UNLABELLED: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.

Our previous studies demonstrated various abnormalities in spermatogenesis after spinal cord injury (SCI) in cord-transected rats. In this study, we examined whether abnormalities in spermatogenesis in SCI rats were related to the degree of SCI. We used spinal cord-contused (SCC) rats as a model. Adult male Sprague-Dawley rats were subjected to various degrees of cord contusion caused by the weight of a rod dropped from different heights (12.5, 25, 50, and 75 mm) using a New York University IMPACTOR. Testicular histology revealed persistent complete spermatogenesis in all SCC rats 4, 8, or 14 weeks after cord contusion regardless of the extent of SCI. Northern blot complementary DNA (cDNA) hybridization revealed transient but significant decreases in the levels of Sertoli cell-specific transcripts in SCC rats. In addition, levels of messenger RNA (mRNA) transcripts for germ cell-specific transition protein-2 and protamine-1 were consistently decreased in these rats. Such effects were related to the height of the weight drop and were associated with reduced levels of mRNA for cyclic adenosine monophosphate (cAMP) responsive element modulator (CREM). These results demonstrated specific effects of SCI on spermiogenesis and were consistent with altered cAMP signaling in testicular cells after SCI. Sperm motility was also significantly decreased in SCC rats and was related to the height of weight drop. Normal sperm motility recovered only in those rats injured by weight drop from 12.5- and 25-mm heights. In summary, current results demonstrate persistent abnormalities in spermiogenesis and sperm motility in rats that suffered spinal cord contusion by weight drop. Such effects were related to the height of the weight drop and thus to the extent of SCI.

INTRODUCTION:Earlier studies demonstrated that the effects of spinal cord injury (SCI) on spermatogenesis were associated with altered Sertoli cell responses to treatment with follicle-stimulating hormone (FSH) and/or testosterone (T). Because of the importance of the cyclic adenosine 3',5'-monophosphate (cAMP) signal pathway in hormonal actions on Sertoli cells and spermatogenesis, the purpose of this study was to determine whether cAMP signaling in testicular cells is altered after SCI.METHODS:Rats with SCI were treated with FSH, T, or FSH + T for 7 or 14 days. Northern blot cDNA hybridization was used to measure testicular levels of Sertoli and germ cell-specific transcripts encoded by genes that contain cAMP responsive element (CRE) and/or steroid hormone responsive element (HRE). Cellular distribution of CRE modulator (CREM) was determined by immunohistochemistry.RESULTS:Treatment of sham control rats with FSH or T + FSH for 2 weeks resulted in decreases in mRNAs for CREM and CRE binding protein (CREB). Concomitantly, levels of mRNA for Sertoli cell inhibin alpha and germ cell-specific protamine 1 (Pm-1), transition protein 2 (TP-2), and lactate dehydrogenase C (LDHC) were all reduced. In contrast, identical FSH and/or T treatments resulted in increases in levels of CREM and CREB mRNAs in the testes of SCI rats; these effects were associated with similar changes in mRNAs for inhibin alpha, Pm-1, TP-2, and LDHC. The effects of SCI on CREM expression were corroborated by similar changes in its distribution in testicular cells.CONCLUSION:SCI is associated with changes in FSH and/or T regulation of cAMP/CRE and HRE signaling in testicular cells. These effects may mediate the effects of SCI on spermatogenesis.

