Leydig cell에서 에스트로젠수용체알파의 기능 연구

Abstract

Leydig cell은 정소에서 남성호르몬을 생성하는 기능을 한다. Leydig cell은 출생 전후로 각각 fetal Leydig cell과 adult Leydig cell로 구분되어 출현한다. Adult Leydig cell의 발생은 세 단계로 나뉜다. 첫째, spindle 형태의 stem Leydig cell에서 spindle 형태의 progenitor Leydig cell로 분화한다. 이때, 3β-hydroxysteroid dehydrogenase를 발현하게 된다. 둘째, progenitor Leydig cell에서 원형의 immature Leydig cell로 변환하다. 이때, 남성호르몬 대사효소의 활성이 증가한다. 셋째, immature Leydig cell에서 adult Leydig cell로 성숙한다. 이때는 세포의 크기가 더 커진다. 에스트로젠은 여성뿐만 아니라 남성에서도 중요한 역할을 하는 것으로 알려져 있다. 정소에서는 aromatase에 의해 남성호르몬이 여성호르몬, 즉 에스트로젠으로 변환한다. 에스트로젠수용체 (ER)는 에스트로젠이 결합하여 다른 유전자의 전사를 조절하는 전사인자이다. 두 종의 ER, 즉 에스트로젠수용체알파 (ERα)와 에스트로젠수용체베타 (ERβ) 모두 남성 생식소에서 발현한다. 생쥐 정소에서 ERα는 Leydig cell에서 발현하는 것으로 알려져 있다. 본 연구에서는 ERα 유전자 형질전환 생쥐모델을 이용하여 Leydig cell에서 ERα의 기능을 이해하고자 하였다. 제 1장에서는 Leydig cell에서 ERα의 세포특이적 기능을 이해하기 위하여 Leydig cell에서 특이적으로 ERα 유전자가 적중된 ERαf/f Cyp17iCre 생쥐를 이용하여 정자형성과 스테로이드생성을 분석하였다. 이들 생쥐에서는 젊은 성체시기에서는 정상 표현형을 보이다가 늙은 성체시기에서 뇌하수체장애와 함께 높은 테스토스테론생성을 보였다. 따라서 Leydig cell에서 ERα는 정자형성에 필수적인 것은 아닌 것으로 보이며, 노화된 Leydig cell의 기능에서 중요한 역할을 하는 것으로 사료된다. 제 2장에서는 생후 발생단계에 따라 Leydig cell에서 ERα의 발현을 살펴보았고, 에스트로젠이 progenitor Leydig cell의 스테로이드생성과 증식에 어떻게 작용하는지 알아보았다. ERα는 Leydig cell의 발생단계에 따라 차등적으로 발현하였다. 따라서 ERα 발현은 Leydig cell 발생의 바이오마커가 될 수 있을 것으로 사료된다. Progenitor Leydig cell에서는 hCG에 의해 상승된 스테로이드생성과 증식이 에스트로젠-ERα 신호에 의해 억제되었다. 제 3장에서는 Leydig cell에서 수분통로의 일종인 aquaporin-5 (AQP5)의 역할을 이해하기 위해 Leydig cell에서 AQP5의 발현과 에스트로젠에 의한 조절을 분석하였다. Leydig cell에서 에스트로젠은 ERα를 통해 AQP5의 발현을 전사수준에서 조절하는 것으로 확인하였다. 또한 aquaporin-5 knockdown 결과, 세포의 면적이 증가하였고, 스테로이드생성이 감소하는 것을 보였다. 따라서 Leydig cell에서 AQP5는 세포용적을 조절하며 스테로이드생성에 관여하는 것으로 사료된다. 마지막으로 제 4장에서는 정소에서 에스트로젠의 활성을 조절하는 것으로 알려진 estrogen sulfotransferase (EST)의 발현조절을 살펴보았다. Microarray 분석 결과, ERαf/f Cyp17iCre 생쥐 정소에서 EST의 발현이 wild type에 비해 낮았다. 또한 wild type 생쥐의 Leydig cell에서 EST의 발현은 에스트로젠에 의해 증가한 반면, ERαf/f Cyp17iCre 생쥐의 Leydig cell에서는 에스트로젠에 의한 영향이 없었다. 따라서 에스트로젠은 ERα를 통해 EST의 발현을 양성적으로 조절하는 것으로 사료된다. 결론적으로 에스트로젠-ERα 신호는 Leydig cell의 발생, 분화 및 스테로이드생성을 내분비적으로 조절함으로써 Leydig cell의 기능을 조절하는 중요한 기작으로 작동하는 것으로 사료된다. |Leydig cells, which are found adjacent to the seminiferous tubules in the testis, produce testosterone in the presence of luteinizing hormone (LH) which is secreted from the pituitary gland. Two populations of Leydig cells, fetal Leydig cells and adult Leydig cells, arise during prenatal and postnatal development, respectively. The development of the adult Leydig cell population consists of three steps. First, spindle-shaped stem Leydig cells differentiate into spindle-shaped progenitor Leydig cells, which express 3β-hydroxysteroid dehydrogenase. Second, the progenitor Leydig cells transform into round-shaped immature Leydig cells, which display high levels of androgen-metabolizing enzyme activity. Third, the immature Leydig cells mature into adult Leydig cells, and this maturation is accompanied by a further increase in cell size. Estrogen plays an important role in both male and female reproduction. In the testis, androgens can be converted into estrogens by aromatase. The estrogen receptor (ER) is a member of the steroid receptor superfamily of ligand-activated transcription factors. Two estrogen receptor isoforms, ERα (ESR1) and ERβ (ESR2), are expressed in male gonad. In mouse testis, ERα is expressed in Leydig cells. To understand the function of ERα in Leydig cells, this study was performed using ERα transgenic mouse models. In Chapter 1, to evaluate the specific function of ERα in Leydig cells, spermatogenesis and steroidogenesis in ERαf/f Cyp17iCre male mice in which ERα was deleted specifically in Leydig cells were explored. These mice showed normal phenotype at young adulthood, and, at old age, showed high testosterone synthesis without pituitary dysfunction. Therefore, ERα in Leydig cells may be not essential for spermatogenesis under control of endogenous estrogens and may have a role in aged Leydig cell function. In Chapter 2, the expression of ERα in Leydig cells of the developing mouse testes and the effects of estrogen on steroidogenesis and proliferation by progenitor Leydig cells were investigated. ERα was differentially expressed during Leydig cell development, suggesting that the ERα level is a potential biomarker of Leydig cell development. In progenitor Leydig cells, hCG-stimulated steroidogenesis and proliferation were negatively regulated by estrogen-ERα signaling. In Chapter 3, to elucidate the role of aquaporin-5 (AQP5), a water channel which plays a role in the fluid homeostasis and cell volume control, in the