Nesfatin-1: Investigating its expression and functional role in mouse reproductive organs

다국어 초록 (Multilingual Abstract)

NUCB2/nesfatin-1 has been known to be expressed in the hypothalamus, playing a role in the regulation of energy metabolism and appetite. Recent studies have revealed its expression has been identified in various organs in addition to the hypothalamus, suggesting diverse physiological functions. However, the precise mechanisms of action and functions in each organ remain poorly understood. In this study, the expression of NUCB2/nesfatin-1 was investigated in the pituitary gland and reproductive organs of mice, including the ovary, uterus, oviduct, testis, and epididymis, aiming to elucidate its functions.
First, the expression of NUCB2/nesfatin-1 in the pituitary gland was confirmed in male and female mice. Subsequently, in castrated mice, the expression of Nucb2 mRNA in the pituitary gland, which had decreased following the procedure, was found to increase due to testosterone. Similarly, in ovariectomized mice, the decrease in Nucb2 mRNA expression in the pituitary gland was observed to be restored by administering estradiol (E2) and progesterone (P4). This demonstrates that each sex steroid hormone directly regulates Nucb2 mRNA expression in the pituitary gland. Moreover, using GH3 pituitary cell line, lacto-somatotrophs in the pituitary gland, it was confirmed that treatment with nesfatin-1 and simultaneous treatment with E2 led to an increase in the mRNA expression of growth hormone (GH) and prolactin (PRL). These results suggest that the hypothalamus-pituitary-gonad axis, which regulates reproductive functions in mice, can also modulate the expression of NUCB2/nesfatin-1 in the pituitary gland. This modulation may potentially impact the production of growth hormone and prolactin.
Next, the expression of NUCB2/nesfatin-1 was examined in the reproductive organs of male and female mice. Specifically, variations in expression were observed in the female reproductive organs, including the ovary, uterus, and oviduct, across the estrous cycle, with the highest expression of Nucb2 mRNA and nesfatin-1 protein during estrus phase. Administration of the PMSG, like a gonadotropin, increased Nucb2 mRNA expression in the female reproductive organs; however, in the uterus and oviduct post-ovariectomy, only E2, not PMSG, elevated the decreased expression of Nucb2 mRNA. In male mice, administration of PMSG and hCG increased NUCB2/nesfatin-1 expression in both the testis and epididymis, but in the epididymis post-castration, only testosterone, not PMSG and hCG, restored the decreased Nucb2 mRNA expression. These results indicate that estradiol and testosterone directly regulate the expression of NUCB2/nesfatin-1 in the reproductive organs of both male and female mice. Furthermore, strong expression of NUCB2/nesfatin-1 was observed in Leydig cells of the testes and their cell line TM3 cells, and treatment with nesfatin-1 led to increased expression of enzymes involved in steroid hormone production in these cells. This suggests that NUCB2/nesfatin-1 expressed in Leydig cells may influence steroid hormone production.
In this study, it was confirmed that NUCB2/nesfatin-1 produced in the pituitary gland and reproductive organs regulates the secretion of growth hormone and prolactin in the pituitary gland via autocrine mechanism. Additionally, NUCB2/nesfatin-1 played a role in the production of sex steroid hormones in the reproductive organs. Moreover, NUCB2/nesfatin-1 may serve as a crucial local regulator in various reproductive and physiological functions, such as fertilization, implantation, pregnancy, embryogenesis, spermatogenesis, and sperm maturation, through the hypothalamus-pituitary-gonad axis.

국문 초록 (Abstract)

