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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, Unitat de Reproducció Animal, Departament de Medicina i Cirurgia Animal, Facultat de Veterinària, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain. |
| Received for publication November 24, 2003; accepted for publication March 18, 2004. |
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Abstract |
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Key words: Testes, follicle-stimulating hormone, luteinizing hormone
Diabetes-related effects on testicular function have been attributed to the lack of insulin. The regulatory action of this hormone is known, and observations of a direct effect on both Leydig cells (Khan et al, 1992; Hurtado de Catalfo et al, 1998) and Sertoli cells (Borland et al, 1984; Mita et al, 1985) have been reported. Nonetheless, the data are confusing, and the exact role that insulin plays in the regulation of the male reproductive function is still unclear. Although STZ induces some side effects, such as renal or hepatic adenoma (Weiss, 1982), STZ-induced diabetes allows a direct comparison between 2 homologous systems, where the main difference is the presence or absence of serum insulin. Additionally, the morphologic alterations observed in the testes of STZ-diabetic rats are not caused by a direct effect of the drug, but rather by diabetes (Oksanen, 1975). This implies that this model of diabetes is a useful tool to study the insulin-related modulation of testicular function.
Testicular function is primarily controlled by pituitary hormones. The follicle-stimulating hormone (FSH) regulates spermatogenesis, whereas the luteinizing hormone (LH) controls Leydig cell function (for a review, see Ward et al, 1991). Decreases in the serum levels of FSH, LH, prolactin, and growth hormone have been reported in diabetes (Hutson et al, 1983; Benítez and Pérez Díaz, 1985). Moreover, the hypophysis of diabetic rats has a blunted response, with a diminished stimuli-induced secretion of FSH and LH (Seethalakshmi et al, 1987). These results indicate that there is a relationship between insulin/glucose and LH/FSH levels in serum. However, the mechanisms by which insulin, glucose, or both control these 2 hormones are unclear. There is a wide discrepancy concerning the effects of insulin treatment on LH and FSH levels in diabetic rats, from a total recovery of LH (Benítez and Pérez Díaz, 1985; Seethalakshmi et al, 1987; Sudha et al, 1999) and FSH (Hutson et al, 1983; Benítez and Pérez Díaz, 1985) to a lack of recovery of LH (Hutson et al, 1983) and FSH (Seethalakshmi et al, 1987; Sudha et al, 1999).
These observations indicate that the regulation of testicular function is the result of multiple mechanisms that include the combined effects of insulin/glucose, LH, and FSH. Nevertheless, to our knowledge, a unified hypothesis to explain the mechanism(s) by which changes in the insulin/glucose axis during diabetes modify the pituitary/testicular function has not yet been formulated, which has hindered the development of a rational approach to the treatment of male reproductive disorders linked to diabetes.
The main objectives of this study were 1) to determine the effects of STZ-induced diabetes on testicular structure and function in rats; 2) to correlate insulin/glucose levels and FSH, LH, and testosterone levels in serum; and 3) to formulate a unified hypothesis, based on our results and those reported in previous studies, to explain the mechanism(s) by which diabetes-induced changes in the insulin/glucose axis alter testicular function. For this purpose, we have determined the amount and localization of the most important biochemical markers of testicular function and serum levels of LH, FSH, testosterone, insulin, and glucose.
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Materials and Methods |
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Determination of the Reproductive Performance
After 10 weeks, individual males were placed in a cage with 1 healthy adult
female (body weight, 250 g). The animals were kept together overnight and were
separated the following morning. Immediately after each separation, a vaginal
scrape was performed to determine the presence of spermatozoa. This nightday
routine was repeated when the vaginal scrape was negative. A vaginal scrape
was considered positive when it showed the presence of a vaginal tap, adhered
spermatozoa, or both. When the vaginal scrape was positive, the night-day
routine was discontinued, and the female was housed individually during the
estimated period of gestation until parturition. Litter size was then
recorded, and the female was anesthetized with diethyl ether and killed by
decapitation; the neonates were killed by CO2 inhalation. When a
rat showed no sexual contact with the female for 9 consecutive days, this
individual was not used for further mating and was labeled
"unable," while those that showed sexual activity were labeled
"able."
The reproductive performance of the male rats was measured using the following parameters:
Analytic Procedures in Serum Samples
Glycemia was measured by the hexokinase method (Glucoquant;
Boehringer-Mannheim) modified for a COBAS Bioautoanalyzer (Roche Biomedica,
Basel, Switzerland). Serum insulin levels were determined by an enzyme-linked
immunosorbent assay (ELISA) using a commercial kit (Crystal Chem, Chicago,
Ill). Serum testosterone levels were measured by another ELISA assay (DRG
Instruments, Marburg, Germany). Serum FSH and LH levels were also determined
by ELISA assays using a specific commercial kit (Amersham, Buckinghamshire,
United Kingdom).
