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Regular Article |
a Population Council, Center for Biomedical Research, New York, New York 10021
ABSTRACT
Results of previous in vitro and in vivo studies have illustrated that the expression of testin by Sertoli cells is tightly associated with the disruption of Sertoli-germ cell junctions. In the present study, treatment of rats with cadmium chloride (CdCl2), which disrupted the inter-Sertoli tight junctions, failed to induce any changes in testicular testin expression. In contrast, lonidamine, an antispermatogenic drug that rearranges the Sertoli cell membrane microfilament structure causing a disruption of Sertoli-germ cell adhesion junctions, induced a drastic increase in testicular testin expression when administered orally. Lonidamine-induced Sertoli cell testin expression involved both ongoing RNA and de novo protein synthesis. Basal testin expression remained stable during the 27-h incubation with actinomycin D but required de novo protein synthesis in vitro. An inhibitor of protein kinase A, Rp-cAMPS, caused a 50% inhibition of Sertoli cell testin expression at 10 µM within 24 h. A biphasic response was noted in testin expression when forskolin was included in the Sertoli cell culture, and high concentrations of cAMP analogues (1 mM) rapidly reduced testin expression. However, lonidamine can abolish the inhibitory effect of cAMP analogues on Sertoli cell testin expression. These results illustrate that the induction of testin expression may involve several signal transduction pathways.
cAMP, Sertoli cells
INTRODUCTION
Development of reversible male contraceptives has been limited to hormonal and immunological approaches targeted at interfering with the hypothalamus-pituitary-testicular axis or sperm motility/sperm-egg interactions, respectively [1]. The lack of new, safer, and innovative male contraceptives largely results from the absence of a thorough understanding of the biochemical and molecular events pertinent to spermatogenesis, mainly because studies performed in this subject area during the past four decades were primarily morphological analyses. Our laboratory has chosen on a new area in which to investigate the biochemistry of germ cell movement in the seminiferous epithelium to understand the mechanism by which germ cells remain attached to Sertoli cells throughout their development. Results of these studies should lead to the development new methods for male contraception. For instance, if germ cells are induced to move faster across the seminiferous epithelium and/or to detach from the epithelium into the tubular lumen prematurely, spermatozoa in the ejaculates will lack the ability to fertilize the egg. Alternatively, if germ cells are retained in the epithelium for an extended period of time, these "aged" germ cells will undergo apoptosis and be removed from the epithelium by Sertoli cells via phagocytosis. In both cases, infertility will result. Such a study to delineate the biology of Sertoli-germ cell interactions, particularly the events leading to the movement of germ cells across the epithelium and the eventual release of spermatids from the epithelium, is of utmost importance to achieve the goal of developing novel male contraceptives. Testin, which is a novel testicular protein produced by Sertoli cells, appears to be a good candidate with which to study these events.
Testin is a putative Sertoli cell secretory protein [2–4] that can become tightly associated with the Sertoli cell surface on its secretion via two interacting plasma membrane proteins of 28 and 45 kDa [5] when specialized inter-Sertoli or Sertoli-germ cell junctions are formed. Immunohistochemistry at both the light and electron microscopy (EM) levels revealed its localization near the Sertoli cell surface in the testis at sites where Sertoli and germ cells interact [5, 6]. Results of tissue distribution studies indicate that testin is expressed in most organs but at a very low level, being virtually undetectable by Northern blot analysis but evident by hot-nested reverse transcription-polymerase chain reaction (RT-PCR). However, its expression is highest in the testis and ovary [7]. When deposition of the testin gene product in the epithelium at different stages of the spermatogenic cycle was examined by immunohistochemistry, a cyclic change in its localization in the epithelium was detected [8]. These results raise the possibility that testin may be involved in spermiation, because an accumulation of testin at the site where germ cells attach to Sertoli cells was found immediately before spermiation. In vivo experiments utilizing agents that induce depletion of germ cells from the testis, such as x-ray radiation, busulfan, and glycerol, can also induce a drastic increase in the testicular testin steady-state mRNA level [5, 6, 9, 10]. However, germ cells per se had no apparent effect in regulating Sertoli cell testin expression in Sertoli-germ cell cocultures in vitro, illustrating that the expression of testin somehow relates only to the disruption of Sertoli-germ cell junctions [5, 6]. This postulate was further strengthened by the observation that hypotonic treatment of Sertoli-germ cell cocultures in vitro to physically lyse germ cells, thereby disrupting the Sertoli-germ cell junctions, can induce a drastic surge in testin expression [6]. These results suggest that testin is a sensitive marker to screen potential male contraceptives targeted at disrupting the Sertoli-germ cell junctions, inducing premature release of germ cells from the epithelium.
Lonidamine (LD; 1-[2,4-dichlorobenzyl]indazole-3-carboxylic acid), which is a derivative of indazole carboxylic acids, is a potent antispermatogenic compound [11]. Its actions on Sertoli cells mimic those on epithelial squamous carcinoma (A431) and melanoma (M14) cells, causing disruption of the cytoskeletal elements [12–14]. In vivo administration of LD causes premature release of germ cells into tubular lumen, making the seminiferous epithelium virtually devoid of elongate and round spermatids as well as spermatocytes [11, 13]. Whereas LD is not cytotoxic to Sertoli cells in vitro [5], its administration to adult rats in vivo at 25 to 50 mg/kg body weight (BW) caused morphological changes in Sertoli cells at the ultrastructural level, as manifested by the vacuolation and retraction of the apical cytoplasm at the site where immature spermatids attach to Sertoli cells [13]. This is followed by enlargement of the Sertoli-germ cell spaces, eventually leading to the release of immature spermatids into the tubular lumen [13]. Interestingly, LD also induces a surge in testicular testin expression within 24 h of administration, which is long before germ cells were seen to deplete from the epithelium at Day 6 [6]. Therefore, when LD exerts its effects on the testicular cytoskeletal elements, it somehow activates the expression of testin.
