Fetal Monkey Research Articles

An adult-like pattern of ocular dominance columns in striate cortex of newborn monkeys prior to visual experience

In macaque monkeys, the geniculocortical afferents serving each eye segregate in layer IVc of striate cortex during early life into a pattern of alternating inputs called ocular dominance columns. It has been disputed whether visual experience is necessary for the formation of ocular dominance columns. To settle this issue, fetal monkeys were delivered prematurely by Caesarean section at embryonic day 157 (E157), 8 d before the end of normal gestation. To avoid light exposure, the Caesarean section and all subsequent feedings and procedures were done in absolute darkness, using infrared night-vision goggles. Tritiated proline was injected into the right eye 1 d after delivery (E158). One week later at postnatal age 0 (P0), the equivalent of a full-term pregnancy (E165/P0), alternate sections of unfolded and flattened visual cortex were prepared for autoradiography or cytochrome oxidase (CO). All three newborns studied at E165/P0 had well segregated ocular dominance columns organized into the characteristic mosaic present in adults. In the upper layers, a mature pattern of CO patches (also known as blobs or puffs) was visible, aligned with the ocular dominance columns in layer IVc. Every other row of patches in layers II, III was labeled by [3H]proline. In V2, a distinct system of alternating thick-pale-thin-pale CO stripes was present. These findings indicate that stimulation of the retina by light is not necessary for the development of columnar systems in the visual cortex. Ocular dominance columns, patches, and V2 stripes all are well formed before visual experience. Even the thalamic input to the patches in the upper layers of striate cortex is segregated by eye in newborns.

Clonage de lymphocytes T infiltrant l'artère temporale et circulant au cours de la maladie de Horton

Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.

Genesis of the retinal pigment epithelium in the macaque monkey.

The development of the retinal pigment epithelium (RPE) was studied in rhesus monkey (Macaca mulatta) fetuses, neonates, and juveniles exposed to a pulse of 3H-thymidine (3H-TdR) between embryonic day (E) 25 and postnatal day (P) 204 and examined at short and long intervals after the injection of the isotope. The RPE develops from the outer layer of the optic cup which by E45 consists of a multistratified epithelium. The outer layer appears immature near the retina's edge and gradually becomes monostratified and more mature centrally. Even at this early stage, all cells contain pigmented melanosomes, although peripherally the pigment is limited to the apical portion of the cells. Examination of autoradiograms from animals allowed to survive for several postnatal months shows that monkey RPE cell genesis begins just after E27, increasing to a peak frequency of 0.38 cells/mm at E43. Between E30 and E85 the density of radiolabelled cells varies within a restricted range of from 0.2 to 0.4 cells/mm (mean = 0.25 +/- 0.09). From the density of radiolabelled cells, and data on the overall density of RPE cells in the juvenile retina, we determined the labelling index. During the first half of gestation, between 0.38% and 0.99% (mean = 0.65 +/- 0.22) of RPE cells are generated during the short interval of isotope availability after pulse injection. Approximately 5% of RPE cells were generated by E33, and 50% by E71. After E85, RPE cytogenesis begins gradually to decrease, and 95% of the cells have been generated by the time of birth. Continued, very low density (0.01 cells/mm) cytogenesis in the RPE is seen at P17, and persists at least until seven months postnatally. RPE cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in both foveal and perifoveal regions by E56, at which time labelled cells first begin to appear peripheral to the equator. Besides the timing differences, RPE genesis in the central retina differs from that in the peripheral retina in that it proceeds at a higher rate, and lasts for a shorter time period. A prolonged postnatal period of low density RPE cell genesis persists in both central and peripheral retina. Comparison of the pattern of expansion of the area containing radiolabelled cells in the RPE and neuroretina demonstrates a remarkable spatial and temporal correspondence. Close analysis suggests that at any point on the retina, the last cells are generated in the neuroretina slightly before the last cells in the RPE.

