Preservation of Spermatogenesis in Spinal Cord Injured Rats With Exogenous Testosterone. Relationship With Serum Testosterone Levels and Cel
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《男科医学杂志》
the Veterans Affairs Medical Center, East Orange, New Jersey; and the Department of Surgery Division of Urology, Neuroscience, and Obstetrics/Gynecology, UMD-New Jersey Medical School, Newark, New Jersey.
Abstract
The current experiment examined the effects of exogenous testosterone (T) on spermatogenesis in rats with spinal cord injury (SCI) and their relationship with the cellular distribution of a cyclic AMP-responsive element modulator (CREM) in testicular cells. Implantation of T-filled Silastic capsules (TCs, 1-20 cm) resulted in dose-dependent, biphasic changes in testicular T levels and spermatogenesis in SCI rats. However, dose responsiveness of spermatogenesis to exogenous T in SCI rats differed from that in sham control rats. Specifically, implantation of 2-cm TCs enhanced the effects of SCI on spermatogenesis, resulting in total regression of the seminiferous epithelium. Although 3-cm TCs maintained complete spermatogenesis in sham control rats, this regimen failed to support complete spermatogenesis in SCI rats. Although complete spermatogenesis was maintained in SCI rats given 5-20-cm TC implants, various abnormalities persisted. Cellular distribution of CREM remained normal in SCI rats but was altered in those SCI rats that received 3- or 5-cm TC implants. Such effects were associated with reduced CREM proteins in testicular tissues. These results were consistent with altered cAMP signaling and its regulation in testicular cells after SCI and provided possible mechanistic explanations for the effects of SCI on spermatogenesis. Conclusion: SCI resulted in changes in the responsiveness of spermatogenesis to exogenous T. These effects were associated with altered cAMP/CREM signaling in testicular cells. Further studies, including a study of the relationship between serum T levels and normalcy of sperm functions and the role of neural-endocrine interactions in mediating the effects of SCI on spermatogenesis and sperm function, are needed so that therapeutic regimens can be designed for clinical use.
Key words: Sertoli cells, CREM, sperm
Male infertility resulting from spinal cord injury (SCI) is associated with abnormal semen parameters, including decreases in sperm count, progressive motility, and sperm with normal morphology (Hirsch et al, 1991; Linsenmeyer and Perkash, 1991; Brackett et al, 1994). In surgically induced SCI rats, abnormal spermiogenesis occurring during the acute phase of SCI was preceded by transient but significant decreases in serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and intratesticular testosterone (ITT) levels (Huang et al, 1995). These results suggest that hormone deprivation might account for the initial effects of SCI on spermatogenesis. Our recent findings demonstrating changes in the responses of mRNA transcripts for a testicular cAMP-responsive element modulator (CREM) and that for Sertoli and germ cell-specific proteins encoded by CRE-containing genes (Huang et al, 2003a) suggest that altered cAMP signaling and its regulation might contribute to the effects of SCI on spermatogenesis. This postulate is based on the facts that cAMP signaling mediates the effects of FSH on Sertoli cells (Means et al, 1976) and that CREM is a functional switch for postmeiotic germ cell differentiation (Sassone-Corsi, 1998). In addition, cAMP signaling might also mediate some of the effects of androgens (Heinline and Chang, 2002).
Testosterone (T) alone, when administered in sufficient amount, is capable of maintaining or restoring qualitatively complete spermatogenesis in hypophysectomized animals (Huang et al, 1987; Santulli et al, 1990) and has been proven effective in preserving spermatogenesis in animals subjected to cytotoxic insults (Delic et al, 1986; Pogach et al, 1988; Meistrich et al, 1999). Our previous studies demonstrated that regression of spermatogenesis after SCI could also be partially prevented by exogenous T (Huang et al, 1999). This result suggested the feasibility of using exogenous T or a related agent to preserve spermatogenesis, and thereby sperm production, in SCI men. To facilitate such application, the current experiment examined whether spermatogenesis in the rat could respond to exogenous T in normal fashions after SCI. Because of the importance of CREM in postmeiotic differentiation (Sassone-Corsi, 1998), we also examined the effects of exogenous T on the expression and cellular distribution of CREM.
Materials and Methods
Animals
Mature Sprague Dawley rats (300-350 gm, Taconic Farm, Taconic, NY) were caged individually in an air-conditioned, light-controlled animal room for 2 weeks prior to the experiment. They were fed Purina rat chow and water ad libitum. The rats were randomly assigned to undergo either SCI or a sham operation. A total of 60 rats underwent surgical transection of the spinal cord at the level of the ninth thoracic vertebra as described previously (Linsenmeyer et al, 1994). Control animals received a sham operation without laminectomy. The SCI procedures were reviewed annually and approved by the Institutional Animal Care and Use Committees at both the East Orange VA Medical Center and UMD-New Jersey Medical School.
Immediately after the SCI surgery, the rats were given subcutaneous implants of various lengths (1, 2, 3, 5, 10, and 20 cm) of T-filled Silastic capsules (TCs, inner diameter = 3.35 mm, outer diameter = 4.65 mm, Dow Corning International, Corning, NY) (Huang et al, 1987) in the flank region. Implantation of TCs resulted in stable serum T levels that were correlated with TC lengths (Huang and Boccabella, 1988; Huang et al, 1991). Sham control animals received 5-cm empty capsules. The animals were killed by decapitation 8 weeks later, and trunk blood was collected for measurement of serum hormones. One half of 1 testis from each rat was fixed in Bouin solution and processed for histology. The remaining tissues were frozen immediately in isohexane immersed in a mixture of methanol and dry ice and then stored at -80°C. In a follow-up experiment, sham control and additional SCI rats were given TC implants of 1, 2, 3, 5, or 10 cm immediately after the surgery and were killed 8 weeks later.
