Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells

博士 === 東海大學 === 生命科學系 === 94 === 英文摘要 I. Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern has been studied extensively in the testis, specific localization of the Esx1 protein (...

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Main Authors: Yueh-Chiao Yeh, 葉月嬌
Other Authors: Neng-Wen Lo
Format: Others
Language:zh-TW
Published: 2006
Online Access:http://ndltd.ncl.edu.tw/handle/83334292052475467743
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description 博士 === 東海大學 === 生命科學系 === 94 === 英文摘要 I. Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern has been studied extensively in the testis, specific localization of the Esx1 protein (ESX1) in the testis remains to be determined. In my study, the ESX1 antiserum was used to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localizations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm and examined the sperm by confocal laser microscopy. Approximately half the sperm population was recognized by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be recognized as a protein marker for X-sperm. 英文摘要 II. Esx1, an X-linked homeobox gene, is highly expressed in adult testis (as a-form Esx1) and in placenta (as b-form Esx1). Surveys of literatures and GenBank sequences identified the other novel isoform (herein referred to as x-form Esx1), which has not been histologically defined. In the present study, two heat-induced stress models, experimentally induced cryptorchidism and heat incubation, were carried out to examine the isoform-specific expression of Esx1 mRNA. The novel Esx1 isoform was revealed without precedent in cryptorchid testes and showed highly stress-inducible. Isoform-specific RT-PCR results revealed that x-form Esx1 replaces the a-form transcript, and maintains comparatively stable transcriptional expressions of Esx1 RNA and ESX1 protein in cryptorchid testes. However, under severe heat challenge, expression of the x-form transcript was delayed. Reduced expression of Esx1 in testes of mice under prolonged incubation at 37oC coincided with deteriorating cellular damage in postmeiotic germ cells. In addition, the effects of thermal insult on the transcriptional regulation of the Esx1 gene in the placenta were studied in pregnant mice. The results demonstrated that exposure of pregnant mice to 37 oC for one hour caused the constitutive b-form in placenta to switch to the a-form transcript. Histological examination of heated placenta revealed that damage had occurred in labyrinthine layer; however, immunofluorescent analysis showed that ESX1 signals were not reduced. The results of isoform switching suggest that the existences of different Esx1 transcripts might be partially due to the usage of alternative promoters in testis and in placenta. To investigate this proposition, the entire 5’-flanking sequences of Esx1 gene were used to study the distinct promoter regions that would regulate the Esx1 expression in testicular germ cells. Transient transfection assays revealed that there were two putatively distinct promoter regions: a distal promoter region between nt -965 and -438 and a proximal promoter region between nt +54 and +227, which were sufficient to retain the promoter activities in round spermatids and primary spermatocytes, and in spermatogonia, respectively. These results will help us to understand the possible promoter regions essential for Esx1 altenative gene expression in testes.
author2 Neng-Wen Lo
author_facet Neng-Wen Lo
Yueh-Chiao Yeh
葉月嬌
author Yueh-Chiao Yeh
葉月嬌
spellingShingle Yueh-Chiao Yeh
葉月嬌
Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
author_sort Yueh-Chiao Yeh
title Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
title_short Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
title_full Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
title_fullStr Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
title_full_unstemmed Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
title_sort differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells
publishDate 2006
url http://ndltd.ncl.edu.tw/handle/83334292052475467743
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AT yèyuèjiāo differentialexpressionofextraembryonictissuespermatogenesishomeoboxgene1esx1inmousetestesandinplacentaeanditspromoteranalysisintesticulargermcells
AT yuehchiaoyeh esx1jīyīnzàixiǎoshǔyìwánjítāipánzhōngzhītèdìngbiǎoxiànyǐjízàiyìwánshēngzhíxìbāozhōngqǐdòngzizhīqūyùfēnxī
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spelling ndltd-TW-094THU001120022015-12-21T04:04:32Z http://ndltd.ncl.edu.tw/handle/83334292052475467743 Differential expression of extraembryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) in mouse testes and in placentae and its promoter analysis in testicular germ cells Esx1基因在小鼠睪丸及胎盤中之特定表現以及在睪丸生殖細胞中啟動子之區域分析 Yueh-Chiao Yeh 葉月嬌 博士 東海大學 生命科學系 94 英文摘要 I. Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern has been studied extensively in the testis, specific localization of the Esx1 protein (ESX1) in the testis remains to be determined. In my study, the ESX1 antiserum was used to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localizations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm and examined the sperm by confocal laser microscopy. Approximately half the sperm population was recognized by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be recognized as a protein marker for X-sperm. 英文摘要 II. Esx1, an X-linked homeobox gene, is highly expressed in adult testis (as a-form Esx1) and in placenta (as b-form Esx1). Surveys of literatures and GenBank sequences identified the other novel isoform (herein referred to as x-form Esx1), which has not been histologically defined. In the present study, two heat-induced stress models, experimentally induced cryptorchidism and heat incubation, were carried out to examine the isoform-specific expression of Esx1 mRNA. The novel Esx1 isoform was revealed without precedent in cryptorchid testes and showed highly stress-inducible. Isoform-specific RT-PCR results revealed that x-form Esx1 replaces the a-form transcript, and maintains comparatively stable transcriptional expressions of Esx1 RNA and ESX1 protein in cryptorchid testes. However, under severe heat challenge, expression of the x-form transcript was delayed. Reduced expression of Esx1 in testes of mice under prolonged incubation at 37oC coincided with deteriorating cellular damage in postmeiotic germ cells. In addition, the effects of thermal insult on the transcriptional regulation of the Esx1 gene in the placenta were studied in pregnant mice. The results demonstrated that exposure of pregnant mice to 37 oC for one hour caused the constitutive b-form in placenta to switch to the a-form transcript. Histological examination of heated placenta revealed that damage had occurred in labyrinthine layer; however, immunofluorescent analysis showed that ESX1 signals were not reduced. The results of isoform switching suggest that the existences of different Esx1 transcripts might be partially due to the usage of alternative promoters in testis and in placenta. To investigate this proposition, the entire 5’-flanking sequences of Esx1 gene were used to study the distinct promoter regions that would regulate the Esx1 expression in testicular germ cells. Transient transfection assays revealed that there were two putatively distinct promoter regions: a distal promoter region between nt -965 and -438 and a proximal promoter region between nt +54 and +227, which were sufficient to retain the promoter activities in round spermatids and primary spermatocytes, and in spermatogonia, respectively. These results will help us to understand the possible promoter regions essential for Esx1 altenative gene expression in testes. Neng-Wen Lo Vie Cheng 羅能文 鄭 葳 2006 學位論文 ; thesis 123 zh-TW