克萊恩費爾特症候群 (Klinefelter syndrome, KS) 被定義為男性的基因體含有兩個以上的X染色體。此病症也是造成男性不孕症的遺傳性主因之一。在患者睪丸內精細胞的缺失目前推測為多出來的X染色體上某些基因過多表現所致。然而，對是哪些致病基因以及調控機制目前仍無所知。TEX11 (testis-expressed 11)是特別表現在生殖細胞的蛋白，而表現該蛋白的基因則是位於X染色體上。Tex11基因剔除鼠的表現型顯示，TEX11在減數分裂的同源重組機制中占有重要地位。然而，TEX11在精原母細胞及早期精母細胞有著大量的表現，則顯示此蛋白必定在減數分裂前有不同的生理功能。我們以酵母雙雜交系統尋找TEX11的結合蛋白，並找到HPIP (Hematopoietic BPX-Interacting Protein)。先前的研究顯示，HPIP可藉由與雌激素受質結合並將之固定於細胞骨架上，進而調控雌激素在細胞內的作用。我們的分析發現老鼠的精原母細胞幹細胞同時表現著TEX11、HPIP以及b型雌激素受質(ERb)，但沒有a型雌激素受質(ERa)的表現。在培養細胞的實驗顯示，TEX11會與ERb競爭和HPIP的結合。當細胞以雌激素17b-estradiol (E2)或是以ERb的專一促效劑(agonist) DPN處理後，發現TEX11可促使ERb進入細胞核內並增加ERb的轉錄能力。此外，細胞在短時間以DPN處理後，可發現TEX11會在細胞質內抑制ERb的非典型基因體活性，此活性可由訊號傳遞蛋白AKT及ERK磷酸化的降低來定義。並且在老鼠精細胞衍生細胞株GC-1及GC-2內過量表現TEX11，可抑制由E2或是DPN促進的細胞增生能力和cFos、Ccnd1及Ccnb1等基因的表現，並同時提高誘導細胞凋亡Bax基因的表現。TEX11抑制細胞增生的能力顯示，過量表現TEX11很可能是造成KS患者生殖細胞缺失的原因之一。

Klinefelter syndrome (KS), characterized by the presence of one extra X chromosome in men, is a major genetic cause of male infertility. Germ cell degeneration in KS patients is thought to be the consequences of over-expression of some genes on the X chromosome. However, the identity of these genes and the underlying mechanisms remain unclear. TEX11 (Testis-expressed 11) is an X-chromosome encoded germ-cell specific protein that is expressed most abundantly in spermatogonia and early spermatocytes in the testes. We used yeast two-hybrid screen to search for TEX11 interacting partners and identified HPIP (Hematopoietic BPX-Interacting Protein) which anchors estrogen receptors (ERs) to the cytoskeleton and modulates their functions. We found that mouse spermatogonial stem cells expressed Tex11, Hpip, and Esr2, but not Esr1. In cultured cells TEX11 competed with ERb for the binding to HPIP. Upon treatment with 17b-estradiol (E2) or an ERb agonist DPN, TEX11 promoted the nuclear translocation of ERb and enhanced its transcriptional activities. On the other hand, TEX11 suppressed the nongenomic activities of ERb in the cytoplasm, as indicated by the reduced phosphorylation of AKT and ERK signaling molecules. Over-expression of TEX11 in mouse germ-cell derived GC-1 and GC-2 cells suppressed the cell proliferation and the expression of cFos, Ccnd1, and Ccnb1 that were stimulated by E2 or DPN, and elevated the expression level of the proapoptotic Bax gene. The negative effect of TEX11 on cell proliferation suggests that increased expression of TEX11 in the germ cells may partially contribute to the spermatogenic defect observed in KS patients.

Contents......................................I
Table lists...................................III
Figure lists..................................IV
Appendixes lists..............................V
Chinese Abstract (中文摘要)....................VI
English Abstract..............................VII

Introduction Spermatogenesis.................................1
Estrogens and Spermatogenesis...................1
Klinefelter syndrome............................6
X-linked Tex11 gene and protein.................7
HPIP and estrogen activities....................8
Objectives......................................8

Materials and Methods Animals................................................10
Generation and Sources of antibodies...................10
Plasmid construction...................................10
Yeast two-hybrid screen................................11
Cell culture and transfection..........................12
Coimmunoprecipitation..................................13
Colocalization of TEX11 and HPIP in transfected cells..13
Purification and gene expression of SSCs...............14
Fractionation of the nuclei and the cytoplasm..........14
Immunofluorescence staining of ERb in transfected GC-1 cells..15
Luciferase reporter assay..............................15
Western analysis on AKT/ERK phosphorylation............16
Cell proliferation assays..............................16
Real-time PCR quantification of gene expression........17

Results
HPIP is a TEX11 interacting protein......................18
Mouse spermatogonial stem cells express Hpip, Tex11, and Esr2....19
TEX11 and ERb compete for binding to HPIP................20
TEX11 enhances the transactivational activities of ERb...20
TEX11 inhibits the nongenomic activities of ERb..........22
TEX11 suppresses E2/DPN-stimulated cell proliferation....22
TEX11 affects the expression of estrogen target genes....23

Discussion...............................................25
Tables...................................................29
Figures..................................................31
References...............................................55
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