Chronic cocaine exposure in the fetal rhesus monkey: consequences for early development of dopamine neurons

The effects of cocaine on the development of neurons containing tyrosine hydroxylase (TH) were investigated in fetal rhesus macaques. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of pregnancy until days 40 or 60. Cocaine concentration in plasma from females thus treated was approximately 800 ng/ml 10-20 min following injection. At the time of C-section, plasma levels of cocaine in fetal blood were 231 +/- 70 ng/ml. The brains from 40 and 60 d old fetuses were examined using immunocytochemistry and in situ hybridization. The appearance of neurons containing TH by day 40 of gestation was not different between control and cocaine-treated fetal monkeys. In both groups (N = 3 each) TH-IR neurons and TH mRNA were located in the mesencephalon and dorsal hypothalamus by day 40, and fiber projections extended to the developing striatum. Also in the day 60 fetuses, the TH-IR neurons were distributed similarly in both groups (N = 5 each), but the TH mRNA content, measured by quantitative in situ hybridization, was reduced in the substantia nigra (SN) and ventral tegmental area (VTA) after cocaine treatment. These data suggest that exposure to cocaine in fetal life does not affect the development of TH or the expression of its mRNA on day 40 of gestation. By day 60, however, the expression of TH mRNA was significantly reduced. This latter effect can be explained by reduced dopamine synthesis in the cocaine-treated fetuses.

Effects of Viral Virulence on Intrauterine Growth in SIV-Infected Fetal Rhesus Macaques (Macaca mulatta)

Studies with a simian immunodeficiency virus (SIV)-infected fetal monkey model were conducted with a focus on fetal growth and viral pathogenesis. Twenty-six fetuses were inoculated in utero via ultrasound guidance with an uncloned pathogenic strain of SIV or vehicle during the second or third trimesters [gestational day (GD) 65, 110, or 130], sonographically monitored weekly (biometrics, blood flow), then necropsied at incremental time points postinfection. Peripheral blood hematologic (complete blood counts, clinical chemistries), immunologic (immunophenotyping), and endocrine studies [insulin-like growth factor (IGF), IGF-binding proteins (IGFBP)] were conducted. Severe intrauterine growth restriction (IUGR), oligohydramnios, and altered lymphocyte counts were noted for fetuses infected on GD 65. Less severe effects were detected for fetuses inoculated at the later time points, with severity dependent upon the length of SIV infection in utero. IGF studies indicated significant reductions in IGF-I and elevated immunoreactive levels of IGFBP-3 in infected fetuses during the third trimester. Parallel studies conducted with four fetuses infected on GD 65 with a nonpathogenic, molecularly cloned virus (SIVmac1A11) resulted in normal fetal growth, with no effects on hematopoiesis or IGF/IGFBP levels, and no evidence of clinical disease. Taken together, these studies show that (1) infection of fetuses during the early second trimester with an uncloned pathogenic strain of SIV results in severe IUGR and a disruption in the molar ratio of IGF:IGFBP-3, and (2) outcome of fetal SIV infection is determined by the timing of infection and the virulence of the viral inoculum.

Neurotransmitter receptors in the proliferative zones of the developing primate occipital lobe.

Film autoradiography was used to investigate the expression of several neurotransmitter receptor subtypes in the transient ventricular and subventricular proliferative zones of the developing occipital lobe in two groups of macaque monkey fetuses. The first group of fetuses were between 60 and 93 days after conception (E60-E93), when the ventricular and subventricular zones of the monkey occipital lobe produce neurons destined for the visual cortex. In the second group, fetuses were between E107 and E128, after generation of cortical neurons has ceased. In the E60-E93 group of fetuses, ventricular and subventricular zones displayed high densities of 5-HT1-serotonergic, D1-dopaminergic, alpha 1- and alpha 2-adrenergic and high affinity kainate receptors. The activation of these receptors has previously been shown to stimulate cell proliferation in other cell systems. The possible involvement of these receptors in regulation of neuronal production is also supported by their absence in the deep laminae of the embryonic cerebral wall after E107, after cortical neurogenesis has been completed. The only exception is a high density of alpha 2-adrenergic receptors maintained near the ventricular surface long after all cortical neurons have been generated. We also found that during neurogenesis, proliferative zones in E66-E90 fetuses displayed virtually no 5-HT2-serotonergic, D2-dopaminergic, beta-adrenergic, M1-muscarinic cholinergic, gamma aminobutyric acid (GABA)A, N-methyl-D-aspartate (NMDA), or alpha-amino-3-hydroxy-5-menthy-4-isoxazole proprionate (AMPA) sites; most of these receptor subtypes have been reported to mediate the suppression of cell proliferation. The present findings suggest that dividing and/or newly generated cortical neurons are capable of receiving specific signals from multiple neurotransmitters present in their environment.

Transplantation of the fetal olfactory placode restores reproductive cycles in female rhesus monkeys (Mucaca mulatta) bearing lesions in the medial basal hypothalamus.