Quantification of Sonication-Resistant Sperm Head
Approximately 500 mg of testicular tissue from each rat was sonicated in 2 mL phosphate-buffered saline (PBS). The sonication-resistant sperm heads were counted with a hemocytometer. Results were expressed as sperm head number per gram of tissue.
Sperm Motility
One caudal epididymides from each rat was immersed in 2 mL 37°C PBS containing 1% bovine serum albumin immediately after it was excised. Subsequently, epididymal sperm were released by puncturing the epididymal duct at 20-30 locations with a 19-gauge needle. The sperm suspensions were kept at 37°C for 10-15 minutes. A drop of sperm suspension was then placed on a prewarmed hemocytometer and examined. Sperm in 10-20 microscopic fields were videotaped, and sperm motility was later determined. A sperm was considered "motile" when it did not remain at the same location during the 5-10-second taping time of each field.
Hormone Measurement
Serum FSH, LH, and testosterone and testicular testosterone (ITT) concentrations were determined by radioimmunoassay as previously described (Huang et al, 1995).
Immunostaining of CREM
Five-micron-thick sections of testicular tissues fixed in Bouin solution were deparaffinized in xylene, rehydrated through graded ethanol, and boiled in 0.01 M citrate buffer (pH 6.0) for 10-15 minutes. After washing in PBS (2 x 5 minutes), the sections were incubated with 1.5% hydrogen peroxide for 10 minutes, washed twice in distilled water followed by 2 changes of PBS (5 minutes each), and blocked in 4% normal goat serum in PBS containing 0.01% Tween 20 for 20 minutes. After 2 washes in PBS, the sections were subsequently incubated with polyclonal anti-CREM antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif, 1:300 dilution in blocking solution) at 4°C overnight. For negative control, CREM antibody was incubated with fivefold excess of purified antigen at 4°C overnight and diluted to 1:300 in blocking solution before use. The sections were subsequently washed in 2 changes of PBS, incubated with Biotin-labeled anti-rabbit IgG (Sigma Chemical, St. Louis, Mo, 1:2000) for 30 minutes, and washed with PBS. The sections were then incubated with Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, Calif) for 30 minutes, washed in 2 changes of PBS, and visualized after adding stable 3-3'-diaminobenzidine tetrahydrochloride solution (Research Genetics, Huntsville, Ala). After counterstaining with fast green, the slides were dehydrated in graded ethanol solution, cleared in xylene, and covered.
Western Blotting of CREM
For Western blotting of CREM, 300-500 mg of testicular tissue was solublized in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS] in PBS) in the presence of protease inhibitors (Phenylmethylsulfonyl fluoride and aprotinin). Polyacrylamide gel electrophoresis and Western blotting were performed according to the procedures described previously (Molina et al, 1993). The anti-CREM polyclonal antibody recognizes all CREM gene products and was used at 1:1000 dilution.
Statistics
All data were evaluated to determine that they were normally distributed, and they were analyzed with analyses of variance with TC dose as the independent variable. When the treatment effects were significant (P < .05), planned a priori comparisons were made using Dunn's tests to determine the statistical significance of differences among treatment groups.
Results
Eight weeks after the SCI surgery, serum T levels of untreated SCI rats were comparable to those of untreated sham control rats and were not affected by implantation of 1-cm TCs (P > 0.1; Figure 1A). Serum T levels of SCI rats were not affected by 2-cm TC implants that significantly suppressed serum T of sham control rats (P < .05). Implantation of 3-20-cm TCs resulted in dose-dependent increases of serum T levels in SCI and sham control rats (P < .05 and .01, respectively). Testicular T concentrations of untreated SCI rats were not different from those of sham control rats (P > .1). Implantation of 1-20-cm TCs in SCI rats resulted in biphasic and dose-dependent decreases in testicular T concentrations (P < .01 and .05, respectively; Figure 1B). A similar pattern also was observed in sham control rats that received 1-10-cm TC implants, but testicular T levels of TC-implanted sham control rats were higher than their SCI counterparts (P < .05). Serum LH and FSH concentrations of SCI and sham control rats decreased in a dose-dependent manner in those that received TC implants (P < .05 and .01, respectively; Figure 1C and D).
The testis weights of untreated SCI rats were reduced by 20%, but the decrease was not statistical significant. Implantation of 1-10-cm TCs resulted in dose-dependent, biphasic changes in testis weights in sham control rats, reaching a nadir in those that received 2-cm TCs (P < .01). Although implantation of 1-cm TCs did not affect the testis weights of SCI rats (P > .1), testis weights of those SCI rats that received 2- or 3-cm TC implants were reduced by >60% compared to untreated SCI rats (P < .01; Figure 2A). Testis weights of SCI rats given 5-cm or longer TC implants rebounded and were greater than those of the 2- or 3-cm groups (P < .05), but the difference remained statistically significant when compared to those of untreated SCI rats (P < .01).
Spermatogenesis
Of the 8 untreated SCI rats, 5 had persisting spermatogenesis in 50%-90% of the tubules examined. However, impaired spermatogenesis, ranging from partial regression of the epithelium to the absence of proliferating spermatogonia, were seen in all rats (Figure 3B). Of the 7 SCI rats given 1-cm TC implants, 1 had a totally regressed seminiferous epithelium in more than 95% of the tubular cross sections. Qualitatively normal spermatogenesis, except for delayed spermiation, was seen in more than 95% of the tubules in the remaining 6 rats. On the other hand, regression of the seminiferous epithelium, ranging from persistence of proliferating spermatogonia to the absence of all spermatogenic cells, including proliferating spermatogonia, was seen in 80%-100% of the tubular cross sections in 5 of the 7 SCI rats that received 2-cm TC implants (Figure 3C). In the other 2 rats, regression of spermatogenesis was also seen in more than 50% of the tubules, but spermatocytes or young spermatids remained in some tubules.
