Haikun Li

Effects of Long-Term Cocaine Exposure on Spermatogenesis and Fertility in Peripubertal Male Rats

PURPOSE This study was conducted to investigate the effects of long-term administration of cocaine on spermatogenesis and fertility in adult male rats. MATERIALS AND METHODS Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg./kg. body weight, corresponding to an average single dose for a heavy cocaine user) either daily or twice weekly (weekend group, cocaine given on Saturday and Sunday) and mated with pregnancy-proven female rats after 100 and 150 days of exposure to the drug. Pregnancy rates and litter birth weights were evaluated. Serum testosterone, follicle stimulating hormone and luteinizing hormone levels were measured in all adult rats. Morphologic analysis of the testis entailed the evaluation of quantitative and qualitative histologic parameters to assess the effect of cocaine on various stages of spermatogenesis. RESULTS After 100 days of treatment, the rats receiving daily cocaine had a pregnancy rate of only 33% versus 86% for the controls (p < 0.05). In rats exposed to cocaine for 150 days the pregnancy rate was 50% compared with 100% for controls (p < 0.05). The birth weights of offspring from the group receiving daily cocaine was 10% less than that of controls (p < 0.05). The weight of the testis and epididymis was not affected by cocaine exposure. Morphometric analysis showed significant differences between the cocaine-treated groups (both the daily cocaine and twice weekly cocaine groups) and their respective controls. The mean diameter of seminiferous tubules in the daily and twice weekly cocaine groups was reduced when compared with their respective controls. These differences between treated groups and their controls were statistically significant (p < 0.05). Similarly the thickness of the germinal epithelium was less in the cocaine-treated groups than in the controls (p < 0.05). Degenerating cells were more numerous in both daily and twice weekly cocaine groups than the controls. Furthermore, the number of step VII spermatids was reduced in both daily and twice weekly cocaine groups, a difference that was statistically significant (p < 0.05). CONCLUSION Our findings demonstrate that chronic administration of cocaine to peripubertal male rats has a profound effect on their testicular function. Even with twice weekly administration there was a significant adverse effect on spermatogenesis although this was not manifested by diminished fertility in this group. These findings confirm that chronic administration of cocaine to male rats can have a deleterious effect on spermatogenesis and fertility.

Long-Term Mechanical Reliability of Multicomponent Inflatable Penile Prosthesis: Comparison of Device Survival

OBJECTIVES To determine the mechanical reliability of multicomponent inflatable penile prosthesis, comparing five different types of devices, as well as the two-piece versus three-piece as a group. METHODS We followed 83 patients with two-piece and 283 patients with three-piece inflatable penile prostheses for a mean time of 66 months. At a cutoff of 63 months, mechanical complication rates were reviewed and statistically analyzed. RESULTS Thirty-one device-related complications occurred, and all were secondary to fluid leakage. The Mentor Alpha-1 prosthesis was significantly better than the Mentor Mark-II in terms of mechanical reliability (P = 0.01). A trend was noted toward the AMS 700 Ultrex inflatable penile prosthesis having fewer mechanical complications than the Mentor Mark-II (P = 0.06). In addition, a trend toward all three-piece prostheses being more mechanically reliable than the two-piece was noted (P = 0.08). The Mentor Alpha-1 device had a higher cumulative proportional survival (0.957) than all other devices (0.842 for AMS 700 Ultrex, 0.839 for AMS 700 CX, 0.783 for Mentor GFS, and 0.750 for Mentor Mark-II). CONCLUSIONS As a group, a trend was noted toward the three-piece prosthesis having better mechanical reliability than the two-piece prosthesis. Comparisons between the individual types of prostheses showed thatthe Mentor Alpha-1 device was significantly more mechanically reliable than the Mentor Mark-II device, and a trend was noted toward the AMS 700 Ultrex device having fewer mechanical complications than the Mentor Mark-II. The Mentor Alpha-1 prosthesis had the highest cumulative proportional survival.