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.

Hematologic and growth-related effects of frequent prenatal ultrasound exposure in the long-tailed macaque ( Macaca fascicularis)

Prior investigations have shown that reduced birth weights and transient neutropenias result from frequent exposure of monkey fetuses to ultrasound. To further explore these findings, 26 animals were studied (16 exposed, 10 controls; “triple mode”; ATL Ultramark 9 with HDI ®; I SPTAd ∼ 645 to 714 mW/cm 2). Exposures were performed daily for 5 days each week from gestational days (GD) 21 to 35 (5 min), three times weekly from GD 36 to 60 (5 min), then weekly from GD 61 to 153 ± 1 (10 min). Fetal blood samples (FBS) were collected for complete blood counts (CBCs), hematopoietic progenitor assay, circulating insulin-like growth factors (IGF-I, IGF-II) and binding proteins (IGFBP-3) (GD 120, 140, 153 ± 1). Animals were delivered by Cesarean section at term (GD 153 ± 1), and body weights, morphometrics, CBCs, and bone marrow aspirates assessed at delivery and postnatally for 3 months. Fetal neutropenias were noted in exposed animals in addition to reduced circulating progenitors (colony forming unit-granulocytemacrophage [CFU-GM]). Growth of CFU-GM from bone marrow was exuberant at term, whereas circulating levels were diminished comparable to prenatal samples. Exposed animals were smaller at birth; marked reductions in IGFBP-3 were noted prenatally. These data suggest that frequent prenatal ultrasound exposure can transiently alter the neutrophil lineage, although these findings may be the result of enhanced margination and organ sequestration. Data also suggest that transient, altered growth patterns may be due to perturbations of the IGF axis.

A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth.

Gastrin-releasing peptide (GRP) is developmentally expressed in human fetal lung and is a growth factor for normal and neoplastic lung but its role in normal lung development has yet to be clearly defined. In this study we have characterized the expression of GRP and its receptor in fetal rhesus monkey lung and determined the effects of bombesin on fetal lung development in vitro. By RNA blot analysis, GRP mRNA was first detectable in fetal monkey lung at 63 days gestation, reached highest levels at 80 days gestation, and then declined to near adult levels by 120 days gestation; a pattern closely paralleling GRP expression in human fetal lung. As in human lung, in situ hybridization localized GRP mRNA to neuroendocrine cells though during the canalicular phase of development (between 63-80 days gestation) GRP mRNA was present not only in classic pulmonary neuroendocrine cells, but also in cells of budding airways. Immunohistochemistry showed that bombesin-like immunoreactivity was present in neuroendocrine cells, but not in budding airways, suggesting that in budding airways either the GRP mRNA is not translated, is rapidly secreted, or a related, but different RNA is present. RNase protection analysis using a probe to the monkey GRP receptor demonstrated that the time course of receptor RNA expression closely paralleled the time course of GRP RNA expression. In situ hybridization showed that GRP receptors were primarily expressed in epithelial cells of the developing airways. Thus GRP would appear to be secreted from neuroendocrine cells to act on target cells in developing airways. This hypothesis was confirmed by organ culture of fetal monkey lung in the presence of bombesin and bombesin antagonists. Bombesin treatment at 1 and 10 nM significantly increased DNA synthesis in airway epithelial cells and significantly increased the number and size of airways in cultured fetal lung. In fact, culturing 60 d fetal lung for 5 d with 10 nM bombesin increased airway size and number nearly to that observed in cultured 80 d fetal lung. The effects of bombesin could be blocked by specific GRP receptor antagonists. Thus this study demonstrates that GRP receptors are expressed on airway epithelial cells in developing fetal lung and that the interaction of GRP with the GRP receptor stimulates airway development.

Effectiveness of spiramycin for treatment of congenital Toxoplasma gondii infection in rhesus monkeys.