In addition, LD can inhibit respiration in condensed mitochondria found in spermatids and spermatocytes but not in Sertoli cells or other somatic cells both in vivo and in vitro [15, 16]. We speculate that the LD-induced changes in germ cell respiration do not play a role in testin gene activation, because germ cells per se had no apparent effects on Sertoli cell testin expression [5, 6]. These earlier results, however, illustrate the novelty of testin in studying Sertoli-germ cell interactions. Thus, we thought it pertinent to examine the mechanism of testin gene activation and regulation in hopes of expanding our knowledge regarding this molecule and its role in Sertoli-germ cell interactions.
MATERIALS AND METHODS
Biochemicals
Adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS, a protein kinase [PKA] A l and ll inhibitor), chelerythrine chloride (CC, a PKC inhibitor), and D-erythro-sphingosine (D-Sph, a PKC and calmodulin [CaM] kinase inhibitor), N62'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br cAMP), forskolin (FSK), cycloheximide, and actinomycin D (AD) were purchased from Sigma (St. Louis, MO). Lonidamine was a gift from the Angelini Pharmaceuticals S.p.A. (Rome, Italy). The Rp-cAMPS was dissolved in sterile water to a stock concentration of 25 mM. Stock solutions of dbcAMP (20 mM), 8-Br cAMP (20 mM), and CC (25 mM) were prepared in water. Stock solutions of cycloheximide (10 mg/ml), AD (0.5 mg/ml), LD (100 µg/ml), and D-Sph (50 mM) were prepared in ethanol. Forskolin was prepared in dimethyl formamide.
In Vivo Disruption of the Tight Junction-Associated Microfilaments in Rat Testis by Cadmium Chloride
Sprague-Dawley rats, approximately 250 g BW, were anesthetized with Metofane (Mallinckrodt Veterinary, Mundelein, IL) and injected i.p. with 1 mg/kg BW of cadmium chloride (CdCl2) as a 0.05% solution in deionized water [17]. Animals were then killed at 4, 24, 48, and 72 h and 7 days after the injection. Three animals were used per time point, and the experiment was performed twice. Testicular RNA was extracted by RNA STAT-60 (Tel Test, Inc., Friendswood, TX) according to the manufacturer's protocols. Testicular cytosols were also prepared in these animals, and the concentrations of testin in these samples were quantified by a testin-specific RIA as described elsewhere [3].
Northern Blot Analysis
Northern blot analysis for testin was performed as described elsewhere [7]. The cDNA probe for connexin (CX)-33 consisted of a 648-base pair (bp) cDNA corresponding to nucleotides 973–1620 of the CX-33 nucleotide sequence [18]. The cDNA probe for CX-43 consisted of a 540-bp cDNA corresponding to nucleotides 282–821 of the CX-43 nucleotide sequence [19]. The cDNA probe for rat ribosomal S16 consisted of a 385-bp cDNA corresponding to nucleotides 15–399 of the S16 nucleotide sequence [20]. All cDNA probes were nick translated using a kit from Promega (Madison, WI) with 
-32P-deoxycytidine triphosphate. Blots were hybridized overnight at 42°C with the corresponding -32P-labeled cDNA probe. To ensure that equal amounts of total RNA were used per lane, each blot was either simultaneously hybridized or rehybridized with an -32P-labeled cDNA for ß-actin or rat ribosomal S16 [7]. Autoradiographs were then densitometrically scanned at 600 nm using an Ultroscan XL Enhanced Laser Densitometer (Amersham Pharmacia Biotech, Uppsala, Sweden), and testin expression was normalized against either ß-actin or S16.