EFFECT OF SURGICALLY INDUCED VARICOCELE ON TESTICULAR BLOOD FLOW AND SERTOLI CELL FUNCTION

Abstract Objectives. To evaluate the effect of varicocele on testicular blood flow and expression by Sertoli cells of transferrin and androgen-binding protein (ABP), to determine whether varicoceles impair Sertoli cell function. Methods. Experimental varicocele was established in male Sprague-Dawley rats by partial ligation of the left renal vein. The control group received a sham operation. At 30 minutes after surgery, rats underwent a xenon-133 washout study, and at 30 days after surgery, transferrin, ABP, and testicular blood flow were evaluated. Expression of transferrin and ABP were evaluated using immunohistochemical techniques. Testicular blood flow was measured using xenon-133 clearance techniques. Statistical analyses were done with an independent t test. Results. The testicular blood flow was 16.7 ± 1.25 mL/100 g/min in varicocele-bearing rats and 21.01 ± 0.46 mL/100 g/min in sham-operated rats 30 minutes after surgery. Testicular blood flow remained decreased at 30 days in varicocele-bearing rats (15.12 ± 1.08 mL/100 g/min) and remained stable in the control group (19.45 ± 0.55 mL/100 g/min). The expression of transferrin and ABP was significantly reduced in varicocele-bearing rats compared with the control group. Conclusions. Our study suggests that a decrease in testicular blood flow may lead to impaired Sertoli cell function in varicocele-bearing rats.

COCAINE INDUCED APOPTOSIS IN RAT TESTES

PURPOSE Exposure of rats to chronic cocaine results in disruption of spermatogenesis including reduction of germ cells. However, the cellular mechanism responsible for the testicular damage in testes is still unknown. We have studied the role of apoptosis in cocaine induced testicular damage. MATERIALS AND METHODS Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg./kg. body weight) subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed at 15, 30, 60, and 90 days of cocaine administration. In situ detection of germ cells with DNA strand breaks in paraffin-embedded testicular section (5 microm.) was achieved by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end-labeling (TUNEL) method. DNA fragmentation was also determined by gel electrophoresis. RESULTS Apoptotic cells were found in the spermatocytes and spermatogonia of germinal epithelium. Less than 7% of seminiferous tubule cross sections showed a high level of apoptosis (> or =3 apoptotic cells per tubule) in control animals compared with experimental group where 25% of the tubules showed a high level of apoptosis (p<0.05). The number of apoptotic cells was significantly increased by 15 days, peaked at 30 days and persisted up to 90 days of cocaine exposure when compared with controls (p<0.05). DNA isolated from the cocaine treated testes displayed a clear ladder pattern whereas the DNA from controls did not. CONCLUSIONS The experimental results presented here suggest that cocaine exposure leads to significant apoptosis in rat testes and the mechanism of cocaine induced testicular injury may be related to the induction of apoptosis.

Assessment of psychosexual adjustment after insertion of inflatable penile prosthesis

OBJECTIVES To evaluate the psychosexual benefit obtained from multicomponent penile implant surgery in patients with erectile dysfunction. METHODS A psychosexual questionnaire was given to 35 patients undergoing penile prosthesis implantation before surgery and at 3 months, 6 months, and 1 year after surgery. The questionnaire consisted of 13 questions scored on a scale from 1 through 5. Results of the questionnaire were statistically analyzed for differences among the preoperative, 3-month postoperative, 6-month postoperative, and 1-year postoperative period. RESULTS The general linear model evaluation showed a significant difference for each overall combination of the following pairs: preoperative versus 3 months postoperative (P=0.0005) and 3 months postoperative versus 6 months postoperative (P=0.002). There was no overall difference between psychosexual total score at 6 months after surgery and 1 year after prosthesis implantation (P=0.85). The patients perceived improvement in their erectile ability and libido. Concern about obtaining and maintaining an erection during intercourse was significantly alleviated. There was an increase in the frequency of sexual activity and an improvement in satisfaction with sex life. A decrease in feelings of sadness, depression, anxiety, anger, frustration, and embarrassment related to sexual activity was also noted. CONCLUSIONS The current study demonstrates significant improvement in the psychosexual well being of multicomponent penile implant recipients, with attainment of a high level of patient satisfaction up to 1 year after surgery.

