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Changes in the protein profiles of sperm cells from adolescents with varicocele
P-129 Changes in the protein profiles of sperm cells from adolescents with varicocele. R. Afonso, A. Cedenho Sr., P. Hassun Sr., R. Bertolla Sr., V. Ortiz Sr., M. Srougi Sr. Federal University of Sao Paulo, Sao Paulo, Brazil. OBJECTIVE: Varicocele is present in 15 to 20% of adolescents. It induces a raise in testicular temperatures, where it may cause progressive lesions, ultimately leading to infertility. Higher testicular temperatures have been shown to lead to a decrease in gene transcription and protein translation, besides changing the proteins synthesized by Leydig and Sertoli cells. There is a protein profile that must be expressed by germ cells and by supporting Leydig and Sertoli cells in order to guarantee fertility potential. Fertilization itself involves a series of protein interactions between the spermatozoon, the environment, and the oocyte. These proteins must be expressed throughout spermatogenesis, and thus produce a protein profile that will allow the mature sperm cell to reach the oocyte, penetrate the zona pellucida and form the male pronucleus. Varicocele may alter this profile and in so potentially affect fertility in many different ways. This study set out to evaluate if sperm from adolescents with clinical diagnosed varicocele display and altered protein profile when compared to adolescents without varicocele by using one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE). DESIGN: Prospective-controlled study. MATERIALS AND METHODS: Varicocele diagnosis was performed by the same physician for both the experimental and the control groups. The study included 15 infertile adolescents with a diagnosed grade II or III bilateral varicocele, and 15 adolescents without varicocele. Standard seminal analysis was performed following two to four days of sexual abstinence. Sperm was isolated by centrifugation, and following washing with PBS to eliminate seminal plasma proteins which could be bound to sperm membranes, proteins were extracted by boiling samples for 5 minutes in an SDS treatment buffer (62.5mM Tris-HCL, pH 6.8, 2% SDS, 5%  -mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue). Protein concentration was determined according to the Lowry protocol, and samples were loaded in triplicate, with decreasing amounts (150mg, 100mg, and 50mg) in each lane, to facilitate band visualization. RESULTS: The varicocele group expressed bands at ⬃86kDa, ⬃52kDa, ⬃22kDa, and ⬃17kDa, which were absent from the control group. On the other hand the control group expressed two bands at ⬃155kDa and ⬃130kDa which were not expressed in the varicocele group. CONCLUSION: The results demonstrate that adolescents with varicocele possess an altered spermatic protein profile, as verified by one-dimensional SDS-PAGE, which may be an early sign of alterations in spermatogenesis. Supported by: None P-130 The association of vascular proximity to cellularity within seminiferous tubules obtained by ultrasonically guided percutaneous biopsies. J. L. Marmar, P. Polyak, C. Millan, S. Benoff. Robert Wood Johnson Medical School at Camden, Camden, NJ; North Shore–Long Island Jewish Research Institute, Manhassett, NY. OBJECTIVE: Acqusition of sperm for ICSI in cases with non-obstructive azoospermia is especially challenging for patients with maturation arrest (MA) and Sertoli cell only (SCO).Several biopsy techniques have been used for these cases including multiple biopsies,testis mapping and microTESE.Although these methods may provide sperm in 10 –20% on these cases,the procedures may require general anasthesia and may be traumatic to the testis.Recently,some investigators reported fine needle aspiration of the testis with ultrasonic control to acquire sperm from the most vascular areas. They hypothesized that sperm may be more abundent in these locations.Although they obtained sperm in some difficult cases,they had no histologic evidence to suggest that increased vascularity was an “oasis” for spermatogenesis.In this report,we present a modified percutaneous testis aspiration biopsy technique with realtime ultrasound to identify vascular areas,and we studied the association of vascularity and cellularity within adjacent seminiferous tubules.Lastly,we present sperm retrival data prior to ICSI in 5 MA and 9 SCO cases with these methods. DESIGN: Diagnostic percutaneous testis aspiration biopsies with ultrasound control were obtained from 95 azoospermic men:obstruction-33,absent vasa-10,non motile sperm-3,hypospermatogenesis-19,MA-9,SCO21.Fourty-nine specimines were stained for CD34 and VEGF (vascular
