Egr1 Knockout Model을 이용한 생쥐 정소에서 전사조절인자 Egr1의 기능 분석

Abstract

Zinc finger transcription factor인 Early Growth Response (EGR) family는 다양한 종류의 mitogen에 의해 신속히 유도되어, 세포의 성장, 분화, 사멸을 조절한다. 본 연구는 생쥐 정소에서 정자형성 (spermatogenesis)과 스테로이드합성 (steroidogenesis)에서 Egr1의 기능에 대해 밝히고자 하였다. 생쥐 정소와 Leydig cell에서 Egr1 mRNA는 출생 직후 가장 많이 발현하였고, 성장하면서 감소하였다. Egr1 immunoreactivity는 출생 직후부터 성체시기 정소의 생식모세포 (gonocytes), 정원세포 (spermatogonia), Leydig cells의 핵에서 나타났다. 교배 실험 결과, Egr1 (-/-) 생쥐와 교배한 암컷의 가임능력이 Egr1 (+/-)와 비교하여 유의적으로 감소하였다. 생후 2 ~ 3 개월령과 1년 이상의 Egr1 (-/-) 수컷 생쥐 모두에서 혈중테스토스테론 농도가 Egr1 (+/-)와 비교하여 유의적으로 낮았다. 정소조직 이미지 분석 결과, 생후 2 ~ 3개월령 생쥐와 1년 이상 Egr1 (-/-) 생쥐에서 Leydig cell의 면적과 수가 Egr1 (+/-) 생쥐와 비교하여 유의적으로 낮았다. 스테로이드합성회로 유전자인 luteinizing hormone (LH) receptor, steroidogenic factor 1 (SF1), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase type 6 (3β-HSD6), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) mRNA는 생후 2 ~ 3개월령과 1년 이상 Egr1 (-/-) 생쥐 정소에서 Egr1 (+/-) 생쥐와 비교하여 유의적으로 감소하였다. 정소와 Leydig cell에 human chorionic gonadotropin (hCG, 50ng/ml) 투여 2시간 후, Egr1 (+/-) 생쥐에서 StAR mRNA는 증가했지만, Egr1 (-/-) 생쥐에서는 Egr1 (+/-) 생쥐에 비해 유의적으로 낮았다. 일일정자생산량 (Daily Sperm Production, DSP)은 Egr1 (-/-) 수컷 생쥐에서 낮았으며, 후기 정자형성과정에 문제가 있는 비정상 세정관의 수와 세정관 내 세포 자살 양성 세포의 수가 유의적으로 증가하였다. In vitro Fertilization (IVF) 결과, 1년 이상 Egr1 (-/-) 생쥐의 미부 부정소 정자의 수정율은 유의적으로 낮았다. hCG와 testosterone propionate (TP) 투여에 의한 Egr1 (-/-) 생쥐의 생식능력 회복 여부를 확인하기 위해 TP (25mg/kg, 30 days)를 투여한 결과 스테로이드합성회로 유전자 mRNA 발현 및 Leydig cell 면적은 유의적으로 감소하였으나, IVF 수정율은 Egr1 (+/-) 생쥐와 유사한 수준으로 회복되었다. hCG (5IU, 14 days) 투여 후 스테로이드합성회로 유전자 mRNA와 Leydig cell의 면적은 회복되었으나, IVF 수정율은 회복되지 않았다. 따라서 Egr1은 어린 수컷 생쥐의 정소에서 정원세포, peritubular cells, Leydig cell의 증식과 생존에 관여하는 것으로 사료된다. Egr1 (-/-) 수컷 성체 생쥐의 생식능력 감소는 뇌하수체에서 LH 감소와 남성호르몬 감소 때문으로 추측되며 Egr1은 LH에 대한 Leydig cell의 반응에 중요한 전자조절인자로 사료된다.| The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulated cell growth, differentiation, and apoptosis. Present study was aimed to verify the role of Egr1 in spermatogenesis and steroidogenesis in mouse testis. Egr1 mRNA levels in mouse testis and Leydig cells were peaked neonatally and decreased thereafter until puberty. Egr1 immunoreactivity was found in the nuclei of gonocytes, spermatogonia, peritubular cells and Leydig cells in fetal through adulthood. Pituitary LHβ mRNA levels of Egr1 (-/-) mouse (2 ~ 3 months old) were significantly lower than those of Egr1 (+/-). The number and size of Leydig cells were significantly decreased in Egr1 (-/-) mouse testes both at young (2 ~ 3 months old) and old (>12 months old) age. Testicular steroidogenic pathway enzyme genes StAR, Cyp11a1, 3β-HSD6, Cyp17a1, and 17β-HSD3 mRNA levels were decreased in Egr1 (-/-) mouse testes both at young (2 ~ 3 months old) and old (>12 months old) age. Serum testosterone levels of Egr1 (-/-) male mice were decreased in young (2 ~ 3 months old) and old (>12 months old) age. When stimulated by hCG for 2 hr StAR mRNA levels were significantly decreased in mouse testes (hCG, 5IU) and Leydig cells (hCG, 50ng/ml) of Egr1 (-/-) mice compared to those of Egr1 (+/-) mice. Daily sperm production (DSP) was significantly decreased in Egr1 (-/-) male mice. Abnormal seminiferous tubules and TUNEL positive cells were significantly increased in Egr1 (-/-) mice testis. Birth rate of female mice mated with Egr1 (-/-) male mice was significantly decreased. IVF using epididymal sperm revealed a significant decrease in sperm fertility of