual cells, were also present.

The similarity of chorionic gonadotrophin and its subunits in term placentae from man, apes, old and New World monkeys and a prosimian.

Chorionic gonadotrophin (CG) was estimated, by bioassay and radioimmunoassay (RIA), in placental extracts from 11 ape and monkey species. There was a significant correlation between the results of the two assay systems (r = 0.903, p less than 0.001). The concentration of CG in most primate term placentae was the same as that in the human placenta at term. Extracts from all placentae cross-reacted with antiserum to ovine LH-beta subunit, and those of the chimpanzee and gorilla also had a significant cross-reaction with an antiserum to the carboxyl terminal peptide of the HCG-beta subunit. Primate placentae chromatographed on Sephadex G-200 had components active in the RIA systems for HCG, HCG-alpha HCG-beta subunits. In general, the elution profiles of all ape and monkey placental extracts resemble those made from human term placentae and of purified HCG and its subunits. The shape of the elution patterns from human and non-human material suggests that there was more than one molecular form of CG-alpha subunit activity. A second, more retarded molecular form having beta subunit activity was found in extracts made from human, gorilla, gibbon and rhesus monkey placentae. The similarity between the structure of ape and monkey placental CG with HCG and its subunits implies a function similar to that of HCG in late pregnancy.

The flow resistance of the spiral artery and the related intervillous space in the rhesus monkey placenta.

Uteroplacental arteries of 4 rhesus monkeys were cannulated at their myometrical portions and perfused with dextran solutions and blood. Pressure-flow curves were obtained for the spiral artery and the related intervillous space, i.e. for the functional cotyledonary unit of the haemochorial villous placenta. For a flow of 3 ml.min-1, which is probably the normal rate of flow through the spiral artery, the pressure at the entry of the spiral artery was found to be 13--18 mm Hg (1.7--2.4kPa) during uterine relaxation (uterine diastole). There was a direct proportional relationship between the spiral artery flow and the pressure difference between the spiral artery and the amniotic fluid; the pressure difference per flow (the resistance of the cotyledonary unit) was found to be 1--3 mm Hg . ml-1 . min (8--24kPa . ml-1 . s). It is concluded that the spiral artery and the related intervillous space do not offer a limiting resistance to maternal blood flow in the absence of uterine contractions.

The cholinergic system in tissues without innervation: Choline acetyltransferase, choline and acetylcholine in the placenta of the rhesus monkey ( Macaca mulatta)

Six near-term placentae from rhesus monkeys ( Macaca mulatta) were analyzed for their contents of choline acetyltransferase (ChAc), acetylcholine (ACh) and choline (Ch). ChAc was measured by a radiometric assay in the presence of [1- 14C]acetylcoenzyme A and choline and distinguished from other acetyltransferases by a differential assay involving acetylcholinesterase or selective ion pair extraction of [1− 14C]ACh with tetraphenylboron. At 150 days of gestation the rhesus placenta synthesized 4.067 ± 0.737 μmoles ACh/g wet weight/hr. Ch and ACh were determined with a radiochemical method based on the phosphorylation of free Ch by choline kinase in the presence of [ 32P]ATP. ACh was first isolated by ion pair extraction and high voltage electrophoresis. Tissue levels of Ch ranged from 737 to 3892 and ACh from 8.8 to 29.0 (nmoles/g wet weight). The rhesus monkey appears to be a suitable animal model to study the physiological significance of ACh in the placenta in vivo.

Duration of chorionic gonadotropin production by the placenta of the rhesus monkey.

Concentrations of macaque chorionic gonadotropin (mCG) in placenta, blood and urine of rhesus monkeys have been measured by both radioimmunoassay and bioassay throughout gestation. mCG was easily detected and quantified in these specimens for a brief period in early pregnancy, but was not detectable between the 40th day of pregnancy and term in placental extracts, serum, or 40-fold urine concentrates. The apparent absence of mCG after the 40th day of pregnancy makes these macaques a valuable model for pregnancy research, where the absence of chorionic gonadotropin is experimentally desirable. Unlike women and some higher primates, the functional status of the fetal, placental and maternal endocrine compartments of macaques can be studied in the absence of circulating chorionic gonadotropin during mid and late gestation.

An electron microscopic study of virus particles in rhesus monkey placenta.

Examination of chorionic villi from rhesus monkey placenta revealed the presence of C-type virus particles budding from syncytial trophoblast, pericytes, Hofbauer cells, and mesenchyme. In addition, particles, were found budding from cells of the cytotrophoblastic cell column and decidual basalis. They measured 30 nm in diameter, had a dense central core, surrounded by a narrow, electron-lucent zone, and were enclosed by an outer unit membrane.

The effect of fetal sex on placental biosynthesis of progesterone.

The concentration of endogenous progesterone (P4) in the rhesus placenta ranged from 1.9 to 9.9 ng/100 mg tissue (Mean ± SE = 4.9 ± .82). There was no correlation between the sex of the fetus and the endogenous P4 in placental tissue. However, when placental slices were incubated without added substrate, female placentas produced significantly more P4 than male placentas. The addition of cholesterol to the incubations did not increase P4 production over control levels. Progesterone biosynthesis from both male and female placentas increased significantly over control levels when pregnenolone (P5) was added to the media. The total production of P4 with P5 tended to be greater for male placentas than for female placentas, but the difference was not significant. However, the net increase over control levels in P4 production with P5 was greater for male placentas than for female placentas. These observations indicate that the step(s) in progesterone biosynthesis that are affected by the fetal genotype lie betw...

In vitro metabolism of palmitic acid and glucose in the developing tissue of the rhesus monkey

Fetal, newborn, and adult tissues of rhesus monkeys metabolize glucose-U- 14 C and palmitate-1- 14 C to lipids and 14 CO 2 in vitro. During growth and development, differences in substrate utilization are detected. In the lung, phospholipid synthesis with the use of either substrate increases as development proceeds. In the brain, lipid synthesis diminishes after birth, though glucose utilization and 14 CO 2 production from glucose-U- 14 C increase. The metabolism of liver lipids varies with age, the term fetus and newborn infant converting less palmitate to lipids than the young fetus or the adult animal. Most of the radioactivity of palmitate is found in the 16 C fatty acid fraction of the various lipids. In the newborn infant, the catabolism of glucose-U- 14 C or palmitate-1- 14 C increases in the brain and lung while palmitate curtails glucose utilization in these tissues. Since palmitate has been shown to cross the rhesus monkey placenta in vitro and in vivo, to curtail glucose utilization, and to be incorporated into fetal lipids, the present data indicate that fatty acids of either maternal or fetal origin can, along with glucose, be utilized for the formation of fetal lipids. In fetal tissues, the conversion of palmitate to carbon dioxide is small. Therefore, a limited but steady flow of fatty acids of maternal origin may account for a large proportion of total fetal tissue lipid deposition. The data indicate that, while glucose metabolism is essential to the primate fetus, lipid synthesis from fatty acids may be more important than it is presently considered.