The effectiveness of spiramycin for the treatment of rhesus monkey fetuses congenitally infected with Toxoplasma gondii was studied. Eight monkeys were infected at day 90 of pregnancy. This is comparable to the second trimester of organogenetic development in humans. Transmission of infection was found prenatally in five of the eight monkeys by detection of the parasite in the amniotic fluid. Treatment with spiramycin (20 mg/kg/day in two intermittent doses given intravenously) was started as soon as fetal infection was proven and was continued until birth. Nine to 14 days after initiation of treatment, the parasite was still detectable in amniotic fluid samples from four of these five cases. However, the parasite was detected only by PCR and not by mouse inoculation. T. gondii was also detected only by PCR in the placenta of one monkey that delivered prematurely. This monkey received spiramycin treatment for only 2 weeks. In the four monkeys that received treatment for about 7 weeks, the parasite was not present at birth in the placenta nor in amniotic fluid or neonatal organs. Spiramycin accumulates mainly in maternal tissues. Although concentrations in neonatal tissue were found to be 5 to 28 times higher than the corresponding concentrations in neonatal serum, the concentrations in neonatal tissue were still 11 to 16 times lower than those found in the mothers. However, no spiramycin was found in the fetal brains. Early treatment with spiramycin may prevent transmission of infection to the fetus but most probably cannot interrupt an existing brain infection, which is the most severe outcome of congenital toxoplasmosis in humans.

An array of early differentiating cones precedes the emergence of the photoreceptor mosaic in the fetal monkey retina.

We previously have demonstrated that approximately 10% of cones in the fetal monkey retina precociously express the red/green opsin. These data suggested the possibility that a subset of cones differentiates prior to their nascent cone neighbors. To further assess this early cone differentiation in the fetal monkey retina, we used monoclonal antibodies proven to be important developmental markers of photoreceptor phenotypes and synaptogenesis (XAP-1, specific to photoreceptor membranes; SV2, specific to synaptic vesicle protein). Although these two antibodies recognize functionally distinct antigens, our analyses revealed that both identify a subset of precociously immunoreactive cones. Further, XAP-1- and SV2-positive cones are distributed in the same pattern as precocious red/green-sensitive cones in immature regions of the fetal monkey retina. These results support the hypothesis that the primate retina possesses a spatially organized protomap that may induce the emergence of the photoreceptor mosaic and trigger the formation of color-specific pathways that include horizontal, bipolar, and retinal ganglion cells.

DNA adducts in human and patas monkey material and fetal tissues induced by platinum drug chemotherapy

Platinum-DNA adducts in placenta and blood from a woman exposed to 200 mg/m 2 of cis-diamminedichloroplatinum(II) (cisplatin) and 300 mg/m 2 diamminecyclobutanedicarboxylatoplatinum(II) (carboplatin) for ovarian cancer have been documented by cisplatin-DNA enzyme-linked immunosorbent assay (ELISA) and atomic absorbance spectrometry (AAS). Apatas monkey model was used to investigate transplacentally induced cisplatin-DNA damage in fetal tissues. During the last trimester of gestation, 5 patas monkeys were given multiple doses of cisplatin to mimic human ovarian cancer treatment. In spite of careful choice of dose and treatment conditions, cumulative toxicity occured in monkeys given doses comparable on a mg/m 2 basis to those received by the human. A total dose of 12 mg/m 2 (0.625 mg/kg body weight), given in the last trimester, supported fetal viability, and multiple tissues, taken by cesarean section, were examined in the fetal monkeys. By cisplatin-DNA ELISA and AAS, maternal tissues from the monkeys receiving the highest dose contained approximately twice as much DNA damage as the fetal tissues. A similar relationship was observed when we compared DNA adduct formation in fetal liver and biopsies of liver taken from the monkey dams at cesarean delivery. In all of the monkey pairs studied there were very significant levels of DNA damage in the placenta, and high adduct levels in brains of fetuses that survived treatment. Thus, cisplatin does cross the placenta in the patas monkey. These observations imply that the human fetus, for which the total maternal dose was approximately 5.4 mg platinum drug/kg body weight, may also have sustained some DNA damage.

Changes in morphology of elastin fibers during development of the tunica intima of monkey aorta.

The normal development of elastin fibers in the thoracic aorta was studied in fetal, young, and adult monkeys. Tissue was examined by scanning electron microscopy (SEM) after NaOH treatment and by transmission electron microscopy (TEM). The NaOH treatment of fixed tissues effectively removed collagen fibers and enabled three-dimensional visualization of the elastin fibers. In intact fetal aortae, the internal elastic lamina (IEL) was situated immediately beneath the endothelium. This IEL consisted of superficial, longitudinally arranged bundles of elastin fibrils and an underlying solid sheet containing round fenestrations. In neonates, diffuse intimal thickening was observed. In the young and young-adult monkeys, the aortae exhibited intimal thickening with slender but split IEL. One of the most important findings of this study was that elastin fibers in the intimal thickening, as well as smooth muscle cells, ran in a longitudinal fashion. This was in contrast with the elastic laminae of the media which were mainly oriented circumferentially. Subendothelial elastin fibers in this intimal thickening combined with longitudinally arranged microfibrils which formed close associations with endothelial stress fibers. In some adult monkey aortae with well-developed intimal thickening, a complex meshwork of slender elastin fibers was also found beneath the endothelium. The development of the intimal elastin fibers is discussed in relation to hemodynamic forces.

