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Doppler ultrasound velocimetry for timing the second intrauterine transfusion in fetuses with anemia from red cell alloimmunization.

OBJECTIVE: Middle cerebral artery peak systolic velocity has been successfully used for timing the first cordocentesis in fetuses who are at risk for anemia because of maternal red cell alloimmunization. The effects on Doppler velocimetry after the intrauterine transfusion of adult blood to these fetuses are unknown. The objective of this study was to assess the applicability of Doppler methods for the prediction of severe anemia in fetuses who had undergone 1 previous intrauterine transfusion. STUDY DESIGN: Doppler examination of middle cerebral artery peak systolic velocity was performed before cordocentesis in 64 fetuses who had undergone 1 previous intrauterine transfusion. Timing of the second intrauterine transfusion was based on traditional criteria. Anemia was defined as mild (hemoglobin value between 0.84 and 0.65 multiples of the median), moderate (hemoglobin value <0.65-0.55 multiples of the median), and severe (hemoglobin value <0.55 multiples of the median). Receiver operator characteristic curves were created to select threshold values to identify the 3 degrees of anemia with a sensitivity of 100%. RESULTS: Gestational age at the Doppler study ranged from 19 to 36 weeks. Forty-six fetuses (72%) were not or mildly anemic; 7 fetuses (11%) were moderately anemic, and 11 fetuses (17%) were severely anemic. Middle cerebral artery peak systolic velocity for the prediction of severe, moderate, and mild anemia at a sensitivity of 100% showed false-positive rates of 6%, 37%, and 70%, respectively. CONCLUSION: In fetuses who have undergone 1 previous intrauterine transfusion because of maternal red cell alloimmunization, timing the second intrauterine transfusion can be determined noninvasively by Doppler ultrasonography on the basis of an increase in the peak velocity of systolic blood flow in the middle cerebral artery.

Anemia↗

Red cell mass responses to daily erythropoietin and iron injections in the ovine fetus.

OBJECTIVE: Anemic ovine fetuses supplemented with intra-amniotic iron undergo rapid expansion of red cell mass. The present study tested the hypothesis that nonanemic fetuses that were supplemented with daily intra-amniotic iron plus intravascular injections of erythropoietin would experience accelerated erythrocyte production. STUDY DESIGN: Nine late gestation ovine fetuses received 100 to 120 units of erythropoietin intravascularly plus 10 mg of iron intra-amniotically daily for 7 days (low erythropoietin dose group). Four additional fetuses received 1000 units of erythropoietin plus 10 mg iron daily for the same period (high erythropoietin dose group). Responses were compared with 9 nonsupplemented time-control fetuses. Statistical testing was by 3-factor repeated measures analysis of variance. RESULTS: Immediately after erythropoietin injection, plasma erythropoietin concentration was elevated approximately 25- and 250-fold in the low and high erythropoietin dose groups, respectively. Erythropoietin returned to basal levels by 24 hours after the injection. Plasma iron concentration increased in the low erythropoietin dose group but not in the control or high erythropoietin dose groups. Reticulocyte index increased in both erythropoietin supplemented groups but not in control fetuses. Hematocrit level increased above control by day 5 in the low erythropoietin dose group and by day 2 in the high erythropoietin dose group. Red cell mass increased significantly on supplement day 7 in the low erythropoietin dose group and on day 5 in the high erythropoietin dose group. Fetal blood gases and pH were unchanged with time in all 3 groups. CONCLUSION: Although daily combined erythropoietin and iron supplements in nonanemic ovine fetuses significantly increased circulating red cell mass in a dose-dependent manner, this increase was small relative to the rapid expansion of red cell mass previously observed after iron supplementation in fetuses with hemorrhage-induced anemia. We speculate that this difference in response may be due to a combination of rapid fetal clearance of erythropoietin plus a relative insensitivity to erythropoietin caused by the absence of other cytokines, which are elevated during fetal anemia.

Amnion↗

The placental transfer of cerium: experimental studies and estimates of doses to the human fetus from 141Ce and 144Ce.

