- Top of page
- Materials and Methods
ABSTRACT: Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate—polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH2-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 × 106 cells/cm2) and high (0.5 × 106 cells/cm2) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli—germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either α2-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.
During spermatogenesis, type B spermatogonia residing in the basal compartment of the seminiferous epithelium outside of the blood-testis barrier must differentiate into preleptotene spermatocytes, while progressively migrating from the basal to the adluminal compartment (for reviews, see Setchell and Waites, 1975; de Kretser and Kerr, 1988; Setchell, 1998). Although the mechanism by which developing germ cells traverse the epithelium is, at present, elusive, previous studies from our and other laboratories have shown that the movement of germ cells likely involves cyclic phases of Sertoli-Sertoli and Sertoli—germ cell junction deadhesion and adhesion (for reviews, see Russell, 1993a,b; Mruk and Cheng, 1999; Cheng and Mruk, 2002). For instance, in vitro studies have demonstrated that there are changes in the expression of several target genes when junctions are being formed in Sertoli cell cultures (Chung et al, 1999; Wong et al, 2000; Lui et al, 2001) or Sertoli—germ cell cocultures (Mruk et al, 1997; Chung et al, 1998a,b; Mruk and Cheng, 1999; Lee et al, 2002), a cellular phenomenon similar to junction assembly during germ cell movement in vivo. These changes were in agreement with those detected when Sertoli cell junctions were disassembled by either transforming growth factor β3 (TGF-β3; Lui et al, 2001) or cadmium chloride (Chung and Cheng, 2001). This suggests that an array of molecules must be temporally up- or down-regulated to bring about junction assembly and disassembly. Yet an extensive literature search revealed that studies examining the participation of proteases and protease inhibitors in the events of junction assembly and disassembly in the testis are limited.
In a previous report, which studied the assembly of adherens junctions between Sertoli and germ cells using an in vitro coculture system, it was shown that adhesion of germ cells onto the Sertoli cell epithelium is associated with an induction of proteases and protease inhibitors (Mruk et al, 1997). Another study showed that chloroquine, a protease inhibitor, could facilitate the assembly and maintenance of the Sertoli cell tight junction barrier (Okanlawon and Dym, 1996). Taken collectively, these results apparently suggest that proteases and protease inhibitors participate in junction dynamics. We herein describe the purification and partial characterization of a putative Sertoli cell protease inhibitor, known as tissue inhibitor of metalloproteases-1 (TIMP-1), from Sertoli cell conditioned medium (SCCM). The purification of TIMP-1 (Mr 31–29 kDa) is an extension of our earlier report on TIMP-2 (Mr 22 kDa) (Grima et al, 1996), because the retention times of both TIMPs overlapped in the preparative anion-exchange and C8 reverse-phase high-performance liquid chromatography (HPLC) steps. Anti-protease activity was also a characteristic of both of these proteins when [125I]-collagen was used as a substrate in a protease assay (Grima et al, 1996). On this note, we have used TIMP-1 as a target molecule to determine whether any changes in its expression could be detected during Sertoli-Sertoli and Sertoli—germ cell junction assembly in vitro, similar to our previous study (Mruk et al, 1997). Because many of the results presented in this report rely heavily on changes in the TIMP-1 mRNA level as detected by reverse transcription-polymerase chain reaction (RT-PCR), we have used an in vitro germ cell adhesion assay to demonstrate that protease inhibitors participate in junction dynamics by facilitating the binding of germ cells onto Sertoli cells.
- Top of page
- Materials and Methods
The function of Sertoli cells in the seminiferous epithelium is dynamic, because these cells secrete an array of proteins required for normal testicular function (for reviews, see Skinner, 1991; Jegou, 1993; Mruk and Cheng, 2000). In the current study, we report the purification of TIMP-1, a Sertoli and germ cell product, from the spent media of primary Sertoli cell cultures having anti-protease activity. To date, 4 members of the TIMP family have been characterized—TIMP-1, TIMP-2, TIMP-3, and TIMP-4 (for reviews, see Gomez et al, 1997; Bode et al, 1999; Brew et al, 2000). Specifically, TIMP-1 and TIMP-2 function by inhibiting the activities of matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent proteases, by maintaining a balance between extracellular matrix deposition and degradation in the testis (for reviews, see Fritz et al, 1993; Dym, 1994). These protease inhibitors have also been shown to have other functions unrelated to their anti-MMP activity, such as inhibiting tumor growth, invasion, and metastasis, modulating cell morphology, controlling growth factor availability, and participating in gonadal steroidogenesis, thus illustrating that they are multifunctional proteins (for reviews, see Brew et al, 2000; Jiang et al, 2002). Although both TIMP-1 (Ulisse et al, 1994) and TIMP-2 (Ulisse et al, 1994; Grima et al, 1996) are produced by Sertoli cells (all 4 TIMPs have been found to be present in the testis; Robinson et al, 2001), the purification of TIMP-1 from SCCM, its presence in immature germ cells, and regulation during junction assembly have not yet been reported. Moreover, the results presented herein are an expansion of our earlier study (Mruk et al, 1997), which demonstrated that protease inhibitors participate in the assembly of adherens junctions.