The detrimental effects of spinal cord injury (SCI) on spermatogenesis in the rat can be attenuated by exogenous testosterone (T) but enhanced by exogenous follicle-stimulating hormone (FSH). These results suggest that T-dependent cellular events may be involved in testicular injury after SCI and that such events may be associated with modification of FSH effects on Sertoli cell function. The current study compared the responses of Sertoli cells to exogenous T and FSH after SCI or sham surgery using steady-state levels of Sertoli cell protein mRNA transcripts as markers of responsiveness. Rats underwent sham surgery or SCI and then were treated for 7 or 14 days with T-filled silastic capsules (2 x 5 cm) and/or daily injections of 0.1 units of porcine FSH. Vehicle-treated control rats received 5-cm empty capsules and daily injections of saline vehicle. Two weeks after sham surgery, levels of mRNA for the androgen receptor (AR), FSH receptor (FSHR), androgen-binding protein (ABP), or sulfated glycoprotein (SGP)-2 in the testis were unaffected by T or FSH alone. Testosterone alone, however, significantly decreased transferrin (Trf) mRNA levels in the testis (P: < 0.01). The combination of T and FSH treatments resulted in significant decreases in levels of the above transcripts (P: < 0.05; P: < 0.01). Seven days after SCI, the testes of vehicle-treated SCI rats had higher levels of AR and SGP-2 mRNA than did those of sham control rats (P: < 0.01); such effects were transient and disappeared by Day 14 post-SCI. Testosterone treatment of SCI rats for 7 days resulted in decreases in mRNA levels for AR and Trf in the testes (P: < 0.01) but increased testicular levels of mRNAs for FSHR and SGP-2 in SCI rats. Follicle-stimulating hormone treatment for 7 days prevented the increase in AR mRNA that was seen in the testis of untreated SCI rats and increased levels of ABP and SGP-2 mRNAs in SCI rats (P: < 0.01). Follicle-stimulating hormone treatment of SCI rats did not affect FSHR mRNA levels by itself, but it blocked the stimulatory effect of T on FSHR and SGP-2 mRNAs. Fourteen days after SCI, testicular AR mRNA levels were not affected by T alone, but they increased in those rats that received FSH with or without concurrent T treatments (P: < 0.05). In contrast to their effects in sham control rats, T or FSH alone or in combination resulted in significant increases in testicular levels of ABP, SGP-2, and FSHR mRNAs (P: < 0.05). At this time, Trf mRNA in the testis of SCI rats was also suppressed by T (P: < 0.05), as it did in sham control rats, but Trf mRNA was increased by the FSH (P: < 0.01) that had inhibited this transcript in the testes of sham control rats. The effects of FSH on the Sertoli cell transcripts in SCI rats were either attenuated or blocked when T was given concurrently. In addition, testicular and serum T levels in those SCI rats that received FSH (alone or in combination with T) for 14 days were significantly increased, an effect that was not seen after sham surgery. These findings demonstrate that hormonal regulation of both Sertoli and Leydig cells was altered during the acute phase of SCI. Such changes may modify the functions of both cell types, thereby affecting the endocrine and/or paracrine microenvironment within the seminiferous epithelium. These effects could impair the functional capacity of Sertoli cells and contribute to impairment of spermatogenesis after SCI.

In the rat, regression of spermatogenesis during the chronic stages of spinal cord injury (SCI) occurs in the presence of normal function of the pituitary-testis hormone axis, thus suggesting that nonendocrine mechanisms might be involved. The current study examined whether disruption of neural input to the testis contributes to the cascade that leads to the regression of spermatogenesis. Four weeks after denervation of the superior spermatic nerve (SSN), testis weight was 25% lower (p < 0.01) than that of the contralateral sham-operated testis. Defects in spermatogenesis including phagocytosis of mature spermatids, vacuolization of spermatid nuclei, delayed spermiation and incomplete cellular associations were observed in >60% of the tubules. In the remaining 30–40% of tubules, the seminiferous epithelium was severely regressed. While cutting the inferior spermatic nerve (ISN) alone did not affect spermatogenesis significantly, it enhanced the effect of SSN denervation on both spermatogenesis and testis weight (p < 0.01). Spermatogenesis was totally regressed in the SSN/ISN-denervated testes. At this time, quantitatively normal spermatogonial proliferation was maintained in SSN- or ISN-denervated testes. Twelve weeks after surgery, regression of the seminiferous epithelium characterized by absence of proliferating spermatogonia, while undifferentiating spermatogonia were present, was observed in all SSN-denervated testes. At this time, regression of the seminiferous epithelia also occurred in >30% of the tubules in ISN-denervated testes. At both times, serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were normal and >60% of normal testicular testosterone concentrations were maintained in the denervated testes. These results indicate that disruption of neural input to the testis is not a cause for the decrease in spermatogonial proliferation during the acute phase of SCI, but may contribute to the chronic effects of SCI on spermatogenesis.

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.

Causes of poor semen quality following spinal cord injury (SCI) are not known. One possible reason, based upon studies that reported improved semen quality in SCI men after several induced ejaculations, is delayed epididymal sperm transport. Our study was designed to establish baseline epididymal sperm transport values in the Sprague Dawley rat and evaluate effects of SCI on this process. Spermatozoa protamine was labeled with tritiated arginine, and the rats were sacrificed various times after injection. Each epididymis was divided into six equal sections from proximal to distal. Sperm tails were dissolved with 8 molar (M) urea in the presence of 2 mM dithiothreitol (DTT); sperm heads were collected by centrifugation (3,000 rpms, 10 min.). The radioactivity in sperm heads from each section was counted and expressed as counts per million sperm heads. To account for different rates of labeled arginine incorporation, the percentage of counts per million sperm heads in each section was calculated relative to the total number of counts in all six sections. Our results showed there was an orderly progression of sperm through the epididymis. It took 8 days for labeled sperm to enter the epididymis and 28 days to peak in the caudal (tail) section in non-SCI rats. Stasis was present 10 days after T-9 SCI in rats compared with transport in sham controls. This was evidenced by a significant increase in the percentage of labeled sperm in proximal sections of the epididymis (sections 1, 2, and 4) in T-9 transected animals (p < 0.01). If similar stasis occurs in SCI men, it could obviously contribute to poor semen quality. However, it remains to be determined how long this stasis persists after SCI in rats.