ANTIBACTERIAL ACTIVITY OF ANTIBIOTIC COATED SILICONE GRAFTS

PURPOSE Postoperative infection remains one of the most serious complications of implantation of penile prostheses. Attempts to reduce the rate of infection by spraying the prosthesis with an antibiotic solution prior to implantation, along with perioperative antibiotics, have failed to eradicate infection. No published studies have evaluated the effect of antibiotic coating of penile prostheses. In this study, we evaluate the antibacterial effect of antibiotic-coated silicone strips as a surrogate for the penile prosthesis. MATERIALS AND METHODS Strips coated with several different antibiotics were dipped in bacterial solutions containing Staphylococcus epidermidis or S. aureus and implanted subcutaneously in adult Sprague-Dawley rats. After a week, the strips were removed, and the number of bacteria on the strips and in the surrounding tissue were determined. The in vitro antibiotic activity of the antibiotic-coated strips against the same organisms was also determined. RESULTS In the group of rats that received silicone strips contaminated in vitro with S. epidermidis, six of nine control rats yielded strips and tissues containing heavy bacterial growth. None of six strips coated with rifampin/minocycline yielded bacterial growth, nor did any of the seven strips coated with vancomycin. One of seven rats that received amikacin-coated strips had infection of the strip. The tissue results were similar to the strip results. In the group using S. aureus as the contaminating bacterium, the strips and tissues from eight of nine control rats yielded bacteria. None of the six rifampin/minocycline-coated strips yielded bacteria, while two of seven vancomycin-treated strips and two of six amikacin-coated strips were infected with S. aureus. The difference in bacterial growth between controls and antibiotic-coated strips reached a level of statistical significance for the rifampin/minocycline and vancomycin groups and was highly significant for the rifampin/minocycline groups. CONCLUSION The experimental results presented here suggest that coating silicone graft material with antibiotics, particularly rifampin/minocycline, can reduce the incidence of graft colonization in contaminated wounds in rats, even in the absence of systemic antibiotics. These graft materials may prove useful in preventing the infection of penile prostheses.

Role of mitochondrial cytochrome c in cocaine-induced apoptosis in rat testes.

OBJECTIVES We have previously demonstrated that cocaine exposure leads to apoptosis in rat testes. To understand further the mechanism of cocaine-induced testicular damage, we studied the effect of cocaine on cytochrome c release from the mitochondria. We also determined the caspase 3, caspase 8, and caspase 9 activities in rat testes after chronic cocaine exposure. METHODS Thirty-day-old male Sprague-Dawley rats received cocaine hydrochloride or equal volumes of normal saline subcutaneously daily for 90 days. The testes were removed at 15, 30, and 90 days of cocaine or saline administration. Mitochondria and cytosolic fractions from testes were isolated. Western blotting was performed in both fractions using anti-cytochrome c antibody. Caspase 3, caspase 8, and caspase 9 activities were determined by fluorometric assay. RESULTS The expression of cytochrome c protein in the cytosolic fraction was increased on day 15 and persisted for up to 90 days after cocaine injection compared with controls. However, the expression of cytochrome c in testes was decreased in the mitochondria fraction on days 15, 30, and 90 after cocaine injections compared with the corresponding controls. The caspase activity study showed caspase 3 and caspase 9 activities increased in cocaine-treated testes at each point of the study compared with the corresponding controls. However, the caspase 8 activity in cocaine-treated testes did not change significantly at each point of the study compared with the corresponding controls. CONCLUSIONS Our results suggest that the release of cytochrome c from mitochondria and its subsequent activation of caspase 9 and caspase 3 in testes play a key role in cocaine-induced germ cell apoptosis. Our findings also indicate that cocaine-induced testicular germ cell apoptosis in rats is at least initiated through a mitochondria-associated pathway.

