Title Page
Contents
Abstract 8
I. Background 11
1. Testis 11
1-1. Germ cells 12
1-2. Sertoli cells 13
1-3. Leydig cells 15
2. Spermatogenesis 15
3. Steroidogenesis 16
3-1. The hypothalamic-pituitary-gonadal axis 16
3-2. Steroidogenesis in Leydig cells 18
II. Materials and methods 20
III. The functional analysis of local steroidogenesis in Leydig cells 31
1. Introduction 31
1-1. Androgen receptor 31
1-2. Steroidogenic enzyme genes 36
1-3. TNF-α 39
1-4. Nur77 40
2. Result 43
2-1. TNF-α□ 43
1) Decreased testicular testosterone level in TNF-α knockout mice 43
2) Reduced expression of steroidogenic enzymes in TNF-α knockout testes 44
3) Delay in the onset of spermatogenesis in TNF-α knockout testes 45
2-2. Nur77 46
1) Androgen suppresses the expression of steroidogenic enzyme genes in Leydig cells. 46
2) AR inhibits the transcription activity of Nur77, and suppresses the promoter activity of steroidogenic enzyme genes. 47
3) Suppression of the transactivation of Nur family members by AR and its specificity. 48
4) AR cotranslocates into the nucleus with Nur77 in an androgen-dependent manner. 48
5) Nur77 physically interacts with AR in vitro and in vivo. 49
6) The inhibitory mechanism of Nur77 by AR through competitive binding with coactivators 50
3. Discussion 62
IV. The Functional analysis of genes highly expressed in germ cell 65
1. Introduction 65
1-1. Androgen in the testis 65
1-2. P-type ATPase in testis 67
1-3. Estrogen receptor 68
1-4. HAKAI 70
2. Result 71
2-1. TSPA 71
1) Expression patterns of TSPA 71
2) Biochemical characterization of TSPA 72
2-2. HAKAI 73
1) Isolation of HAKAI from mouse testis 73
2) Functional analysis of HAKAI as a repressor to estrogen receptor 74
3) HAKAI physically interacts with ERα in vitro and in vivo 74
4) DNA-binding activity of ERα was not affected by HAKAI 75
5) The inhibitory mechanism of ERα by HAKAI through competitive binding with coactivators 75
3. Discussion 84
V. Reference 87
국문초록 106
Acknowledgement 109
The functional analysis of local steroidogenesis in leydig cells 6
Fig. 1. Reduced testicular testosterone levels in TNF-α knockout mice. 52
Fig. 2. Reduced mRNA levels of steroidogenic genes in TNF-α knockout testes. 53
Fig. 3. Reduced testis weights and sperm production in TNF-α knockout mice. 54
Fig. 4. Delay in the onset of spermatogenesis in TNF-α knockout mice. 55
Fig. 5. Steroidogenic enzyme genes are decresed by androgen in Leydig cells. 56
Fig. 6. Suppression of Nur77 transactivation by AR in mLTC-1 Leydig cells. 57
Fig. 7. Suppression of the transactivation of Nur family members by AR and its specificity. 58
Fig. 8. Nur77 interacts and colocalizes with AR in vivo. 59
Fig. 9. Physical interaction between Nur77 and AR in vitro and in vivo. 60
Fig. 10. AR competes with coactivator for the modulation of Nur77 transactivation. 61
Functional analysis of genes highly expressed in germ cell 6
Fig. 1. The expression of Testis-Specific P-type ATPase (TSPA). 77
Fig. 2. The ATPase activity and the formation of the phosphoenzyme intermediate of TSPA. 79
Fig. 3. Function of Hakai as a transcriptional repressor. 80
Fig. 4. HAKAI specifically interacts with ERα□. 81
Fig. 5. DNA-binding activity of ERα was not affected by HAKAI. 82
Fig. 6. HAKAI competes with coactivators for the modulation of ERα transactivation. 83