Histologic Techniques for Optic Microscope Observation
Formaldehyde-fixed samples were embedded in paraffin and then sliced (slice
thickness, 3–4 µm) on silane-precoated slides. They were further
deparaffined with xylol, and histologic observations were performed after
staining by the hematoxylin-eosin method
(Stevens, 1982).
Morphometric Analyses
The morphometric analyses of testes were performed in tissue slices treated
as described. Morphometric measurements were carried out following the
technique described by Anderson and Thliveris
(1986). Between 250 and 300
seminiferous tubulae and interstitial spaces were measured in each rat. The
analyses were performed from images obtained at 200x (seminiferous
tubulae) or 400x augmentations (interstitial spaces) and digitalized
using a Sony 3CCD digital camera (Sony Europe, Berlin, Germany). The images
were processed by the computerized image analysis system analySIS 2.1 (Soft
Imaging System GmbH, Münster, Germany).
Immunologic Techniques
For Western blot analysis, frozen samples were processed in 2 ways:
Western blot analysis was based on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (Laemmli, 1970) following transference to nitrocellulose (Burnett, 1981) and tested with the antibodies, which were used at a dilution of 1:200 (vol/vol), except for the antibody against phosphotyrosine, which was used at a dilution of 1:1000 (vol/vol). Immunoreactive proteins were tested by peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat secondary antibodies (Amersham), and the reaction was developed with an electrogenerated chemilunminescent detection system (Amersham). A single testes sample was run for each animal for all of the Western blots performed. Moreover, each lane was loaded in all cases with 50.0 ± 0.1 µg of total protein (mean ± SEM of all of the performed blots) to normalize the results obtained. Total protein was determined by the Bradford method (1976) using a commercial kit (Bio-Rad Laboratories, Hercules, Calif).
To perform immunohistochemical analysis, formaldehydefixed and paraffin-embedded samples were sliced (3–4 µm thick) and placed onto silane-treated slides. The samples were deparaffined with xylol and permeabilized with 0.2% (wt/vol) Triton X-100 in a phosphate-buffered saline (PBS) solution containing 9 mM Na2HPO4, 1.5 mM NaH2PO4, and 150 mM NaCl (pH 7.4). These samples were next blocked in the solution included in the commercial detection kits for immunohistochemistry (ABC Staining System; Santa Cruz Biotechnology, Santa Cruz, Calif). Tissues were then incubated with the distinct primary antibodies at 1:100 to 1:200 dilutions in PBS for 8 hours at 4°C. Samples were thereafter washed with PBS, and primary antibodies were detected by the ABC Staining System for 1 minute in all cases to standardize the results. Finally, contrast hematoxylin staining was performed, and samples were mounted as described above.
Antibodies
The anti-rabbit androgen, IGF-I
, insulin, and FSH receptors, the
anti-rabbit c-kit, and the SCF were from Santa Cruz Biotechnology.
The anti-rabbit GLUT-3 was obtained from Chemicon International (Temecula,
Calif), and the anti-mouse phosphotyrosine (PY20) was purchased from
Transduction Laboratories (Lexington, Ky).
Oligonucleotide Primers
Two pairs of 20-mer oligonucleotide primers were synthesized. They were
from the complementary DNA (cDNA) sequence of the rat FSH receptor
(Sprengel et al, 1990) and the
rat glucose-6-phosphate dehydrogenase (Ho
et al, 1988); the latter was used as an internal control. The
primers for PCR analysis for the FSH receptor were the following: the
5'-primer (5'-ATCTGGATGTCATCACTGGCT-3') corresponded to
nucleotides 45–65 of the FSH receptor cDNA, and the 3'-primer
(5'-AATGCATCTGGCTTTGGTGAG-3') corresponded to nucleotides
1035–1055. This amplified an approximately 1000-kilobase fragment of the
FSH receptor, localized between the extracellular and transmembrane domains.
The 5'-primer for the glucose-6-phosphate dehydrogenase
(5'-GACCTGCAGCTCCAATCAAC-3') and the 3'-primer
(5'-CACGACCCTCAGTACCAAAGGG-3') amplified an approximately 150-kd
fragment of the cDNA sequence for this enzyme.