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stains).Germ cell scores or cellularity (scale 0 – 4) within seminiferous tubules were correlated with the intensity of vascular staining.Cellularity was also evaluated on the basis of arteriole proximity (near ⬍25 microns, far 25 microns). Sperm retrival rates were reported for 5 MA and 9 SCO cases who had therapeutic biopsies from vascular areas in connection with ICSI. MATERIALS AND METHODS: All biopsies were performed with local anasthesia and an #18 gauge angiocath connected to a 20 cc syringe for negative pressure.Diagnostic biopsies were performed areas away from major vessels, but therapeutic biopsies were adjacent to the vascular areas. RESULTS: The diagnostic biopsies from all azospermic men in the study group had sperm in at least 15% of the tubules,except for 9 of 9 MA and 19 of 21 SCO patients.There was a significant correlation between CD34/ VEGF staining and tubular cellularity (r⫽0.452 and 0.468, respectively) (p⬍0.001). The mean cellularity score for tubules near vessels was 2.87 vs. 2.077 for tubules far from vessels (p⬍0.02).Fourteen patients with no sperm on the diagnostic biopsy had a therapeutic biopsy for ICSI near a vascular area.One of 5 MA(20%) and 3 of 9 SCO(33.3%) patients had sucessful sperm retrival with the modified method. CONCLUSION: 1.A minimally invasive diagnostic, percutaneous aspiration testis biopsy with ultrasonic control reveals the extent of spermatogenesis in azoospermic men,and these findings may be discussed prior to ICSI for a realistic assesment of their chances for sperm retrival. 2.Cellularity within the seminiferous tubule was corellated with vascular staining,and the cellularity was greatest in tubules closest to vessels. 3.Repeat therapeutic biopsies from vascular areas yielded sperm for ICSI in 20% MA and 33.3% SCO patients who had no sperm on diagnostic biopsies. 4.The findings suggest that increased vascularity may have an “oasis” effect on spermatogenesis. Supported by: NIH grant No. ES 10496 to SHB
P-131 Absence of heat shock protein 90 (HSP90) mutations in the varicoceleassociated infertility. P. Hassun Sr., A. Cedenho Sr., S. Lima, R. Bertolla Sr., V. Ortiz Sr., M. Srougi. Federal University of Sao Paulo, Sao Paulo, Brazil. OBJECTIVE: Varicocele induces a raise in intra-testicular temperature and may affect spermatogenesis. Heat shock 90 kD protein (HSP90) is a molecular chaperone expressed during spermatogenesis, and is related to sperm motility and both heat and oxidative stress. Possible alterations in the HSP90 protein may lead to increased susceptibility to the varicoceleassociated detrimental effects of heat and oxidative stress to spermatogenesis. Single nucleotide polymorphisms (SNPs) were screened for in patients with varicocele in order to investigate if they are associated with infertility. DESIGN: Prospective-controlled study. SNPs in the HSP90 gene were screened for in patients with varicocele and altered spermatogenesis, in normozoospermic patients with varicocele, and in normozoospermic patients without varicocele. MATERIALS AND METHODS: Varicocele diagnosis was performed by the same physician for both the experimental and the control groups. The study included 12 infertile patients with a diagnosed grade II or III bilateral varicocele, 10 normozoospermic patients with a diagnosed grade II or III bilateral varicocele, and 15 normozoospermic men without varicocele. Standard seminal analysis was performed according to the World Health Organization criteria and morphology was evaluated by strict criteria. Polymerase chain reaction (PCR) primer sets were designed, and exons sequences were amplified from genomic DNA. Single strand conformation polymorphism (SSCP) was performed in these men, and samples showing an altered band pattern in the PCR-SSCP were further characterized through DNA sequencing. Blood samples were obtained by venipuncture for karyotype, Y chromosome microdeletion scan and hormonal analysis (FSH, LH and testosterone). RESULTS: Two SNPs were identified: a 3774 CT polymorphism in exon 4 of patient P11 (varicocele with altered spermatogenesis), and a 6192G⬎A polymorphism in exon 10 of patient P5 (normozoospermic with varicocele). Both polymorphisms did not lead to amino acid substitution (silent polymorphisms). CONCLUSION: Mutations in the HSP90 gene do not seem to play a role in increasing testicular germ cell susceptibility to the varicocele-associated alterations in spermatogenesis. Supported by: None.
