臺灣博碩士論文加值系統

中文摘要 (OSR1/SPAK)
睪丸內的支持細胞Sertoli cells (SCs)對於男性生殖與精子生成扮演重要的角色。Sertoli-cell-only syndrome (SCOS)在男性不孕症中，並非少見。其睪丸切片在曲細精管主要剩下Sertoli cells而無生殖細胞。其病理機轉至今仍不清楚。Oxidative stress-responsive kinase-1 (OSR1) and STE20 (sterile 20) and SPS1-related proline/alanine-rich kinase (SPAK)是屬於STE20 superfamily of MAPK-like 蛋白激酶且在睪丸有高的表現量。我們藉由小鼠模式來分析OSR1與SPAK在男性的角色。在分析公小鼠的生殖力中，我們發現全身性的SPAK基因剔除與Sertoli cell 專一性OSR1基因剔除的公小鼠有正常的生殖力。但全身性的OSR1基因異合子(OSR1+/–)的公小鼠有生殖力下降的現象。此外我們發現在SPAK基因剔除的睪丸組織中，磷酸化的OSR1蛋白表現量上升，說明OSR1與SPAK在男性生殖中有其互補與代償的角色。為證實此代償效應，我們藉由Sertoli cell 專一的OSR1基因剔除的小鼠與全身SPAK基因剔除的小鼠配種以製作Sertoli cell 同時失去OSR1與SPAK基因的小鼠(雙基因剔除小鼠，DKO mice)。這些成熟的公小鼠無生殖能力且睪丸切片顯示曲細精管內主要剩下Sertoli cells而無生殖細胞，表徵如同Sertoli-cell-only syndrome。藉由分析DKO小鼠的第一波精子生成發現：精子細胞生成出現問題同時合併生殖細胞凋亡的現象。另外在DKO小鼠的睪丸組織發現NKCC1的總量與磷酸化表現有下降的現象。這些結果說明在Sertoli cells中，OSR1與SPAK 可協同地藉由活化NKCC1的功能來調控精子的生成。

中文摘要 (DcR3)
T細胞對於維持免疫系統的恆定扮演重要角色。在重複性流產的病人周邊血液與蛻膜組織可發現有較高Th17細胞表現量，說明Th17細胞對此疾病有一定的病生理角色。我們的研究是探討第三號誘餌受體對重複性流產小鼠的治療調控角色。藉由水動力學的方式，給予懷孕的流產小鼠靜脈注射第三號誘餌受體的質體，我們分析第三號誘餌受體對於治療重複性流產的角色。我們發現：第三號誘餌受體可減少流產小鼠的流產率。在治療的組別中，其子宮Th17細胞的表現量也有下降的現象。此實驗主要流產小鼠的治療為主，能否應用在重複性流產的病人需要更多的研究。我們希望未來有機會能應用在重複性流產病人的治療。

ABSTRACT (OSR1/SPAK)

Somatic Sertoli cells (SCs) play a key role in orchestrating the development of sperm cells through the stages of spermatogenesis and their function is important for male fertility. Sertoli-cell-only syndrome (SCOS), is not an uncommon disorder in male infertility, characterized with a condition of the testes in which only Sertoli cells line the seminiferous tubules. The etiology and mechanism of this syndrome are currently unknown. Oxidative stress-responsive kinase-1 (OSR1) and STE20 (sterile 20) and SPS1-related proline/alanine-rich kinase (SPAK), belong to STE20 superfamily of MAPK-like protein kinases, and express in testis tissues. We investigated the roles of OSR1 and SPAK in mouse models of male fertility. Male global SPAK knockout (SPAK–/–) and Sertoli cell-specific OSR1 gene knockout (SC-OSR1–/–) mice are fertile. But male global OSR1+/– gene mutations cause subfertility. Our data revealed that increased the phosphorylated (p) OSR1 expression in SPAK–/– testis tissue, suggested the compensatory effects of OSR1 and SPAK in male fertility. To dissect this effect, we generated SC-OSR1–/– and SPAK–/– double knockout (DKO) male mice. These adult male mice are infertile with defective spermatogenesis, presenting a SC-only-like syndrome. Disrupted meiotic progression and increased germ cell apoptosis occurred in the first wave of spermatogenesis. Impaired NKCC1 activity was noted. These results indicate that OSR1 and SPAK cooperatively regulate an NKCC1-dependent spermatogenesis a SC-restricted manner.