Of the 5 SCI rats that received 3-cm TC implants, 1 had a regressed epithelium in all tubules. In the remaining 4, incomplete spermatogenesis, characterized by the presence of spermatogonia, spermatocytes, and young spermatids, but without elongated spermatids, was seen in more than 95% of the tubules (Figure 3D). Complete spermatogenesis, characterized by the presence of mature spermatids at the lumenal edges of stages VII-VIII epithelia, was maintained in 4 of 7, 2 of 4, and 5 of 5 SCI rats that received TC implants of 5, 10, and 20 cm, respectively (Figure 3E and F). Spermatogenesis was partially maintained or regressed in the remaining rats from these groups. Spermatogenesis in sham control rats given various lengths of TC implants in the follow-up experiments (not shown) was comparable to those reported previously (Huang and Boccabella, 1988; Huang et al, 1991).
Sperm Head Number
Figure 2B shows that the sonication-resistant sperm head in SCI rats was reduced by 20% in those given 1-cm TCs and was undetectable in those given 2-cm TCs (P < .001). It reappeared in the testes of those rats given 3-cm TCs and rebounded to the level of untreated rats in rats given 10-cm TCs (P < .01).
Sperm Motility
Percent motility of sperm recovered from the caudal epididymides of SCI rats was significantly lower than that of sham control rats (P < .05; Figure 2C). Implantation of 10- or 20-cm TCs resulted in a slight decrease of sperm motility in sham control rats (P < .05). Despite the presence of residual sperm in those SCI rats that received 2- or 3-cm TC implants, and significant number of sperm in those that received 5-cm TCs, none of these sperm were motile (Figure 2C). Although a large number of sperm was present in the caudal epididymides of those SCI rats given 10- or 20-cm TC implants, only less than 20% of these sperm were motile (P < .05).
Western Blotting of CREM
Western blotting of testicular lysates revealed a general decrease in levels of CREM (the activator isoform, see "Discussion") in the testes of SCI rats (Figure 4A). This decrease was not affected by implantation of 1-cm TCs but was further suppressed in those SCI rats that received 2-5-cm TC implants. CREM levels in the SCI rats that received 10- or 20-cm TC implants were not different from those of untreated SCI rats. Implantation of TCs of different lengths in sham control rats did not affect CREM significantly (Figure 4B).
Cellular Distribution of CREM
In sham control rats, CREM immunostaining was localized primarily in maturing pachytene spermatocytes and young spermatids; presence of CREM in spermatogonia and prepachytene spermatocytes was rare (Figure 5A). In untreated SCI rats, CREM was also detected in young spermatids, but the intensity of CREM immunostaining was less pronounced (Figure 5B). Implantation of 1-cm TCs resulted in a further decrease in CREM staining in spermatids (not shown). Although CREM was also detected in some young spermatids in those SCI rats receiving 3-cm TC implants, it appeared primarily in spermatogonia and prepachytene spermatocytes in 3 of 5 of the rats in this dose group (Figure 5C and D). Of note, in spite of persistence of complete spermatogenesis, CREM was not detected in young spermatids in those SCI rats given 5-cm TC implants (Figure 5E). In 5 of the 6 rats in this group, CREM was present in spermatogonia and primary spermatocytes, including preleptotene spermatocytes, in most of the tubules (Figure 5F). These results were confirmed 2-3 times with slides from each animal immunostained at different dates. CREM reappeared in young spermatids in most of the tubules of SCI rats given 10- or 20-cm TC implants, regardless of the status of spermatogenesis (Figure 5G). However, young spermatocytes in many of the tubules, specifically those with partially regressed epithelia, also contained immunoactive CREM. Immunostaining of CREM in the testes of sham control rats given different lengths of TC implants in the follow-up experiment revealed normal cellular distribution of CREM (Figure 5H), despite variations in staining intensity. We also retrospectively examined CREM cellular distribution in archived testicular tissues fixed in Bouin solution of intact and hypophysectomized rats given various lengths of TC implants from previous studies (Huang and Boccabella, 1988; Huang et al, 1991). CREM was distributed normally in maturing spermatocytes and young spermatids in all tissues, regardless of the status of spermatogenesis and variations in the intensity of immunostaining (not shown). Specificity was demonstrated by the absence of CREM immunostaining when the sections were stained with antigen preadsorbed antiserum (Figure 5I).
Discussion
Our recent report demonstrating preservation of spermatogenesis in SCI rats by exogenous T (Huang et al, 1999) suggests the feasibility of using androgen or related therapeutic means to preserve spermatogenic function and perhaps fertility in SCI men. In order to validate such regimens for clinical use, optimizing the effective doses and understanding the mechanisms underlying the beneficial effects of exogenous T on spermatogenesis after SCI are essential.
The effects of increasing doses of exogenous T on serum LH, FSH, and testicular T and biphasic changes in spermatogenesis in SCI rats were consistent with those seen in intact rats (Zirkin et al, 1989; Santulli et al, 1990; Huang et al, 1991). However, dose effects of T on spermatogenesis seen in SCI rats were different from those seen in intact rats. Specifically, although implantation of 1-cm TCs resulted in a 40% reduction of testis weight and incomplete spermatogenesis in non-SCI intact rats (Huang and Boccabella, 1988), identical treatment maintained testis weight and qualitatively complete spermatogenesis in SCI rats. Total regression of the seminiferous epithelium in SCI rats that received 2-cm TC implants, characterized by the absence of proliferating spermatogonia in many tubular cross sections, also differed from their sham control counterpart. These changes resembled those that occurred in chronic SCI rats (Huang et al, 1995) and after testicular denervation (Chow et al, 2000) and were far more severe than that occurred in chronic hypophysectomized rats (Huang et al, 1987) and in SCI rats that received 3-cm TC implants and had nearly identical testicular T levels. The putative neurogenic mechanisms contributing to the loss of proliferating spermatogonia after SCI and testicular denervation must have been exaggerated specifically by the 2-cm TC regimen. Although such an effect was overcome by higher doses of exogenous T (3-20-cm TC implants), failure to maintain complete spermatogenesis in SCI rats given 3-cm TC implants also differed from their sham control rat counterparts. Such differences cannot be ascribed to hormone status, suggesting that local factors might be involved in neural-endocrine interactions in the control of spermatogenesis. Maintenance of qualitatively complete spermatogenesis and relatively normal sperm motility in SCI rats that received 1 cm-TC implants nevertheless suggests potential applicability of specific low doses of exogenous T in preservation of spermatogenesis and sperm function in SCI men.
