Delayed Long Bone Formation in Hyperthermia-exposed Mouse Embryos / 체질인류학회지

Maternal hyperthermia, which is currently confirmed as one of major causative factors inducing growth retardation, congenital anomalies and abortion, is known to influence normal development of CNS and various organ system. In addition, maternal hyperthermia could induce severe developmental defects including development of the limb. However, it is not clearly identified how maternal hyperthermia affects the expression of chondrogenesis-related proteins in developing limb of mouse. Thus, this study is aimed to investigate the effects of the maternal hyperthermia on the expression of a various proteins in developing upper limb. To elucidate it, ICR mice were used in this study, and the animals were divided into control and heat shock groups. The heat shock treatment was given to embryonic day (ED) 8. The animals were sacrificed on ED 11, 13, 15 and 17, and the humerus were removed. Chondrogenesis-related factors such as FGF8, SOX9 and collagen II were detected on ED 11, 13 and 15 using western blot and immunohistochemistry. Developing humerus on ED 17 was stained with alizarin red S and alcian blue. The expression of FGF8 of heat shock groups was continued even though the development was succeeded. SOX9 expression in heat shock groups was significantly elevated on ED 13 compared to the control embryos. In addition, collagen II expression of heat groups was significantly higher than that of the control group on ED 13 and 15. The results of this study suggest that hyperthermia causes delayed endochondral ossification in long bone through continuous expression of FGF8, SOX9 and collagen II proteins even though the endochondral ossification is succeeded.

Delayed Long Bone Formation in Hyperthermia-exposed Mouse Embryos / 체질인류학회지

Maternal hyperthermia, which is currently confirmed as one of major causative factors inducing growth retardation, congenital anomalies and abortion, is known to influence normal development of CNS and various organ system. In addition, maternal hyperthermia could induce severe developmental defects including development of the limb. However, it is not clearly identified how maternal hyperthermia affects the expression of chondrogenesis-related proteins in developing limb of mouse. Thus, this study is aimed to investigate the effects of the maternal hyperthermia on the expression of a various proteins in developing upper limb. To elucidate it, ICR mice were used in this study, and the animals were divided into control and heat shock groups. The heat shock treatment was given to embryonic day (ED) 8. The animals were sacrificed on ED 11, 13, 15 and 17, and the humerus were removed. Chondrogenesis-related factors such as FGF8, SOX9 and collagen II were detected on ED 11, 13 and 15 using western blot and immunohistochemistry. Developing humerus on ED 17 was stained with alizarin red S and alcian blue. The expression of FGF8 of heat shock groups was continued even though the development was succeeded. SOX9 expression in heat shock groups was significantly elevated on ED 13 compared to the control embryos. In addition, collagen II expression of heat groups was significantly higher than that of the control group on ED 13 and 15. The results of this study suggest that hyperthermia causes delayed endochondral ossification in long bone through continuous expression of FGF8, SOX9 and collagen II proteins even though the endochondral ossification is succeeded.

Immunohistochemical Study on Early Odontogenesis in Hsp70 Knock-out Mice Fetuses Exposed by Maternal Hyperthermia / 대한해부학회지

To investigate the effects of maternal hyperthermia on early odontogenesis,pregnant Hsp70 knock-out and wild type mice at embryonic day (ED)8.5 were immersed in a 43 degrees C water bath until their core body temperature reached that temperature,and then given a further 5 min of hyperthermia.Untreated Hsp70 WT mice fetuses were used as the control group.Fetuses were collected at EDs 13.5,15.5 and 17.5.Developing teeth in the mandible were processed for histological and immunohistochemical studies.Tissue sections were immunostained for FGF-8 and FGF -4 and observed using light microscopy.In the controls, FGF-8 immunolocalization was observed in cells within the dental lamina and in apically located dental epithelium at ED 13.5.However,a few cells were immunopositive in the heat shocked (HS)group.At EDs 15.5 and 17.5 of the control group,the basal lamina adjacent to the dental pulp showed positive immunostaining.In contrast,most of the dental epithelium was immunopositive at ED 15.5 in the HS group and inner and outer dental epithelial cells were continuously immunopositive by ED 17.5.FGF-4 immunolocalization was found in apical dental epithelium at ED 13.3 in the control group,but no such positive reaction was observed in the HS group.At ED 15.5 in the controls,basal lamina and dental epithelium near the cervical loop were immunopositive.In contrast,early cap-stage teeth had cells near the mouth of the dental bud and cervical loop that were immunopositive to FGF-4 in the HS group.In controls at ED 17.5,cells near the future secondary enamel knot were immunopositive,whereas most of the dental epithelium except for cells in the mouth of the dental lamina was negative in the HS group.Thus,maternal hyperthermia may inhibit normal odontogenesis through sustained production of FGF-8 and downregulation of FGF-4.