ABSTRACT (DcR3)
T cells play a central role in immune system, including immunoregulation and immunostimulation. The peripheral blood and decidua in unexplained recurrent spontaneous abortion patients revealed increased prevalence of T helper 17 (Th17) cells, suggesting an important role of The 17 cells in the pathogenesis of RSA. In this study, we investigated the modulatory effects of decoy receptor 3 (DcR3) in recurrent spontaneous abortion (RSA). We used hydrodynamic-based intravenous (IV) administration of DcR3 plasmid into female CBA/J mated male DBA/2 as the abortion-prone model to elucidate the physiological role of DcR3 on RSA. Our results revealed that DcR3 significantly reduced abortion rate in pregnant female CBA/J mice on d13.5. The expression of IL-17-producing CD4 T cells was decreased in the uterus of DcR3-treated mice. Our data suggested that DcR3 has potential as a suppressor of uterus inflammation in the abortion-prone model, which may be attributed to either direct inhibition of uterus inflammation or suppression of abortive Th17 cells. This study is mainly performed in the abortion-prone model. Application of DcR3 in unexplained recurrent spontaneous abortion patients needs further studies. This study provides a therapeutic effect of DcR3 in the abortion-prone model, suggesting its potential for treating women RSA.

CONTENTS
CONTENTS III
LIST OF TABLES IX
LIST OF FIGURES X
中文摘要 (OSR1/SPAK) XII
ABSTRACT (OSR1/SPAK) XIV
中文摘要 (DcR3) XVI
ABSTRACT (DcR3) XVII
第一篇 OSR1 and SPAK collaboratively regulate Sertoli cell support of spermatogenesis in mice through modulation of NKCC1 1
第一章 INTRODUCTION (OSR1/SPAK) 2
Introduction to infertility 2
Sertoli cells and Sertoli cell-only syndrome 2
OSR1 and SPAK 3
NKCC1 and fertility 4
Germinal centre kinase-3 and fertility 5
Hypothesis 5
第二章 SPECIFIC AIMS (OSR1/SPAK) 6
In global OSR1+/- mice 6
Generation of Sertoli cell (SC)-specific OSR1 KO mice 6
Generation of SC-OSR1–/– and SPAK–/– double knockout (DKO) mice 6
第三章 MATERIALS AND METHODS (OSR1/SPAK) 7
Mice 7
Generation of tissue-specific KO mice and genotyping 8
Fertility evaluation 9
Tissue histology 9
Serum testosterone assessment 9
Semen analysis 10
Acrosome reaction assessment 11
In vitro fertilization (IVF) assessement 12
An NKCC inhibitor (Furosemide) on fertilization 13
RT-PCR and quantitative RT-PCR 14
Immunofluorescence stain and western blot 14
TUNEL assay 17
Statistics 17
第四章 RESULTS (OSR1/SPAK) 18
Genotyping of OSR1+/– and SPAK–/– mice 18
Expression of OSR1 and SPAK expression on mouse Sertoli TM4 cell lines, mouse testis tissues and Sertoli cells 18
Fertility assessment in global OSR1+/– and SPAK–/– mice 19
Gross evaluation and histology analysis of reproductive tissues in global OSR1+/– mice 20
Semen analysis in global OSR1+/– mice 21
Effect of OSR1 haploinsufficiency on sperm function focusing on acrosome reaction and in vitro fertilization 22
Abundance of OSR1, SPAK, and NKCC1 and their phosphorylation status in testicular tissues and mature spermatozoa 23
The effect of furosemide, an NKCC inhibitor on fertilization 24
Failed generation of germ cell-specific OSR1 KO mice 25
Generation of Sertoli cell-specific OSR1 KO mice and fertility assessment 26
Increased p-OSR1 expression in the absence of SPAK in testis tissues 28
Generation of SC-OSR1–/– and SPAK–/– DKO mice and validation of their genotypes 29
Fertility and phenotype evaluation in DKO mice 31
Loss of OSR1 and SPAK in Sertoli cells of DKO mice manifested as a Sertoli cell-only appearance 31
Analysis of the first wave spermatogenesis in DKO mice 33
Assessment apoptosis events in the first wave spermatogenesis of DKO mice 34
Analysis of total and phosphorylated NKCC1 abundance in testicular tissues of DKO mice 34
第五章 DISCUSSION (OSR1/SPAK) 37
第六章 CONCLUSION (OSR1/SPAK) 48
第二篇 Investigation of the therapeutic potentials of decoy receptor 3 in the abortion-prone mouse model and analysis of its mechanism 49
第一章 INTRODUCTION (DcR3) 50
Pregnancy and immune tolerance 50
Th 17 cells 50
DcR3 51
Hypothesis 52
第二章 SPECIFIC AIMS (DcR3) 53
Verification of DcR3 plasmid 53
Generation of the abortion-prone model 53
Treatment of the abortion-prone model by DcR3 plasmid 53
Dissection of the mechanism after treatment 53
第三章 MATERIALS AND METHODS (DcR3) 54
Mice 54
Abortion rate 54
DcR3 plasmid 55
Western blot 55
Hydrodynamics-based gene delivery 55
Preparation of splenocytes 56
Isolation of uterus cell suspensions 57
Antibodies and flow cytometry 57
Quantitative RT-PCR 58
Statistical analysis 59
第四章 RESULTS (DcR3) 60
Validation of DcR3 protein in vitro 60
Serum level of DCR3 protein in treated female CBA/J mice. 60
Female CBA/J mated with male DBA/2 as an abortion-prone model 60
Delivery of DcR3 plasmid reduced abortion rates in the abortion-prone model 61
Administration of DcR3 plasmid decreased Th17 cells in uteri 62
第五章 DISCUSSION (DcR3) 63
第六章 CONCLUSION (DcR3) 65
REFERENCES 66



