Changes in aquaporin 5 in the non-ciliated cells of mouse oviduct according to sexual maturation and oestrous cycle

Nah, Won Heum,Oh, Yeong Seok,Hwang, Jung Hye,Gye, M. C. Commonwealth Scientific and Industrial Research Or 2017 Reproduction, fertility, and development Vol. No.

<P> Aquaporin (AQP) water channels play an important role in fluid homeostasis and the control of epithelial cell volume. To understand the oviductal fluid homeostasis, the expression of aqp5 was examined in mouse oviduct. In the oviduct of cycling females, aqp1, aqp3, aqp4, aqp5, aqp6, aqp7, aqp8, and aqp11 mRNA were detected. Of these, expression of aqp5 mRNA increased significantly from the early prepubertal period to puberty. Epithelial AQP5 immunoreactivity was markedly increased during the same period and was most notable in the infundibulum. In immature female mice (3 weeks old), gonadotropin (pregnant mare’s serum gonadotropin (5 IU/head) and human chorionic gonadotropin (5 IU/head), single intraperitoneal injection) significantly increased oviductal aqp5 mRNA and AQP5 immunoreactivity in oviduct epithelia. In adult mouse oviduct epithelia, AQP5 was primarily found in the apical membrane, subapical cytoplasm and basolateral membrane of secretory non-ciliated cells, whereas weak to negligible immunoreactivity was found in β-tubulin-positive ciliated cells. Taking into account the fact that non-ciliated cells are well developed with subapical secretory vesicles as well as endosomes, AQP5 may also participate in the secretion and endocytosis in addition to water movement through non-ciliated secretory cells. AQP5 immunoreactivity was also found in the isthmic muscle and lamina propria beneath the epithelia. In cycling females, oviductal aqp5 mRNA levels were the highest at oestrus and the lowest at di-oestrus. AQP5 immunoreactivity in non-ciliated cells was notable in the infundibulum, where AQP5 immunoreactivity was relatively high at oestrus but low at dioestrus and pro-oestrus, indicating synchrony between aqp5 gene activation and the ovarian cycle. Together, the findings of the present study indicate that aqp5 specific to non-ciliated cells is activated during sexual maturation, supporting fluid homeostasis in mouse oviduct. </P>

Aquaporin 5 expression is regulated by estrogen receptor α in mouse oviductal epithelium

Won Heum Nah,Myung Chan Gye,Jung Wook Kim,Jung Hye Hwang 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

Aquaporin5 (AQP5), a water channel plays an important role in the fluid homeostasis and cell volume control in epithelial cells. In an effort to understand fluid homeostasis in the oviduct, tissue specific expression of AQP 5 was examined together with hormonal regulation of AQP5 in the mouse oviduct. To understand the oviductal fluid homeostasis and its regulation by sex steroids, We examined AQP5 expression in mouse oviduct during developmental stage and estrous cycle, and in estrogen receptor α (ERα) knockout mice oviduct. In immature mouse oviduct, expression of AQP5 expression was examined after stimulation with gonadotropins. The effect of ERα agonist (PPT) and ERβ agonist (DPN) on the oviductal expression of AQP5 was examined in ovariectomized mouse. All samples were subjected to realtime-PCR and immunohistochemistry analysis. In oviduct epithelium, AQP5 was largely found in the apicolateral membrane and cytoplasm of ERα-positive non-ciliated cells but weakly expressed in the ciliated cells. Interstitial cells, muscle cells and blood vessels were also weakly positive for AQP5 immunoreactivity. In cyclic female mice oviductal AQP5 mRNA levels were the highest at estrous. In immature mouse oviduct AQP5 mRNA and epithelial immunoreactivity were increased by PMSG, and followed by a decrease after hCG. In ERα KO mice oviduct, AQP5 mRNA levels were significantly lower than those of WT females at diestrous stage. In immature and OVX mouse oviducts, AQP5 mRNA and epithelial immunoreactivity were significantly increased by E2 and PPT. Together, our results suggest the pivotal role of AQP5 in fluid secretion and absorption of water in non-ciliated cells in oviduct. AQP5 gene is tightly activated by estrogen – ERα signaling in non-ciliated cells in oviductal epithelium, mediating the effect of estrogen on gamete transport, fertilization and early embryo development via regulating the fluid homeostasis in oviduct.

Expression and role of endocytic receptor amnionless in preimplantation mouse embryos