A decrease in sperm motility in untreated SCI rats was consistent with that in SCI men. Such effects could be attributed to abnormal sperm transport, maturation in the epididymis, or both (Linsenmeyer et al, 1999) as a result of impaired epididymal function after denervation (Billups et al, 1990). Although sperm motility in SCI rats was not affected by the 1-cm TC implant, it was totally eliminated in those rats that received 5-cm TC implants but rebounded slightly in those that received 10- or 20-cm TC implants. Because epididymal function of these SCI rats would have been better stimulated than in those that received 1-cm TC implants because of the elevated serum T level, the loss and rebound of sperm motility in SCI rats that received 5-20-cm TC implants might reflect functional status of the sperm motile apparatus. In addition, because sperm motility also was decreased in the sham control rats that received 5- or 10-cm TC implants, a difference in the metabolic states of sperm secondary to changes in epididymal function under different serum T conditions might also contribute to differences in sperm motility.
Testosterone modulates spermatogenesis through its effects on Sertoli cells. These effects were initiated by the binding of T to, and activation of, androgen receptors (AR) that transactivate androgen-dependent genes to induce changes in Sertoli cell functions (Zhou et al, 1994). Such effects thus mediate the effects of androgen on spermatogenesis. However, presence of AR and androgen-binding protein (ABP) in spermatogenic cells (Vornberger et al, 1994; Joseph et al, 1997), stimulation of protein synthesis in spermatocytes by the T/ABP complex (Gerard, 1995), and presence of a hormone-responsive element (HRE) in the promoter of genes that encode germ cell-specific proteins (Bonny et al, 1998; Ha et al, 1997) suggest that T might also affect spermatogenic cells directly. In addition, recent studies have demonstrated that cAMP signaling might also mediate some effect of androgen, especially in those cells lacking functional AR (Heinline and Chang, 2002).
CREM is a nuclear transcription factor that modulates the function of cAMP-responsive genes (Foulkes et al, 1991; Habener et al, 1996). Alternative gene splicing results in different CREM isoforms that either activate or repress the function of cAMP (Foulkes et al, 1991). In prepubertal animals, CREM repressor isoforms (CREM and ?) were detected in spermatogonia, early meiotic cells, or both (Foulkes et al, 1992). In adult testes, the activator isoform (CREM) was detected mainly in maturing spermatocytes and young spermatid and was a functional switch for postmeiotic germ cell differentiation (Foulkes et al, 1992; Sassone-Corsi, 1998). Modulation of the expression of CREM by FSH and T (Foulkes et al, 1993; West et al, 1994) suggests its role in mediating the effects of hormones on spermatogenesis. Recently, we reported changes in the short-term effects of FSH and T on testicular CREM transcripts and transcripts for Sertoli and germ cell-specific proteins encoded by cAMP-responsive genes in SCI rats (Huang et al, 2003a). Current observation of abnormal cellular localization of CREM in SCI rats that received 3- and 5-cm TC implants further illustrated changes in the timing of CREM translation during spermatogenic differentiation. Decreased localization of CREM in spermatids in these SCI rats was consistent with a reduction of CREM protein in their testicular tissues. Because such effects were only seen in SCI rats that received 3- or 5-cm TC implants, and not in their sham control counterparts, they were most likely related to changes in cellular function attributable to a specific neural-endocrine interaction. These findings were consistent with changes in hormonal regulations of cAMP signaling in testicular cells after SCI. Precocious expression of CREM in spermatogonia and young spermatocytes has also been observed in acute SCI rats following cord contusion (Huang et al, 2003b). Such changes could disturb cAMP signaling in spermatogenic cells; impede normal spermatogenesis; and result in reduced sperm count or production of sperm with abnormal functions or both. Persistence of CREM in spermatids in SCI rats given 1-cm TCs and its reappearance in spermatids in those rats that were given 10- and 20-cm TC implants and had qualitatively complete spermatogenesis support our postulate. Further studies with reverse transcription PCR techniques (Peri et al, 1998) to characterize properties of CREM in isolated spermatogenic cells of SCI rats could provide new insights into the functional roles of different CREM isoforms in mediating the neural-endocrine interaction in the control of spermatogenesis. In this regard, premature expression of Pm-1 has been linked to spermatogenic arrest (Lee et al, 1995), and reduced CREM expression has been found in human testes with spermatid maturation arrest (Peri et al, 1998; Steger et al, 1999).
The results of this study demonstrate changes in the effects of exogenous T on spermatogenesis in the rat after SCI. Preservation of spermatogenesis and sperm motility in SCI rats that received 1-cm TC implants suggests potential efficacy of low doses of exogenous T in the maintenance of sperm production and function in SCI men. These effects were associated with changes in the expression of testicular CREM and its distribution in spermatogenic cells. These results support the notion that altered cAMP signaling and its regulation in testicular cells might be mediating the effects of SCI on spermatogenesis. Such changes might affect timely and sequential progression of spermatogenesis and might contribute to the production of sperm with abnormal morphology and function after SCI.