LIST OF TABLES
Table 1 Primers used for SYBG-based qPCR assays. 77



LIST OF FIGURES
Figure 1 Genotyping of OSR1+/– and SPAK–/– mice. 78
Figure 2 OSR1 and SPAK expression on Sertoli TM4 cell lines. 79
Figure 3 OSR1 and SPAK expression on testis tissues and Sertoli cells. 80
Figure 4 Fertility analysis. 81
Figure 5 Evaluation and histology analysis of reproductive tissues in global OSR1+/– mice. 82
Figure 6 Semen analysis of global OSR1+/– mice. 84
Figure 7 Sperm function focusing on acrosome reaction and IVF in global OSR1+/– mice. 86
Figure 8 Abundance of OSR1, SPAK, and NKCC1 and their phosphorylation status in testicular tissues. 88
Figure 9 Abundance of OSR1, SPAK, and NKCC1 and their phosphorylation status in mature spermatozoa. 89
Figure 10 The effect of furosemide, an NKCC inhibitor on fertilization 91
Figure 11 Failed generation of germ cell-specific OSR1KO mice. 92
Figure 12 Generation of Sertoli cell-specific OSR1 KO mice and fertility assessment. 93
Figure 13 Increased p-OSR1 expression in the absence of SPAK in testis tissues of SPAK-null mice. 95
Figure 14 Generation of SC-OSR1–/– and SPAK–/– DKO mice. 96
Figure 15 Validation of genotypes of DKO mice by PCR and RT-qPCR. 97
Figure 16 Validation of OSR1 and SPAK expression in testis tissues of DKO mice by IF staining. 99
Figure 17 Fertility and phenotype evaluation of DKO mice. 101
Figure 18 Loss of OSR1 and SPAK in Sertoli cells of DKO mice manifested as a Sertoli cell-only appearance. 103
Figure 19 Characterization of the first wave spermatogenesis in testicular tissues of DKO mice. 105
Figure 20 Assessment of TUNEL assays in testicular tissues of DKO mice. 107
Figure 21 Total and phosphorylated OSR1, SPAK, and NKCC1 abundance in testis tissues of DKO mice. 108
Figure 22 Total and phosphorylated NKCC1 abundance in testis tissues of DKO mice. 110
Figure A1 Overexpression of DcR3 by TE671 cells in vitro. 112
Figure A2 Serum level of DCR3 protein in treated female CBA/J mice. 113
Figure A3 Establishment of the abortion-prone model. 114
Figure A4 Delivery of DcR3 plasmid reduced abortion rates in abortion-prone models. 115
Figure A5 Administration of DcR3 plasmid decreased Th17 cells at the maternal-fetal interface. 117

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