Won Heum Nah,Yeong Seok Oh,Myung Chan Gye 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Amnionless (AMN) is a plasma membrane protein that binds to cubilin and megalin in various epithelia and mediates endocytosis of extracellular ligands. This function has been studied in the kidney where it plays a key role in vitamin B12 and vitamin D homeostasis. Present study aimed to elucidate developmental pattern of expression of AMN during the peri-implantation period in mouse embryos. In an effort to understand functional role of AMN in the histiotropic nutrition in blastocyst, endocytotic function of AMN for apoplipoprotein was examined in blastocyst. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. To obtain embryos, females were mated with males. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG by flushing oviducts and uterus, and we also obtained gestation day 6.5, 7.5 and 8.5 embryos in uterus. All samples were subjected to quantitative RT-PCR, whole-mount immunofluorescence and immunohistochemistry analysis. To analyze endocytotic function of AMN, we examined uptake experiment of FICT labeled apolipoprotein A-I (ApoA-I-FITC) following functional blocking of AMN in blastocysts. AMN and cubilin mRNA was expressed in all developmental stages of mouse embryos. Megalin was the first detected at morula stage. AMN protein was expressed in trophectoderm (TE) and inner cell mass (ICM). AMN and cubilin were expressed in visceral endoderm of GD 6.5 and 7.5 embryos and visceral yolk sec of GD 8.5 embryos. In normal IgG treated embryos, ApoA-I-FITC was detected in intracellular vesicles of TE and ICM. However, in the presence of anti-AMN antibody, ApoA-I-FITC was weakly detected in apical surface of plasma membrane of TE. To date, AMN has been believed to be expressed in visceral endoderm of post implantation embryos. Our results demonstrated that AMN is the important molecular partner of cubilin and megalin in the preimplantation embryos and that AMN mediates endocytosis of apoplipoprotein, which may play a crucial role in embryonic development and normal growth via supporting histiotropic nutrition during peri-implantation period.

Expression and role of CAR in preimplantation mouse embryos

Won Heum Nah,Yeong Seok Oh,Myung Chan Gye 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Coxsackievirus and adenovirus receptor (CAR) is a member of the Ig-type superfamily of cell adhesion molecules. In polarized epithelial cells CAR is expressed at the tight junction. The mouse CAR gene is composed of at least eight exons, and CAR splice variants that differ at the end of the cytoplasmic tail have been identified in a number of tissues. The present study aimed to examine the expression of (CAR), a TJ protein sealing the muticellular contact point during preimplantation embryos and role of CAR in the formation and integrity of the blastocoel. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG in oviducts and uterus, and we also obtained gestation day 5 and 6.5 embryos in uterus. All samples were subjected to RT-PCR, immunofluorescence and immunohistochemistry analysis. To analyze epithelial permeability of CAR, we examined permeability of FITC-labeled dextran (MW 40 kDa) following functional blocking of CAR in blastocysts. Long isoform of CAR mRNA was expressed from throughout the preimplantation development and markedly increased at morulae stage onward. Small amount of short isoform CAR mRNA was expressed at blastocyst stage. On Western blot, 64 kDa protein was detected together with 43 kDa protein corresponding to short and long forms CAR, respectively in blastocysts. CAR immunoreactivity was found in cell contacts between blastomeres from 4-cell stage onward. Under Ca2+ switching condition blocking antibodies for CAR increased the permeability of blastocysts to FITC-dextran, a permeability tracer. At 5 dpc, trophoblasts of the implanting embryos were immunoreactive with anti-CAR IgG. At 6.5 dpc, the egg cylinder stage in mouse, the visceral and parietal endoderm were immunoreactive with anti-CAR IgG. Our results suggest that alternative splicing of CAR transcript is highly dependent on the development of expanding blastocyst. CAR may play a crucial role as a barrier to adenovirus infection and adhesion molecule for epithelial permeability during peri-implantation period.

RE-05 : Aquaporin 5 expression is regulated by estrogen receptor α in mouse oviductal epithelium

( Won Heum Nah ),( Jeong Wook Kim ),( Jung Hye Hwang ),( Myung Chan Gye ) 대한산부인과학회 2012 대한산부인과학회 학술대회 Vol.99 No.-

Aquaporin5 (AQP5), a water channel plays an important role in the fluid homeostasis and cell volume control in epithelial cells. In an effort to understand fluid homeostasis in the oviduct, tissue specific expression of AQP 5 was examined together with hormonal regulation of AQP5 in the mouse oviduct. To understand the oviductal fluid homeostasis and its regulation by sex steroids, we examined AQP5 expression in mouse oviduct during developmental stage and estrous cycle, and in estrogen receptor α (ERα) knockout mice oviduct. In immature mouse oviduct, expression of AQP5 expression was examined after stimulation with gonadotropins. The effect of ERα agonist (PPT) and ERβ agonist (DPN) on the oviductal expression of AQP5 was examined in ovariectomized mouse. All samples were subjected to realtime-PCR and immunohistochemistry analysis. In oviduct epithelium, AQP5 was largely found in the apicolateral membrane and cytoplasm of ERα-positive non-ciliated cells but weakly expressed in the ciliated cells. Interstitial cells, muscle cells and blood vessels were also weakly positive for AQP5 immunoreactivity. In cyclic female mice oviductal AQP5 mRNA levels were the highest at estrous. In immature mouse oviduct AQP5 mRNA and epithelial immunoreactivity were increased by PMSG, and followed by a decrease after hCG. In ERα KO mice oviduct, AQP5 mRNA levels were significantly lower than those of WT females at diestrous stage. In immature and OVX mouse oviducts, AQP5 mRNA and epithelial immunoreactivity were significantly increased by E2 and PPT. Together, our results suggest the pivotal role of AQP5 in fluid secretion and absorption of water in non-ciliated cells in oviduct. AQP5 gene is tightly activated by estrogen - ERα signaling in non-ciliated cells in oviductal epithelium, mediating the effect of estrogen on gamete transport, fertilization and early embryo development via regulating the fluid homeostasis in oviduct.