RT-PCR Procedures
RNA from the testes was extracted using the TriPure Isolation Reagent,
1-step method (Roche). Three micrograms of the total testicular RNA was mixed
with the PCR reaction buffer (10 mM Tris-HCl [pH 8.3], 1.5 mM
MgCl2, 50 mM KCl, 200 µM deoxynucleotide triphosphates, and 1 mM
of each primer) and added to 40 U of RNase inhibitor (RNasin; Promega,
Madison, Wis) to a final volume of 50 µL. A total of 12
U of avian
myeloblastosis virus RT (Promega) was added to the PCR mixture, and samples
were placed in a thermal cycler in the following sequence:
Semiquantitative analyses of RT-PCR results were carried out using 2-µL aliquots of the RT-PCR reaction mixture and 5-µL aliquots of a 5:1 (vol/vol) formamide loading buffer containing 25 mM EDTA and 50 mg of dextran blue per milliliter (pH 8). A further 0.5 µL of molecular-weight markers (GeneScan 2500 ROX Size Standard ABI PRISM; Applied Biosystems, Foster City, Calif) was added to the mixture. The resultant sample was denatured by heating at 95°C for 2 minutes.
Suppliers
All reagents were of analytic grade and were purchased from Sigma Chemical
Co (St Louis, Mo), Merck (Darmstadt, Germany), Bio-Rad Laboratories, and EMS
(Fort Washington, Pa).
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Results |
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Effects of Diabetes on the Reproductive Performance of Male Rats
The number of unable males was significantly higher in diabetic rats than
in their healthy littermates, whereas the mating index, fertility, and libido
were significantly (P < .05) reduced in diabetic animals
(Table 1). Prolificacy was also
lower in diabetic rats (Table
1).
Histologic Analysis of Testes
STZ-diabetic rats had smaller testes than their healthy littermates (1.27
± 0.12 g in controls vs 0.76 ± 0.07 g in STZ-diabetic rats).
However, this decreased size was not related to changes in the seminiferous
tubules, since morphometric analysis did not show any significant differences
in the size and density of these structures (data not shown). This observation
indicates that the reduced size was mainly due to a loss of interstitial
tissue. This is supported by the results from histologic analysis. Diabetic
rats had functional testes, and their seminiferous tubules showed all the
distinct developmental stages of spermatogenesis, including spermatozoa in the
lumen of the tubules (Figure
1C). In contrast, the interstitial tissue of these rats was less
compact than that of the healthy controls and showed an increase in amorphous
material and a significant decrease in the total number of Leydig cells per
interstitial space, which went from 14 ± 4 in healthy rats to 4
± 1 in diabetic animals (mean ± SEM) (see
Figure 1D;
Table 1).
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Changes in the Intensity and Distribution Pattern of Tyrosine Phosphorylation
Testicular function is related to the tyrosine phosphorylation levels in
testes (Arad-Dann et al, 1993).
Western blot analysis of testicular extracts showed a specific pattern of
phosphorylation, with 2 main bands of about 50 and 66 kd
(Figure 2). This pattern was
similar in the 2 groups, and no significant differences in the intensity of
the band patterns between healthy and diabetic rats were observed
(Figure 2).
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Immunohistochemical analyses detected phosphotyrosine in all of the testicular cell types of healthy rats, with a lower intensity in interstitial tissues (Figure 3C). Tyrosine phosphorylation was practically absent in the interstitial cells of diabetic rats (Figure 3F).
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Changes in the Expression and Distribution Pattern of GLUT-3 Hexose Transporters
The presence of the GLUT-3 hexose transporter is a useful marker for
mammalian spermatogenic function (Burant
and Davidson, 1994). Western blot analysis of GLUT-3 showed a
specific band of 40–45 kd, which is consistent with previous reports
(Gerhart et al, 1992;
Burant and Davidson, 1994).
There were no significant differences in the intensity of this transporter
between groups (Figure 4A).
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Immunohistochemistry showed a diffused distribution of GLUT-3 in the testes of healthy rats (Figure 4C; data not shown). In sharp contrast, a significant decrease in this transporter in the interstitial tissue of STZ-diabetic rats was observed (Figure 4D).
Expression and Distribution Pattern of IGF-I and Insulin Receptors
IGF-I (Sharpe et al, 1990;
Skinner, 1991;
Hull and Harvey, 2000) and
insulin (Sharpe et al, 1990;
Khan et al, 1992;
Hurtado de Catalfo et al,
1998) are crucial modulators of testicular function. STZ-diabetic
rats showed a marked decrease in testicular IGF-I receptor content, measured
as a single 160-kd band (Figure
4B). On the other hand, immunocytochemical analysis showed IGF-I
receptors in almost all testicular cell types
(Figure 4E; data not shown).