Semiquantitative RT-PCR
Semiquantitative RT-PCR analysis was performed essentially as described elsewhere [6] to examine the changes of testicular protein zonula occludens-1 (ZO-1) expression after treatment with CdCl2. The ZO-1 was coamplified with rat ribosomal S16, which was used as an internal control to monitor the sample-to-sample variation in the RT and PCR results. In a series of preliminary experiments (data not shown), increasing concentrations of template RNA were used to verify the linearity of the assay regarding the amount of products. The amount of PCR products was also examined over a range of 15 to 30 amplification cycles for a given amount of template RNA to select the optimal number of amplification cycles in the linear range. Briefly, 1 µg of RNA was reverse transcribed into cDNAs using 2 µg of oligo(thymidine)-15 primer and avian myeloblastosis virus reverse transcriptase in a final reaction volume of 25 µl according to the manufacturer's protocols (Promega). Approximately 2.5 µl of the RT product was used as a template for PCR. Approximately 0.3 µg of the sense and antisense ZO-1 primers were used in the PCR reaction in combination with primer pairs for rat ribosomal S16. In addition to the unlabeled primers, approximately 200 ng of the sense primers for both ZO-1 and S16 were 5'-end-labeled with 
-32P-ATP (specific activity = 2500 Ci/mmol; Amersham Pharmacia Biotech) by using T4 polynucleotide kinase (Promega) and added to the PCR reaction. The nucleotide sequences for the ZO-1 primers used for amplification were as follows: ZO-1 sense primer corresponding to nucleotides 61–78 (5'-GCCTCTGCAGTTAAGCAT-3'), and ZO-1 antisense primer corresponding to nucleotides 292–309 (5'-AAGAGCTGGCTGTTTTAA-3') [21]. The nucleotide sequences for the rat ribosomal S16 primers used in conjunction with the ZO-1 primers were as follows: S16 sense primer corresponding to nucleotides 15–38 (5'-TCCGCTGCAGTCCGTTCAAGTCTT-3'), and S16 antisense primer corresponding to nucleotides 376–399 (5'-GCCAAACTTCTTGGATTCGCAGCG-3') [20]. The cycling parameters for the PCR were 94°C for 1 min, with annealing at 56°C for 2 min and extension at 72°C for 2 min. A total of 22 cycles were performed. Aliquots (10 µl) of the PCR reaction were resolved on a 5% polyacrylamide gel and visualized by ethidium bromide staining and autoradiography using X-OMAT AR film (Eastman Kodak, Rochester, NY).
Preparation of Testicular Cell Cultures
Sertoli cells were isolated from 20-day-old Sprague-Dawley rats as described elsewhere [22]. Cells were cultured in F12/Dulbecco modified Eagle medium (DMEM) supplemented with gentamicin (20 mg/L), sodium bicarbonate (1.2 g/L), 15 mM HEPES, bovine insulin (10 µg/ml), human transferrin (5 µg/ml), epididymal growth factor (5 ng/ml), and bacitracin (5 µg/ml, a protease inhibitor) and incubated at 35°C in a humidified atmosphere of 95% air/5% CO2 (v/v). Cells were plated at high cell density at 0.5 x 106 cells/cm2 on six-well Matrigel (diluted 1:5 with F12/DMEM)-coated plates unless described otherwise. Low-cell-density cultures were plated on 100-mm, Matrigel (Collaborative Biochemicals, Bedford, MA) (diluted 1:5 with F12/DMEM)-coated dishes at 0.5 x 105 cells/cm2. In studies examining the changes in Sertoli cell testin steady-state mRNA level over time, the day of Sertoli cell isolation was designated Day 0, and no hypotonic treatment was performed on these cultures. The isolation procedure routinely took 5–6 h. In all other cases, Sertoli cells (plated on Day 0) were hypotonically treated with 20 mM Tris (pH 7.4) for 2.5 min to remove residual germ cells 24 h later on Day 1 [23]. Cells were then allowed to recover for a 24-h period before receiving any treatments that began on Day 2. Cells were pretreated with either cycloheximide or AD for 3 h before the addition of LD. In experiments utilizing signal transduction inhibitors, cells were pretreated with Rp-cAMPS, CC, and D-Sph for 6 h before the addition of LD.
Germ cells were isolated from adult rat testes by a mechanical procedure that removed elongated spermatids as previously elsewhere [6] and immediately used for coculture with a Sertoli:germ cell ratio of 1:3 (Sertoli cells at 0.5 x 105 cells/cm2). These Sertoli-germ cells were cocultured for 2 days to allow formation of specialized Sertoli-germ cell junctions [24, 25] before the addition of increasing doses of LD. Thereafter, these cultures were incubated with LD for 24 h before termination. The LD was dissolved in ethanol to a stock concentration of 1 mg/ml. Control experiments contained cells incubated with vehicle (i.e., ethanol) alone.
In the experiments used to assess the effects of LD on maintenance of the inter-Sertoli tight junctions, Sertoli cells were plated in the apical chamber of a nitrocellulose bicameral culture unit coated with Matrigel (1:7 dilution) at a density of 2 x 106 cells/cm2 after their isolation. Tight junctions were allowed to form over a period of 5 days as described elsewhere [4, 5]. Thereafter, different doses of LD were added to both the apical and basal chambers and incubated with the cells for 24 h. Cells were then rinsed twice with fresh F12/DMEM, and the integrity of the inter-Sertoli tight junctions was assessed by measuring the diffusion of 125I-BSA as described elsewhere [4]. Briefly, 1 x 104 cpm of 125I-BSA was added to the apical chamber, and its diffusion was determined by sampling media from both the apical and basal chambers over time. Radioactivity was measured in a Hewlett Packard Cobra II -counter (Wilmington, DE) at 95% efficiency. Transepithelial electrical resistance (TER) across the Sertoli cell epithelia were also determined using a Millicell Electrical Resistance system (Millipore, Bedford, MA) as detailed elsewhere [5], and similar results were obtained compared with those of the 125I-BSA or 3H-inulin diffusion studies. Each tissue culture experiment consisted of two to three wells, and each the experiment was repeated twice.
General Methods
Protein estimation was performed by the Coomassie blue-dye binding assay using BSA as a standard [26]. Statistical analysis was performed using ANOVA by the Tukey honestly significant difference with GB Stat Version 3.0 software (Silver Spring, MD) or by the Student t-test using Jandel Scientific Sigmaplot Version 3.0 (SPSS Inc., Chicago, IL).
