Ai Takeshita

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.

Maternal protein restriction that does not have an influence on the birthweight of the offspring induces morphological changes in kidneys reminiscent of phenotypes exhibited by intrauterine growth retardation rats.

Severe restriction of maternal protein intake to 6–8% protein diet results in intrauterine growth retardation (IUGR), low birthweight and high risk of metabolic syndrome in the adult life of the offspring. However, little information is available on the effects of maternal protein restriction on offspring under the conditions that does not have an influence on their birthweight of the offspring,. In the present study, pregnant rats were kept on a diet consisting of either 9% (low‐protein, Lp rats) or 18% (normal‐protein, Np rats) protein by weight/volume/etc. After birth, both Lp and Np rats were kept on a diet containing 18% protein. Neonatal body weight was significantly lower in Lp rats compared to Np rats from 4 days to 5weeks after birth. While glomerular number per unit volume (1 mm3) of the kidney (Nv) was comparable between Lp and Np rats 4 weeks after birth, the Nv was significantly decreased in Lp rats at 20 weeks after birth. Four and 20 weeks after birth, glomerular sclerosis index, interstitial fibrosis score, and ratio of ED1‐positive cell ratio were all significantly higher in Lp compared to Np rats. Transforming growth factor‐β1‐positive cells were observed in the distal tubules in the kidney of 4‐ and 20‐week‐old Lp rats kidneys, but not in those of age‐matched Np rats. Altogether, these findings revealed that maternal protein restriction that does not have an influence on the birthweight of the offspring, induces similar changes as those seen in the kidneys of IUGR neonates.

Effects of Transient Forebrain Ischemia on the Hippocampus of the Mongolian Gerbil (Meriones unguiculatus): An Immunohistochemical Study

In the Mongolian gerbil, bilateral common carotid artery occlusion (BCCAO) for several minutes induces ischemia and delayed neuronal cell death in the CA1 region of the hippocampus due to their incomplete Circle of Willis. In the present study, the expression of fibroblast growth factor 2 (FGF2), its receptors (FGFR1 and FGFR2), glial fibrillary acidic protein (GFAP), and isolectin B4 (ISLB4) was investigated by immunohistochemical and lectin-binding methods after BCCAO was performed for 5 min in gerbils. One day after BCCAO, the pyramidal cells of the CA1 region of the hippocampus showed degenerative changes and lowered expression of FGF2, FGFR1, and FGFR2. Three days after BCCAO, there was an increase in GFAP-positive astrocytes and ISLB4-positive microglial cells. From five to 10 days after BCCAO, intense neuronal cell death in the stria pyramidale of the hippocampal CA1 region was observed, as well as an increase in GFAP-positive astrocytes and decrease in ISLB4-positive microglial cells. These results indicate that transient forebrain ischemia induces neuronal cell death with lowered expression of FGF2 and its receptors, and that the activation of glial cells may not directly lead to neuronal cell death.

Effects of low protein intake on the development of the remaining kidney in subtotally nephrectomized immature rats: expression of inducible and endothelial NO synthase

We examined the effects of low protein intake on the development of the remaining kidney in subtotally (5/6) nephrectomized immature rats. Three-week-old rats were kept on a diet containing either 12% protein (Lp rats) or 18% protein (Np rats) for 4 or 8 weeks after subtotal nephrectomy (SUNx). In Western blot analysis, the endothelial NO synthase (eNOS) protein expression of the Lp rats was significantly higher than that of the Np rats at 4 weeks after SUNx. Immunohistochemically, more inducible NO synthase (iNOS)-positive cells were observed in the Np rats than in the Lp rats 4 weeks after SUNx in the distal tubules. In semiquantitative RT-PCR, the expression of renin mRNA was significantly lower in the Lp rats than in the Np rats at 4 and 8 weeks after SUNx. These findings reveal that protein restriction is effective in preventing renal failure of immature rats and that the changes in the expression levels of renin, eNOS, and iNOS is involved in the process of this prevention.

Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J×DBA/2 mice.

The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J×DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J×DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin.

An increase in apoptosis and reduction in αB-crystallin expression levels in the lens underlie the cataractogenesis of Morioka cataract (MCT) mice

We examined the morphological changes in fibers, localization of apoptotic cells, and protein expression of αB-crystallin in the lens of Morioka cataract (MCT) mice, a novel cataract model. Using a scanning electron microscope, swollen lens fibers and enlarged spaces between lens fibers were observed in the lens of 3-week-old MCT mice. At 2 weeks of age (before cataract), the single-strand DNA (ssDNA)-positive (indicating apoptosis) cell ratio of the lens epithelium was significantly higher in MCT than in wild-type ddY mice. At 2 and 4 weeks of age, αB-crystallin protein expression of the lens in MCT mice was significantly lower than that in wild-type ddY mice. These findings suggest that increase in apoptosis and reduction in αBcrystallin level are involved in the cataractogenesis of MCT mice.

