Basic evaluation of a novel 4D target and human body phantom.

Stereotactic body radiation therapy (SBRT) is usually verified with a dynamic phantom or solid phantom, but there is a demand for phantoms that can accurately simulate tumor dynamics within an individual that would allow customized validation in every patient. We developed a new 4D dynamic target phantom (multi-cell 4D phantom) that allows simulation of tumor movement in patients. The basic quality and dynamic reproducibility of this new phantom was verified in this investigation. The newly developed multi-cell 4D phantom comprises four main components: soft tissue, bones, lungs, and tumor (target). The phantom structure was based on computed tomography (CT) data of a male. In this study, we investigated the basic performance of a multi-cell 4D phantom. All the CT numbers of the phantom were very close to those of human data. The geometric maximum amplitudes were 4.57 mm in the lateral direction, 4.59 mm in the ventrodorsal direction, and 3.68 mm in the cranio-caudal direction. Geometric errors were 0.84, 0.58, and 0.40 mm, respectively. Movements of the abdominal surface were stable for 60 s. Repeated measurements show no actual differences in target movements between multiple measurements and indicated high reproducibility (r > 0.97). End-to-end tests using Gafchromic film revealed a gamma pass rate of 98% or above (2 mm/3%). Although our phantom performed limited reproducibility in the movement of the patient tumor at present, a satisfactory level of precision was confirmed in general. This is a very promising device for use in the verification of radiation therapy for moving targets.

Intravenous dexmedetomidine for cesarean delivery and its concentration in colostrum.

BACKGROUND: Dexmedetomidine is a sedative agent with high α2-adrenoreceptor selectivity. We investigated intravenous dexmedetomidine administration during scheduled cesarean delivery under neuraxial anesthesia; and its concentration in the colostrum. METHODS: Twenty-seven participants having elective cesarean delivery under combined spinal-epidural anesthesia were enrolled. After delivery and cord clamping, 6µg/kg/h of intravenous dexmedetomidine was administered for 10minutes, followed by a dose of 0.7µg/kg/h until peritoneal closure. Sedation, vital signs and side effects were recorded. Blood and colostrum samples were collected from each participant at 6, 12, and 24h after dexmedetomidine administration. Samples were analysed using liquid chromatography tandem-mass spectroscopy. RESULTS: Colostrum samples were collected from 10 patients. The median [95% CI] plasma dexmedetomidine concentration was 333 [303-534] pg/ml at 0h and 19.7 [13.5-25.8] pg/ml at 6h. The colostrum concentration was 12.3 [8.1-20.1] pg/ml at 6h. The dexmedetomidine completely disappeared from both within 24h. The calculated milk-to-plasma ratio at 6h was 0.76 [0.57-0.86]. The relative infant dose was 0.034% [0.020-0.062%]. At dexmedetomidine discontinuation, the Richmond Agitation-Sedation Scale score was -2 (range,-4 to -1). During surgery, no patients complained of nausea, peritoneal irritation or afterbirth pain. CONCLUSIONS: The dexmedetomidine milk-to-plasma ratio did not exceed 1 in any participant, and the relative infant dose was very low. Maternal sedation using dexmedetomidine is unlikely to be harmful for the infant.

The expression and function of histamine H3 receptors in pancreatic beta cells.

BACKGROUND AND PURPOSE: Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin. EXPERIMENTAL APPROACH: The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H3 receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H3 receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting. KEY RESULTS: Histamine H3 receptors were expressed in pancreatic beta cells. A selective H3 receptor agonist, imetit, and a selective inverse H3 receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H3 receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H3 receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation. CONCLUSIONS AND IMPLICATIONS: Histamine H3 receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.

Low BMI at age 20 years predicts gestational diabetes independent of BMI in early pregnancy in Japan: Tanaka Women's Clinic Study.

