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b Departments of Pharmacology
c Physiology, Louisiana State University Medical Center-Shreveport, Shreveport, Louisiana 71130
d College of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803
e Division of Molecular Biochemistry & Molecular Biology, University of Glasgow, Scotland G12 8QQ, United Kingdom
f Hematology & Oncology, University of Alabama, Birmingham, Alabama 35294
g Department of Psychiatry, Boston University, Boston, Massachusetts 02215
h Division of Reproductive Biology, Stanford University, Stanford, California 94305
i Department of Pharmacology, University of Tennessee, Memphis, Tennessee 38163
ABSTRACT
The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.
cyclic adenosine monophosphate, gametogenesis, gene regulation, phosphodiesterases, signal transduction, spermatogenesis
INTRODUCTION
Mammalian spermatogenesis typically occurs at a temperature a few degrees lower than core body temperature, which is attained by testicular descent into the scrotal sac. Cryptorchidism, i.e., the failure of the testes to descend from the abdomen to the scrotal sac, exposes the testis to the higher body temperature. The abdominal positioning of the cryptorchid testis is detrimental to spermatogenesis and associated with degeneration of the germinal epithelium [1–4]. By contrast, the steriodogenic functions of the somatic cells (Leydig and Sertoli cells) appear to be normal when exposed to the core body temperature [5]. The elevated testicular temperature affects the normal functioning of all major cell types (germ, Leydig, and Sertoli cells) to some extent. Changes are first detected in the pachytene spermatocytes and early spermatids in cryptorchid testes [6]. Significant morphological, degenerative, and physiological changes have been observed in both spontaneous and experimental cryptorchid testes [5, 7, 8]. Such changes in the abdominal testis also influence the physiology and morphology of the scrotal testis in experimental unilateral cryptorchidism [9, 10]. However, molecular and cellular mechanisms of these changes in cryptorchidism are not fully understood. Various studies indicate that cryptorchidism-induced testicular cell degeneration is mediated by apoptosis. Multifaceted insults such as increased oxidative stress [11], loss of germ cell-specific glucose transporters [4], and differential response to gonadotropins [5, 10] may contribute to gonadal cell demise. Although the physiological significance of these changes has been elucidated in some cases, their exact role in testicular degeneration is largely obscure. Elevated levels of cAMP have been reported in cryptorchid testes mainly due to up-regulation of FSH and LH receptor function [10]. Furthermore, cAMP-specific phosphodiesterase (PDE4) inhibitors and increased cAMP levels can induce apoptosis in many cell lines including granulosa cells of the ovarian follicle [12–14]. What role elevated cAMP levels play in gonadal cell demise in cryptorchidism is yet to be determined.
Intracellular concentrations of cAMP are regulated either at the level of synthesis by alterations in adenylyl cyclase activity or at the level of degradation by cyclic nucleotide phosphodiesterase (PDE). Recent studies characterizing PDEs suggest that cAMP-dependent regulatory pathways are indeed involved in cell differentiation and spermatogenesis [15–17]. Spermatogenesis is accompanied by expression of a varied repertoire of PDE enzymes at different stages of cell differentiation that presumably perform specialized functions [16]. There are 21 different PDE genes, classified into 11 families based on substrate specificity, requirements for enzyme activity, tissue distribution, and sensitivity to various pharmacological inhibitors [18, 19]. In rat testes, calcium-calmodulin-dependent PDE1 and low Km, cAMP-specific PDE4 are the most abundant PDEs [20]. The PDE4 enzymes are encoded by four genes designated as PDE4A, B, C, and D; the genes exhibit multiple splice variants resulting in an even larger number of distinct PDE4 enzymes. Among the various PDE4A variants, only PDE4A8 and an uncharacterized 88-kDa PDE4A variant are expressed in rat testes [17].
The therapeutic management of the undescended testis is limited to hormonal treatment or surgical intervention to reduce the risk of infertility and testicular cancer [7, 21, 22]. Experimental data from rats seem to indicate that early orchidopexy and hormonal therapies, including hCG and LHRH analogues, are useful in preventing testicular damage [23]. In humans, prepubertal orchidopexy generally restores sperm counts and motility parameters better than postpubertal surgical correction of the undescended testis [24].
To study the regulation of PDE4 in the testicular response to elevated temperature, we used an in vivo model of surgically induced unilateral cryptorchidism in rats. In this model, normal descent of the right testis was surgically blocked, while the contralateral left testis was allowed to descend into the scrotum. The advantage of using such an experimental model was that the contralateral scrotal testis served as a euthermic contralateral control for studying temporal changes in PDE4 expression [9].