RESULTS
Disruption of Inter-Sertoli Tight Junctions in Rat Testis by CdCl2 In Vivo
Results of earlier studies have shown that LD can cause a dramatic increase in testicular testin expression within 24 h after its administration in vivo [6]. However, whether this outcome results from the damage of inter-Sertoli tight junctions, anchorage, or gap communicating junctions found between Sertoli cells and/or between Sertoli and germ cells is not known. We sought to examine whether a disruption of inter-Sertoli tight junctions, specifically by CdCl2, could also affect testin expression as well as other junctional complex proteins in vivo. Cadmium has long been recognized as being a testicular toxin [27], and at high doses, it can cause dramatic vascular damage to the testis [28]. However, at low doses, CdCl2 can specifically disrupt inter-Sertoli cell tight junctions in rats in vivo, resulting in breakdown of the blood-testis barrier without vascular damage [17]. Adult Sprague-Dawley rats received a low dose of 1 mg/kg BW CdCl2 i.p. as described elsewhere [17]. At specific time points, animals in groups of three to six were killed, and RNA was extracted for Northern blot and semiquantitative RT-PCR analyses. Although CdCl2-induced damage to the Sertoli cell microfilaments became apparent by 24 h and increased in severity with time [17], no changes were detected in testin expression in the testis when examined by Northern blot analysis, as shown in Figure 1, A and B. In addition, no changes in testin concentration were detected in the testicular cytosol from CdCl2-treated animals when quantified by RIA (Fig. 1C). This indicates that CdCl2 treatment affected neither testin expression nor its secretion. Thus, CdCl2 and its inability to affect testin expression contrasts sharply with LD, which causes a drastic increase testin expression in the testis that is accompanied by a massive loss of germ cells. These results illustrate that only a disruption of either anchorage or gap junctions (or both) can elicit a surge in testin expression. We next examined whether CdCl2 treatment would affect ZO-1. Because the expression of ZO-1 in these samples was less than the detection limit of Northern blot analysis as observed in preliminary experiments, we used semiquantitative RT-PCR to analyze any changes in expression, as shown in Figure 1D. The S16 ribosomal RNA was coamplified with ZO-1 to monitor sample-to-sample variation in each reaction (described earlier). This study indicates that the steady-state ZO-1 mRNA level in testes from all CdCl2-treated rats were not different from that in control rats at time 0 (Fig. 1D). In addition, no significant changes in testicular expression after CdCl2 treatment were noted for two gap junction proteins, CX-43 and CX-33, as shown in Figure 1, E and F, respectively, versus S16 (Fig. 1G).
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Effect of LD on Testin Expression by Sertoli Cells and Sertoli-Germ Cell Cocultures In Vitro
Because LD induces testicular testin expression almost 50-fold in 24 h [6], we next examined its effect on Sertoli cells in vitro. Sertoli cells cultured at 0.5 x 106 cells/cm2 were incubated with increasing doses of LD from 0 to 1000 ng/ml for 24 h, and the changes of testin expression were examined by Northern blot analysis. We found that LD caused a mild, dose-dependent increase in Sertoli cell testin expression in vitro (Fig. 2A). It should be noted that a greater induction of testin expression by LD may not have been observed, because testin expression by Sertoli cells in culture is already stimulated 24 h after the culture procedure (see Fig. 4). The secretion of testin by Sertoli cells also increased in a dose-dependent manner with LD treatment (Fig. 2C). Interestingly, none of the doses of LD that stimulated testin expression could affect the inter-Sertoli tight junction permeability barrier in vitro when assessed by diffusion of 125I-BSA (1 x 104 cpm) from the apical chamber into the basal chamber over time when Sertoli cells were cultured in bicameral culture chambers at 2 x 106 cells/cm2 (Fig. 2D). Similar results were obtained when the TER across the Sertoli cell epithelia was monitored (data not shown), illustrating that LD did not disrupt the tight junctional microfilaments involved in the Sertoli cell tight junction permeability barrier. Only the control chambers without any cells permitted diffusion of 125I-BSA into the basal chamber and reached equilibrium by 24 h. Figure 2E is a phase-contrast micrograph of the Sertoli cells plated at 2 x 106 cells/cm2 in the apical chamber of a bicameral culture unit, illustrating the morphology of the Sertoli cells used for diffusion studies. These data indicate that LD specifically altered the Sertoli cell testin expression without affecting the inter-Sertoli tight junction permeability barrier. In addition, these studies illustrate that CdCl2 and LD had different effects on the Sertoli cell microfilament elements.