PURPOSE: To measure the transfer of cerium from mother to fetus in experimental animals and estimate doses to the human fetus following intakes of radioisotopes of Ce. MATERIALS AND METHODS: Cerium-141 in chloride solution was administered intravenously to rats at different stages of pregnancy (days 9.5, 12.5 or 18.5), and retention in the embryo/fetus and associated tissues was measured 3 days later in each case. Retention in rat fetal tissues on day 21.5 (shortly before birth) was also measured after administration of 141Ce chloride 1 month prior to conception or 141Ce citrate on day 18.5. Cerium-141 chloride was administered to guinea pigs on day 50 for measurements of fetal retention on day 57 (shortly before birth). RESULTS: Retention of 141Ce in the rat embryo/fetus, measured at 3 days after administration to the mother, increased from about 0.00002% of injected activity per embryo/fetus on day 12.5 to about 0.014% on day 21.5 of gestation. However, the relative concentrations of 141Ce in the embryo/fetus and mother (CF:CM ratio) were between 0.005 and 0.01 in each case. After 141Ce administration prior to conception, retention by the rat fetus on day 21.5 was substantially lower than after short-term administration. Comparison of retention of 141Ce on day 21.5 after administration on day 18.5 as either chloride or citrate showed similar levels in maternal tissues but greater transfer to the fetus (CF:CM ratio of 0.03). Retention in the guinea pig fetus in late gestation at 7 days after administration of (141)Ce chloride was about 0.05% injected activity per fetus, corresponding to a CF:CM ratio of about 0.02. CONCLUSION: These results and other published animal data have been used to specify CF:CM ratios for use in the calculation of doses to the human fetus. The values used were 0.05 for intakes during pregnancy and 0.01 for intakes prior to conception. Doses to the offspring after maternal ingestion of 141Ce or 144Ce are largely due to irradiation from activity in the maternal colon and are insensitive to CF:CM. After inhalation, however, absorption of Ce to blood is much greater and doses to the offspring are dominated by the contribution from activity in the fetus, and therefore dependent on the CF:CM ratio used.

Animals↗

Increased chromosome aberration levels in cells from mouse fetuses after zygote X-irradiation.

Cell cultures derived from skin biopsies of mouse fetuses X-irradiated at the zygote stage (2 Gy) were studied for the presence of chromosomal aberrations and micronuclei. In comparison with cells from control, unirradiated fetuses significantly higher spontaneous frequencies of aberrations per 100 metaphases and aberrant metaphases were found in cells which were obtained from normal fetuses and fetuses with gastoschisis after irradiation of the zygote. The proportion of aberrant metaphases in cells from fetuses with gastroschisis but not the frequency of aberrations per 100 cells was significantly higher than in cells from normal fetuses. Spontaneous aberrations were mainly chromatid and chromosome fragments. A significantly higher proportion of cells with micronuclei was observed in cells from fetuses with gastroschisis than in cells from control or normal fetuses after irradiation. The induction of chromosome instability in fetal fibroblasts after zygote irradiation has not previously been reported.

Animals↗

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation.

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.

Animals↗

Genome-wide epigenetic alterations in cloned bovine fetuses.

To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.

5-Methylcytosine↗

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses.

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.

Animals↗

Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses.

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

5-Methylcytosine↗

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.

Animals↗

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies.

OBJECTIVE: To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. DESIGN: Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis. PROCEDURE: The results of identification of 99 bacterial strains as determined by conventional phenotyping or by polymerase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis; the remaining strains were field isolates putatively identified as C fetus. In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling. RESULTS: The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories. CONCLUSION: Our results indicate that misidentification of C fetus in routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.

Animals↗

The effect of pancreatectomy on the uptake of metabolites by the sheep fetus.

The effects of fetal pancreatectomy on the uptake of oxygen, glucose and lactate by the fetus, uterus and uteroplacental tissues were investigated in chronically catheterized sheep during the last third of gestation. Pancreatectomy reduced the uptake of glucose by the fetus but had little apparent effect on glucose uptake by the uterus or uteroplacental tissues. The mean umbilical uptake of glucose by the pancreatectomized fetuses was 10.4 +/- 1.1 mumol/kg . min (n = 4) which was significantly less than the value observed in the intact fetuses (24.5 +/- 2.7 mumol/kg . min, n = 4, P less than 0.01). When all the data from the pancreatectomized and intact fetuses were combined, there was a significant positive correlation between the plasma insulin concentration in utero and the umbilical uptake of glucose (r = 0.95, n = 14, P less than 0.01). The glucose/oxygen quotient in the pancreatectomized fetuses (0.32 +/- 0.02, n = 9) was significantly less than the value observed in the intact fetuses (0.59 +/- 0.04, n = 6, P less than 0.01). There was also a significant positive correlation between the glucose/oxygen quotient and the plasma concentration of insulin in the fetus (r = 0.73, n = 59, P less than 0.01). Pancreatectomy had no significant effect on the fetal lactate/oxygen quotient or on the uptake of oxygen or lactate by any of the tissues. These observations demonstrate that the endogenous concentration of insulin is a physiological regulator of glucose uptake by the fetal tissues.

Animals↗

Effect of carotid denervation on plasma vasopressin levels during acute hypoxia in the late-gestation sheep fetus.