Effects of Maternal Hyperthermia on the Development of C57/BL6 Strain Hsp70 Knock-out Mice Fetuses / 체질인류학회지

To investigate the effects of maternal hyperthermia on the formation of congenital anomalies, pregnant Hsp70 knock-out and wild type mice at gestational day (GD) 8.5 were immersed in 43degrees C water bath until their body core temperature reached at 43degrees C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Pregnant mice were killed at GD 15.5, and fetuses were photographed for external appearance analysis. Fetuses with congenital anomalies such as anophthalmia and exencephaly were 72.6% (53 out of 73) in KO group and 28.2% (26 out of 90) in WT group, respectively. Histological findings showed exencephaly, eye abnormalities such as eyeball with retina only or buried eyeball or absence of eye structure, numerous apoptotic cells in the retina and inner ear neuroepithelium, cleft palate, and delayed endochondral ossification. The results of this study suggest that Hsp70 may have a protective function against heat shock.

Effects of Maternal Hyperthermia on the Palatal Mesenchyme Development of Hsp70 Knock-out Mice Fetuses / 체질인류학회지

To investigate the effects of maternal hyperthermia on the development of the palate, pregnant Hsp70 knock-out mice at gestational day (GD) 8.5 were immersed in 43degrees C water bath until their body core temperature reached at 43degrees C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Heat-untreated Hsp70 WT mice fetuses were used as the control group. Fetuses were collected at embryonic day 13.5, 14.5 and 15.5 (E13.5, E14, 5 and E15.5). Heads followed by removal of the mandible and the tongue were obtained and photographed for palatal development. Developing palates were processed for histological and immunohistochemical studies. Tissue sections were immunostained for TGF-beta2, FGF-8 and fibronectin, and observed with light microscope. The obtained results were as follows: Cleft palate was formed in heat-treated Hsp70 KO fetuses at E14.5 and E15.5. Immunohistochemical findings indicated that TGF-beta2 expression of the experimental fetuses were more delayed than that of the control fetuses. Mesenchyme under the medial edge epithelium (MEE) and cells of MEE showed continuously strong positive TGF-beta2 reactivity at E15.5. FGF-8 was revealed in both of the mesenchyme and the epithelium at the same time. FGF-8 immunoreactivity in the mesenchyme and the epithelium of the heat-treated fetuses showed strong reactivity at E15.5. In the experimental fetuses fibronectin was revealed the mesenchyma and basal lamina at E15.5. Taken together, it is suggested that maternal hyperthermia induces continuous expression of TGF-beta2 and FGF-8 in the mesenchyme and delayed expression of fibronectin. These should affect the normal palatogenesis and result in cleft palate.

Delayed Endochondral Ossification in Hsp70 Knock-out Mice Fetuses due to Maternal Hyperthermia / 대한해부학회지

To investigate the effects of maternal hyperthermia on the endochondral ossification, pregnant Hsp70 knock-out and wild type mice at gestational day (GD)8 were immersed in 43 degree C water bath until their body core temperature reached at 43 degree C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Fetuses were collected at GD 15 and photographed for external appearance analysis. In addition, heat treated Hsp70 fetuses with external congenital anomalies and heat untreated wild type fetuses were used as experimental and control animals, respectively. Developing humeri at GD 17 were stained with alizarin red S and alcian bue according to the method of Kimmel and Trammell (1981).Developing upper limbs were immunostained for FGF-8, and observed with light and transmission electron microscope. 1.The proportion of average length of the humerus in heat untreated group to heat treated group was 1 :0.58.The proportion of average length of calcified part to average whole length was 1 :0.46 in control and 1 :0.18 in experimental group, respectively. 2.Apical ectodermal ridge was positively reacted to FGF-8 immunohistochemistry in the control group, but not in the experimental group. The humerus of experimental group showed more delayed endochondrial ossification than that of control group. The chondrocytes in the proliferating zone did not react in the experimental group. 3.Collagen fibers were loosely arranged in the experimental group. Mitochondria possessed early staged mitochondrial cristae, i.e.vesicular type in the experimental group. The results of this study suggest that maternal hyperthermia may inhibit the expression of FGF-8 in the developing upper limb, resulting in delayed endochondral bone growth of the humerus.