There were no clear changes in the intensity of the immunocytochemical signal
in seminiferous tubules between healthy and diabetic rats (data not shown). On
the contrary, a perceptible decrease in IGF-I receptors was detected in the
interstitial tissues of diabetic rats
(Figure 4F).
Western blot analysis of the insulin receptor showed 2 specific bands of 105 and 160 kd (Figure 5A). No significant differences in the intensity of the bands between healthy and diabetic rats were observed (Figure 5A).
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Immunohistochemical analysis of the testicular insulin receptor in healthy rats showed a specific signal in practically all testicular cell types. Diabetes induced a slight decrease in insulin receptor expression, which was perceptible in interstitial tissue (Figure 5C and D).
Expression and Distribution Pattern of Androgen Receptors
In testes, androgen receptors play a key role in the control of
spermatogenesis and spermiogenesis
(Vornberger et al, 1994).
Western blot analysis of androgen receptors showed 2 specific bands of 105 and
160 kd (Figure 5B). No
significant differences in the testicular expression of these bands were
observed between groups (Figure
5B).
Immunohistochemical analysis showed that androgen receptors were widely distributed in testicular tissue and were present in all germ cells of healthy rats (data not shown). STZ-diabetic rats showed a significant decrease in androgen receptors in Leydig cells (Figure 5E and F).
Localization and Expression of c-kit and SCF
The c-kit/SCF system is one of the most important regulatory
mechanisms of germ cell development
(Yoshinaga et al, 1991) and
Leydig cell replication in testes (Feng et
al, 1999). Western blot analysis with the
anti–c-kit antibody showed a band of about 90 kd
(Figure 6A), consistent with
the apparent molecular weight of c-kit, as previously reported
(Sandlow et al, 1997). There
were no significant differences in c-kit expression in testicular
extracts (Figure 6A).
Furthermore, diabetes induced a robust increase in the phosphorylation of
testicular c-kit (Figure
6B).
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Immunolocalization of c-kit indicated that, in healthy rats, the receptor was mainly localized in the basal and peribasal cells of seminiferous tubules (spermatogoniae and spermatocytes) and in Leydig cells (Figure 6D). Diabetes induced a lack of expression in interstitial tissue, clearly related to the loss of Leydig cells (Figure 6E). Moreover, c-kit expression in seminiferous tubules changed to a more homogeneous presence of the protein, even in spermatidae and spermatozoa (Figure 6E).
Western blot analysis of the effector for c-kit, the SCF, showed a faint band of 45 kd. Diabetes did not modify the intensity of this band compared to healthy controls (data not shown). This factor was present in the epithelium of seminiferous tubules and interstitial tissue, while diabetes induced a clear decrease in the SCF signal only in Leydig cells (data not shown).
Expression of FSH Receptors
Western blot analysis using anti-FSH receptor antibodies showed a 70-kd
band consistent with the FSH receptor. The amount of this receptor slightly
decreased in diabetic rats (Figure
6C). Immunocytochemical analysis showed that this receptor was
evenly localized in the cells of seminiferous tubules and that it was
intensely localized in Leydig cells (Figure
6F and G). The signal for the FSH receptor was not modified in
Leydig cells, and there was a slight decrease in the intensity mark in the
epithelium of seminiferous tubules in diabetic rats
(Figure 6G). However, this
decrease was difficult to quantify in the photographs. Semiquantitative RT-PCR
analysis of messenger RNA (mRNA) expression for the FSH receptor showed a
60.3% decrease in the mRNA content of diabetic rats
(Figure 7).
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Discussion |
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However, the changes in Leydig cell number and function observed in STZ-diabetic rats may not only be the result of a decrease in serum LH. The almost complete suppression of serum insulin in these animals also has a direct effect on interstitial tissue. The effect of insulin on Leydig cells has been previously described and is related to the control of cell proliferation and metabolism. Addition of insulin to the medium increased the incorporation of [3H]thymidine into DNA in cultured Leydig cells (Khan et al, 1992). In this regard, LH mediates the proliferation of Leydig cells through a mechanism that involves insulin and IGF-I signaling (Feng et al, 1999). Moreover, insulin partially restored alterations in lipid metabolism 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 biosynthesis (Romanelli et al, 1995), the recovery of this metabolism leads to a concomitant effect on testosterone biosynthesis. The remarkable decrease in the expression of interstitial tissue insulin receptors in diabetic rats, together with the virtual absence of serum insulin, is expected to lead to a total loss of the insulin-mediated stimulation of androgen biosynthesis and cell proliferation, which is consistent with the morphologic alterations in the interstitial tissue observed in our experimental model.