Our previous studies have demonstrated that impaired spermatogenesis during the acute phase of spinal cord injury (SCI) is preceded by a transient (but significant) suppression of serum FSH, LH, and testosterone (T) concentrations. It is hypothesized that hormonal deprivation may impair Sertoli cell function, leading to the loss of spermatogonia, degeneration of spermatogenic cells, and eventual regression of the seminiferous epithelium. The current study examined the efficacy of exogenous T and FSH in the maintenance of spermatogenesis and Sertoli cell functions in SCI rats. Implantation of T capsules (TC, 2 x 5 cm) attenuated some of the spermatogenic lesions and maintained qualitatively complete spermatogenesis in all SCI rats 4 weeks after the surgery. In contrast, daily injections of 0.1 U of FSH alone, or in combination with TC implants, paradoxically enhanced the regression of spermatogenesis in SCI rats. At this time, the numbers of Aal, A1, and B spermatogonia and preleptotene spermatocytes in SCI rats have decreased by 25-30%. Though not prevented by TC implants, the decrease in Aal and A1 spermatogonia was attenuated by FSH alone but was further enhanced when FSH-treated rats also received TC implants. The intratesticular T concentration in untreated and FSH-treated SCI rats was not different from that of sham control rats, but it decreased by more than 95% in those SCI rats given TC implants alone. These results demonstrate that impairment of spermatogenesis during the acute phase of SCI is not related to the availability of FSH and/or T. Northern blot analysis revealed an increase in androgen receptor messenger RNA (mRNA) in the testis of SCI rats; this increase was prevented by TC implants but persisted when FSH was also given. In contrast, the levels of FSH-receptor, androgen binding protein, and transferrin mRNA were not affected by SCI but were significantly higher in those SCI rats given FSH alone or in combination with TC. TC implants alone suppressed mRNA levels of transferrin in testes of SCI rats, without concomitant change in those for FSH-receptor and ABP. The changes in Sertoli cell responses to FSH and T, and perhaps other hormones, may alter signal events elicited by these hormones, thus contributing to abnormal epithelial environments and regression of spermatogenesis. Maintenance of spermatogenesis in SCI rats by exogenous T suggests the feasibility of using exogenous hormones to impede the detrimental effects of SCI on spermatogenesis. This approach may have clinical applicability for the preservation of spermatogenic functions in SCI men.

Recently, we reported that changes in spermatogenesis in adult rats during acute phase (within 2 weeks) of spinal cord injury (SCI) were associated with a suppression of pituitary-testis hormone axis, and these effects mimic those that occur after hormone deprivation. In this study, we examined the long-term (>4 weeks) effects of SCI on spermatogenesis and its recovery. Results of this study reveal that while serum follicle stimulating hormone, luteinizing hormone, and testosterone levels in SCI rats recovered within 1 month after the injury, their spermatogenesis continued to regress. By 3 months, spermatogenesis in 70% of SCI rats has totally regressed, characterized by the absence of proliferating spermatogonia; these effects could not be prevented by an otherwise effective regimen of testosterone treatment. Sertoli cells in the regressed seminiferous tubules exhibited unusual behavior, characterized by the formation of multiple cell layers and/or aggregates that extended into the tubular lumen. Active spermatogenesis was observed in nine of the 19 SCI rats by 6 months, seven of which had complete spermatogenesis, but with persisting abnormalities. These results demonstrate that SCI results in total, but reversible, regression of spermatogenesis. Failure to prevent such effects by an otherwise effective exogenous testosterone regimen suggests that non-endocrine factors are involved in the SCI effects on spermatogenesis. The unusual Sertoli cell localization in the regressed testes may have been triggered by the loss of proliferating spermatogonia and may be involved in subsequent spermatogenic recovery.