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (30 days of age) were maintained on a 12-h/day photoperiod and provided free access to food and water. All procedures performed on rats were approved by the Institutional Animal Care and Use Committee, Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA, and conformed to the NIH guidelines for the care and use of laboratory animals.
Surgical Procedures
Surgical cryptorchidism of the right testis and sham operations were performed as described previously [4]. A 2-cm right paramedial incision was made through the skin, beginning 3–4 cm caudal to the prepuce and extended cranially. The right internal inguinal ring was exposed and closed to prevent the normal descent of the right testicle. Sham operations were conducted the same way, but the inguinal ring was not closed. Following 10, 20, or 30 days, both testes were removed and decapsulated; each testis was divided into two pieces for histological/electron microscopic examination and immunoblot analyses. Three rats were maintained without any treatment and killed at comparable ages serving as intact controls. In a separate experiment, six rats were surgically rendered unilaterally cryptorchid. After 30 days, orchidopexy was performed on three of these rats that were then allowed to recover for an additional 50 days. These rats were killed with age-matched intact control rats, and testes were removed and processed as above.
Immunocytochemistry and Confocal Microscopy
Immediately after rats were killed, the tissue samples were immersed in ice-cold Zamboni fixative [25], coarsely chopped, and fixed for at least 24 h. The tissue was then removed from fixative, dipped in PBS, trimmed, and stored at 4°C until immunohistochemistry was performed [4]. Frozen sections were prepared after the tissue was equilibrated in 30% sucrose for cryoprotection, embedded in OTC, and frozen. The sections were permeabilized using 100% dimethyl sulfoxide, three times for 10 min each. Nonspecific staining was blocked by incubating in normal donkey serum (10% in antibody diluent; Biogenex, San Ramon, CA) for 1 h at room temperature, and rinsed with PBS three times for 10 min. The primary antibodies against PDE4A and PDE4B were diluted 1:50 in antibody diluent, applied, and incubated overnight at room temperature in humidified chambers. After incubations, the samples were washed in PBS, and the secondary antibody, diluted 1:100 in antibody diluent, was applied. Secondary antibodies, fluorescein isothiocyanate, or Cy3-coupled donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were incubated at 1:100 dilution for 60 min; following incubation, the sections were washed in glycerol for 2 h at room temperature. The sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) to minimize photobleaching. The negative controls were prepared by substituting a nonbinding primary antibody (1:100 dilution) for the primary antibody. Samples were imaged with a Bio-Rad MRC 1024 scanning laser confocal system (Bio-Rad Laboratories, Hemel Hempstead, UK) equipped with a krypton/argon laser.
Sodium Dodecyl Sulfate-PAGE and Western Blotting
Homogenates of testes were prepared in 10 mM Tris, pH 7.4, containing protease inhibitors (10 µg/ml each of leupeptin, pepstatin, aprotinin, and antipain; 50 µg/ml benzamidine; and 1 mM PMSF). Aliquots containing 50 µg protein were mixed with 2x SDS-polyacrylamide gel sample loading buffer (62.5 mM Tris-Cl, pH 6.8; 10% glycerol; 2% SDS; 0.7 M 2-mercaptoethanol; and 0.005% bromophenol blue). The samples were boiled for 3 min and subjected to SDS-PAGE electrophoresis. The proteins were transferred to nitrocellulose, and the blots were blocked in Tris-buffered saline (TBS; 25 mM Tris, pH 7.4, 500 mM NaCl) containing 5% BSA and 0.5% normal goat serum (NGS) for 1 h. After washing in TBS, the blots were incubated with PDE4 antibodies (FabGennix Inc., Shreveport, LA) for 90 min. Blots were washed three times with TBST (TBS containing 0.05% Tween-20) for 10 min and incubated with secondary antibody, goat antirabbit IgG conjugated with alkaline phosphatase or horseradish peroxidase (FabGennix Inc.). The immunoreactive bands were visualized by either colorimetric method using nitroblue tetrazolium/5-bromo-4-chloro-3-indoylphosphate toluidinium as a substrate or by enhanced chemiluminiscence (ECL; Amersham, Piscataway, NJ). The PDE4A selective antibody K53 was raised against a C-terminal peptide ([C]-T-P-G-R-W-G-S-G-G-D-P-A) common to all PDE4A subtypes [26, 27]. The PDE4B-selective antibody was raised against recombinant PDE4B protein [28]; it recognizes PDE4B1, B2, and B3 variants [29]. Immunoblots of testicular homogenates developed with K53 antibody labeled a 109-kDa and an 88-kDa band (Fig. 