Altered expression of ZO-1 and ZO-2 in Sertoli cells and loss of blood-testis barrier integrity in testicular carcinoma in situ

Abstract

Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.

Figure 1

Schematic drawing of the current model of the tight junction structure in the seminiferous epithelium. Tight junctions are formed between adjacent Sertoli cells near the basal lamina, constituting the blood-testis barrier and separating the seminiferous epithelium into a basal compartment and an adluminal compartment. SG, spermatogonium; pSP, preleptotene/leptotene spermatocyte; SP, pachytene spermatocyte; rSp, round spermatid; Sp, elongated spermatid; SC, Sertoli cells; JAM, junctional adhesion molecules (modified from Lui et al. [14]).

Figure 2

Immunohistochemical localization of ZO-2 in human seminiferous epithelium. (A) Cross section of a normal seminiferous tubule. Immunoreactive ZO-2 is localized between Sertoli cells at the basal compartment, consistent with the site of the blood-testis barrier (arrows). (B) Cross section of a tubule with CIS cells and RSP. In regions with RSP, ZO-2 immunostaining is concentrated at the basolateral region of Sertoli cells (arrows), whereas in regions containing CIS cells, ZO-2 immunostaining became weak and diffuse (arrowheads). (C) Cross section of a seminiferous tubule containing CIS cells, and cross section of a part of a normal tubule. Within the CIS-only tubule, ZO-2 immunostaining became even weaker and more diffuse around the basolateral site, but spread to stain the lateral site of Sertoli cells up to the adluminal compartment, as well as their cytoplasm (arrows). Bar = 50 µm.

Figure 3

Western blot analysis for ZO-1 and ZO-2 of testis homogenates. Western blot analysis revealed ZO-1 immunoreactivity by a triple band between approximately 220 and 240 kDa in testes with NSP and by a weaker triple band at the same level in testes with CIS. ZO-2 immunoreactivity was revealed by a band at approximately 160 kDa in testes with NSP and by a weaker band at the same level in testes with CIS.

Figure 4

RT-PCR analysis for ZO-1 and ZO-2 mRNA of testis homogenates. By RT-PCR, an amplification product of expected size (370 bp for ZO-1 and 306 bp for ZO-2) is obtained in testes with NSP and in testes showing CIS, confirming the presence of ZO-1 and ZO-2 mRNA.

Figure 5

Electron micrograph of a seminiferous tubule showing NSP. Lanthanum tracer, which appears as an electron-dense material (arrow), impregnates spermatogonia on the basal lamina. Bar = 5 µm. Enlargement of inset: Inter-Sertoli cell junctional complex, where tracer (arrow) penetration is stopped by tight junctions. Bar = 0.5 µm.

Figure 6

Electron micrograph of a seminiferous tubule containing CIS cells. Lanthanum tracer (arrow) surrounds CIS cells on the basal lamina. Bar = 10 µm. Enlargement of inset: Inter-Sertoli cell junctional complex, typified by subsurface bundles of actin filaments and associated cisternae of endoplasmic reticulum. Lanthanum tracer (arrows) passes through the junctional complex, illustrating that tight junctions were functionally disrupted. Bar = 0.5 µm.

Figure 7

Electron micrograph of a seminiferous tubule containing CIS cells and RSP. Lanthanum tracer (black arrow) surrounds the CIS cell on the basal lamina and the neighboring round spermatid (inset a), illustrating local disruption of the blood-testis barrier. In the area of residual spermatogenesis, the intercellular space of a round spermatid is free of lanthanum (inset b), indicating an intact blood-testis barrier. Bar = 10 µm. Enlargement of inset a: Round spermatid with lanthanum in the intercellular space (black arrow). Bar = 1 µm. Enlargement of inset b: Round spermatid without lanthanum in the intercellular space (transparent arrow). Black arrow shows lanthanum in the intercellular space of two Sertoli cells. Bar = 1 µm.