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Tungstate Treatment Improves Leydig Cell Function in Streptozotocin-Diabetic Rats

JORGE DOMÍNGUEZ

M. CARMEN MUñOZ

JOAN J. GUINOVART

From the ^* Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain; and the Department of Biochemistry and Molecular Biology and IRBB, Barcelona Science Park University of Barcelona, Barcelona, Spain.

Correspondence to: Dr Joan E. Rodríguez-Gil, Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.

Received for publication October 5, 2004; accepted for publication May 24, 2005.

	Abstract

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Oral administration of sodium tungstate to adult male streptozotocin-diabetic rats for 3 months normalized serum levels of glucose, insulin, luteinizing hormone, and follicle-stimulating hormone. These effects were accompanied by an increase in reproductive performance, which was related to a strong improvement in Leydig cell function markers, such as the recovery of the number of Leydig cells and serum testosterone levels. Moreover, this in vivo recovery was related to a concomitant increase in the cell expression of insulin receptors. Tungstate treatment did not modify Leydig cell function in healthy rats. Furthermore, the addition of tungstate or insulin to the mTLC-1 cell line from Leydig cell origin increased the phosphorylation states of MAP-kinase and glycogen synthase kinase-3. Our results indicate that tungstate treatment in diabetic rats leads to a recovery of reproductive performance by increasing the number of Leydig cells. This increase contributes to the recovery of their functionality, thereby improving the overall function of these cells. We propose that this improvement is caused by the combined effect of the tungstate-induced normalization of insulin glucose and luteinizing hormone serum levels and a direct action of the effector on Leydig cells through modulation of at least MAP-kinase and glycogen synthase kinase-3 activities.

     Key words: Diabetes, male, reproductive performance

One of the mammalian systems that is clearly impaired in diabetes is the male reproductive function. Diabetes-induced alterations of Leydig cell functions include a decrease in androgen synthesis and in the total number of these cells (Foglia et al, 1969). Together, these effects cause an impairment of male libido (Foglia et al, 1969). The diabetes-induced alterations of Leydig cells are related to concomitant alterations in the control mechanisms that modulate the proliferation, differentiation, and overall function of these cells (Oksanen, 1975; Tesone et al, 1980; Lin et al, 1986; Feng et al, 1999). Furthermore, it is noteworthy that diabetes-related alterations in Leydig cells are also related to changes in the pituitary-testicular axis (Steger and Rabe, 1997). Thus, this disease induces a decrease in the serum levels of luteinizing hormone (LH), which is responsible for normal Leydig cell function (Benítez and Pérez-Díaz, 1985).

Long-term tungstate treatment can prevent the onset of the detrimental effects caused by diabetes in several tissues, such as eyes or kidney (Barberà et al, 2001). Here we determine whether tungstate administration can restore Leydig cell function in insulin-dependent diabetes. Oral tungstate was administered to adult male streptozotocin-induced type I diabetic rats for 3 months. In addition, we performed complementary experiments using a cell line obtained from Leydig cells to gain further insight about a putative direct effect of tungstate on these cells.

	Materials and Methods

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Animals and Research Design

All the procedures described here were reviewed and approved by the Animal Research Committees of the Autonomous University of Barcelona and the University of Barcelona and were performed in accordance with the Animal Welfare Law of the Catalan Government (Generalitat de Catalunya, Spain). Adult male Wistar rats (200 g) were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink ad libitum. When stated, diabetes was induced by a single intraperitoneal injection of streptozotocin (70 mg/kg of body weight) in 0.9% NaCl with 100 mM sodium citrate buffer (pH 4.5). Diabetes was confirmed by measuring glucosuria and glycemia (glucose and glycemia strips; Boehringer Mannheim, Mannheim, Germany). Under these parameters, rats were classified as diabetic when they showed detectable levels of glucosuria and blood glucose levels above 400 g/dL. No rat died as a direct consequence of the streptozotocin injection. Five to 7 days poststreptozotocin injection, all the rats inoculated became diabetic according to glucosuria and blood glucose level parameters. At the beginning of the experiment, rats were divided into 4 groups, each of which contained 15 animals. The first 2 groups, the healthy (UH) and diabetic (UD) groups, received a solution of 0.9% NaCl as drinking water. The remaining 2 groups, the healthy (TH) and diabetic (TD) groups, were given a solution of 2 mg/mL sodium tungstate in 0.9% NaCl. The treatment was carried out for 3 months. During this period, we measured glycemia and body weight regularly. The time period of 3 months was chosen to allow tungstate to exert a complete effect on testicular function. At the end of the experiment, all the rats in the UH, TH, and TD groups were alive, while only 8 rats survived in the UD group. All the animals were then anesthetized with diethyl ether and sacrificed by decapitation between 0900 and 1100 hours. Blood was collected immediately to measure serum parameters. Testes were prepared in 3 ways: 1) Tissues were immediately fixed in 3% formaldehyde in a buffered solution containing 54 mM NaH₂PO₄ and 28 mM Na₂HPO₄ (pH 7.4) at 4°C (buffered formaldehyde). This material can be stored for up to 5 weeks. These tissues were used for optical microscope histology and immunohistochemistry; 2) Tissues were immediately fixed in 2.5% glutaraldehyde in ice-cold 0.1 M sodium cacodylate buffer (pH 7.4). These tissues were used for transmission electronic microscope histology; and 3) Tissues were immediately frozen in liquid N₂ and stored at -90°C until Western blot analyses were performed.