testis, the expression and the regulation of AQP5 were examined in mouse Leydig cells. The AQP5 expression was closely coupled with differentiation of adult Leydig cells and was transcriptionally regulated by estrogen via ERα. In addition, AQP5 knockdown resulted in decreased steroidogenesis, suggesting that AQP5 is involved in cell volume control and in steroid synthesis in Leydig cells. Finally, in Chapter 4, the regulation of estrogen sulfotransferase (EST) expression in Leydig cells by estrogen was investigated. EST modulates the estrogen activity in the testis. Microarray analysis revealed that EST was down-regulated in ERαf/f Cyp17iCre mouse testis. In wild type Leydig cells, the expression of EST was induced by estrogen, while the expression was not affected in ERαf/f Cyp17iCre Leydig cells. This suggests that estrogen regulates the EST expression in Leydig cells via ERα. In conclusion, estrogen-ERα signaling should be considered as a mechanism of the endocrine control of Leydig cell development and function in the testis.; Leydig cells, which are found adjacent to the seminiferous tubules in the testis, produce testosterone in the presence of luteinizing hormone (LH) which is secreted from the pituitary gland. Two populations of Leydig cells, fetal Leydig cells and adult Leydig cells, arise during prenatal and postnatal development, respectively. The development of the adult Leydig cell population consists of three steps. First, spindle-shaped stem Leydig cells differentiate into spindle-shaped progenitor Leydig cells, which express 3β-hydroxysteroid dehydrogenase. Second, the progenitor Leydig cells transform into round-shaped immature Leydig cells, which display high levels of androgen-metabolizing enzyme activity. Third, the immature Leydig cells mature into adult Leydig cells, and this maturation is accompanied by a further increase in cell size. Estrogen plays an important role in both male and female reproduction. In the testis, androgens can be converted into estrogens by aromatase. The estrogen receptor (ER) is a member of the steroid receptor superfamily of ligand-activated transcription factors. Two estrogen receptor isoforms, ERα (ESR1) and ERβ (ESR2), are expressed in male gonad. In mouse testis, ERα is expressed in Leydig cells. To understand the function of ERα in Leydig cells, this study was performed using ERα transgenic mouse models. In Chapter 1, to evaluate the specific function of ERα in Leydig cells, spermatogenesis and steroidogenesis in ERαf/f Cyp17iCre male mice in which ERα was deleted specifically in Leydig cells were explored. These mice showed normal phenotype at young adulthood, and, at old age, showed high testosterone synthesis without pituitary dysfunction. Therefore, ERα in Leydig cells may be not essential for spermatogenesis under control of endogenous estrogens and may have a role in aged Leydig cell function. In Chapter 2, the expression of ERα in Leydig cells of the developing mouse testes and the effects of estrogen on steroidogenesis and proliferation by progenitor Leydig cells were investigated. ERα was differentially expressed during Leydig cell development, suggesting that the ERα level is a potential biomarker of Leydig cell development. In progenitor Leydig cells, hCG-stimulated steroidogenesis and proliferation were negatively regulated by estrogen-ERα signaling. In Chapter 3, to elucidate the role of aquaporin-5 (AQP5), a water channel which plays a role in the fluid homeostasis and cell volume control, in the testis, the expression and the regulation of AQP5 were examined in mouse Leydig cells. The AQP5 expression was closely coupled with differentiation of adult Leydig cells and was transcriptionally regulated by estrogen via ERα. In addition, AQP5 knockdown resulted in decreased steroidogenesis, suggesting that AQP5 is involved in cell volume control and in steroid synthesis in Leydig cells. Finally, in Chapter 4, the regulation of estrogen sulfotransferase (EST) expression in Leydig cells by estrogen was investigated. EST modulates the estrogen activity in the testis. Microarray analysis revealed that EST was down-regulated in ERαf/f Cyp17iCre mouse testis. In wild type Leydig cells, the expression of EST was induced by estrogen, while the expression was not affected in ERαf/f Cyp17iCre Leydig cells. This suggests that estrogen regulates the EST expression in Leydig cells via ERα. In conclusion, estrogen-ERα signaling should be considered as a mechanism of the endocrine control of Leydig cell development and function in the testis.