Acknowledgments
We thank Norihiro Chinen for performing Western blotting of CREM, William Giglio for hormone assays, and Robert Anesetti for animal care.
Footnotes
Supported by V.A. Rehabilitation R&D Services (B885-2RA).
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Abstract
The current experiment examined the effects of exogenous testosterone (T) on spermatogenesis in rats with spinal cord injury (SCI) and their relationship with the cellular distribution of a cyclic AMP-responsive element modulator (CREM) in testicular cells. Implantation of T-filled Silastic capsules (TCs, 1-20 cm) resulted in dose-dependent, biphasic changes in testicular T levels and spermatogenesis in SCI rats. However, dose responsiveness of spermatogenesis to exogenous T in SCI rats differed from that in sham control rats. Specifically, implantation of 2-cm TCs enhanced the effects of SCI on spermatogenesis, resulting in total regression of the seminiferous epithelium. Although 3-cm TCs maintained complete spermatogenesis in sham control rats, this regimen failed to support complete spermatogenesis in SCI rats. Although complete spermatogenesis was maintained in SCI rats given 5-20-cm TC implants, various abnormalities persisted. Cellular distribution of CREM remained normal in SCI rats but was altered in those SCI rats that received 3- or 5-cm TC implants. Such effects were associated with reduced CREM proteins in testicular tissues. These results were consistent with altered cAMP signaling and its regulation in testicular cells after SCI and provided possible mechanistic explanations for the effects of SCI on spermatogenesis. Conclusion: SCI resulted in changes in the responsiveness of spermatogenesis to exogenous T. These effects were associated with altered cAMP/CREM signaling in testicular cells. Further studies, including a study of the relationship between serum T levels and normalcy of sperm functions and the role of neural-endocrine interactions in mediating the effects of SCI on spermatogenesis and sperm function, are needed so that therapeutic regimens can be designed for clinical use.
Key words: Sertoli cells, CREM, sperm
Male infertility resulting from spinal cord injury (SCI) is associated with abnormal semen parameters, including decreases in sperm count, progressive motility, and sperm with normal morphology (Hirsch et al, 1991; Linsenmeyer and Perkash, 1991; Brackett et al, 1994). In surgically induced SCI rats, abnormal spermiogenesis occurring during the acute phase of SCI was preceded by transient but significant decreases in serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and intratesticular testosterone (ITT) levels (Huang et al, 1995). These results suggest that hormone deprivation might account for the initial effects of SCI on spermatogenesis. Our recent findings demonstrating changes in the responses of mRNA transcripts for a testicular cAMP-responsive element modulator (CREM) and that for Sertoli and germ cell-specific proteins encoded by CRE-containing genes (Huang et al, 2003a) suggest that altered cAMP signaling and its regulation might contribute to the effects of SCI on spermatogenesis. This postulate is based on the facts that cAMP signaling mediates the effects of FSH on Sertoli cells (Means et al, 1976) and that CREM is a functional switch for postmeiotic germ cell differentiation (Sassone-Corsi, 1998). In addition, cAMP signaling might also mediate some of the effects of androgens (Heinline and Chang, 2002).
Testosterone (T) alone, when administered in sufficient amount, is capable of maintaining or restoring qualitatively complete spermatogenesis in hypophysectomized animals (Huang et al, 1987; Santulli et al, 1990) and has been proven effective in preserving spermatogenesis in animals subjected to cytotoxic insults (Delic et al, 1986; Pogach et al, 1988; Meistrich et al, 1999). Our previous studies demonstrated that regression of spermatogenesis after SCI could also be partially prevented by exogenous T (Huang et al, 1999). This result suggested the feasibility of using exogenous T or a related agent to preserve spermatogenesis, and thereby sperm production, in SCI men. To facilitate such application, the current experiment examined whether spermatogenesis in the rat could respond to exogenous T in normal fashions after SCI. Because of the importance of CREM in postmeiotic differentiation (Sassone-Corsi, 1998), we also examined the effects of exogenous T on the expression and cellular distribution of CREM.
Materials and Methods
Animals
Mature Sprague Dawley rats (300-350 gm, Taconic Farm, Taconic, NY) were caged individually in an air-conditioned, light-controlled animal room for 2 weeks prior to the experiment. They were fed Purina rat chow and water ad libitum. The rats were randomly assigned to undergo either SCI or a sham operation. A total of 60 rats underwent surgical transection of the spinal cord at the level of the ninth thoracic vertebra as described previously (Linsenmeyer et al, 1994). Control animals received a sham operation without laminectomy. The SCI procedures were reviewed annually and approved by the Institutional Animal Care and Use Committees at both the East Orange VA Medical Center and UMD-New Jersey Medical School.
Immediately after the SCI surgery, the rats were given subcutaneous implants of various lengths (1, 2, 3, 5, 10, and 20 cm) of T-filled Silastic capsules (TCs, inner diameter = 3.35 mm, outer diameter = 4.65 mm, Dow Corning International, Corning, NY) (Huang et al, 1987) in the flank region. Implantation of TCs resulted in stable serum T levels that were correlated with TC lengths (Huang and Boccabella, 1988; Huang et al, 1991). Sham control animals received 5-cm empty capsules. The animals were killed by decapitation 8 weeks later, and trunk blood was collected for measurement of serum hormones. One half of 1 testis from each rat was fixed in Bouin solution and processed for histology. The remaining tissues were frozen immediately in isohexane immersed in a mixture of methanol and dry ice and then stored at -80°C. In a follow-up experiment, sham control and additional SCI rats were given TC implants of 1, 2, 3, 5, or 10 cm immediately after the surgery and were killed 8 weeks later.
Quantification of Sonication-Resistant Sperm Head
Approximately 500 mg of testicular tissue from each rat was sonicated in 2 mL phosphate-buffered saline (PBS). The sonication-resistant sperm heads were counted with a hemocytometer. Results were expressed as sperm head number per gram of tissue.