1. We measured plasma concentrations of arginine vasopressin (AVP), arterial, venous and amniotic pressures, and carotid and femoral blood flows in fifteen chronically instrumented fetal sheep at 119-125 days of gestation. In eight of the fetuses the carotid sinus nerves were cut (denervated fetuses); the other seven remained intact and served as controls (intact fetuses). 2. In the intact fetuses during hypoxia there was an increase in plasma [AVP] and in perfusion (arterial-venous) pressure, a transient bradycardia, and an increase in carotid and a decrease in femoral blood flow. Whilst femoral vascular resistance (perfusion pressure/femoral blood flow) increased, there were no changes in carotid vascular resistance during hypoxia. 3. In the denervated fetuses no significant bradycardia, fall in femoral blood flow or increase in femoral vascular resistance was present soon after the onset of hypoxia but plasma AVP increased to similar concentrations to those observed in intact fetuses during hypoxia. 4. We conclude that carotid denervation does not affect plasma [AVP] during hypoxia in fetal sheep. This suggests that (1) AVP release during hypoxia is not mediated by a carotid chemoreflex and (2) AVP does not play an important role in these initial fetal cardiovascular responses. Furthermore, we previously reported that intact fetuses survive acute hypoxia better than denervated fetuses following phentolamine treatment, and we believe this to be due to the action of a non-alpha-adrenergic vasoconstrictor released in part via a carotid chemoreflex. The present results suggest that this vasoconstrictor is not AVP.

Acute Disease↗

Effects of prevailing hypoxaemia, acidaemia or hypoglycaemia upon the cardiovascular, endocrine and metabolic responses to acute hypoxaemia in the ovine fetus.

Although it is established that the fetus can successfully withstand a single, acute hypoxaemic challenge during gestation, little is known about what effects prevailing adverse intrauterine conditions might have on the fetal response to acute hypoxaemia. The aims of this study were therefore: (1) to characterise the effects of prevailing and sustained hypoxaemia, acidaemia or hypoglycaemia on the fetal cardiovascular responses to an episode of acute hypoxaemia; and (2) to determine the effects of these adverse intrauterine conditions on mechanisms mediating these cardiovascular responses. Thirty-three Welsh Mountain sheep fetuses were chronically instrumented (1-2 % halothane) between 117 and 125 days of gestation (term is ca 145 days) with amniotic and vascular catheters and with a transit-time flow probe around a femoral artery. The animals were divided retrospectively into four groups based upon post-surgical, sustained, basal blood oxygen (chronically hypoxaemic; P(a,O2), 17.3 +/- 0.5 mmHg; n = 8), glucose (chronically hypoglycaemic; blood glucose, 0.49 +/- 0.03 mmol l(-1); n = 6) and acid-base (chronically acidaemic; pH(a), 7.25 +/- 0.01; n = 5) status. Values for compromised fetuses were -2 S.D. from a group of control (n = 14) fetuses. At 130 +/- 4 days, a 1 h episode of acute, isocapnic hypoxaemia (9 % O(2) in N(2), to reduce carotid P(a,O2) to 12 +/- 1 mmHg) was induced in all fetuses by reducing the maternal inspired O(2) fraction (F(I,O2)). Fetal cardiovascular variables were recorded at 1 s intervals throughout the experimental protocol and arterial blood samples taken at appropriate intervals for biophysical (blood gases, glucose, lactate) and endocrine (catecholamines, vasopressin, cortisol, ACTH) measures. During acute hypoxaemia all fetuses elicited hypertension, bradycardia and femoral vasoconstriction. However, prevailing fetal compromise altered the cardiovascular and endocrine responses to a further episode of acute hypoxaemia, including: (1) enhanced pressor and femoral vasoconstriction; (2) greater increments in plasma noradrenaline and vasopressin during hypoxaemia; and (3) basal upward resetting of hypothalamic-pituitary-adrenal axis function. Only chronically hypoxaemic fetuses had significantly elevated basal concentrations of noradrenaline and enhanced chemoreflex function during acute hypoxaemia. These data show that prevailing adverse intrauterine conditions alter the capacity of the fetus to respond to a subsequent episode of acute hypoxaemia; however, the partial contributions of hypoxaemia, acidaemia or hypoglycaemia to mediating these responses can vary.

Acid-Base Equilibrium↗

Nitric oxide plays a role in the regulation of adrenal blood flow and adrenocorticomedullary functions in the llama fetus.