To determine whether changes in the TIMP-1 mRNA level correlated with the events of junction assembly and/or disassembly, such as during the formation of the blood-testis barrier or the first wave of spermiation, we relied on semiquantitative RT-PCR. The increase in testicular TIMP-1 expression from 40 to 60 days of age is suggestive of its participation in the events of spermiation. However, the increase in testicular TIMP-1 expression cannot be ascribed to germ cells, because we failed to detect TIMP-1 mRNA in germ cells isolated from rats at 45 to 120 days of age, which is similar to a previously published report by Gronning et al (2000). Subsequently, the increase in testicular TIMP-1 expression was justified when the Sertoli cell TIMP-1 mRNA level was found to increase from 45 to 90 days of age. The increase in testicular TIMP-1 may also be assigned to Sertoli—germ cell interactions or to peritubular myoid, but not Leydig cells, because they were also found to express TIMP-1 (Gronning et al, 2000). Taking these results collectively, it is possible that an elevated level of Sertoli cell TIMP-1 may be required to neutralize endogenous MMP activity immediately after spermiation. As such, TIMP-1 may function by promoting junction assembly and, in turn, maintaining the integrity of the epithelium. It is also likely that other TIMP family members participate in these events as well.
On the other hand, we were able to detect TIMP-1 expression in 10-, 15-, and 20-day-old germ cells, consisting of spermatogonia and primary spermatocytes. The presence of TIMP-1 mRNA in germ cells was not due to somatic cell contamination because, in a series of preliminary experiments by RT-PCR, we did not amplify testin (a Sertoli cell product; Cheng et al, 1989; Zong et al, 1994), 3β-hydrosteroid dehydrogenase (a Leydig cell product; Zwain et al, 1991), and fibronectin (a peritubular myoid cell product; Tung et al, 1984), as described elsewhere (Lee et al, 2002). Although we do not know the immediate reason for the presence of TIMP-1 mRNA in 10-to 20-day-old germ cells, an elevated level of TIMP-1 may need to be restricted to the basal compartment of the epithelium to facilitate the assembly of the blood-testis barrier, which takes place at 15 to 18 days of age in the rat. Despite the fact that germ cells per se do not contribute directly to the assembly of the Sertoli cell tight junction, it is very likely that these cells participate indirectly via a yet-to-be-identified mechanism. Second, we failed to detect an increase in testicular TIMP-1 expression from 10 to 20 days of age, probably because other events, unrelated to the assembly of the blood-testis barrier, are taking place, thereby masking the up-regulation in TIMP-1 mRNA. Third, these germ cells, which reside in the basal compartment adjacent to the basal lamina, may also regulate the remodeling of the extracellular matrix. Last, TIMP-1 expression was not detected in 45- to 120-day-old germ cells, which suggests that secondary spermatocytes and round spermatids (elongate spermatids and spermatozoa were removed from all of our germ cell isolations) do not synthesize this protease inhibitor.
More important, when Sertoli cells were cultured at high density, TIMP-1 expression was found to increase approximately threefold during junction assembly, followed by a significant decline from days 3 to 5 after junctions assembled. At present, it is difficult to target changes in the TIMP-1 mRNA level to the assembly of a single junction type, because the assembly of tight, anchoring, and gap junctions in epithelial cells is interdependent. We also do not know whether similar changes in expression could be detected with other protease inhibitors, such as α2-MG, cystatin C, or plasminogen activator inhibitor. However, we do anticipate that an array of protease inhibitors functions during junction assembly with a majority of the inhibitors being up-regulated, although some may be down-regulated.
Similar changes in TIMP-1 expression were detected when germ cells were cocultured with Sertoli cells, co-inciding with the adhesion of germ cells onto the Sertoli cell epithelium. The increase in TIMP-1 expression as early as 5 minutes suggests that this protease inhibitor may be required to limit endogenous MMP activity at the site of Sertoli—germ cell interaction. This study did not determine, however, whether a higher level of TIMP—anti-protease activity can be detected in apical chambers of bicameral units. In control cultures, we failed to detect changes in TIMP-1 expression when Sertoli cells were cultured alone without the addition of germ cells.
The results presented in this study demonstrate that the inclusion of protease inhibitors can facilitate the adhesion of fluorescently-labeled germ cells onto the Sertoli cell epithelium, whereas anti-α2-MG antibody was able to perturb adhesion when compared with control cocultures. We speculate that the inclusion of TIMP-1 into this in vitro adhesion assay would also improve germ cell binding. Ideally, an experiment should be performed in which inhibitors that block serine, cysteine, aspartic, and metalloprotease activity are added into this in vitro assay and the adhesion of germ cells assessed; commercially available protease inhibitors, however, are toxic to cells. To circumvent this technical difficulty, we are presently attempting to localize proteases and protease inhibitors by fluorescent microscopy to the site of Sertoli—germ cell interaction using cross-sections of Sertoli—germ cell co-cultures that have been cultured on bicameral units. This experiment should demonstrate the importance of these molecules in junction assembly.
It is also possible that other yet-to-be-defined factors, such as cytokines, are functioning in this in vitro system, in addition to proteases and protease inhibitors. For instance, α2-MG can bind growth factors, such as TGF-β3, rendering TGF-β3 inactive (O'Connor-McCourt and Wakefield, 1987; Huang et al, 1988). In the testis, TGF-β3 can regulate Sertoli cell tight junction dynamics, because its presence can perturb the tight junction permeability barrier in vitro by modulating occludin and ZO-1 levels (Lui et al, 2001). On the other hand, basic fibroblast growth factor β and TGF-β3 can affect proteases, such as plasminogen activator, a serine protease, in Sertoli cells in vitro (Jaillard et al, 1987; Nargolwalla et al, 1990). Taken collectively, the results presented in the present study suggest the participation of protease inhibitors in cell junction dynamics.