생쥐 정소에서 급성 전신성 염증반응에 의한 혈액정소장벽 구조와 기능의 변화

Abstract

혈액정소장벽은 Sertoli 세포간의 밀착결합으로써 세정관과 Sertoli 세포 사이의 물리적 장벽이다. 이러한 혈액정소장벽은 정자형성과정 동안 세정관내에서 물리적 장벽 유지와 면역체계로부터 생식세포를 보호한다. 사람에서 전신 감염과 만성 염증반응은 일시적으로나 영구적인 불임을 초래할 정도로 정소의 스테로이드 합성과 정자형성과정에 손상을 입힐 수 있다. 본 연구는 급성 염증반응이 혈액정소장벽에 미치는 변화를 밝히기 위해 lipopolysaccharide (LPS) 투여 이후 생쥐 정소에서 일어나는 밀착결합 유전자 발현과 확산장벽기능의 변화를 조사하였다. 성체 생쥐에 LPS를 투여한 후 12, 24시간 뒤에 정소와 부정소를 적출하였다. 정소와 부정소 중량은 LPS투여군에서 12시간경과시에는 큰 변화가 없었으나 24시간 뒤에는 유의적으로 증가하였다. 급성 염증반응이 일어난 증거로 realtime RT-PCR 결과 TNF-α가 12시간 경과시 유의적으로 증가하였고, TNF-α, IL-4, IL-5가 LPS 투여 24시간에 유의적으로 증가하였다. 조직 재구성인자인 MMP2, MMP9 mRNA 또한 LPS 투여 24시간 경과 생쥐 정소에서 유의적으로 증가하였다. 12시간 경과시 밀착결합유전자 발현에 유의적 변화가 없었으나, 24시간 경과 후 Claudin 1, 2, 4, 5, JAM 3, CAR long form, Tricellulin long form, mid form은 유의적으로 증가하였고, Claudin 3, ZO-1 mRNA는 유의적으로 감소하였다. Occludin, Claudin 11, JAM 1, JAM 2 mRNA는 LPS 투여 이후 유의적인 변화를 보이지 않았다. Western Blot 결과 Claudin 1은 확연하게 증가하였으나, Claudin 3, 11, ZO-1은 감소하였다. Occludin은 크게 변화가 없었다. Biotin tracer 주입을 통해 혈액정소장벽의 투과성을 조사한 결과 대조군에서는 세정관 안에서 biotin이 검출되지 않았지만, LPS를 투여한 생쥐에서는 세정관내의 정모세포 주변 및 Sertoli 세포 사이에서 biotin이 검출되었다. 결론적으로 LPS에 의한 급성 염증반응이 Sertoli 세포 사이의 밀착결합의 분자구조를 변화시키는 것으로 사료된다. 이러한 변화에는 염증성 cytokine과 조직재구성요소인 MMP9의 변화에 의해 매개되는 것으로 사료된다. 전신성 염증반응은 혈액정소장벽의 변화를 야기하므로 정자형성과정에 영향을 미치는 것으로 사료된다.|Blood testis barrier (BTB) is a inter-Sertoli tight junction (TJ) and functions as a physical barrier between seminiferous tubules and Sertoli cells. In an effort to elucidate the changes in BTB in acute inflammation, the change in the expression of TJ genes and paracellular diffusion barrier property were examined in mouse testis following the lipopolysaccharide (LPS) injection. Mouse testes were isolated 12hr and 24hrs after LPS injection. In result, testis and epididymis weight were significantly increased in LPS mice. Realtime RT-PCR revealed that TNF-α and IL-4, IL-5 mRNA levels were significantly increased following LPS injection for one day, indicating the acute inflammation. MMP2 and MMP9 mRNA levels were significantly increased in LPS mouse testes. CLDN 1, 2, 4 5, JAM 3, CAR long form, and tricellulin long form and mid form mRNA levels were significantly increased in LPS mouse testes. In contrast, CLDN 3 and ZO-1 mRNA levels were significantly decreased in LPS mouse testes. Occludin, CLDN 11, JAM 1, and JAM2 mRNA levels were not significantly different between LPS and control mouse testes. On western blot, CLDN 1 was markedly increased but CLDN 3, 11, and ZO-1 were decreased in LPS injected mouse testes. Occludin was not significantly different between LPS and control mice. The permeability of BTB was examined by tail vein injection of biotin tracer. In control mice, biotin was not detected in the adluminal part of seminiferous epithelium. In contrast, biotin was detected at the vicinity of spermatocytes in LPS mouse testis. Together, acute inflammation by LPS may alter the molecular structure of inter-Sertoli TJs via increasing the CLDN1 and decreasing CLDN 3, 11, and ZO-1 in testis. Furthermore, MMP2 and 9 as a tissue remodeling factors may participate in alteration of BTB in LPS injected mouse testis. This changes in the structure of inter-Sertoli TJs may alter the function of BTB, contributing the spermatogenesis dysfunction under inflammation.; Blood testis barrier (BTB) is a inter-Sertoli tight junction (TJ) and functions as a physical barrier between seminiferous tubules and Sertoli cells. In an effort to elucidate the changes in BTB in acute inflammation, the change in the expression of TJ genes and paracellular diffusion barrier property were examined in mouse testis following the lipopolysaccharide (LPS) injection. Mouse testes were isolated 12hr and 24hrs after LPS injection. In result, testis and epididymis weight were significantly increased in LPS mice. Realtime RT-PCR revealed that TNF-α and IL-4, IL-5 mRNA levels were significantly increased following LPS injection for one day, indicating the acute inflammation. MMP2 and MMP9 mRNA levels were significantly increased in LPS mouse testes. CLDN 1, 2, 4 5, JAM 3, CAR long form, and tricellulin long form and mid form mRNA levels were significantly increased in LPS mouse testes. In contrast, CLDN 3 and ZO-1 mRNA levels were significantly decreased in LPS mouse testes. Occludin, CLDN 11, JAM 1, and JAM2 mRNA levels were not significantly different between LPS and control mouse testes. On western blot, CLDN 1 was markedly increased but CLDN 3, 11, and ZO-1 were decreased in LPS injected mouse testes. Occludin was not significantly different between LPS and control mice. The permeability of BTB was examined by tail vein injection of biotin tracer. In control mice, biotin was not detected in the adluminal part of seminiferous epithelium. In contrast, biotin was detected at the vicinity of spermatocytes in LPS mouse testis. Together, acute inflammation by LPS may alter the molecular structure of inter-Sertoli TJs via increasing the CLDN1 and decreasing CLDN 3, 11, and ZO-1 in testis. Furthermore, MMP2 and 9 as a tissue remodeling factors may participate in alteration of BTB in LPS injected mouse testis. This changes in the structure of inter-Sertoli TJs may alter the function of BTB, contributing the spermatogenesis dysfunction under inflammation.