NUCB2/nesfatin-1은 시상하부에서 발현하면서 식욕과 에너지 대사를 조절하는 것으로 잘 알려져 있으며 최근 연구에서는 시상하부 이외에도 체내 다양한 기관에서 nesfatin-1이 발현하면서 다양한 생리적 기능을 하는 것으로 밝혀졌다. 그러나 아직까지 각 기관에서의 정확한 작용 기전과 기능에 대해서는 잘 알려져 있지 않다. 이에 본 연구에서는 생쥐의 뇌하수체와 생식 기관인 난소, 자궁, 난관, 정소, 부정소에서 NUCB2/nesfatin-1 발현을 조사하고 그 기능을 확인했다.
먼저 수컷과 암컷 생쥐의 뇌하수체에서 NUCB2/nesfatin-1의 발현을 확인했다. 이후 수컷 생쥐에서는 정소 절제술 이후 testosterone에 의해, 암컷 생쥐에서는 난소 절제술 이후 에스트라디올(E2)과 프로게스테론 (P4)에 의해, 절제술 이후 감소되었던 뇌하수체에서의 Nucb2 mRNA 발현이 다시 증가하는 것을 확인하였다. 이를 통해 각각의 성 스테로이드 호르몬이 뇌하수체에서의 Nucb2 mRNA 발현을 직접적으로 조절한다는 것을 입증하였다. 또한, 뇌하수체의 성상자극세포주인 GH3 세포를 사용하여 nesfatin-1 처리 및 E2와의 동시처리 후 성장호르몬과 프로락틴의 mRNA 발현이 증가하는 것을 확인하였다. 이러한 결과는 생쥐에서 생식기능을 조절하는 시상하부-뇌하수체-생식선 축이 뇌하수체의 NUCB2/nesfatin-1 발현도 조절할 수 있으며 이는 성장호르몬과 프로락틴 생성에도 영향을 미칠 수 있음을 제시한다.
다음으로, 수컷과 암컷 생쥐의 생식 기관에서 NUCB2/nesfatin-1의 발현을 확인하였다. 특히 암컷 생식 기관인 난소, 자궁, 난관에서 발정 주기에 따라 발현에 변화가 있었고, 특히 발정기에 Nucb2 mRNA와 nesfatin-1 단백질 발현이 가장 높은 것을 확인하였다. 생식소자극호르몬인 PMSG 투여에 의해 암컷 생식 기관에서의 Nucb2 mRNA 발현이 증가하는 것을 확인하였으나, 난소 절제술 이후의 자궁과 난관에서는 PMSG가 아닌 E2만이 절제술로 인해 감소되었던 Nucb2 mRNA 발현을 증가시켰다. 수컷 생쥐에서도 PMSG와 hCG 투여에 의해 정소와 부정소에서 NUCB2/nesfatin-1 발현이 증가하였으나, 정소 절제술 이후의 부정소에서는 PMSG와 hCG가 아닌 테스토스테론만이 절제술 이후 감소되었던 Nucb2 mRNA 발현을 증가시켰다. 이러한 결과는 에스트라디올과 테스토스테론이 암컷과 수컷 생쥐의 생식기관에서 NUCB2/nesfatin-1 발현을 직접적으로 조절함을 의미한다. 뿐만 아니라 정소의 라이디히 세포와 그 세포주인 TM3 세포에서도 NUCB2/nesfatin-1이 강하게 발현하였으며 nesfatin-1을 처리하였을 때 각 세포에서 스테로이드 호르몬 생성에 관여하는 효소의 발현이 증가하였다. 이러한 결과는, Leydig 세포에서 발현되는 NUCB2/nesfatin-1이 스테로이드 호르몬 생성에 영향을 미칠 수 있음을 시사한다.
본 연구에서는, 뇌하수체 및 생식기관에서 생성되는 NUCB2/nesfatin-1이 자가분비 조절 방식을 통해 뇌하수체에서 성장호르몬과 프로락틴 생성을 조절하고 생식기관에서 성 스테로이드 호르몬 생성에 관여한다는 것을 확인하였다. 뿐만 아니라 NUCB2/nesfatin-1이 시상하부-뇌하수체-생식선 축을 통해 수정과 착상, 임신, 배아 발달, 정자 형성 및 성숙 등의 다양한 생식∙생리적 기능에 중요한 국부조절인자로서 역할을 할 수 있음을 시사한다.

목차 (Table of Contents)