To explain the effects of LH and insulin on Leydig cell function, we need to invoke the regulation of serum LH levels by insulin. An alteration in Leydig cell function mediated by an LH-linked mechanism in diabetes has been described (Dinulovic and Radonjic, 1990; Steger and Rabe, 1997; Sudha et al, 1999). The relationship between LH and insulin has also been shown in transgenic mice that lack brain insulin receptors. This model showed a significant decrease in Leydig cell number, and it was suggested that this effect was related to a defect in the regulation of LH secretion (Brüning et al, 2000). However, although this study supports our hypothesis, it did not show whether insulin regulates serum LH levels directly or indirectly. Our results indicate that insulin regulates serum LH levels by an indirect mechanism, since there was no direct correlation between serum levels of insulin and LH. In addition, the strong correlations between serum insulin and FSH levels and serum FSH and LH support this hypothesis. These concatenate correlations indicate that the diabetes-induced decrease in insulin levels indirectly decreases serum LH levels, probably through an FSH-linked mechanism, which ultimately affects Leydig cell function.
On the basis of the above considerations, we can now formulate a unified hypothesis to explain the alterations observed in the testes of insulin-dependent diabetic rats. This hypothesis is based on the observation that the regulatory effects of LH on Leydig cell number and function are reinforced by a direct effect of insulin. Thus, there are 2 parallel mechanisms that cause diabetes-related alterations in Leydig cells: 1) the effect of hypoinsulinemia on serum LH levels, and 2) the combined effects of the decreased levels of LH and insulin on Leydig cells.
Our results indicate that insulin-dependent diabetes alters spermatogenesis, primarily by an FSH-related mechanism. Lack of insulin in the STZ-diabetic rats did not affect spermatogenesis via a direct effect on the epithelium of seminiferous tubules, but rather by an alteration in serum FSH levels. A similar mechanism has been reported by Hutson et al (1983) and Sudha et al (1999). Sperm production is an FSH-regulated process that requires normal Sertoli cell function (Ward et al, 1991). In our experimental model, the decrease in FSH was also accompanied by a decrease in tubular FSH receptors. Therefore, as expected, the response of the epithelium of seminiferous tubules to FSH stimulation was significantly diminished, although it was not completely lost, as indicated by the maintenance of tyrosine phosphorylation and a significant level of spermatogenesis. The diabetes-linked decrease in the response to FSH explains the subsequent tubular alterations observed, especially the changes in the expression of c-kit, which controls spermatogonia multiplication and differentiation into meiotic cells (Mauduit et al, 1999). The ultimate result of these changes would be the diabetes-related decrease in male fertility and prolificacy. However, in contrast to the observations on Leydig cell function, there were no apparent changes in the expression of insulin receptors in the seminiferous tubules, indicating that germ cells conserved their capacity to respond to insulin in diabetes. Therefore, diabetes-related hypoinsulinemia may have a major effect on tubular function by altering serum FSH levels. This hypothesis is consistent with the strong correlation found between FSH and insulin levels in serum. Similar studies (Hutson et al, 1983; Sudha et al, 1999) indicate that one of the most important regulatory roles of insulin on spermatogenesis is the modulation of serum FSH levels. This strong correlation indicates a direct effect of insulin and/or glucose on the pituitary biosynthesis and/or secretion of FSH. In this regard, one of the most striking features of transgenic mice that lack brain insulin receptors is a strong impairment of fertility that is concomitant with impaired spermatogenesis (Brüning et al, 2000).
In summary, our results, combined with those of previous studies, allow us, for the first time, to formulate a unified hypothesis to explain the changes observed in the testes of diabetic rats. This hypothesis postulates that the testicular alterations in insulin-dependent diabetes are explained by several mechanisms that affect the 2 main testicular functions: 1) Leydig cell function and testosterone production are diminished in insulin-dependent diabetes due to the absence of the stimulatory effect of insulin on Leydig cells and to an insulin-dependent decrease in FSH, which, in turn, decreases LH levels; and 2) sperm output and fertility are reduced due to a decrease in FSH caused by a decrease in insulin. A better understanding of the mechanisms that underlie the changes in the testes of insulin-dependent diabetic patients would allow the rational design and development of more specific therapeutic strategies to overcome these alterations.
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Acknowledgments |
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Footnotes |
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 I.M. Agbaje, D.A. Rogers, C.M. McVicar, N. McClure, A.B. Atkinson, C. Mallidis, and S.E.M. Lewis Insulin dependant diabetes mellitus: implications for male reproductive function Hum. Reprod., July 1, 2007; 22(7): 1871 - 1877. [Abstract] [Full Text] [PDF]
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