AIMS: Maternal obesity and weight gain since early adulthood are known predictors of gestational diabetes in Western countries. However, their impact has not been evaluated well in Asia, where mean BMI levels are generally lower than in Western countries. We therefore examined the associations of BMI at age 20 years and BMI change since age 20 years with the risk of gestational diabetes in Japanese pregnant women. METHODS: Six hundred and twenty-four consecutive pregnant women without recognized diabetes before pregnancy, whose initial obstetric clinic visit was before 13 weeks' gestation, were prospectively observed. Weight at age 20 years was self-reported. Baseline height and weight measurements were obtained at the initial obstetric visit. Multivariate logistic regression analysis estimated the risk of incident gestational diabetes for BMI change since 20 years and BMI at age 20 years. RESULTS: Twenty-eight women developed incident gestational diabetes. By multivariate logistic regression analysis that adjusted for maternal age, parity and baseline BMI, we observed a statistically significant inverse association between BMI at age 20 years and incidence of gestational diabetes (odds ratio 0.68, 95% CI 0.51-0.92). Similarly, when we assessed the association of BMI change since age 20 years, adjusted for maternal age and parity, BMI change was associated with an increased risk of gestational diabetes (odds ratio 1.26, 95% CI 1.03-1.53). When we focused on the threshold of risk of gestational diabetes, women with BMI at 20 years of less than 18 kg/m(2) had a 6.30-fold (2.26-17.59) greater risk than women with both BMI at age 20 years of 18 kg/m(2) or more and BMI change since age 20 years of less than 1.85. CONCLUSIONS: Both low BMI at age 20 years and BMI change since age 20 years were significantly associated with increased risk of incident gestational diabetes.

HbA(1c) variability and the development of microalbuminuria in type 2 diabetes: Tsukuba Kawai Diabetes Registry 2.

AIMS/HYPOTHESIS: The aim of this study was to examine the association between HbA(1c) variability and the development of microalbuminuria as defined by an albumin/creatinine ratio ≥ 3.4 mg/mmol (≥ 30 mg/g) in at least two of three consecutive urine samples in Japanese patients with type 2 diabetes. METHODS: HbA(1c) level was measured in 812 serially registered normoalbuminuric adults aged 21-79 years with type 2 diabetes. After registration, a 1-year period to establish baseline values for mean HbA(1c) and HbA(1c) variability (measured as the intrapersonal SD of serially collected HbA(1c)) was decided upon. The association between HbA(1c) variability and the development of microalbuminuria was determined by Cox regression analysis after adjustment for other risk factors for microalbuminuria. RESULTS: Microalbuminuria occurred in 193 patients during the observation period of (mean ± SD) 4.3 ± 2.7 years. Even after adjustment for mean HbA(1c), HbA(1c) variability was a significant predictor of microalbuminuria independently of the mean HbA(1c); the HR for every 1% (95% CI) increase in mean HbA(1c) was 1.22 (1.06, 1.40) (p = 0.005), and that for HbA(1c) variability was 1.35 (1.05, 1.72) (p = 0.019). The effects of these two variables were quite similar when 1 SD was used; the HR for every 1 SD increase (95% CI) in HbA(1c) was 1.23 (1.07, 1.43) (p = 0.005), and that for HbA(1c) variability was 1.20 (1.03, 1.39) (p = 0.019). CONCLUSIONS/INTERPRETATION: HbA(1c) variability affects the development of microalbuminuria independently of mean HbA(1c) in type 2 diabetes. Further studies should be performed to evaluate the influence of HbA(1c) variability on other complications and in individuals of other ethnicities with type 2 diabetes.

Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice.

AIMS/HYPOTHESIS: Hyperlipidaemia is an independent risk factor for the progression of diabetic nephropathy, but its molecular mechanism remains elusive. We investigated in mice how diabetes and hyperlipidaemia cause renal lesions separately and in combination, and the involvement of Toll-like receptor 4 (TLR4) in the process. METHODS: Diabetes was induced in wild-type (WT) and Tlr4 knockout (KO) mice by intraperitoneal injection of streptozotocin (STZ). At 2 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Functional and histological analyses were carried out 6 weeks later. RESULTS: Compared with treatment with STZ or HFD alone, treatment of WT mice with both STZ and HFD markedly aggravated nephropathy, as indicated by an increase in albuminuria, mesangial expansion, infiltration of macrophages and upregulation of pro-inflammatory and extracellular-matrix-associated gene expression in glomeruli. In Tlr4 KO mice, the addition of an HFD to STZ had almost no effects on the variables measured. Production of protein S100 calcium binding protein A8 (calgranulin A; S100A8), a potent ligand for TLR4, was observed in abundance in macrophages infiltrating STZ-HFD WT glomeruli and in glomeruli of diabetic nephropathy patients. High-glucose and fatty acid treatment synergistically upregulated S100a8 gene expression in macrophages from WT mice, but not from KO mice. As putative downstream targets of TLR4, phosphorylation of interferon regulatory factor 3 (IRF3) was enhanced in kidneys of WT mice co-treated with STZ and HFD. CONCLUSIONS/INTERPRETATION: Activation of S100A8/TLR4 signalling was elucidated in an animal model of diabetic glomerular injury accompanied with hyperlipidaemia, which may provide novel therapeutic targets in progressive diabetic nephropathy.