• General abstract 1
• Chapter I. Introduction 6
• 1.1. General introduction 7
• 1.2. Nucleobindins 15
• 1.3. Nucleobindin-2 and Nesfatin-1 17
• 1.4. Tissue-specific expression of nesfatin-1 in mammals 19
• 1.4.1. Nesfatin-1 expression and its function in the brain 19
• 1.4.2. Nesfatin-1 expression and its function in digestive organs 21
• 1.4.3. Nesfatin-1 expression and its function in adipose tissues 23
• 1.4.4. Nesfatin-1 expression and its function in the heart 23
• 1.4.5. Nesfatin-1 expression and its function in reproductive system 24
• 1.5. Hypotheses and Specific Objectives 28
• Chapter II. Expression of NUCB2/nesfatin-1 and its function in pituitary gland 29
• 2.1. Introduction 30
• 2.2. Materials and methods 34
• 2.2.1. Animals 34
• 2.2.2. Castration and ovariectomy 34
• 2.2.3. Sex steroid hormones treatment 34
• 2.2.4. Culture of the hypothalamus and pituitary gland 35
• 2.2.5. Cell culture and hormone treatment 35
• 2.2.6. Immunohistochemistry and fluorescence microscopy 36
• 2.2.7. RNA extraction, conventional RT-PCR, and qRT-PCR 37
• 2.2.8. Western blot 37
• 2.2.9. Small interfering RNA 38
• 2.2.10. Statistical analysis 38
• 2.3. Results 40
• 2.3.1. Expression of Nucb2 mRNA and nesfatin-1 protein in the hypothalamus and pituitary gland of male mice 40
• 2.3.2. Nucb2 mRNA expression in the hypothalamus and pituitary gland after testosterone injection in castrated mice 43
• 2.3.3. Nucb2 mRNA expression in the cultured hypothalamus and pituitary gland after testosterone treatment in male mice 45
• 2.3.4. Nucb2 mRNA expression in the pituitary gland of ovariectomized mice after E2 and P4 injection 47
• 2.3.5. Nucb2 mRNA expression after E2 and P4 treatment in the cultured pituitary gland of female mice 49
• 2.3.6. Expression co-localization of nesfatin-1 and growth hormone or prolactin in pituitary gland 51
• 2.3.7. Effect of E2 and P4 on Nucb2 mRNA expression in GH3 cells 56
• 2.3.8. Effect of E2, P4, and nesfatin-1 on Gh and Prl mRNA expression in GH3 cells 58
• 2.4. Discussion 61
• Chapter III. Expression of NUCB2/nesfatin-1 and its function in female reproductive organs 67
• 3.1. Introduction 68
• 3.2. Materials and methods 71
• 3.2.1. Animals and samples 71
• 3.2.2. RNA extraction, conventional RT-PCR, and qRT-PCR 71
• 3.2.3. Western blot 72
• 3.2.4. Immunohistochemical and fluorescence microscopy 72
• 3.2.5. Ovariectomy and hormone treatment 73
• 3.2.6. In situ hybridization 74
• 3.2.7. Statistical analysis 74
• 3.3. Results 75
• 3.3.1. Nucb2 mRNA and nesfatin-1 protein expression in reproductive organs of female mice 75
• 3.3.2. Localization of Nucb2 mRNA and nesfatin-1 protein in ovary and uterus tissues of mouse 77
• 3.3.3. Expression of Nucb2 mRNA and nesfatin-1 protein in the ovary and uterus of mice during the estrous cycle 81
• 3.3.4. Effect of PMSG and 17β-estradiol on Nucb2 mRNA expression in the ovary and uterus 85
• 3.3.5. Nucb2 mRNA and nesfatin-1 protein expression in the mouse oviduct 88
• 3.3.6. Nucb2 mRNA expression in the mouse oviduct during the estrous cycle 91
• 3.3.7. Effect of gonadotropins and sex steroid hormones on Nucb2 mRNA expression in the mouse oviduct 93
• 3.4. Discussion 96
• Chapter IV. Expression of NUCB2/nesfatin-1 and its function in male reproductive organs 103
• 4.1. Introduction 104
• 4.2. Materials and methods 107
• 4.2.1. Animals and samples 107
• 4.2.2. Hormone administration in mice 107
• 4.2.3. Castration and hormone treatment 108
• 4.2.4. Isolation and culture of mouse Leydig cells 108
• 4.2.5. TM3 cell culture 109
• 4.2.6. Hormone treatment in cultured cells 109
• 4.2.7. RNA extraction, conventional RT-PCR, and qRT-PCR 109
• 4.2.8. Western blot 110
• 4.2.9. Immunofluorescence staining 111
• 4.2.10. In situ hybridization 112
• 4.2.11. Statistical analysis 112
• 4.3. Results 113
• 4.3.1. Expression and localization of NUCB2/nesfatin-1 in mouse testis and Leydig cells 113
• 4.3.2. Localization of nesfatin-1 protein and nesfatin-1 binding sites in isolated Leydig cells and TM3 cells 116
• 4.3.3. Nucb2 mRNA expression in the testis and Leydig cells after PMSG and hCG treatment 118
• 4.3.4. Effect of nesfatin-1 on the expression of steroidogenesis-related enzyme genes in isolated Leydig cells and TM3 cells 120
• 4.3.5. Expression of Nucb2 mRNA and nesfatin-1 protein in the epididymis 123
• 4.3.6. Effects of gonadotropins or testosterone on Nucb2 mRNA and nesfatin-1 protein expression in the mouse epididymis 126
• 4.4. Discussion 129
• Chapter V. General discussion 134
• 5.1. Contribution 1: Expression of nesfatin-1 and its function in pituitary gland 135
• 5.2. Contribution 2: Expression of nesfatin-1 and its function in female reproductive organs 136
• 5.3. Contribution 3: Expression of nesfatin-1 and its function in male reproductive organs 137
• 5.4. Conclusions 138
• References 140
• 국문요약 156
• 저작물활용동의서 159