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The effect of increasing doses of LD on testin expression by Sertoli-germ cell cocultures was then examined. Sertoli cells were cocultured with germ cells (Sertoli:germ cell) at a ratio of 1:3 (Sertoli cells at 0.5 x 106 cells/cm2) for 2 days at the time when specialized junctions such as desmosome-like anchorage and gap junctions are known to form between Sertoli and germ cells [24, 25]. Cultures were then incubated with increasing doses of LD (10–1000 ng/ml) for an additional 24 h. We found that LD stimulated testin expression and secretion in a dose-dependent manner (Fig. 2, F and H). Notably, total testin accumulation in the medium of Sertoli-germ cell cocultures without any LD (controls), as shown in Figure 2H, is less than that of the Sertoli cell only controls, as shown in Figure 2C (125 ± 5 ng/well vs. 225 ± 8 ng/well). This probably resulted from more testin being bound to the site where Sertoli and germ cells interact, as reported elsewhere [7]. As such, less testin is available in the medium. It should also be noted that the relative increase in testin expression after LD treatment in the Sertoli cell cultures was comparable to the Sertoli-germ cell cocultures, which contrasts with expectations (Fig. 2A vs. Fig. 2F). Taking these results collectively, they seem to support the postulate that LD exerts its effect directly on Sertoli cells by increasing testin expression. However, this effect on Sertoli cells may ultimately result in the intracellular space between Sertoli and germ cells to increase subsequent to LD treatment in vivo, thereby disrupting Sertoli-germ cell junctions, as found in earlier morphological studies [13]. In other words, an alteration of Sertoli cell membrane components induced by LD treatment may, therefore, be responsible for the massive depletion of germ cells.
Effects of Cycloheximide and AD on LD-Induced Testin Expression by Sertoli Cells
The requirement of ongoing RNA and protein synthesis for the stimulation of testin mRNA by LD was also examined. Sertoli cells were pretreated for 3 h with either 50 µg/ml of cycloheximide (i.e., a protein synthesis inhibitor) or 5 µg/ml of AD (i.e., an RNA synthesis inhibitor) before the addition of LD (100 ng/ml) and then incubated for an additional 24 h. Cycloheximide and AD at these concentrations inhibit Sertoli cell protein and RNA synthesis by 96% and 98%, respectively [29]. We found that cycloheximide caused a significant decrease in the Sertoli cell basal testin expression compared with the control (Fig. 3, A and C), indicating that the basal testin expression by Sertoli cells requires ongoing protein synthesis. This result also implies that Sertoli cells may produce an activator of the testin gene to regulate its expression. In contrast, AD alone did not affect basal testin expression after 27 h of exposure (Fig. 3C), suggesting that testin mRNA is very stable. Regarding cultures in which Sertoli cells were treated with both LD and cycloheximide, LD failed to induce testin expression, indicating that this response requires ongoing protein synthesis. Similarly, LD in the presence of AD also failed to induce an increase in testin expression, illustrating that it also requires RNA synthesis. Treatment of cells with AD, however, caused a significant inhibition of Sertoli cell testin secretion into the medium, which contrasts with the testin expression (Fig. 3D vs. Fig. 3, A and C). This may result from the ability of AD to inhibit RNA synthesis of certain proteins that are needed for protein secretion. Expression data in these experiments were not normalized to ß-actin or S16, because AD inhibited expression of these mRNAs, indicating that AD was, indeed, effective in inhibiting ongoing RNA expression in this culture.
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Expression of Testin and Its Secretion by Sertoli Cells When Cultured at Different Cell Densities In Vitro
Because the basal testin expression appears to be maintained by the presence of an activator, we examined the pattern of testin expression by Sertoli cells in vitro under different culture conditions. Sertoli cells were cultured at high (0.5 x 106 cells/cm2) and low (0.5 x 105 cells/cm2) cell densities. Cells maintained at high density on Matrigel-coated dishes mimic the in vivo morphology of Sertoli cells within the testis compared with low-cell-density monolayers [4]. Testin expression and secretion were analyzed from Day 0 (corresponding to the time of cell plating) to Days 9 to 10, as shown by Figure 4, A and D. Testin expression was hardly detectable in Sertoli cells immediately after their isolation (Day 0; Fig. 4A) from the testes, indicating that its expression under in vivo conditions is normally very low, which is consistent with results from two earlier reports [2, 7]. However, within a 24-h culture period, the level of testin expression increased by as much as 10-fold and remained high thereafter in both high- and low-density cultures (Fig. 4, A and C). Testin expression began to decline on Day 8 and was more drastic in the low-density cultures (Fig. 4, A and C). When the immunoreactive testin secreted by Sertoli cells in these experiments was quantified by RIA, the amount of testin secreted into the medium for each 24-h interval was consistently higher in the low-cell-density cultures throughout the entire experimental period, even though only the densities of the cultures differed (0.5 x 106 cells/cm2 vs. 0.5 x 105 cells/cm2). The reason is because a greater amount of cell contact occurs in high-density cultures, which form a columnar monolayer compared with the low-density cultures. Therefore, these data are consistent with those of an earlier study illustrating that testin is reabsorbed onto the inter-Sertoli cell interface through two interacting proteins [5].