Measure of Reproductive Performance

To evaluate reproductive performance, after 10 weeks of treatment, individual males were placed in a cage with 1 healthy adult female (body weight: 250 g). The animals were kept together overnight, and the following morning they were separated. Immediately after separation, a vaginal examination and scrape were performed to determine whether sexual intercourse had occurred. When intercourse was positive (presence of a vaginal tap with or without spermatozoa), the night routine was discontinued. When the male showed no sexual contact with the female during 9 consecutive days, this individual was not used for further mating and was termed "unable," while those that showed sexual activity, as determined above, were termed "able." The reproductive performance of the male rats was determined by calculating the percentage of "able" males out of the total number tested.

Analytical Procedures in Serum Samples

Glycemia was measured by the hexokinase method (Glucoquant; Boehringer Mannheim), which was adapted to a COBAS Bio autoanalyzer (Roche Biomedica, Basel, Switzerland). Serum insulin levels were analyzed by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Cristal Chem, Chicago, Ill). Serum testosterone levels were determined by ELISA (DRG Instruments, Marburg, Germany). Serum levels of follicle-stimulating hormone (FSH) and LH were also measured using a commercial ELISA kit (Amersham, Buckinghamshire, United Kingdom).

Culture of a Cellular Line of Leydig Cell Origin

We used the mLTC-1 cell line, which is of mouse Leydig cell tumor origin, kindly provided by Dr Ilpo Huhtaniemi (University of Turku, Finland). Cells were thawed, transferred to a 75-cm² culture flask, and diluted in Dulbecco's modified essential medium (DMEM)/F12 medium supplemented with 9% human serum, 4.5% fetal calf serum, 20 mM HEPES, and 50 µg/mL gentamycin (HD medium). Growing cells were transferred to 60-mm² plates to perform the experiments.

For incubation with insulin or tungstate, confluent 60-mm² plates of mLTC-1 cells were left overnight in HD medium without serum (HDP medium). After this, medium was replaced by fresh HDP medium containing the appropriate concentrations of either tungstate or insulin. After the adequate incubation times, medium was removed and plates were immediately frozen in liquid N₂ until analysis. Formaldehyde- and paraformaldehyde-fixed samples were embedded in paraffin and sliced (thickness: 3-4 µm) onto slides precoated with silane. Slices were deparaffinized with xylol, and histological studies were performed using the hematoxylin-eosin staining method (Stevens, 1984). For long storage, slides were mounted with a commercial mounting medium (Adh CLINIC; Clinic Services, Barcelona, Spain).

Histological Techniques for Transmission Electronic Microscopy

Glutaraldeyde-fixed samples were postfixed in 1% OsO₄ in cacodylate buffer and embedded in Spurr ERL 4026 resin. After thin sectioning, the specimens were contrasted with uranyl-acetate and lead citrate and were then observed under a Zeiss 910 EM electron microscope.

Immunological Techniques

Western blot analyses were performed either to test the usefulness of the anti-insulin receptor antibody for further immunohistochemistry analyses or to determine MAP-kinase and GSK-3 functionality in cultured mLTC-1 cells. For these purposes, frozen samples were treated in accordance with their origin. Testicular samples were homogenized (proportion 1:8, wt/vol) in a 25-mM HEPES buffer (pH 7.4) containing 4 mM EDTA, 250 mM sucrose, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin A, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Final supernatants were discarded, and the resultant pellets were resuspended with 200-300 µL of the above homogenation buffer before Western blot analyses.