Upregulation of Ugt1a genes in placentas and fetal livers in a murine model of assisted reproduction.

Genes from Ugt1a family in placenta and fetal liver are responsible for hormone, nutrient and chemical balance during pregnancy. Assisted reproduction technologies (ART) i.e. intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) alter steroid homeostasis in pregnancy through increased glucuronidation. Here we show that ART (particularly ICSI) upregulates Ugt1a1, 1a2, 1a6 and 1a9 expression in murine placentas and fetal livers with higher mRNA related to lower progesterone (1a1) and cholesterol (1a2, 1a6) in placentas. Greater steroid clearance in ART through transcriptional upregulation of Ugt1a in the placental-fetal unit may decrease the availability of essential molecules, mediating negative reproductive outcomes.

Twin-electron biprism.

In order to obtain a large deflection angle without increasing the applied voltage to an electron biprism, we have developed a 'twin-electron biprism' (TBP), which is composed of two filament electrodes and a pair of ground plates. The observed interference-fringe spacing revealed that the deflection angle created by a TBP was about twice larger than that by a 'conventional electron biprism'. Also, we have suggested, in a double-electron biprism interferometry, the optimal disposition of a TBP for reducing the intensity of Fresnel fringes recorded in an electron hologram.

Factors associated with inappropriate weight loss attempts by early adolescent girls in Japan.

Attempting to lose weight by normal or underweight adolescent girls is a serious issue in many countries. It has been reported that the mode of attempted weight loss does not differ between normal weight and overweight girls. These inappropriate weight loss attempts (IWLA) by normal or underweight adolescent girls is associated with various health issues, but factors associated with IWLA have only been marginally elucidated. In this study, we applied a single multivariate regression analysis to clarify independent factors for IWLA. Study subjects were 134 pairs of early adolescent girls (aged 12-15) and their mothers. In addition to IWLA, many factors including height, weight, body image, perceived weight status, depressive symptoms, media influence and self-esteem were surveyed in both mothers and daughters and subjected to multivariate analysis. Approximately half of girls surveyed had IWLA, even though all were of normal weight and 62.9% knew that they were of normal weight. IWLA were independently associated with depressive symptoms (OR (95% CI); 2.80 (1.21-6.50), p=0.016) independent of actual or perceived weight status. Factors significantly associated with IWLA by the girls were percentage deviation of weight from standard weight (%DW) and media influence on the girls themselves, and media influence on and self-esteem of their mothers. IWLA, which were frequently observed among early adolescent girls even among those of normal weight, were closely related to depressive status. IWLA were significantly associated with not only factors related to the girls (1.09 (1.04-1.14), p=0.001), but also with maternal psychological factors (1.06 (1.00-1.13), p=0.035) conveyed by the media. Future prospective or interventional studies are required to clarify whether these factors could be targeted in an effort to prevent IWLA.

Novel effects of beraprost sodium on vasculatures.

Beraprost sodium (BPS) is a stable orally active prostacyclin analogue with vasodilatory and anti-platelet effects, and has been widely used as therapeutics for pulmonary artery hypertension and chronic arterial obstruction. In order to elucidate its effects on endothelium, we first examined the short-term effects of BPS on nitric oxide (NO) production and endothelial NO synthase (eNOS) activation using bovine aortic endothelial cells. Short-term treatment of BPS induced NO production as well as eNOS phosphorylation at Ser-1179 mediated via cAMP/protein kinase A (PKA) pathway. The effects of BPS on capillary-like tube formation were next determined using human umbilical vein ECs (HUVECs)/normal human dermal fibroblasts co-culture system. BPS was observed to induce capillary-like tube formation mediated via cAMP/PKA pathway, but not via NO generation. Finally, we performed DNA microarray analyses using RNA extracted from BPS treated HUVECs. Interestingly, BPS up-regulated several genes involved in angiogenesis, anti-atherosclerosis, and endothelial function, while down-regulated several genes involved in atherosclerosis. Our data therefore indicate that BPS may be useful not only for patients with pulmonary artery hypertension and chronic arterial obstruction, but also for general atherosclerotic patients complicated with endothelial dysfunction. Further studies are needed to clarify molecular mechanisms of these BPS effects including the involvement of peroxisome proliferator-activated receptor-delta.

Assisting the diagnosis of Graves' hyperthyroidism with Bayesian-type and SOM-type neural networks by making use of a set of three routine tests and their correlation with free T4.