Effects of Rp-cAMPS, CC, and D-Sph on Basal and LD-Induced Sertoli Cell Testin Expression
We next examined the effects of different PK inhibitors on the basal and LD-induced testin expression in Sertoli cells to assess its mechanism of induction. In preliminary experiments, Sertoli cells were treated with 1, 10, and 100 µM of Rp-cAMPS, CC, and D-Sph to determine the optimal concentration of each inhibitor to elicit an effect and as suggested by the manufacturers and other published studies [30–32]. High concentrations (100 µM) of CC and D-Sph caused cells to die. Therefore, Sertoli cells were pretreated for 6 h with 100 µM of Rp-cAMPS, an inhibitor of cAMP-dependent PKA I and II; 10 µM CC, an inhibitor of PKC; or 10 µM D-Sph, an inhibitor of PKC and CaM-dependent kinases, before the addition of 100 ng/ml of LD or without LD (i.e., kinase inhibitor alone) for an additional 24 h. We found that LD alone induced testin expression significantly compared with control (Fig. 5, A and B), and Rp-cAMPS caused a significant decrease in the basal testin expression, indicating that the putative testin activator is PKA dependent (Fig. 5, A and B). However, Rp-cAMPS only mildly inhibited the LD-induced expression of Sertoli cell testin, indicating that LD may not exert its effects totally via the cAMP pathway. In addition, CC, an inhibitor of PKC, and D-Sph, an inhibitor of PKC and CaM-dependent kinases, had no apparent effects on the basal and LD-induced testin expression. These results illustrate that basal Sertoli cell testin expression may be regulated via the cAMP pathway, which may be in contrast to the mechanism used by LD. To ensure that the Rp-cAMPS inhibitory effect is, indeed, specific, Sertoli cells (0.5 x 106 cells/cm2) were incubated for 24 h with 1, 10, and 100 µM Rp-cAMPS; testin expression decreased in a dose-dependent manner, as illustrated in Figure 5, C and D.
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Dose-Dependent Effects of FSK, dbcAMP, and 8-Br cAMP on Testin Expression by Sertoli Cells
The effects of FSK, dbcAMP, and 8-Br cAMP on testin expression by Sertoli cells was also examined, because results of earlier studies indicated that basal testin expression may be regulated via the cAMP pathway. Forskolin activates the catalytic subunit of adenylate cyclase and raises cAMP levels. As indicated by the data in Figure 6, A and B, increasing concentrations of FSK caused a biphasic response of testin expression. This suggests that low levels of cAMP may stimulate testin expression, whereas high concentrations decrease testin expression. A mild stimulation of testin expression, similar to that of FSK, was also noted using low concentrations (0.01–1 µM) of dbcAMP (data not shown). Notably, a greater induction of testin expression by FSK and cAMP analogues may not be observed, because testin expression by Sertoli cells remains stimulated 24 h after the culture procedure, as previously shown in Figure 4. When Sertoli cells were incubated with elevated levels (0.01–1 mM) of dbcAMP (Fig. 6, C and D) and 8-Br cAMP (Fig. 6, E and F), a dose-dependent inhibition of Sertoli cell testin expression was noted.
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Effects of dbcAMP and 8-Br cAMP on LD-Induced Expression of Testin by Sertoli Cells
We next investigated the combined effects of cAMP analogues along with LD on testin mRNA expression. Elevated levels of cAMP analogues, such as dbcAMP (1 mM) and 8-Br cAMP (1 mM), caused a marked decrease in the basal testin steady-state level (Fig. 7, A and C). In contrast, when LD was added in conjunction with the cAMP analogues, testin expression remained significantly higher than control values, indicating that LD could still induce testin expression, prevailing over the inhibitory effects of the cAMP analogues (Fig. 7).
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Effects of Cycloheximide, AD, Rp-cAMPS, CC, and D-Sph on Inhibition of Sertoli Cell Testin Expression by dbcAMP
We next investigated whether the inhibition of testin expression by dbcAMP required ongoing RNA and protein synthesis. In addition, we examined whether dbcAMP inhibition of Sertoli cell testin expression could be reversed by inhibiting PKA, PKC, and CaM kinases using Rp-cAMPS, CC, and D-Sph, respectively. We found that cycloheximide could not reverse the inhibitory effects of dbcAMP on Sertoli cell testin expression, because cycloheximide alone at this concentration causes a decrease in the basal testin expression (Fig. 8C). In contrast, AD had no effects on Sertoli cell testin expression alone (Figs. 3 and 8), but it could abolish the inhibitory effect of dbcAMP (Fig. 8, A and C). This indicates that ongoing RNA synthesis is a requirement for testin inhibition by dbcAMP. Similar to cycloheximide, Rp-cAMPS had an inhibitory effect on the basal Sertoli cell testin expression alone because of the biphasic expression pattern exhibited in response to varying concentrations of cAMP analogues. However, Rp-cAMPS did not abolish the inhibitory effect of 1 mM dbcAMP on testin expression (Fig. 8, A and C), because the 100 µM concentration of Rp-cAMPS used might not have been potent enough. Neither CC nor D-Sph had any effect on the basal Sertoli cell testin expression. They also failed to reverse the inhibitory effect of dbcAMP on Sertoli cell testin expression, indicating that the effect of dbcAMP on Sertoli cell testin expression does not work via PKC or CaM kinases.
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Time-Dependent Effects of dbcAMP and LD on Sertoli Cell Testin Expression
The time-dependent action of dbcAMP and LD on Sertoli cell testin expression was also investigated. Sertoli cells were cultured at 0.5 x 106 cells/cm2 on Matrigel-coated dishes for 3 days to allow the testin steady-state mRNA level to stabilize (Fig. 4). Thereafter, Sertoli cells were treated with either 1 mM dbcAMP or LD (100 ng/ml) for 6 and 24 h and then compared with controls cultured for 24 h without any treatment. The LD-treated cultures showed no apparent change in testin expression by 6 h (Fig. 9, A and C). However, LD induced a 100% increase in testin expression by 24 h (Fig. 9, A and C). In contrast, dbcAMP caused an 80% decrease in testin expression by 6 h and completely abolished its expression by 24 h, indicating that the induction of testin mRNA by LD is much slower than the inhibition by dbcAMP.