Characterization of Cocaine Binding Sites in the Rat Testes

PURPOSE An estimated 29 million individuals use cocaine in the United States. Studies have shown a high affinity for dose dependent binding of cocaine in the testes. Recent work done in our laboratory has shown that chronic administration of cocaine to male rats has an adverse effect on fertility and spermatogenesis by producing extensive morphological changes in the testes, leading to reduction in sperm production. As a first step toward understanding this process, we characterized and identified the pharmacological properties of [3H]cocaine binding sites in the testes. MATERIALS AND METHODS Crude membranes from the testes were prepared from 35 days old male Sprague-Dawley rats. [3H]cocaine binding was measured by using the method of Madras et al. (1989) with modifications. The data from saturation binding assays were analyzed by Inplot (GraphPad Software, San Diego, CA) to determine the Kd and Bmax. RESULTS Specific binding of [3H]cocaine was linearly dependent on membrane protein concentrations ranging from 0.2 to 8 mg./ml. The pooled data from three independent experiments revealed a mean affinity of 36 +/- 2.0 nM and Bmax of 1.84 +/- 0.13 pmol/mg. The present study demonstrates that testicular tissue has receptor protein that binds [3H]cocaine saturably and specifically. Competition displacement experiments revealed a shallow displacement curve for (-)cocaine and Win 35,428 with r2 = 0.96, indicative of multiple binding components. Computer analysis confirmed that a two component binding model was preferred statistically over a one component model in all three experiments (p < 0.001). CONCLUSION The results from these studies suggest that the testicular tissue contains a protein that binds [3H]cocaine in a saturable and specific manner. It has a different sensitivity from the [3H]cocaine binding protein in the brain and placenta. Further clarification of the relationship between cocaine and its recognition site is necessary to understand the mechanism of testicular damage after cocaine exposure.

Lipid peroxidation and antioxidant activities in rat testis after chronic cocaine administration

OBJECTIVES Our recent study has shown that cocaine has adverse action on spermatogenesis and fertility in male rats. Adverse effects of cocaine on the testes may be mediated by oxidative damage and subsequent lipid peroxidation. Glutathione is a cellular antioxidant and is found in high concentrations in the rat testes. In this study, the effects of chronic cocaine administration on the activities of glutathione peroxidase, the level of testicular reduced glutathione, and lipid peroxidation were investigated. METHODS Thirty-day-old male Sprague-Dawley rats were given cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed at 15, 30, 60, and 90 days after cocaine injection. Tissues were washed and homogenized in ice-cold metaphosphoric acid solution or Tris-HCI buffer. Reduced glutathione, glutathione peroxidase, and malonaldehyde levels were determined by colorimetric assay. Statistical analysis was performed using analysis of variance. RESULTS Testicular reduced glutathione and glutathione peroxidase were significantly decreased in the treatment testes 15, 30, 60, and 90 days after chronic cocaine injection compared with controls (P <0.05). The testicular malonaldehyde level was 20.8% (P <0.05), 22.1% (P <0.05), 31.2% (P <0.05), and 24.7% (P <0.05) above the control value on days 15, 30, 60, and 90, respectively. CONCLUSIONS Our findings demonstrate that chronic administration of cocaine to male rats induces a depletion of reduced glutathione and glutathione peroxidase. Adverse effects of cocaine on the testes are at least in part due to impairment of the function of the antioxidant defense and further enhancement of lipid peroxidation.

EXPRESSION OF cAMP RESPONSIVE ELEMENT MODULATOR (CREM) IN THE RAT TESTES FOLLOWING CHRONIC COCAINE ADMINISTRATION

OBJECTIVE c-AMP-responsive element modulator (CREM), one of the nuclear factors involved in the regulation of gene expression by cAMP, has an important role in spermatogenesis. Our recent study has shown that chronic administration of cocaine to male rats results in disruption of spermatogenesis, including reduction of germ cells. As a further step toward understanding this process, we have studied the role of CREM in cocaine-induced testicular damage. MATERIALS AND METHODS Sprague-Dawley rats were administered cocaine hydrochloride subcutaneously daily for 90 days. Control animals received equal volumes of normal saline daily for 90 days. Testes were removed after 15, 30, 90 days of cocaine administration. Total RNA was extracted from the testes and subjected to RT-PCR. Testicular tissue was also homogenized in a lysis buffer, and Western blotting was performed using anti-CREM antibody. RESULTS RT-PCR analysis detected a single fragment of approximately 520 base pairs (bp) in control testes at all time points. The cocaine-treated testes showed reduced expression of CREM fragment. Western blot analysis using CREM antibodies confirmed the RNA data. There were reduced CREM proteins in the cocaine-treated testes compared with controls. CONCLUSIONS The CREM gene is essential for spermatogenesis. Our results indicate that the reduction in testicular CREM expression may be one of the mechanisms responsible for disruption or impairment of spermatogenesis in the testes following chronic cocaine administration.