The prostate is one of the male accessory sex glands that produce fluid components of the seminal plasma. In addition to androgen, a normal innervation of the prostate is believed to be important for maintaining normal function of the prostate. Previously we noted that, in the rat, the weight of the prostate decreased following surgically induced spinal cord injury (SCI). This observation suggests that growth, and possibly function, of the prostate may be compromised after SCI. To explore this possibility, we examined the effects of SCI on the androgen-related biochemical properties and morphology of the prostate in the rat at various times after surgically induced SCI. SCI resulted in an acute decrease in prostate weight and an increase in steady state level of mRNA for testosterone-repressed prostate message 2 (TRPM 2) during the first 2 weeks postinjury. These changes perhaps relate to an increase in cell death or a decrease in secretory activity due to an acute suppression of serum testosterone after the injury. Concomitantly, there was a transient, but significant, decrease in the steady state level of androgen receptor (AR) mRNA in the prostate during the first 2 weeks after SCI, an indication of an altered autoregulation of AR by its own ligand. Despite the fact that growth of the prostate, as indicated by weight increase, in SCI rats resumed 2 weeks postinjury, prostate weights were persistently lower in SCI rats than sham-operated controls for at least 3 months. Furthermore, prostate TRPM 2 mRNA levels remained elevated throughout the recovery period even after a normal prostate weight had been restored. In addition, a decrease in the height of ventral prostate epithelial cells was noted in SCI rats 28 and 90 days postinjury. These results demonstrate a prolonged effect of SCI on prostate function. These findings and our unreported observation of persistently smaller seminal vesicles in the same groups of SCI rats suggest that functions of male accessory sex glands may also be compromised after SCI. These changes may affect biochemical properties of the secretory products of these glands and may provide some explanation for the reported changes in the composition of the seminal plasma and abnormal sperm motility seen in the semen of SCI men.

The present study investigated the time course of the onset of the abnormalities in spermatogenesis following spinal cord injury, and their relationship to changes in the pituitary testicular hormonal axis and Sertoli cell function. Spinal cord injury (SCI) was induced in adult male rats by surgical transection of the spinal cord at the level of T9 and L1 vertebrae. Animals were killed 3, 7, and 14 days after the operation. As early as 3 days following SCI, abnormalities in spermatogenesis, including delayed spermiation and vacuolization of the nucleus of spermatids, were noted in both the T9 and L1 animals. By 14 days, other lesions, including phagocytosis of mature spermatids, incomplete cellular associations, and total regression of seminiferous epithelium, became apparent. Concurrently a transient but significant (P < 0.05) suppression of serum follicle-stimulating hormone (FSH) occurred in the T9 animals, and a suppression of serum luteinizing hormone (LH) occurred in both the T9 and the L1 animals 3 days after the surgery. This was accompanied by a suppression of testicular and serum testosterone levels (P < 0.05, P < 0.01, respectively). Most of the hormonal parameters had recovered and were not different from those of sham-operated animals by 14 days (P > 0.10). Northern blot analysis of testicular poly(A)+ RNA revealed a transient but significant reduction in the steady-state level of the 2.7-kilobase (kb) Sertoli cell transferrin mRNA transcript in both the T9 and the L1 animals 3 days after the operation (P < 0.05). On the other hand, the 1.7-kb androgen binding protein (ABP) mRNA remained unaffected during the 2-week study period. The steady-state level of mRNA transcripts for spermatogenic cell-specific hemiferrin and spermatid specific transition protein 2 and protamine 1 also remained unchanged. These results suggest that spinal cord injury will result in a temporary, but profound, effect on the pituitary-testicular hormone axis. These changes may impair certain aspects of Sertoli cell function that could render these cells incapable of supporting normal spermatogenesis. However, the severity of spermatogenic lesions and the disparate responses of the two major Sertoli cell proteins make it unlikely that hormone deficiency is the only mechanism responsible for the impaired spermatogenesis following spinal cord injury.

Male infertility frequently occurs after spinal cord injury (SCI). However, little is known about the acute effects of SCI on male reproductive function. This study evaluated the effects of SCI on spermatogenesis and testicular-pituitary function in rats 2 and 4 weeks after injury. Spinal cord injury was produced in rats by T9 spinal cord transection. Controls received similar surgery without transection. Complete spermatogenesis was seen 2 weeks after SCI; however, abnormalities were present in the seminiferous tubules. Hormone levels were similar in the two groups. Four weeks after SCI, incomplete spermatogenesis was noted in 3 of 9 rats, 4 others had delayed spermiation, and the last 2 had nonspecific regression of seminiferous epithelium. Serum testosterone levels were lower at 4 weeks in SCI rats than in controls, but testicular testosterone content was not. Plasma gonadotropin levels were similar in the two groups 4 weeks after SCI. Quantitative analysis revealed a 26 to 33% decrease in the number of spermatogenic cells in stage VII seminiferous tubules at 4 weeks in SCI rats (p < 0.01). This study demonstrated that qualitative and quantitative impairments of spermatogenesis occur during the acute phase of SCI in rats.