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DISCUSSION
In the present study, regulation of testin expression by the cAMP/PKA pathway was examined. Our data indicate that testin expression is under multifaceted regulation. The basal Sertoli cell testin expression in vitro requires ongoing protein synthesis. It appears to be partially regulated through the cAMP pathway via PKA, because Rp-cAMPS at 10 µM, an inhibitor of PKA l and ll, caused a 50% reduction in basal testin expression by Sertoli cells in culture. Forskolin and cAMP analogues induce a biphasic response of basal Sertoli cell testin expression, causing a mild stimulation at low levels and a rapid reduction with high levels of cAMP analogues. Incubation of Sertoli cells with low concentrations of adenylate cyclase stimulators only induce a slight, but statistically significant, increase in testin expression, possibly because it has already been induced by the culture procedure itself, as illustrated in Figure 4. Whether the culture environment may affect the intracellular cAMP levels, which may be responsible for the increase in testin expression, is not known. A similar biphasic response to cAMP was shown for the 117 antigen mRNA in Dictyostelium discoideum. Interestingly, the 117 antigen mRNA is induced by low concentrations of pulsed cAMP (10-7 M) through a mechanism that does not involve a measurable increase in intracellular cAMP, whereas high concentrations of cAMP (1 mM) elicit a rapid decrease [33]. This quick decline is also associated with mRNA destabilization of the 117 antigen mRNA and numerous other mRNAs in different systems, such as cultured brown adipocytes [34], rat skeletal muscle [35, 36], and T-lineage cells [37] via the cAMP/PKA pathway. Although mRNA stability studies have not been done to prove that testin mRNA is, indeed, rapidly degraded by the production of PKA-dependent destabilization protein(s), the rapid reduction of testin expression, by as much as 70% within 6 h after treatment with 1 mM of dbcAMP, may be indicative of such an event. In addition, the cAMP inhibitory effect on testin expression depends on an ongoing RNA synthesis, which is required for the production of mRNA destabilizing proteins [33–35]. However, Rp-cAMPS and cycloheximide could not reverse the effect of the cAMP analogues. Although no mRNA destabilizing proteins are produced when cycloheximide is included in the culture media, no increase in testin expression was noted, because cycloheximide incubated with the cells alone at 50 µg/ml causes a decrease in testin expression. In essence, cycloheximide (50 µg/ml) cannot be used to evaluate the actions of cAMP, because it can affect the basal concentration of testin expression. Similarly, the concentration of Rp-cAMPS (100 µM) used in our culture experiments might not be potent enough to obliterate the effects of 1 mM dbcAMP. In addition, testin mRNA is extremely stable in Sertoli cells under in vitro conditions. Our data indicate that even after a 27-h exposure to AD, testin mRNA expression remains unchanged.
In the present study, basal testin expression by Sertoli cells was induced dramatically during the first 24 h of culture. This increase can be provoked in several different ways. It may be associated with the disruption of Sertoli-germ cell junctions [6] simply by the culture procedure alone. Alternatively, testin expression may normally be suppressed by an inhibitor produced by cell types removed during isolation, resulting in its induction such as peritubular myoid cells [22, 38, 39]. Then again, testin induction at Day one of culture may also result from a mild increase in intracellular cAMP that activates the PKA signal transduction pathway.
Our findings indicate that LD-induced stimulation of testin expression could dominate over the inhibitory effects of cAMP analogues. However, it remains to be determined exactly how LD works in stimulating testin mRNA. It may stimulate testin expression by 1) inducing changes in Sertoli cell intracellular cAMP, 2) inducing mRNA stabilizing proteins, 3) inhibiting mRNA destabilizing proteins, or 4) using of another signal transduction pathway. Furthermore, LD-induced Sertoli cell testin expression requires both ongoing RNA and protein synthesis and is not regulated via PKA, PKC, or CaM kinases. Therefore, LD-induced testin expression is mediated by a mechanism different from the basal expression.
That LD has such a dramatic effect on Sertoli cell testin expression seemingly suggests that the Sertoli cell is the target of this drug, as illustrated by the results of other studies. For instance, LD or its analogue, tolidamine, can induce a drastic decline in testicular androgen bindings protein concentration [40, 41]. However, our data indicate that increasing doses of LD had no apparent effects on the integrity of the Sertoli cell tight junctional barrier in vitro. To further confirm that LD-induced testin expression is not associated with any damage to the tight junctional complex microfilaments, testin expression was monitored after CdCl2 treatment to disrupt the inter-Sertoli tight junctions [17]. Cadmium toxicity in the testis was recognized as early as 1919 [27]. High doses of CdCl2 (3.7–10 mg/kg BW) can cause vascular damage to the testis [42, 43]. Low doses of CdCl2 (0.5–1 mg/kg BW) cause failure of spermiation [44] and stage-specific disruption of tight junction-associated microfilaments in Sertoli cells within 24 h and without visible vascular lesions, as shown by the results of confocal microscopy studies [17]. In addition, administration of CdCl2 to Sertoli cells in vitro causes damage to tight junctional microfilaments [45]. When rats were treated with low doses of CdCl2 to specifically damage tight junctional-associated microfilaments, no effect on testicular testin expression was noted. Thus, the increase in Sertoli cell testin expression after treatment with LD apparently does not associate with changes in the Sertoli cell tight junction microfilament elements. Interestingly, CdCl2-induced disruption of the inter-Sertoli tight junctions also did not change the expression of ZO-1, which is an authentic tight junction-associated cytoplasmic protein. These observations are expected, because one would not anticipate any changes in expression during the breakdown of junctional complexes. Other studies have shown an increase in ZO-1 expression when inter-Sertoli tight junctions were being formed in vitro [46, 47], because their establishment likely requires new tight junction component building blocks.