In our previous paper, we proposed a novel screening method that assists the diagnosis of Graves' hyperthyroidism via two types of neural networks by making use of routine test data. This method can be applied by non-specialists during physical check-ups at a low cost and is expected to lead to rapid referrals for examination and treatment by thyroid specialists, that is, to improve patients' QOL. In this report, the amount of female sample data was increased and routine test data (14 parameters) from 120 subjects with a known diagnosis (35 patients with Graves' hyperthyroidism and 85 healthy volunteers) were adopted as training data, before 171 individuals who had also undergone the same routine tests at the Tohoku University Hospital were screened by the network for Graves' hyperthyroidism. The present re-examination of the screening method showed its high screening ability with the set of parameters used (low serum creatinine was added to the established measures of elevated alkaline phosphatase and low total cholesterol that appear in the Graves' hyperthyroidism guidelines) and robustness due to the increase of the training sample data. It was also found that there is a strong correlation between the three parameters and serum free thyroxine (FT4) in Graves' hyperthyroidism, which supports the usefulness of our screening method.

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P<0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.

Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice.

Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes.

Transgenic overexpression of brain natriuretic peptide prevents the progression of diabetic nephropathy in mice.

AIMS/HYPOTHESIS: Brain natriuretic peptide (BNP) is a potent vasorelaxing and natriuretic peptide that is secreted from the heart and has cardioprotective properties. We have previously generated hypotensive transgenic mice (BNP-Tg mice) that overproduce BNP in the liver, which is released into the circulation. Using this animal model, we successfully demonstrated the amelioration of renal injury after renal ablation and in proliferative glomerulonephritis. Glomerular hyperfiltration is an early haemodynamic derangement, representing one of the key mechanisms of the pathogenesis of diabetic nephropathy. Based on the suggested involvement of increased endogenous natriuretic peptides, the aim of this study was to investigate their role in the development and progression of diabetic nephropathy. MATERIALS AND METHODS: We evaluated the progression of renal injury and fibrogenesis in BNP-Tg mice with diabetes induced by streptozotocin. We also investigated the effect of BNP on high glucose-induced signalling abnormalities in mesangial cells. RESULTS: After induction of diabetes, control mice exhibited progressively increased urinary albumin excretion with impaired renal function, whereas these changes were significantly ameliorated in BNP-Tg mice. Notably, diabetic BNP-Tg mice revealed minimal mesangial fibrogenesis with virtually no glomerular hypertrophy. Glomerular upregulation of extracellular signal-regulated kinase, TGF-beta and extracellular matrix proteins was also significantly inhibited in diabetic BNP-Tg mice. In cultured mesangial cells, activation of the above cascade under high glucose was abrogated by the addition of BNP. CONCLUSIONS/INTERPRETATION: Chronic excess of BNP prevents glomerular injury in the setting of diabetes, suggesting that renoprotective effects of natriuretic peptides may be therapeutically applicable in preventing the progression of diabetic nephropathy.

Peroxisome proliferator-activated receptor gamma in human breast carcinoma: a modulator of estrogenic actions.

It has been reported that agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibit proliferation of breast carcinoma cells, but the biological significance of PPARgamma remains undetermined in human breast carcinomas. Therefore, we immunolocalized PPARgamma in 238 human breast carcinoma tissues. PPARgamma immunoreactivity was detected in 42% of carcinomas, and was significantly associated with the status of estrogen receptor (ER) alpha, ERbeta, progesterone receptor, retinoic X receptors, p21 or p27, and negatively correlated with histological grade or cyclooxygenase-2 status. PPARgamma immunoreactivity was significantly associated with an improved clinical outcome of breast carcinoma patients by univariate analysis, and multivariate analysis demonstrated that PPARgamma immunoreactivity was an independent prognostic factor for overall survival in ERalpha-positive patients. We then examined possible mechanisms of modulation by PPARgamma on estrogenic actions in MCF-7 breast carcinoma cells. A PPARgamma activator, 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2)), significantly inhibited estrogen-responsive element-dependent transactivation by estradiol in MCF-7 cells, which was blocked by addition of a PPARgamma antagonist GW9662. Subsequent study, employing a custom-made microarray focused on estrogen-responsive genes, revealed that mRNA expression was significantly regulated by estradiol in 49 genes, but this significance vanished on addition of 15d-PGJ(2) in 16 out of 49 (33%) genes. These findings were confirmed by real-time PCR in 11 genes. 15d-PGJ(2) significantly inhibited estrogen-mediated proliferation of MCF-7 cells, and caused accumulation of p21 and p27 protein. These results suggest that PPARgamma is mainly expressed in well-differentiated and ER-positive breast carcinomas, and modulates estrogenic actions.

Development of rat tetraploid and chimeric embryos aggregated with diploid cells.

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.