Expression of claudin-11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testes. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the non-breeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonular occludens-1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those of non-breeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the BTB where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJ’s expressing CLDN11 at the BTB in amniotes.

Testicular expression of CLDN11 (claudin-11), a tight junction protein was examined together with spermatogenesis and circulating testosterone levels in Korean soft-shelled turtle (Pelodiscus maackii). Spermatogenesis started during the breeding season in May and peaked in August when the breeding season ended. Spermiation started in July and peaked in October, showing the typical pattern of spermatogenesis in temperate zone reptiles. Deduced amino acid sequences of P. maackii CLDN11 was highly homologous to those of avian and mammals, suggesting the conserved nature of CLDN11 in amniotes. During the non-breeding season when the spermatogenesis was active and circulating testosterone levels elevated, testicular CLDN11 mRNA and protein (19kDa) levels were high. Strong, wavy CLDN11 immunoreactive strands run parallel to basement membrane in the basal part of the seminiferous epithelium, delaminating the spermatogonia and early spermatocytes in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm perpendicular to basement membrane. In double labeling experiment, punctate ZO-1 immunoreactivity was found within the CLDN11 strands run parallel to the basement membrane as well as at the most periphery of seminiferous epithelium where ZO-1 and CLDN11 in Sertoli cells were mostly cytoplasmic and perpendicular to basement membrane. Together, recruit of CLDN11 and ZO-1 to the inter-Sertoli TJs was tightly coupled with spermatogenic stage. At the breeding season when the circulating testosterone levels and spermatogenic activity remained low, testicular CLDN11 mRNA and protein levels were low. CLDN11 was found at apicolateral contacts between adjacent Sertoli cells devoid of the postmeiotic germ cells, suggesting that CLDN11 between adjacent Sertoli cells also participates in the maintenance of seminiferous lumen. In P. maackii testis, CLDN11 as a structural element of the blood-testis barrier dynamically changed according to spermatogenic activity and circulating androgen levels. This is the first study on the CLDN TJs at the BTB in reptilian testis.

정소 내 생식세포 분화가 이루어지는 세정관과 혈관 사이에 존재하는 blood-testis barrier (BTB)는 다양한 밀착결합(tight junction) 단백질로 이루어져 있다. Claudin-11은 이러한 밀착결합을 구성하는 단백질 중 하나로 잘 알려져 있다. 본 연구에서는 토끼(domestic rabbit; Oryctolagus cuniculus domesticus), 꿩(pheasant; Phasianus colchicus), 자라(soft shelled turtle; Pelodiscus sinensis)의 정소에서 claudin-11의 발현을 관찰하고, 성적 성숙 또는 번식기에 따른 변화를 확인하고자 하였다. 이를 위해 각 동물의 claudin-11 partial cDNA 염기서열을 확보하고 이를 통해 RT-PCR을 수행하였다. 또한 정소 조직 내에서 claudin-11 발현 부위를 확인하기 위해 면역염색을 시행하였으며, western bolt을 통해 단백질발현을 확인하였다. 토끼와 꿩의 정소에서 claudin-11은 성적으로 성숙한 이후에, 자라의 경우 생식세포의 분화가 활발히 일어나는 비번식기에 mRNA 발현량의 급격히 증가하였으며, 이는 단백질의 발현량과 일치하는 것을 확인하였다. 성적 성숙이 일어나지 않은 토끼와 꿩 및 번식기 자라의 정소내에서 claudin-11 발현은 주로 Sertoli cells 및 그 사이에서 확인되었다. 그러나 생식세포 분화가 활발한 시기의 세정관 내에서 claudin-11은 Sertoli cells 및 그 사이 뿐만 아니라 Sertoli cell이 spermatogonia와 접하는 바닥면에 물결형태로 분포하는 것이 확인되었다. 이는 포유류뿐만 아니라 조류에서도 claudin-11이 생식세포 분화를 진행하는 세정관 내에서 BTB를 구성하고 있음을 의미한다. 또한, 계절에 따라 생식세포 군집의 변화를 보이는 자라의 정소에서 생식세포 분화가 활발한 시기에 claudin-11이 BTB 구성에 참여하는 것이 확인되었다. 꿩의 정소 및 정소(organ culture)에서 분리해 낸 Sertoli cells(primary culture)를 이용한 실험에서 testosterone 처리에 따라 claudin-11 mRNA가 증가하는 것이 확인되었으며, 자라의 호르몬을 분석한 결과, 세정관내 생식세포 분화가 활발한 시기에 testosterone 농도가 높아지는 것을 확인하였다. 이는 claudin-11이 남성호르몬의 조절을 받으며, 생식세포의 분화가 활발한 시기의 높은 농도의 남성호르몬은 세정관내부 환경을 독립적으로 유지할 수 있도록 claudin-11이 포함된 BTB를 발달시키는 것으로 사료된다. 본 연구를 통해 현재까지 잘 알려져 있지 않은 조류와 파충류 정소에서의 claudin-11 발현을 확인하였으며, 정소 내 고유환경 조성에 참여하는 것은 claudin-11이 진화선상에서 갖는 공통된 기능임을 확인하였다.