1, lane 3). The 109-kDa band comigrated with recombinant PDE4A8 protein (Fig. 1, lane 1). The identity of the 109-kDa PDE4A8 band was further confirmed using a PDE4A8-specific antibody K34 (FabGennix Inc.). The polyclonal antibody K34 was raised in rabbit against purified glutathione-S-transferase fusion protein from the unique N-terminal region from PDE4A8. The antibody K34 does not cross react with recombinant PDE4A5 or PDE4A1 proteins (data not shown). The K53 blot was stripped in stripping buffer (Tris HCl, pH 6.8; 10 mM ß-mercaptoethanol; 2% SDS) at 50°C for 1 h and reprobed with PDE4A8-selective antibody K34 that labeled a single 109-kDa band corresponding to PDE4A8 band labeled with K53 (Fig. 1B). Preincubation of antibody K53 with antigenic peptide (500 µg peptide/25 µl antiserum) abolished the labeling of all PDE4A bands (Fig. 1C). The PDE4B-selective antibody labeled two major bands (78 kDa and 66 kDa) in testes (Fig. 1D). The electrophoretic mobility of testicular PDE4B proteins was compared with recombinant PDE4B3 or with rat brain cortical PDE4B proteins. The 78-kDa PDE4B bands in testes correspond to the PDE4B2 variant and the 66-kDa variant corresponds to an uncharacterized rat brain PDE4B protein [29]. Preincubation of the PDE4B antibody with antigenic recombinant protein (500 µg peptide/25 µl antiserum) abolished the labeling of all PDE4B bands (Fig. 1E).
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The densitometric scanning data for each PDE were statistically analyzed by ANOVA, followed by paired Student t-test. Statistical significance was accepted at the 95% confidence level.
RESULTS
Histology and Ultrastructural Morphology of Scrotal and Abdominal Testes
Marked degenerative changes in the lining of the seminiferous tubules were observed 30 days following surgically induced unilateral abdominal cryptorchid testes (Fig. 2, A and B) and as reported earlier [4]. The cryptorchidism-induced changes included a marked reduction in the size of the seminiferous tubules, extensive cell disruption, apoptosis, and fragmentation of nuclear DNA of the primary spermatocytes. In contrast, the scrotal testis showed no significant changes in histological features when compared to intact testes. Rats that had undergone orchidopexy 30 days following unilateral cryptorchidism and were allowed to recover for 50 days showed marked regeneration of spermatogonia, mature spermatozoa, and few apoptotic cells (Fig. 2C). There was no morphological difference between the left and right testes of control rats. Electron microscopic examination revealed that the remaining atrophic spermatogonia showed vacuolation and scattered cellular degeneration, irregular nuclei with fissures, and cytoplasmic pockets (data not shown). Sertoli cells contained large amounts of lipid globules of various densities and a thickening and undulation of the basement membranes in the seminiferous tubules.
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Cellular Distribution of PDE4A and PDE4B Proteins in Rat Testes
Immunocytochemical localization of PDE4A protein using confocal microscopy with the PDE4-selective antibody K53 revealed the presence of PDE4A in the abluminal compartment of the seminiferous tubule where postmeiotic round spermatids were present (Fig. 3A). In contrast, PDE4B expression was evident throughout the seminiferous epithelium, which is consistent with the labeling of somatic cells (Fig. 3B). No fluorescence signal above the background level was detected when sections were incubated with either preimmune serum or with normal rabbit serum (data not shown). The differential localization of PDE4A8 and the 88-kDa PDE4A variant was further analyzed using immunofluorescence microscopy and antibodies K34 and K53. The immunofluorescence from both antibodies suggest that PDE4A8 and 66-kDa PDE4A variants are localized in a subset of germ cells (Fig. 4, A and B).
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Effect of Cryptorchidism on PDE4 Expression
Figure 5 shows the effect of 10, 20, and 30 days cryptorchidism on the expression of PDE4A8 and 88-kDa PDE4A variants in abdominal and scrotal testes. Compared with contralateral scrotal testes, the undescended abdominal testis exhibited lower expression of both PDE4A8 and 88-kDa PDE4A proteins. An approximately 70% and 80% (P < 0.01) reduction in PDE4A8 and 88-kDa PDE4A, respectively, in the abdominal testis was observed relative to the expression in contralateral scrotal testes. By contrast, 30-day cryptorchidism marginally reduced PDE4A8 and 88-kDa PDE4A proteins in contralateral scrotal testes (Fig. 5). There was no significant difference in PDE4A8 or 88-kDa DE4A levels between 40- and 80-day-old rat testes (data not shown).