Sperm Motility
One caudal epididymides from each rat was immersed in 2 mL 37°C PBS containing 1% bovine serum albumin immediately after it was excised. Subsequently, epididymal sperm were released by puncturing the epididymal duct at 20-30 locations with a 19-gauge needle. The sperm suspensions were kept at 37°C for 10-15 minutes. A drop of sperm suspension was then placed on a prewarmed hemocytometer and examined. Sperm in 10-20 microscopic fields were videotaped, and sperm motility was later determined. A sperm was considered "motile" when it did not remain at the same location during the 5-10-second taping time of each field.
Hormone Measurement
Serum FSH, LH, and testosterone and testicular testosterone (ITT) concentrations were determined by radioimmunoassay as previously described (Huang et al, 1995).
Immunostaining of CREM
Five-micron-thick sections of testicular tissues fixed in Bouin solution were deparaffinized in xylene, rehydrated through graded ethanol, and boiled in 0.01 M citrate buffer (pH 6.0) for 10-15 minutes. After washing in PBS (2 x 5 minutes), the sections were incubated with 1.5% hydrogen peroxide for 10 minutes, washed twice in distilled water followed by 2 changes of PBS (5 minutes each), and blocked in 4% normal goat serum in PBS containing 0.01% Tween 20 for 20 minutes. After 2 washes in PBS, the sections were subsequently incubated with polyclonal anti-CREM antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif, 1:300 dilution in blocking solution) at 4°C overnight. For negative control, CREM antibody was incubated with fivefold excess of purified antigen at 4°C overnight and diluted to 1:300 in blocking solution before use. The sections were subsequently washed in 2 changes of PBS, incubated with Biotin-labeled anti-rabbit IgG (Sigma Chemical, St. Louis, Mo, 1:2000) for 30 minutes, and washed with PBS. The sections were then incubated with Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, Calif) for 30 minutes, washed in 2 changes of PBS, and visualized after adding stable 3-3'-diaminobenzidine tetrahydrochloride solution (Research Genetics, Huntsville, Ala). After counterstaining with fast green, the slides were dehydrated in graded ethanol solution, cleared in xylene, and covered.
Western Blotting of CREM
For Western blotting of CREM, 300-500 mg of testicular tissue was solublized in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS] in PBS) in the presence of protease inhibitors (Phenylmethylsulfonyl fluoride and aprotinin). Polyacrylamide gel electrophoresis and Western blotting were performed according to the procedures described previously (Molina et al, 1993). The anti-CREM polyclonal antibody recognizes all CREM gene products and was used at 1:1000 dilution.
Statistics
All data were evaluated to determine that they were normally distributed, and they were analyzed with analyses of variance with TC dose as the independent variable. When the treatment effects were significant (P < .05), planned a priori comparisons were made using Dunn's tests to determine the statistical significance of differences among treatment groups.
Results
Eight weeks after the SCI surgery, serum T levels of untreated SCI rats were comparable to those of untreated sham control rats and were not affected by implantation of 1-cm TCs (P > 0.1; Figure 1A). Serum T levels of SCI rats were not affected by 2-cm TC implants that significantly suppressed serum T of sham control rats (P < .05). Implantation of 3-20-cm TCs resulted in dose-dependent increases of serum T levels in SCI and sham control rats (P < .05 and .01, respectively). Testicular T concentrations of untreated SCI rats were not different from those of sham control rats (P > .1). Implantation of 1-20-cm TCs in SCI rats resulted in biphasic and dose-dependent decreases in testicular T concentrations (P < .01 and .05, respectively; Figure 1B). A similar pattern also was observed in sham control rats that received 1-10-cm TC implants, but testicular T levels of TC-implanted sham control rats were higher than their SCI counterparts (P < .05). Serum LH and FSH concentrations of SCI and sham control rats decreased in a dose-dependent manner in those that received TC implants (P < .05 and .01, respectively; Figure 1C and D).
The testis weights of untreated SCI rats were reduced by 20%, but the decrease was not statistical significant. Implantation of 1-10-cm TCs resulted in dose-dependent, biphasic changes in testis weights in sham control rats, reaching a nadir in those that received 2-cm TCs (P < .01). Although implantation of 1-cm TCs did not affect the testis weights of SCI rats (P > .1), testis weights of those SCI rats that received 2- or 3-cm TC implants were reduced by >60% compared to untreated SCI rats (P < .01; Figure 2A). Testis weights of SCI rats given 5-cm or longer TC implants rebounded and were greater than those of the 2- or 3-cm groups (P < .05), but the difference remained statistically significant when compared to those of untreated SCI rats (P < .01).
Spermatogenesis
Of the 8 untreated SCI rats, 5 had persisting spermatogenesis in 50%-90% of the tubules examined. However, impaired spermatogenesis, ranging from partial regression of the epithelium to the absence of proliferating spermatogonia, were seen in all rats (Figure 3B). Of the 7 SCI rats given 1-cm TC implants, 1 had a totally regressed seminiferous epithelium in more than 95% of the tubular cross sections. Qualitatively normal spermatogenesis, except for delayed spermiation, was seen in more than 95% of the tubules in the remaining 6 rats. On the other hand, regression of the seminiferous epithelium, ranging from persistence of proliferating spermatogonia to the absence of all spermatogenic cells, including proliferating spermatogonia, was seen in 80%-100% of the tubular cross sections in 5 of the 7 SCI rats that received 2-cm TC implants (Figure 3C). In the other 2 rats, regression of spermatogenesis was also seen in more than 50% of the tubules, but spermatocytes or young spermatids remained in some tubules.