Effects of maternal subtotal nephrectomy on the development of the fetal kidney: A morphometric study

The present study was designed to explore if maternal subtotal (5/6) nephrectomy affects the development of fetal rat kidneys using morphometric methods and examining whether there are any apoptotic changes in the fetal kidney. To generate 5/6 nephrectomized model rats, animals underwent 2/3 left nephrectomy on gestation day (GD) 5 and total right nephrectomy on GD 12. The fetal kidneys were examined on GDs 16 and 22. A significant decrease in fetal body weight resulting from maternal 5/6 nephrectomy was observed on GD 16, and a significant decrease in fetal renal weight and fetal body weight caused by maternal nephrectomy was observed on GD 22. Maternal 5/6 nephrectomy induced a significant increase in glomerular number, proximal tubular length, and total proximal tubular volume of fetuses on GD 22. Maternal 5/6 nephrectomy resulted in an increase in the number of apoptotic cells in the metanephric mesenchyme of the kidney on GD 16, and in the collecting tubules on GD 22. These findings suggest that maternal 5/6 nephrectomy stimulates the development of the fetal kidney while suppressing fetal growth.

Phagocytosis mechanism of apoptotic granulosa cells regulated by milk-fat globule-EGF factor 8

In the process of ovary sexual maturation, most immature ovarian follicles degrade into atretic follicles accompanied by apoptosis in granulosa cells. Macrophages can recognize apoptotic cells through specific binding with phosphatidylserine (PS), exposed on the surface of apoptotic cells, which is mediated by milk-fat globule-EGF factor 8 (MFG-E8). In the present research, we examined the involvement of the MFG-E8-dependent phagocytosis system in the atretic follicles of developing mouse ovaries. The number of atretic follicles and DNA-fragmented granulosa cells significantly increased in B6C3F1 mice during 2 to 6 weeks. Chromatin-condensed granulosa cells were engulfed by macrophages, which existed in the stroma or atretic follicles, or by neighboring normal granulosa cells. MFG-E8 mRNA increased in ovaries during 2 to 6 weeks, and immunoreactivity of MFG-E8 was detected at the surface of apoptotic cells existing around the antrum. Immunoelectron microscopic study revealed MFG-E8-positive signals on the membrane of apoptotic cells near macrophages, but apoptotic cells engulfed by neighboring granulosa cells showed few signals. Anti-Fas antibody elevated the annexin-V-positive reaction in isolated granulosa cells from 3-week-old mouse ovaries. MFG-E8 seems to act on the phagocytosis of apoptotic granulosa cells via macrophages and contribute to the regression process of atretic follicles.

Expression of transforming growth factor β and fibroblast growth factor 2 in the lens epithelium of Morioka cataract mice

In the Morioka cataract (MCT) mice, lens opacity appears at 6 to 8 weeks of age, and swollen lens fiber is electron‐microscopically observed at 3 weeks after birth. The present study was designed to characterize the expression of transforming growth factor β (TGFβ) and fibroblast growth factor 2 (FGF2) in the lens epithelium of the MCT mice. Immunohistochemical analysis showed that the expression of TGFβ in the lens epithelium of the MCT mice was stronger than that of the wild‐type ddY mice at 2 and 4 weeks after birth. The expression of TGFβ receptors (TGFβRI and TGFβRII) and FGF2 in the lens epithelium of the MCT mice was stronger than that of the wild‐type ddY mice at 4 weeks and weaker than that of the wild‐type ddY mice at 15 weeks after birth. Using real time polymerase chain reaction (PCR), quantitative RT‐PCR analysis showed that expression of TGFβ1 and TGFβ2 mRNA in the lens of 2‐week‐old MCT mice was significantly higher compared to age‐matched wild‐type ddY mice. These findings indicate that the lens epithelium of MCT mice has increased expression of TGFβ before cataract affection and that changes in the expression of FGF2 as well as TGFβ may contribute to the progression of the cataract in the mice.

Characteristic patterns of maternal and fetal arterial construction in the rabbit placenta

We studied vascular structure of the rabbit placenta, especially on three-dimensional morphological patterns and developmental process. Basic structure of maternal arterial system was re-constructed during day 13–18 of pregnancy, forming main routes for blood supply through the arterial sinuses and radial arteries. Intra-villous spaces were drastically developed showing as branches from the terminal radial arteries. Fetal arterial system was generated accompanied with maternal vascular development, showing characteristic features such as the perforating linear artery, hairpin flexion, and circular anastomoses in the capillaries. From the correlation of maternal and fetal blood currents, gas-exchange style in the rabbit placenta was considered as counter-current and pool mixed patterns. These data demonstrated an original feature for the placental arterial systems in rabbits, which differed from other animals having a property for discoid placenta.