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In contrast to PDE4A, the expression of 66-kDa PDE4B was significantly increased in abdominal testes after experimentally induced cryptorchidism (1.5-fold, 1.4-fold, and 4.5-fold increases relative to contralateral scrotal testes after 10, 20, and 30 days of experimental cryptorchidism, respectively; Fig. 6). By contrast, the levels of 66-kDa PDE4B in contralateral scrotal testes was reduced by 10%, 30%, and 53% after 10-, 20-, and 30-day experimental cryptorchidism, respectively, compared to control values (Fig. 6). There was no significant difference in either PDE4A or PDE4B expression between left and right testes in control rats (data not shown).
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Effect of Orchidopexy on PDE4 Expression
To investigate the effect of orchidopexy on PDE4 expression, the 30-day cryptorchid rat testis was surgically descended into the scrotum and allowed to recuperate for an additional 50 days. PDE4 expression was measured in both surgically descended testes and in testes from age-matched control rats. The cryptorchidism-induced decrease in abdominal PDE4A8 and 88-kDa PDE4A levels were normalized to scrotal levels in rats subjected to orchidopexy (Fig. 7). The levels of PDE4A variants were similar in both testes from age-matched controls. The effects of orchidopexy on PDE4B expression are shown in Figure 7. The elevated levels of PDE4B4 in abdominal testes of 30-day cryptorchid rats (P < 0.001) were reduced to normal levels (i.e., to that of age-matched control rats; Fig. 7). The recuperated animals exhibited an increased expression of the PDE4B2 variant in both contralateral control and corrected testes.
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DISCUSSION
It has been recognized for many years that raising the ambient temperature of the testis from 32 to 37°C (core body temperature) resulted in degenerative changes in the germinal epithelium. Suprascrotal temperatures, such as in varicocele, warm water baths, or warm weather and experimental cryptorchidism [30, 31], results in impaired spermatogenesis, decreased fertility, and apoptotic damage to germ cells. However, the mechanisms mediating these degenerative changes are not clear. The present study using surgically induced unilateral cryptorchidism, demonstrates a clear temporal relationship between germ cell loss and cAMP-dependent PDEs. These data demonstrate that degenerative changes in abdominal cryptorchidism are associated with altered expression of PDE4. The testicular PDE4 proteins are expressed in a cell-specific manner; i.e., PDE4A variants are predominantly localized in germ cells of postmeiotic stages of differentiation (secondary spermatids), while PDE4B is localized mainly in somatic cells (Sertoli and Leydig cells). The expressions of these PDE4 proteins are differentially regulated in germ and somatic cells in cryptorchid testes. Finally, the degenerative changes and changes in the PDE4 expression in abdominal testes are normalized after 50 days following orchidopexy.
The surgical unilateral cryptorchidism model allowed the evaluation of temperature effects in abdominal testes while providing a euthermic contralateral scrotal testis as a control under similar hormonal and systemic factors [9]. Such an experimental model also is beneficial when biochemical data are correlated with histological findings. There was a marked increase in the apoptotic activity and degeneration of the seminiferous tubules in the operated, abdominal testis compared with the contralateral, scrotal, control testis of unilaterally cryptorchid animals [4, 31, 32]. The present results, consistent with earlier observations, indicate that cryptorchidism-induced degeneration of the testis is associated with apoptotic demise of the germ cells [31, 33]. Various noxious agents can initiate apoptosis, but unlike necrosis, it occurs primarily after a physiological stimulus such as a surge or withdrawal of hormone [34–36]. It has been established that testicular testosterone concentrations are reduced in cryptorchidism [37, 38] and that removal of testosterone can induce germ cell apoptosis [39]. However, determining whether germ cell apoptosis at abdominal temperature is secondary to decreased testosterone levels requires further examination. Apoptotic activity has been observed in normal rat testes [40]; however, abdominal positioning of the testis results in increased apoptosis and fragmentation of nuclear DNA [27, 30, 31].