Of the 5 SCI rats that received 3-cm TC implants, 1 had a regressed epithelium in all tubules. In the remaining 4, incomplete spermatogenesis, characterized by the presence of spermatogonia, spermatocytes, and young spermatids, but without elongated spermatids, was seen in more than 95% of the tubules (Figure 3D). Complete spermatogenesis, characterized by the presence of mature spermatids at the lumenal edges of stages VII-VIII epithelia, was maintained in 4 of 7, 2 of 4, and 5 of 5 SCI rats that received TC implants of 5, 10, and 20 cm, respectively (Figure 3E and F). Spermatogenesis was partially maintained or regressed in the remaining rats from these groups. Spermatogenesis in sham control rats given various lengths of TC implants in the follow-up experiments (not shown) was comparable to those reported previously (Huang and Boccabella, 1988; Huang et al, 1991).
Sperm Head Number
Figure 2B shows that the sonication-resistant sperm head in SCI rats was reduced by 20% in those given 1-cm TCs and was undetectable in those given 2-cm TCs (P < .001). It reappeared in the testes of those rats given 3-cm TCs and rebounded to the level of untreated rats in rats given 10-cm TCs (P < .01).
Sperm Motility
Percent motility of sperm recovered from the caudal epididymides of SCI rats was significantly lower than that of sham control rats (P < .05; Figure 2C). Implantation of 10- or 20-cm TCs resulted in a slight decrease of sperm motility in sham control rats (P < .05). Despite the presence of residual sperm in those SCI rats that received 2- or 3-cm TC implants, and significant number of sperm in those that received 5-cm TCs, none of these sperm were motile (Figure 2C). Although a large number of sperm was present in the caudal epididymides of those SCI rats given 10- or 20-cm TC implants, only less than 20% of these sperm were motile (P < .05).
Western Blotting of CREM
Western blotting of testicular lysates revealed a general decrease in levels of CREM (the activator isoform, see "Discussion") in the testes of SCI rats (Figure 4A). This decrease was not affected by implantation of 1-cm TCs but was further suppressed in those SCI rats that received 2-5-cm TC implants. CREM levels in the SCI rats that received 10- or 20-cm TC implants were not different from those of untreated SCI rats. Implantation of TCs of different lengths in sham control rats did not affect CREM significantly (Figure 4B).
Cellular Distribution of CREM
In sham control rats, CREM immunostaining was localized primarily in maturing pachytene spermatocytes and young spermatids; presence of CREM in spermatogonia and prepachytene spermatocytes was rare (Figure 5A). In untreated SCI rats, CREM was also detected in young spermatids, but the intensity of CREM immunostaining was less pronounced (Figure 5B). Implantation of 1-cm TCs resulted in a further decrease in CREM staining in spermatids (not shown). Although CREM was also detected in some young spermatids in those SCI rats receiving 3-cm TC implants, it appeared primarily in spermatogonia and prepachytene spermatocytes in 3 of 5 of the rats in this dose group (Figure 5C and D). Of note, in spite of persistence of complete spermatogenesis, CREM was not detected in young spermatids in those SCI rats given 5-cm TC implants (Figure 5E). In 5 of the 6 rats in this group, CREM was present in spermatogonia and primary spermatocytes, including preleptotene spermatocytes, in most of the tubules (Figure 5F). These results were confirmed 2-3 times with slides from each animal immunostained at different dates. CREM reappeared in young spermatids in most of the tubules of SCI rats given 10- or 20-cm TC implants, regardless of the status of spermatogenesis (Figure 5G). However, young spermatocytes in many of the tubules, specifically those with partially regressed epithelia, also contained immunoactive CREM. Immunostaining of CREM in the testes of sham control rats given different lengths of TC implants in the follow-up experiment revealed normal cellular distribution of CREM (Figure 5H), despite variations in staining intensity. We also retrospectively examined CREM cellular distribution in archived testicular tissues fixed in Bouin solution of intact and hypophysectomized rats given various lengths of TC implants from previous studies (Huang and Boccabella, 1988; Huang et al, 1991). CREM was distributed normally in maturing spermatocytes and young spermatids in all tissues, regardless of the status of spermatogenesis and variations in the intensity of immunostaining (not shown). Specificity was demonstrated by the absence of CREM immunostaining when the sections were stained with antigen preadsorbed antiserum (Figure 5I).
Discussion
Our recent report demonstrating preservation of spermatogenesis in SCI rats by exogenous T (Huang et al, 1999) suggests the feasibility of using androgen or related therapeutic means to preserve spermatogenic function and perhaps fertility in SCI men. In order to validate such regimens for clinical use, optimizing the effective doses and understanding the mechanisms underlying the beneficial effects of exogenous T on spermatogenesis after SCI are essential.
The effects of increasing doses of exogenous T on serum LH, FSH, and testicular T and biphasic changes in spermatogenesis in SCI rats were consistent with those seen in intact rats (Zirkin et al, 1989; Santulli et al, 1990; Huang et al, 1991). However, dose effects of T on spermatogenesis seen in SCI rats were different from those seen in intact rats. Specifically, although implantation of 1-cm TCs resulted in a 40% reduction of testis weight and incomplete spermatogenesis in non-SCI intact rats (Huang and Boccabella, 1988), identical treatment maintained testis weight and qualitatively complete spermatogenesis in SCI rats. Total regression of the seminiferous epithelium in SCI rats that received 2-cm TC implants, characterized by the absence of proliferating spermatogonia in many tubular cross sections, also differed from their sham control counterpart. These changes resembled those that occurred in chronic SCI rats (Huang et al, 1995) and after testicular denervation (Chow et al, 2000) and were far more severe than that occurred in chronic hypophysectomized rats (Huang et al, 1987) and in SCI rats that received 3-cm TC implants and had nearly identical testicular T levels. The putative neurogenic mechanisms contributing to the loss of proliferating spermatogonia after SCI and testicular denervation must have been exaggerated specifically by the 2-cm TC regimen. Although such an effect was overcome by higher doses of exogenous T (3-20-cm TC implants), failure to maintain complete spermatogenesis in SCI rats given 3-cm TC implants also differed from their sham control rat counterparts. Such differences cannot be ascribed to hormone status, suggesting that local factors might be involved in neural-endocrine interactions in the control of spermatogenesis. Maintenance of qualitatively complete spermatogenesis and relatively normal sperm motility in SCI rats that received 1 cm-TC implants nevertheless suggests potential applicability of specific low doses of exogenous T in preservation of spermatogenesis and sperm function in SCI men.