Results of the CdCl2 experiment together with those of earlier in vitro and in vivo studies overwhelmingly suggest that testin is a sensitive marker for disruption of the Sertoli-germ cell junctions, but not of the inter-Sertoli tight junctions [3, 5–7, 10]. However, results of one experiment presented here indicate otherwise. In our culture system, a similar induction of testin expression between LD-treated Sertoli-germ cell cocultures and Sertoli cell cultures alone was noted, which contrasted with expectations. If testin were a marker of Sertoli-germ cell junction disruption, a greater induction of testin expression would be expected from the Sertoli-germ cell coculture. Several explanations for this discrepancy exist. The ratio of Sertoli cells to germ cells used in vitro was 1:3, whereas the ratio in vivo would actually be 1:50, which is unattainable with the in vitro system. On the other hand, testin expression is already induced by Day 1 of culture, as shown in Figure 4, and a greater increase in testin expression may be unattainable after further induction by LD. Alternatively, a greater induction of testin expression was not observed, because the isolated germ cell population used for the culture does not contain elongated spermatids. Elongated spermatids are released from the Sertoli cell between Day 2 and 3 after LD administration in vivo [6]. In addition, testin is localized at the site where elongated spermatids attach to Sertoli cells. Therefore, our culture system cannot mimic the in vivo morphology, which would ultimately be more sensitive to the effects of LD treatment. Based on all this information, we believe that testin is a sensitive marker of the Sertoli-germ cell junctions. This conclusion is reached based on several lines of evidence. First, the disappearance of germ cells from the seminiferous epithelium in vivo induced by busulfan [3, 9] and x-ray radiation [10] can induce a surge in testin expression; however, germ cells per se had no apparent effect on Sertoli cell testin expression in Sertoli-germ cell cocultures [5, 6]. Second, intratesticular injection of glycerol [48] also failed to induce a surge in testin expression at 2 wk after treatment, at the time when the inter-Sertoli tight junctions were disrupted [5]. Third, disruption of Sertoli-germ cell junctions by hypotonic treatment in Sertoli-germ cell cocultures induced a drastic increase in testin expression that was comparable to the results obtained by treating rats with LD [6]. Fourth, disruption of the inter-Sertoli tight junctions by Ca2+ depletion causes no alterations in testin expression [5]. Taking these results collectively, testin clearly is a novel marker in studying the events of Sertoli-germ cell interactions, particularly the events involving disruption of Sertoli-germ cell junctions.
Results of previous studies have shown that the cell membrane is a target of LD. Treatment with LD can cause changes to the F-actin molecules, with disappearance of the stress fibers in epithelial squamous carcinoma cells [12]. In addition, perinuclear patches of tubulin were also detected after LD treatment. Intermediate filaments are affected differentially in several cell types. These changes in membrane configuration may possibly be the cause of the profound effects on the energy metabolism machinery of condensed mitochondria that are found in certain types of germ cells such as spermatids and spermatocytes, tumors, and during embryo development [11]. Results of further studies have shown that the site of action by LD is not on the respiratory chain per se but, rather, at the level of dehydrogenases [11]. An interesting point to be considered is that LD only inhibits dehydrogenases found in intact mitochondria and is inactive on purified dehydrogenase. Results of these studies, therefore, imply that LD causes some rearrangement of the membrane proteins that impairs the respiration process. A mild change in membrane morphology can have dramatic effects on mitochondrial respiration and, possibly, on the adhesion properties between cells. Results of immunohistochemistry studies have shown that testin is localized between Sertoli and germ cells in the basal compartment of the tubule, which is consistent with its localization at the junctional complexes, and is also stage specifically localized in the ectoplasmic specializations, where spermatids attach to Sertoli cells [8]. Testin localization to this junctional specialization was only detected during the early phase of stage VIII in the seminiferous epithelium at the time of spermiation, which then rapidly disappeared [8]. These data, taken together with the fact that LD can induce testin expression approximately 50-fold within 24 h of treatment [6], which is just before the premature release of spermatids, strongly suggest a correlation between spermiation and testin expression. However, the exact function of testin in the release of spermatids remains to be determined, because testin is neither a protease nor a protease inhibitor [7, 9].
FOOTNOTES
First decision: 28 February 2000.
1 Supported in part by grants from CONRAD (CIG-96-05-A), the Rockefeller Foundation (PS9721, PS9815), National Institutes of Health (U54HD-13541-20S), and the Noopolis Foundation. 
2 Correspondence: C. Yan Cheng, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733; yan{at}popcbr.rockefeller.edu
Accepted: July 7, 2000.
Received: January 14, 2000.
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250 g BW) were injected i.p. with a single dose of CdCl2 at 1 mg/kg BW, and testes were removed at specific time points. A) Northern blot analysis illustrating testicular testin expression at different times after CdCl2 treatment. D, Day; HR, hour. B) The ethidium bromide-stained gel shown in A indicating that similar amounts of total RNA (