Acute effects of perinatal hypoxic insult on concentrations of dopamine, serotonin, and metabolites in fetal monkey brain

Seven monkeys (Macaca mulatta) were laparotomized under general anesthesia (halothane, nitrous oxide, oxygen). Fetal hypoxia was induced in four monkeys by occlusion of the umbilical cord with a hydraulic occluder for 5–6 min. Three sham-operated fetuses served as controls. After unclamping, the fetuses were allowed to reperfuse for 20–30 min. To monitor hypoxia, the fetal electrocardiogram was recorded continuously. Hypoxic insult was associated with a decrease in fetal heart rate during the occlusion. After reperfusion, fetuses were immediately sacrificed and neocortex regions dissected on ice, frozen on dry ice and stored at −70°C. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid were assayed by high performance liquid chromatography with electrochemical detection (HPLC/EC) in hippocampus, caudate nucleus and cortical regions. In the hippocampus, there was a significant increase in 5-hydroxyindoleacetic acid concentration. In prefrontal cortex, there was a trend toward an increase in serotonin but no effects on dopamine and homovanillic acid concentrations. Dopamine, serotonin and metabolites were not altered in the caudate nucleus. These data demonstrate that fetal hypoxia followed by reperfusion produced an increase in serotonin concentration measured within the hippocampus and selected cortical areas known to be targets of hypoxic injury.

Developmental changes in the distribution of acetylcholinesterase in the extrastriate visual cortex of the monkey

In the fetal and neonatal monkey, periodically organized regions of high activity of acetylcholinesterase were found in the visual cortical area V2 (Area 18). The acetylcholinesterase bands, like the thin and thick stripes of cytochrome oxidase, were found to run orthogonal to the area 17/18 border. During neonatal development these bands progressively narrow and finally disappear shortly after four months of age.

Neurochemical development of the hippocampal region in the fetal rhesus monkey. II. Immunocytochemistry of peptides, calcium-binding proteins, DARPP-32, and monoamine innervation in the entorhinal cortex by the end of gestation.

Material for the study came from one 126 day-old rhesus monkey fetus and two 3 day-old neonates. The immunocytochemical detection of somatostatin, neurotensin (NT), parvalbumin, calbindin D-28K, DARPP-32 as well as tyrosine hydroxylase (TH), dopamine-beta-hydroxylase and serotonin (5-HT), was carried out on serial cryostat sections of the entorhinal cortex. The authors reported in a previous paper the precocious differentiation of the entorhinal cortex in rhesus monkey fetuses and featured the conspicuous expression of calbindin D-28K, somatostatin, neurotensin, and the monoaminergic innervation during the first half of gestation. The present study shows distinct temporal profiles of neurochemical development during the second half of gestation: the dense neuropeptidergic innervation remained a constant feature; the three aminergic systems gradually increased in density; parvalbumin, unlike calbindin D-28K, was primarily expressed during the last quarter of gestation. Three other prominent features of the last quarter of gestation are illustrated: the refinement of the modular neurochemical organization of the lamina principalis externa, the delayed chemoanatomical development of the rhinal sulcus area, and the establishment of a distinct rostrocaudal pattern of neurochemical distribution. In correspondence with the cluster-like organization of the lamina principalis externa, the authors observed in the olfactory, rostral, and intermediate fields of the neonate monkey entorhinal cortex, a particular subset of pyramidal-shaped neurons: located in layer III, they were characterized by fasciculated apical dendrites ascending between the cellular islands of the discontinuous layer II and the coexpression of calbindin D-28K and DARPP-32. Besides, most of the other chemical systems displayed a distinct, area-specific, patchy distribution, except for the homogeneously distributed noradrenergic innervation. In the olfactory and rostral fields, TH positive dopaminergic fibers accumulated on the neuronal islands of layers II-III, and parvalbumin labeled fibers on those of layer III, whereas patches of 5-HT and NT-like reactive terminals were segregated between the cellular islands, overlapping the DARPP-32/calbindin D-28 K labeled dendritic bundles. At the opposite, in the intermediate field, 5-HT positive terminals overlapped the cellular islands of layer II and thin fascicles of dopaminergic fibers ran in the inter island spaces. The somatostatin-LIR innervation was apparently too dense to reveal a patchy distribution that existed at earlier developmental stages. In the caudal field, the patchy pattern was replaced by a predominant bilaminar type of distribution of NT, 5-HT, and TH-like positive afferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Fetal tissue collection from spontaneous abortions: a report from a single centre.