Vol. 82, Suppl. 2, September 2004
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stains).Germ cell scores or cellularity (scale 0 – 4) within seminiferous tubules were correlated with the intensity of vascular staining.Cellularity was also evaluated on the basis of arteriole proximity (near ⬍25 microns, far 25 microns). Sperm retrival rates were reported for 5 MA and 9 SCO cases who had therapeutic biopsies from vascular areas in connection with ICSI. MATERIALS AND METHODS: All biopsies were performed with local anasthesia and an #18 gauge angiocath connected to a 20 cc syringe for negative pressure.Diagnostic biopsies were performed areas away from major vessels, but therapeutic biopsies were adjacent to the vascular areas. RESULTS: The diagnostic biopsies from all azospermic men in the study group had sperm in at least 15% of the tubules,except for 9 of 9 MA and 19 of 21 SCO patients.There was a significant correlation between CD34/ VEGF staining and tubular cellularity (r⫽0.452 and 0.468, respectively) (p⬍0.001). The mean cellularity score for tubules near vessels was 2.87 vs. 2.077 for tubules far from vessels (p⬍0.02).Fourteen patients with no sperm on the diagnostic biopsy had a therapeutic biopsy for ICSI near a vascular area.One of 5 MA(20%) and 3 of 9 SCO(33.3%) patients had sucessful sperm retrival with the modified method. CONCLUSION: 1.A minimally invasive diagnostic, percutaneous aspiration testis biopsy with ultrasonic control reveals the extent of spermatogenesis in azoospermic men,and these findings may be discussed prior to ICSI for a realistic assesment of their chances for sperm retrival. 2.Cellularity within the seminiferous tubule was corellated with vascular staining,and the cellularity was greatest in tubules closest to vessels. 3.Repeat therapeutic biopsies from vascular areas yielded sperm for ICSI in 20% MA and 33.3% SCO patients who had no sperm on diagnostic biopsies. 4.The findings suggest that increased vascularity may have an “oasis” effect on spermatogenesis. Supported by: NIH grant No. ES 10496 to SHB
P-131 Absence of heat shock protein 90 (HSP90) mutations in the varicoceleassociated infertility. P. Hassun Sr., A. Cedenho Sr., S. Lima, R. Bertolla Sr., V. Ortiz Sr., M. Srougi. Federal University of Sao Paulo, Sao Paulo, Brazil. OBJECTIVE: Varicocele induces a raise in intra-testicular temperature and may affect spermatogenesis. Heat shock 90 kD protein (HSP90) is a molecular chaperone expressed during spermatogenesis, and is related to sperm motility and both heat and oxidative stress. Possible alterations in the HSP90 protein may lead to increased susceptibility to the varicoceleassociated detrimental effects of heat and oxidative stress to spermatogenesis. Single nucleotide polymorphisms (SNPs) were screened for in patients with varicocele in order to investigate if they are associated with infertility. DESIGN: Prospective-controlled study. SNPs in the HSP90 gene were screened for in patients with varicocele and altered spermatogenesis, in normozoospermic patients with varicocele, and in normozoospermic patients without varicocele. MATERIALS AND METHODS: Varicocele diagnosis was performed by the same physician for both the experimental and the control groups. The study included 12 infertile patients with a diagnosed grade II or III bilateral varicocele, 10 normozoospermic patients with a diagnosed grade II or III bilateral varicocele, and 15 normozoospermic men without varicocele. Standard seminal analysis was performed according to the World Health Organization criteria and morphology was evaluated by strict criteria. Polymerase chain reaction (PCR) primer sets were designed, and exons sequences were amplified from genomic DNA. Single strand conformation polymorphism (SSCP) was performed in these men, and samples showing an altered band pattern in the PCR-SSCP were further characterized through DNA sequencing. Blood samples were obtained by venipuncture for karyotype, Y chromosome microdeletion scan and hormonal analysis (FSH, LH and testosterone). RESULTS: Two SNPs were identified: a 3774 CT polymorphism in exon 4 of patient P11 (varicocele with altered spermatogenesis), and a 6192G⬎A polymorphism in exon 10 of patient P5 (normozoospermic with varicocele). Both polymorphisms did not lead to amino acid substitution (silent polymorphisms). CONCLUSION: Mutations in the HSP90 gene do not seem to play a role in increasing testicular germ cell susceptibility to the varicocele-associated alterations in spermatogenesis. Supported by: None.
Vol. 82, Suppl. 2, September 2004