Effects of early prepubertal exposure to bisphenol A on the onset of puberty, ovarian weights, and estrous cycle in female mice

Nah, Won-Heum,Park, Mi-Jung,Gye, Myung-Chan The Korean Society for Reproductive Medicine 2011 Clinical and Experimental Reproductive Medicine Vol.38 No.2

Objective: Bisphenol A (BPA) is a chemical used extensively to manufacture plastics and epoxy resin liners for food and beverage cans. BPA, with properties similar to estrogen, has endocrine-disrupting effects. In the present study, we examined the effects of early prepubertal BPA exposure on the onset of puberty and reproductive parameters such as estrous cycle and reproductive organ weights in female mice. Methods: Female mice were injected subcutaneously at postnatal day (PND) 8 with BPA (0.1, 1, 10, 100 mg/kg) in sesame oil or with sesame oil alone. Body weight was measured from PND 10 to 70. Vaginal opening and estrous cycle were monitored from PND 20 to 29. Animals were sacrificed at PND 25, 30, and 70, and the ovary and uterus weights were measured. Results: Early prepubertal exposure to BPA (10 and 100 mg/kg) significantly decreased body weight from PND 18 to 30. BPA treated mice at testing dose levels showed early opening of the vagina compared to the control group. The number of estrous cycle and days of estrus were significantly decreased in high dose (100 mg/kg) BPA treated mice. The ovary weight at PND 25 and 30 was significantly decreased in all BPA treatment groups. Conclusion: Early prepubertal exposure to BPA accelerated the onset of puberty but decreased reproductive parameters in female mice.

총설 : 파라벤류의 독성과 내분비계장애 효과

안혜선 ( Hae Sun Ahn ),나원흠 ( Won Heum Nah ),이재은 ( Jae Eun Lee ),오영석 ( Yeong Seok Oh ),계명찬 ( Myung Chan Gye ) 한국환경생물학회 2009 환경생물 : 환경생물학회지 Vol.27 No.4

파라벤은 p-하이드록시 벤조산(p-hydroxybenzoic acid)의 알킬에스테르로, 비교적 빠르게 흡수, 대사 및 배설되는 살균성 보존제로 식품, 화장품, 약품 등에 널리 사용되고 있다. 실제 인체는 파라벤 복합물에 노출된다. 파라벤의 안전성에 관한 연구결과들에 대한 고찰 결과 파라벤 종류에 따라 다양한 독성종말점을 대상으로 파라벤의 급성, 아급성 및 만성독성 영향은 비교적 적은 것으로 나타났다. 파라벤은 에스트로젠 유사활성을 가지며 화장품을 통한 경피흡수를 통해 유방암과의 상관성이 보고되었으나, 이와 상반된 견해도 있다. 파라벤의 항안드로젠성은 남성생식기계의 장애를 유발할 수 있으나 이와 상반된 견해도 있다. 파라벤은 정자의 미토콘드리아 기능 및 남성호르몬 생성을 저해할 수 있으나 이와 상반된 견해도 있다. 배아발달에는 독성이 없는 것으로 나타났다. 세포독성으로는 세포용혈, 미토콘드리아 막투과성변화, 세포사멸 등을 유발할 수 있다. 수환경에서 파라벤은 환경에스트로젠으로 작동하여 어류에서 내분비장애 효과를 발휘한다. 결론적으로 파라벤은 저독성물질로 분류할 수 있으나, 인체 및 수생동물들에서 파라벤의 노출경로 및 농도, 사용기간 등에 따른 독성과 내분비계장애 효과에 대하여는 다양한 종말점을 대상으로 좀 더 구체적인 독성자료들이 요구된다. Parabens are alkyl esters of p-hydroxybenzoic acid, which are widely used in foods, cosmetics, and pharmaceutic products as preservatives. Absorbed parabens are metabolized fastly and excreted. Actually human body is exposed to complex mixture of parabens. Safety assessment at various toxicological end points revealed parabens have a little acute, subacute and chronic toxicities. Some reports have argued that as parabens have estrogenic activity, they are associated with the incidence of breast cancer through dermal absorption by cosmetics. There is an inference that antiandrogenic activity of parabens may give rise to a lesion of male reproductive system, but also there is an contrary. At cellular level, parabens may inhibit mitochondrial function of sperms and androgen production in testis, but also there is an contrary. Parabens seem to have little or no toxicity in embryonic development. Parabens can cause hemolysis, membrane permeability change in mitochondria and apoptosis, suggesting cellular toxicity of parabens. Parabens evoked endocrine disruption in several fish species and have toxic effect on small invertebrates and microbes. Therefore, the toxicity of parabens should be considered as a potentially toxic chemical in the freshwater environment. In conclusion, though parabens may be considered as a low toxic chemical, more definite data are required concerning the endocrine disrupting effect of parabens on human body and aquatic animals according to route and term of exposure as well as the residual concentration of parabens.