Due to immunological immaturity, the fetus is the ideal recipient as well as donor of haemopoietic stem cells (HSCs); thus intrauterine therapy may prove to be effective in all major haemopoietic disorders when early prenatal diagnosis is available. In man, "fetus-to-fetus" transplantation has demonstrated the possibility of grafting donor HSCs and reconstituting immunodeficient fetuses. The limitations of fetal tissue use for transplantation derive from the origin of tissues from elective abortions. Early and late live spontaneous abortions may constitute an alternative to elective abortions, but are widely considered as unsuitable for fetal tissue collection because of rapid loss of viability and/or infections. The aim of this retrospective study was to assess the number of live abortions in a population of women who underwent spontaneous abortion in a single centre. In a 19-month period, 9 spontaneous abortions alive at the moment of delivery and 8 with a heart beat at the last ultrasound scan before abortion were recorded. In 1 case, fetal liver (FL) harvesting was easily performed and the tissue was cryopreserved, subsequently thawed and injected into a monkey fetus. This case shows the feasibility of this approach. The majority of cases reviewed consisted of late abortions. These findings clearly show that fetal tissue collection from spontaneous abortions is feasible for research purposes, for postnatal FL transplantation and for intrauterine transplantation, postnatal FL transplantation and for intrauterine transplantation, provided that depletion of more mature cells is performed when FL of later gestational age are used.(ABSTRACT TRUNCATED AT 250 WORDS)

Cluster Formation of Basal-Granulated Cells in the Intestinal Villi of the Fetal Japanese Monkey (&lt;i&gt;Macaca fuscata fuscata&lt;/i&gt;)

Segi's cap, an aggregation of the basal-granulated cells in the intestinal villi of the fetal Japanese monkey was investigated by means of light microscopy and immunocytochemical techniques. Characteristic Segi's caps were observed at the tips of the villi in the duodenum and upper jejunum in fetal Japanese monkeys, aged 120 and 140 days. The Segi's cap of Japanese monkeys is characterized by the following morphological features: 1) There are relatively fewer caps per section in comparison with humans. 2) The number of basal-granulated cells of the cap is also less than in humans. 3) The characteristic indentation at the center of the Segi's cap, reported in humans, cattle and pigs is only rarely present. 4) Argentaffin cells, which are now known as serotonin-containing cells, are present in the monkey Segi's cap. 5) Somatostatin-, motilin-and CCK-immunoreactive cells are found in the monkey Segi's cap.

Effects of exogenous androgen on brain androgen receptors of the fetal rhesus monkey.

Testosterone secreted by the fetal testes masculinizes and defeminizes the nonhuman primate brain during a defined prenatal critical period. We previously demonstrated the presence of high-affinity, specific androgen receptors (AR) in the developing rhesus monkey brain, but did not present data concerning their capacity for activation. To achieve this end, we analyzed the AR content in brains from intact and gonadectomized rhesus monkey fetuses at approximately 125 days of gestation, 2 h after injection of either 500 micrograms dihydrotestosterone (DHT) or vehicle directly into the fetus. After treatment, plasma DHT concentrations increased five-fold in the fetal circulation. In gonad-intact fetuses, cytosolic AR decreased in preoptic area, medial basal hypothalamus, and septum following DHT treatment. No significant effect of DHT treatment on nuclear AR was seen. In contrast, the increased level of DHT in the maternal circulation decreased cytosolic AR and increased nuclear AR of the maternal myometrium. In gonadectomized fetuses, DHT treatment decreased cytosolic AR as it did in the intact group. In contrast, a significant increase in nuclear AR was seen in preoptic area, medial basal hypothalamus, and tegmentum of these fetuses. Thus AR in fetal rhesus brain can be activated by DHT when the gonads are removed, but not in the intact fetuses. These data suggest that AR in the developing nervous system of rhesus macaques can be activated by exogenous androgen and hence are probably functional.

A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.