The changes observed in cyclic nucleotide PDEs during spermatogenic cell differentiation have suggested that unique forms of PDEs are expressed during spermatogenesis [16]. The Western blotting data on PDE4 enzymes reported here are consistent with the pattern of mRNA expression studies using in situ hybridization [17]. The PDE4A variants are mostly expressed in round spermatids where the PDE4A mRNA is expressed at the highest level. Developmental studies are also consistent with the presence of PDE4A8 mRNA and the induction of PDE4A8 and 88-kDa PDE4A protein at 20–30 days of age [17], which coincides with the first appearance of spermatids in the seminiferous tubules [41]. The most important finding in the present study was reduced expression of PDE4A in abdominal cryptorchid testes. To our knowledge, this is the first demonstration that a cryptorchid testis expresses little or no PDE4A variants, which under normal physiological conditions allows for the rapid and efficient hydrolysis of cAMP produced by Gs-coupled receptor stimulation [42]. At least three possibilities could explain the observed decrease in the PDE4A expression. First, the decreased levels of PDE4A reflect the overall degeneration of the abdominal testis. Second, the increase in the ambient temperature to core body temperature (32.5–37°C) caused by cryptorchidism may inhibit PDE4A expression directly. Third, spermatogenic damage may be responsible for altered PDE4A gene expression. The influence of spermatogenic damage on Sertoli and Leydig cell functions has been reported earlier [12]. The aberrations in Sertoli and Leydig cell function in cryptorchid testes are not due to direct effects of elevated temperature but rather are secondary to the spermatogenic damage [12]. The decreased levels of PDE4A are unlikely to be due to generalized necrosis of testicular tissue mainly because PDE4B expression is significantly elevated in the abdominal testis; further, few degenerative changes were observed in cryptorchid testes on Day 10 despite significant down-regulation of PDE4A.
The observation that PDE4A expression is mainly localized in secondary spermatocytes and is decreased in cryptorchidism suggests that down-regulation of PDE4A might be involved in initiating signals for apoptosis. Inhibition of cAMP-dependent PDEs by rolipram has been shown to induce apoptosis in various cell lines [21, 43]. Moreover, increased cAMP concentrations are known to induce apoptosis in granulosa cells [13]. The mechanism by which cAMP can promote apoptosis is not yet clear. It is possible that increased levels of cAMP may augment the transport of exogenous calcium or induce release of calcium from internal stores that may activate the caspase cascade and calcium-dependent endonucleases responsible for DNA fragmentation [44]. Alternatively, increased cAMP concentrations may alter the expression of p53, a key modulator of apoptosis in many systems; consistent with this possibility, increased expression of p53 is observed in cryptorchid testes [2, 30].
There have been reports indicating that cryptorchidism-induced defects in spermatogenesis are not permanent, particularly if the contributing factors are corrected before the onset of peritubular fibrosis [21, 24]. Orchidopexy performed on young mutant trans-scrotal rats with congenital unilateral undescended testes results in a complete normalization of seminiferous tubules [12]; this suggests that germ cell deficiency is secondary to elevated testicular temperature caused by abdominal location of the testis. In the present study, the expression of both PDE4A and PDE4B enzymes were restored to normal levels, similar to those of the scrotal testis. The mechanism for temperature-dependent regulation of PDE4 genes is not known. The presence of heat shock proteins and heat shock factor (HSF2) that regulates the expression of heat shock proteins in testis has been localized in high levels in round spermatids and spermatocytes of some seminiferous tubules [45]. Interestingly, a sharp increase in heat shock proteins was observed in chromatid bodies and round spermatids following hyperthermic treatment [46]. Whether heat shock proteins, HSF2, or other similar factors regulate the expression of the PDE4 gene in a temperature-dependent manner is yet to be determined.
In conclusion, this study has demonstrated that abdominal heat stress induces time-dependent degeneration of germ cells of the rat testis. The findings that these changes are associated with altered expression of PDE4A in abdominal testes of unilateral cryptorchid rats provides a potential model for the study of regulatory mechanisms of the cell-specific apoptotic signals initiated by altered cAMP metabolism.
ACKNOWLEDGMENTS
Authors acknowledge Ms. Victoria L. Specian for her assistance with confocal microscopy and Ms. Shiru Q. Farooqui for her technical help.
FOOTNOTES
First decision: 7 August 2000.
1 Correspondence: Shakeel M. Farooqui, FabGennix Int. Inc., P.O. Box 53062, Shreveport, LA 71135-3062. FAX: 318 798 1849; sfaroo{at}fabgennix.com 
Accepted: January 22, 2001.
Received: June 26, 2000.
REFERENCES
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