In addition, mLTC-1 cells were scraped and mechanically broken with 400 µL/plate of cold 30-mM Tris-HCl buffer (pH 7.4) with 25 mM NaCl, 1% (vol/vol) Triton X-100, 0.1% sodium dodecyl sulphate (SDS), 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ethylene glycolbis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 20 nM okadaic acid, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 µg/mL pepstatin. After 10 minutes on ice, extracts were centrifuged for 10 minutes at 4°C at 13 000 x g and supernatants were used for Western blots. In extracts from both testes and mLTC-1 cells, protein concentration was measured using the Bio-Rad Protein Assay (München, Germany).

View larger version (64K): [in this window] [in a new window]   Figure 1. Interstitial tissue of rat testes: untreated healthy (A), untreated diabetic (B), and tungstate-treated diabetic (C) rats, showing the morphology of Leydig cells. Hematoxylin-eosin staining. Magnification: 1000x. Arrows indicate the location of Leydig cells. Asterisk shows the presence of amorphous lymphatic content.

For immunohistochemistry, samples were deparaffinized with xylol and were then permeabilized with 0.2% (wt/vol) Triton x-100 in a phosphate-buffered saline (PBS) containing 9 mM Na₂HPO₄, 1.5 mM NaH₂PO₄, and 150 mM NaCl (pH 7.4). Permeabilized samples were blocked in a commercial blocking solution (ABC Staining System; Santa Cruz Biotechnology, Santa Cruz, Calif). They were then incubated with the distinct primary antibodies at dilutions of 1/100 to 1/200 in PBS for 8 hours at 4°C. They were then washed with PBS, and the primary antibodies were detected using the ABC Staining System, which uses diaminobenzidin tetrahydrochloride as a revealing substrate. Finally, contrast hematoxylin staining was performed, and samples were mounted as described above.

View larger version (94K): [in this window] [in a new window]   Figure 2. Ultrastructural morphology of Leydig cells in rats: untreated healthy (A), untreated diabetic (B), and tungstate-treated diabetic (C) rats. Arrows indicate the localization of mitochondria and nuclear chromatin. Asterisks indicate the lipidic vacuoles. Magnification: 7000x.

Suppliers

All chemical reagents were of analytical grade and were obtained from Sigma (St Louis, Mo), Merck (Darmstadt, Germany), Bio-Rad Laboratories (Hercules, Calif), and EMS (Fort Washington, Pa).

	Results

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Effects of Tungstate Treatment on Body Weight Gain and Serum Parameters

The administration of tungstate had distinct effects on healthy and diabetic rats. Thus, the former showed a smaller increase in body weight than did healthy controls (Table 1). On the contrary, tungstate treatment counteracted the negative body weight variations of UD rats, although it did not lead to the complete recovery of body weight gain when compared with UH rats (an increase of 27.2 ± 4.2 g during the treatment in TD rats vs an increase in 234.1 ± 7.5 g in UH animals; see Table 1).

Diabetic rats showed very high glycemia and low levels of serum insulin, testosterone, FSH, and LH (Table 1). Tungstate treatment counteracted these alterations. Thus, the TD group showed a practical normalization of glycemia and a recovery of serum insulin, FSH, testosterone, and LH concentrations (Table 1).

Effects of Tungstate on Reproductive Performance

Diabetes induced a clear impairment of reproductive performance, with a low percentage of "able" males (Table 1). This effect was almost completely counteracted after tungstate treatment (50.0% "able" UD vs 93.3% "able" TD) (Table 1). Tungstate administration did not alter the reproductive performance of healthy animals (Table 1).

Microscopic Morphology of Leydig Cells After Tungstate Treatment

Leydig cells in UH rats presented a characteristic appearance, with a very active nucleus, as shown by the presence of a decondensed chromatin and several nucleoli (Figure 1A). This was confirmed by electron microscopy (Figure 2A). The cytoplasm of Leydig cells from these rats showed a uniform granular appearance and were filled mainly by lipid vacuoles and mitochondria; the space between cells was filled by capillari and fibroblasts and a small amount of an amorphous matrix (Figure 1A). In contrast, in UD rats the intracellular space was mainly occupied by an amorphous matrix with few fibroblasts (Figure 1B). Leydig cells were scarce and showed a highly vacuolized cytoplasm (Figure 1B). The ultrastructural image showed the presence of altered mitochondria and very few lipid vacuoles (Figure 2B). Furthermore, the nuclei of the cells were of irregular shape, and the chromatin content had a distinct appearance (Figure 2B). TD rats showed some interstitial spaces similar to those of their untreated counterparts; however, most of these spaces were similar in appearance to those of the UH group. Thus, TD rats showed a noticeable increase in the number of Leydig cells per interstitium, which also showed an ultrastructural morphology similar to that of UH rats (Figures 1C and 2C; Table 2). Tungstate administration to healthy rats did not modify the appearance or number of Leydig cells (data not shown).