A decrease in sperm motility in untreated SCI rats was consistent with that in SCI men. Such effects could be attributed to abnormal sperm transport, maturation in the epididymis, or both (Linsenmeyer et al, 1999) as a result of impaired epididymal function after denervation (Billups et al, 1990). Although sperm motility in SCI rats was not affected by the 1-cm TC implant, it was totally eliminated in those rats that received 5-cm TC implants but rebounded slightly in those that received 10- or 20-cm TC implants. Because epididymal function of these SCI rats would have been better stimulated than in those that received 1-cm TC implants because of the elevated serum T level, the loss and rebound of sperm motility in SCI rats that received 5-20-cm TC implants might reflect functional status of the sperm motile apparatus. In addition, because sperm motility also was decreased in the sham control rats that received 5- or 10-cm TC implants, a difference in the metabolic states of sperm secondary to changes in epididymal function under different serum T conditions might also contribute to differences in sperm motility.
Testosterone modulates spermatogenesis through its effects on Sertoli cells. These effects were initiated by the binding of T to, and activation of, androgen receptors (AR) that transactivate androgen-dependent genes to induce changes in Sertoli cell functions (Zhou et al, 1994). Such effects thus mediate the effects of androgen on spermatogenesis. However, presence of AR and androgen-binding protein (ABP) in spermatogenic cells (Vornberger et al, 1994; Joseph et al, 1997), stimulation of protein synthesis in spermatocytes by the T/ABP complex (Gerard, 1995), and presence of a hormone-responsive element (HRE) in the promoter of genes that encode germ cell-specific proteins (Bonny et al, 1998; Ha et al, 1997) suggest that T might also affect spermatogenic cells directly. In addition, recent studies have demonstrated that cAMP signaling might also mediate some effect of androgen, especially in those cells lacking functional AR (Heinline and Chang, 2002).
CREM is a nuclear transcription factor that modulates the function of cAMP-responsive genes (Foulkes et al, 1991; Habener et al, 1996). Alternative gene splicing results in different CREM isoforms that either activate or repress the function of cAMP (Foulkes et al, 1991). In prepubertal animals, CREM repressor isoforms (CREM and ?) were detected in spermatogonia, early meiotic cells, or both (Foulkes et al, 1992). In adult testes, the activator isoform (CREM) was detected mainly in maturing spermatocytes and young spermatid and was a functional switch for postmeiotic germ cell differentiation (Foulkes et al, 1992; Sassone-Corsi, 1998). Modulation of the expression of CREM by FSH and T (Foulkes et al, 1993; West et al, 1994) suggests its role in mediating the effects of hormones on spermatogenesis. Recently, we reported changes in the short-term effects of FSH and T on testicular CREM transcripts and transcripts for Sertoli and germ cell-specific proteins encoded by cAMP-responsive genes in SCI rats (Huang et al, 2003a). Current observation of abnormal cellular localization of CREM in SCI rats that received 3- and 5-cm TC implants further illustrated changes in the timing of CREM translation during spermatogenic differentiation. Decreased localization of CREM in spermatids in these SCI rats was consistent with a reduction of CREM protein in their testicular tissues. Because such effects were only seen in SCI rats that received 3- or 5-cm TC implants, and not in their sham control counterparts, they were most likely related to changes in cellular function attributable to a specific neural-endocrine interaction. These findings were consistent with changes in hormonal regulations of cAMP signaling in testicular cells after SCI. Precocious expression of CREM in spermatogonia and young spermatocytes has also been observed in acute SCI rats following cord contusion (Huang et al, 2003b). Such changes could disturb cAMP signaling in spermatogenic cells; impede normal spermatogenesis; and result in reduced sperm count or production of sperm with abnormal functions or both. Persistence of CREM in spermatids in SCI rats given 1-cm TCs and its reappearance in spermatids in those rats that were given 10- and 20-cm TC implants and had qualitatively complete spermatogenesis support our postulate. Further studies with reverse transcription PCR techniques (Peri et al, 1998) to characterize properties of CREM in isolated spermatogenic cells of SCI rats could provide new insights into the functional roles of different CREM isoforms in mediating the neural-endocrine interaction in the control of spermatogenesis. In this regard, premature expression of Pm-1 has been linked to spermatogenic arrest (Lee et al, 1995), and reduced CREM expression has been found in human testes with spermatid maturation arrest (Peri et al, 1998; Steger et al, 1999).
The results of this study demonstrate changes in the effects of exogenous T on spermatogenesis in the rat after SCI. Preservation of spermatogenesis and sperm motility in SCI rats that received 1-cm TC implants suggests potential efficacy of low doses of exogenous T in the maintenance of sperm production and function in SCI men. These effects were associated with changes in the expression of testicular CREM and its distribution in spermatogenic cells. These results support the notion that altered cAMP signaling and its regulation in testicular cells might be mediating the effects of SCI on spermatogenesis. Such changes might affect timely and sequential progression of spermatogenesis and might contribute to the production of sperm with abnormal morphology and function after SCI.
Acknowledgments
We thank Norihiro Chinen for performing Western blotting of CREM, William Giglio for hormone assays, and Robert Anesetti for animal care.
Footnotes
Supported by V.A. Rehabilitation R&D Services (B885-2RA).
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