Egr1 (-/-) mice. To test recovery of Egr1 (-/-) male mice fertility by hormonal treatment, hCG (5IU, 14 days) and/or testosterone propionate (TP, 25mg/kg, 30 days) were given to Egr1 (-/-) male mice. Following hCG treatment the number and size of Leydig cells, testicular sterodiogenic pathway enzyme genes mRNA were recovered but IVF fertilization rate was not. In TP injected Egr1 (-/-) male mice, the number and size of Leydig cells, testicular steroidogenic pathway enzyme genes mRNA levels were significantly decreased but IVF fertilization rate was recovered. In conclusion, Egr1 may participate in the survival and proliferation early male germ cells, peritubular cells and fetal Leydig cells. The decrease in male reproductive potential in Egr1 (-/-) male might be attributable to decrease in pituitary LH synthesis and Leydig cell testosterone synthesis, and the Leydig cells insensitivity to LH in male mice.; The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulated cell growth, differentiation, and apoptosis. Present study was aimed to verify the role of Egr1 in spermatogenesis and steroidogenesis in mouse testis. Egr1 mRNA levels in mouse testis and Leydig cells were peaked neonatally and decreased thereafter until puberty. Egr1 immunoreactivity was found in the nuclei of gonocytes, spermatogonia, peritubular cells and Leydig cells in fetal through adulthood. Pituitary LHβ mRNA levels of Egr1 (-/-) mouse (2 ~ 3 months old) were significantly lower than those of Egr1 (+/-). The number and size of Leydig cells were significantly decreased in Egr1 (-/-) mouse testes both at young (2 ~ 3 months old) and old (>12 months old) age. Testicular steroidogenic pathway enzyme genes StAR, Cyp11a1, 3β-HSD6, Cyp17a1, and 17β-HSD3 mRNA levels were decreased in Egr1 (-/-) mouse testes both at young (2 ~ 3 months old) and old (>12 months old) age. Serum testosterone levels of Egr1 (-/-) male mice were decreased in young (2 ~ 3 months old) and old (>12 months old) age. When stimulated by hCG for 2 hr StAR mRNA levels were significantly decreased in mouse testes (hCG, 5IU) and Leydig cells (hCG, 50ng/ml) of Egr1 (-/-) mice compared to those of Egr1 (+/-) mice. Daily sperm production (DSP) was significantly decreased in Egr1 (-/-) male mice. Abnormal seminiferous tubules and TUNEL positive cells were significantly increased in Egr1 (-/-) mice testis. Birth rate of female mice mated with Egr1 (-/-) male mice was significantly decreased. IVF using epididymal sperm revealed a significant decrease in sperm fertility of Egr1 (-/-) mice. To test recovery of Egr1 (-/-) male mice fertility by hormonal treatment, hCG (5IU, 14 days) and/or testosterone propionate (TP, 25mg/kg, 30 days) were given to Egr1 (-/-) male mice. Following hCG treatment the number and size of Leydig cells, testicular sterodiogenic pathway enzyme genes mRNA were recovered but IVF fertilization rate was not. In TP injected Egr1 (-/-) male mice, the number and size of Leydig cells, testicular steroidogenic pathway enzyme genes mRNA levels were significantly decreased but IVF fertilization rate was recovered. In conclusion, Egr1 may participate in the survival and proliferation early male germ cells, peritubular cells and fetal Leydig cells. The decrease in male reproductive potential in Egr1 (-/-) male might be attributable to decrease in pituitary LH synthesis and Leydig cell testosterone synthesis, and the Leydig cells insensitivity to LH in male mice.