Effect of Diabetes and Tungstate Treatment on the Expression of Insulin Receptors of Leydig Cells

Diabetes decreased the intensity of the signal for the insulin receptor (Figure 3B). Tungstate treatment clearly recovered the signal for this receptor in these cells (Figure 3C). However, administration of this compound to healthy rats did not modify the expression of this receptor (data not shown).

View larger version (119K): [in this window] [in a new window]   Figure 3. Immunohistochemistry against the insulin receptor that shows the signal in rat interstitial tissues: untreated healthy (A), untreated diabetic (B), and tungstate-treated diabetic (C) rats. Magnification: 1000x. Arrows indicate the location of Leydig cells.

Incubation with 1 mM tungstate induced a clear increase in the phosphorylation signal of MAP-kinase of these cells, which reached a maximum after 10 minutes of incubation (Figure 4A). Similar results were observed after incubation with 100 nM insulin (Figure 4A). No effect of 1 mM tungstate or 100 nM insulin was observed on the levels of total MAP-kinase (Figure 4B).

View larger version (73K): [in this window] [in a new window]   Figure 4. Effects of tungstate and insulin in MAP-kinase phosphorylation and MAP-kinase total amount of mLTC-1 cells. The mLTC-1 cells were incubated, at the indicated times, with either 1 mM tungstate (W) or 100 nM insulin (I), and cellular extracts were subjected to Western blot for detecting total (A) or phosphorylated (B) MAP-kinase. The 1 Western blot for the total MAP-kinase was used as an internal standard to detect variations in the phosphorylation state of the enzyme. The figure shows representative results from 7 experiments. M indicates molecular weight markers.

View larger version (63K): [in this window] [in a new window]   Figure 5. Effects of tungstate and insulin in GSK-3 phosphorylation and GSK-3 total amount of mLTC-1 cells. The mLTC-1 cells were incubated, at the indicated times, with either 1 mM tungstate (W) or 100 nM insulin (I), and cellular extracts were subjected to Western blot for detecting total (A) or phosphorylated (B) GSK-3. The 1 Western blot for the total GSK-3 was used as an internal standard to detect variations in the phosphorylation state of the enzyme. The figure shows representative results from 7 experiments. M indicates molecular weight markers.

	Discussion

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The recovery of the number and function of Leydig cells in the TD group can be explained by 2 mechanisms. First, tungstate administration significantly increases serum levels of LH and insulin and also the Leydig cell expression of insulin receptors. Second, this compound exerts a direct effect on Leydig cells. Serum LH levels regulate not only the number of these cells but also their testosterone production (Hall, 1994). The alteration of the LH-related Leydig cell function in diabetes has been reported (Dinulovic and Radonjic, 1990; Steger and Rabe, 1997; Sudha et al, 1999) and, thus, the recovery of insulinemia in TD rats induces the recovery of this function. One of the most prominent alterations in transgenic mice that lack the expression of the insulin receptor in brain is a reduction in Leydig cell population (Brüning et al, 2000). This reduction is related to an alteration in the regulation of LH secretion from the hypophysis (Brüning et al, 2000), thereby linking insulin and LH. The capacity of tungstate to recover insulinemia in TD rats is striking. In this regard, previous experiments showed that administration of tungstate to neonatally streptozotocin-induced diabetic rats for 15 days induced a recovery of islet insulin secretion and ß cell/pancreatic exocrine tissue ratio, indicating a recovery of pancreatic islet function (Barberà et al, 1997). This recovery is linked to a stable and functional regeneration of pancreatic ß cells in these rats (Fernández-Álvarez et al, 2004). Thus, our results are consistent with these data, since our streptozotocin model of diabetes did not lead to a complete lack of ß-cell function (Rossi and Hekdstab, 1981; Cossel et al, 1985; Van Gompel et al, 1993), and tungstate would act on the remnant pancreatic islets tissue by inducing their stable, long-term recovery and stabilization. Moreover, another mechanism related to the counteraction of the body weight loss induced by diabetes may contribute to the recovery action of tungstate. In this regard, the severe weight loss associated with diabetes is a direct cause of reproductive alterations (Steger, 1990). In our experimental design, tungstate administration counteracted this loss. Thus, the alterations in weight loss related to diabetes were avoided. However, tungstate administration did not lead to the complete recovery of body weight in comparison with healthy animals. In fact, TH rats showed a much lower body weight gain than that the UH group. This observation indicates that TD rats have much lower body weight gain than do healthy animals, and, thus, tungstate does not seem to completely recover the body weight gain-induced reproductive alterations. In conclusion, the effects of tungstate on body weight do not appear be the main mechanism of action of this compound on Leydig cells function, although they most certainly play an additive, cooperative role, together with the other mechanisms involved in tungstate action.