The hypothesis that nitric oxide plays a key role in the regulation of adrenal blood flow and plasma concentrations of cortisol and catecholamines under basal and hypoxaemic conditions in the llama fetus was tested. At 0.6-0.8 of gestation, 11 llama fetuses were surgically prepared for long-term recording under anaesthesia with vascular and amniotic catheters. Following recovery all fetuses underwent an experimental protocol based on 1 h of normoxaemia, 1 h of hypoxaemia and 1 h of recovery. In nine fetuses, the protocol occurred during fetal I.V. infusion with saline and in five fetuses during fetal I.V. treatment with the nitric oxide synthase inhibitor L-NAME. Adrenal blood flow was determined by the radiolabelled microsphere method during each of the experimental periods during saline infusion and treatment with L-NAME. Treatment with L-NAME during normoxaemia led to a marked fall in adrenal blood flow and a pronounced increase in plasma catecholamine concentrations, but it did not affect plasma ACTH or cortisol levels. In saline-infused fetuses, acute hypoxaemia elicited an increase in adrenal blood flow and in plasma ACTH, cortisol, adrenaline and noradrenaline concentrations. Treatment with L-NAME did not affect the increase in fetal plasma ACTH, but prevented the increments in adrenal blood flow and in plasma cortisol and adrenaline concentrations during hypoxaemia in the llama fetus. In contrast, L-NAME further enhanced the increase in fetal plasma noradrenaline. These data support the hypothesis that nitric oxide has important roles in the regulation of adrenal blood flow and adrenal corticomedullary functions during normoxaemia and hypoxaemia functions in the late gestation llama fetus.

Adrenal Cortex↗

Serological response of the bovine fetus to bovine viral diarrhea virus.

Susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 days of gestation and earlier. Fetuses were surgically collected at selected times after inoculation. Serum immunoglobulins were quantitated, and the presence of specific antibodies was determined. In fetuses from heifers inoculated at 150 days, immunoglobulin M (IgM) appeared approximately 2 weeks after inoculation and was followed in 7 days by IgG1. Later IgG2 was detected in the sera of three fetuses. Serum-neutralizing and complement-fixing antibodies were first detected in a fetus taken at 206 days of gestation. Fetuses taken at later times also had specific serum antibodies. Possible explanations for the appearance of serum immunoglobulin substantially before specific bovine viral diarrhea antibodies include the viral alteration of host tissues rendering them antigenic viral activation of polyclonal B cells, and viral modulation of virus-specific lymphocytes causing specific interference with the appearance of antiviral antibodies. In one of the fetuses having IgG2, the serum also contained IgA. Placental leakage of material immunoglobulins was thought to be responsible for the presence of IgA and IgG2 in this fetus. Small quantities of IgM were found in the serum of two fetuses taken from heifers inoculated between 65 and 95 days of gestation, but specific antibodies were found in none.

Animals↗

Antigenic differences among Campylobacter fetus S-layer proteins.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.

Amino Acid Sequence↗

Creatinine exchange between mother, fetus, and amniotic fluid.

Creatinine exchange between mother, fetus, and amniotic fluid was investigated in rhesus monkeys in late pregnancy in which a different radioactive-labeled form of creatinine was injected simultaneously into either of two of the three possible compartments. Creatinine specific activity was then determined for each isotope in each compartment over the ensuing 4-6 h. A rapid and extensive bidirectional exchange occurred between all three compartments. Maternal-fetal flux, while bidirectional, seemed to be predominately from mother to fetus. Approximately 35% of labeled creatinine originally injected into the fetus and 25% of labeled creatinine originally injected into the mother were present in the amniotic sac within 2 h. Creatinine pool size was calculated to be approximately 6 mg/kg in both mother and fetus. Comparison of experiments involving living had dead fetuses indicated that the route of creatinine transfer from mother to amniotic fluid was through the living fetus and directly across the chorioamnion in the dead fetus. In addition, the time required for 'mixing' of creatinine injected into the amniotic sac was longer with a dead than with a living fetus but its half-life averaged 4.5 h regardless of fetal status.

Amnion↗

Histochemistry of connective tissue cells in subcutaneous adipose tissue of normal and decapitated pig fetuses.

Connective tissue cells that are histochemically and morphologically distinct from 'fibroblasts' are localized around developing hair follicles in the pig and rat. Immature adipose tissue is limited to small areas immediately around fully descended hair follicles in the rat hypodermis. In the present study, connective tissue around large nerves and blood vessels in fetal pig subcutaneous tissue was examined for the presence of enzymes typical of adipocytes. Samples from decapitated pig fetuses were studied so that the effects of an altered hormonal profile could be examined. Samples of dorsal subcutaneous adipose tissue were obtained from fetuses at 65, 70, 85, 90, 110, and 112 days of gestation. Fetuses were decapitated in utero at 45 days of gestation, and adipose tissue samples were obtained from these fetuses at 110 days of gestation. A close spatial relationship was observed between the growth of large blood vessels and nerves and fat cell cluster development in the older (greater than 70 days) fetuses. Connective tissue cells that were contiguous with fat cell clusters were histochemically identical to adipocytes. The lipid histochemistry of the reactive connective tissue cells (histochemically identical to adipocytes) was variable in young fetuses. In all 110-and 112-day-old fetuses, the reactive cells contained lipid, whereas the reactive cells in decapitated fetuses were devoid of lipid. The reactive connective tissue cells were not associated with capillaries and did not contain basement membranes. The histochemistry of these cells suggests that they respond to a particular hormonal or metabolic profile as do adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