Although LH is the most important regulatory factor of Leydig cell function, insulin exerts a direct effect on these cells. These cells have insulin receptors, and we showed that tungstate treatment recovers insulinemia and insulin receptor expression in diabetic rats. The direct role of insulin in Leydig cell function may be related to the control of cell multiplication and energy metabolism. In this way, the addition of insulin to the medium increases the incorporation of [³H]thymidine to DNA in cultured Leydig cells (Khan et al, 1992). Moreover, insulin partially recovers the malfunction of lipid metabolism observed in cultured Leydig cells from diabetic rats (Hurtado de Catalfo et al, 1998). Since the lipid metabolism of these cells is strongly related to androgen synthesis (Romanelli et al, 1995), the recovery of this metabolism leads to a concomitant effect on testosterone synthesis. Therefore, the recovery of Leydig cells in TD rats may be the result of the accompanying action of LH and insulin. Our results indicate a joint effect of these 2 hormones. This observation is not surprising, since LH mediates the proliferation of Leydig cells through a mechanism that involves insulin and IGF-I signaling (Sharpe et al, 1990). This implies that these 2 hormones modulate Leydig cell proliferation by interdependent mechanisms.

The insulin-mediated effect of tungstate does not preclude a direct effect of the compound on Leydig cells. Our results in cultured cells show a direct action of tungstate on these cells, as demonstrated by the phosphorylation, and thus the activation of MAP-kinase concomitantly with phosphorylation, and thus inactivation of GSK-3 observed in mTLC-1 cells. In this respect, MAP-kinase and GSK-3 play key roles in the control of insulin signaling pathway (Downward, 1996). Therefore, tung-state action on MAP-kinase and GSK-3 in Leydig cells may lead to a direct effect of the compound on these cells. Moreover, MAP-kinase activity is essential in the maintenance of overall testicular function (Luconi et al, 1998). Thus, some types of male infertility are associated with alterations in this activity (Luconi et al, 1998). In this regard, the maintenance of optimal MAP-kinase activity after tungstate treatment could contribute to the recovery of Leydig cell function in diabetes.

Our results indicate that tungstate recovers testosteronemia in diabetic rats. However, it must be stressed that testosterone itself does not appear to be instrumental in the tungstate-induced effects. In fact, alterations in copulatory behavior are related to much greater decreases in testosteronemia (Damassa et al, 1977). Moreover, testosterone replacement does not restore normal sex behavior in diabetic rats (Steger, 1990). This indicates that the variations of testosterone levels observed should be considered purely an indicator of the overall functional state of Leydig cells, which are only loosely related to the overall recovery of reproductive performance of TD rats.

One of the most remarkable characteristics of tungstate is its lack of effects on healthy animals. TH rats did not show hypoglycemia or any alteration in Leydig cell function, as shown by serum testosterone levels. This finding is difficult to reconcile with the direct, insulin-like effects observed in cell culture. We propose that "in vivo" tung-state is more like an antidiabetic than an insulin-mimetic agent. This property makes it an optimal agent to counteract all the diabetes-related alterations detected "in vivo," regardless of the presence or absence of insulin treatment.

	Conclusion

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In summary, on the basis of our results, we conclude that tungstate treatment dramatically improves male reproductive performance in diabetic rats by increasing the number of Leydig cells and recovering their function. The mechanisms involved in this recovery are mainly related to a normalization of the insulin and, hence, the LH signaling pathways in Leydig cells. Moreover, a direct effect of tungstate may also contribute to the recovery of Leydig cell function observed.

	Acknowledgments

 
We thank Anna Adrover (University of Barcelona) and Alejandro Peña and the Clinical Biochemistry Service (Autonomous University of Barcelona) for their excellent technical assistance. We also thank Tanya Yates for assistance in preparing the English manuscript. J.B. is the recipient of a Doctoral Fellowship from the Interdepartmental Commission for Research and Technology (Autonomous Government of Catalonia).

	Footnotes

 
Supported by grant 97/2040 from the Health Research Fund (Ministry of Health, Spain).

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