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Screening of an adult, human, testis-specific cDNA library for putative binding partners of the protamine 1 protein
P-790
P-792
ULTRASONOGRAPHICALLY MEASURED TESTICULAR VOLUMES IN 0 TO 6 YEAR OLD BOYS. E. A. Kuijper, J. van Kooten, J. I. Verbeke, M. van Rooijen, C. B. Lambalk. Reproductive Medicine, VUmc, Amsterdam, Noord-Holland, Netherlands; Radiology, VUmc, Amsterdam, Noord-Holland, Netherlands; Infant Welfare Center, GG&GD, Amsterdam, Noord-Holland, Netherlands.
MONO-(2-ETHYLHEXYL) PHTHALATE RAPIDLY DECREASES CLAUDIN-11 IN SERTOLI CELLS. Y. Kondo, T. Ishikawa, K. Yamaguchi, T. Haraguchi, Y. Sakamoto, M. Fujisawa. Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
OBJECTIVE: Aside from converted orchidometer measurements, there are no referential values for testicular ultrasound measurements in children available. Therefore, the aim of the current study was to obtain ultrasonographically measured normative data for testicular volumes in 0 to 6 year old boys. DESIGN: Cohort Study. MATERIALS AND METHODS: A total of 344 healthy, term born, singleton boys of different ethnical origin (Caucasian, Mediterranean, African, Asian) ranging in age from 1 to 69 months, were included in the study. RESULTS: No significant differences were found neither between the various ethnical groups, nor between the left and right testicle. Therefore, data were pooled and the mean testicular volume was used as a unit of analysis per child. Mean testicular volume was compared between the different age categories. Testicular volume increases significantly in the first 5 months after birth from 0.27 (0.02) to 0.44 (0.03) after which the volume decreases to 0.31 (0.02) at 9 months. Over the following years testicular volume remains stable. CONCLUSIONS: This study provides normal values for ultrasonographically measured testicular volumes in 0 to 6 year old boys. Ultrasound is a valid method for measuring small pre-pubertal testicles as it is able to detect minor changes in volume in relation to established physiological changes in the first year of life. Supported by: None.
P-791 SCREENING OF AN ADULT, HUMAN, TESTIS-SPECIFIC cDNA LIBRARY FOR PUTATIVE BINDING PARTNERS OF THE PROTAMINE 1 PROTEIN. B. R. Emery, D. T. Carrell. Andrology And IVF Laboratories, Univ. of Utah School of Medicine, Salt Lake City, UT. OBJECTIVE: The human protamines are rapidly emerging as a significant indication of malefactor infertility. These proteins are incorporated into the sperm nucleus in a multi-step process as the majority of nuclear histones are removed. It is also clear that substantial phosphorylation is required for this step of spermiogenesis. There has been much attention given to the coding and translation of protamine 1 and 2 with little focus on the post-translational modifications necessary for their proper integration into the chromatin. The purpose of this study is to elucidate novel interacting partners of protamine 1. DESIGN: A yeast two hybrid library screen was used to determine potential protein – protein interactions present between the human protamie 1 and the human testis proteome, represented by a commercial pre-transformed adult human testis cDNA library. MATERIALS AND METHODS: A clone containing the full-length cDNA of human protamine 1 was obtained from Open Biosystems. The cDNA was inserted into the PGBKT7 (Clontech) vector, next to the GAL4 binding domain, and transformed into AH109 yeast. The AH109 yeast strain was mated to Y187 yeast pretransformed with the testis cDNA library, which had been inserted into the PACT2 vector (Clontech) adjacent to the GAL4 activation domain. The mated cell culture was plated onto quadruple dropout agar containing the substrate X a GAL for 14–21 days. Blue colonies were picked and replicated twice. The PACT2 vector was isolated from each colony and the cDNA library insert sequenced. RESULTS: A total of 48 colonies, representing 32 unique cDNA sequences were obtained from two separate screens of the testis library. The 32 cDNA sequences represent mostly previously identified proteins with a known or putative function. Approximately a third of these proteins are in frame with the GAL4 AD, suggesting they may be real binding partners of protamine 1. CONCLUSIONS: Our results suggest that protamine 1 may interact with several proteins. These proteins may play a role in post-translational modification of protamine 1 or the incorporation of the protamines into the sperm chromatin. Co-immunoprecipitation assays are underway to help determine which of the identified proteins are true interacting partners with protamine 1. Supported by: None.
FERTILITY & STERILITYÒ
OBJECTIVE: Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Claudin-11 is involoved in membrane interactions at Sertoli cells tight-junction and with the extracellular matrix. The function of tight-junction is under the control of paracrine and endocrine as well as physiochemical factors in various tissues. So, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and claudin-11 in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP by activating mitogen-activated protein kinases (MAPK) signaling pathways. DESIGN: Controlled animal experiment in vitro. MATERIALS AND METHODS: Sertoli cells were isolated and purified from the testes of 18-d-old Sprague Dawley rats and incubated at 34 C. On day 3 ex vivo, Sertoli cells were treated with MEHP (1 mM, 10 mM, or 100 mM). At 0.5, 1, 3, 6, and 24 h after either vehicle (control) or MEHP, whole cell lysates for protein and total RNA were isolated from each replicate. Claudin-11 mRNAs were evaluated by quantitative real-time RT-PCR analysis and phosphorylated proteins (p38, p44/42, and SAPK/JNK) were assayed by Western blot analysis. We also examined nitric oxide synthases (NOSs), the members of the tumor necrosis factor (TNF) ligands (Fas L and TRAIL) and its receptor (Fas) expressions by RT-PCR and Western blot analysis. RESULTS: MEHP treatment led to a significant time- and dose-dependent decrease in Claudin-11 mRNA. Fas, Fas L, TRAIL, and NOSs protein and mRNA were not changed. The MEHP exposure induced the phosphorylation of p44/42, not p38 and SAPK/JNK. When p44/42 MAPK activity inhibitors are used, decrease of claudin-11 by MEHP is prevented, indicating that these effects depend on activation of enzymes. CONCLUSIONS: These data show that MEHP exposure rapidly alters claudin-11 in Sertoli cells, which might contribute to defects in intact junctional complex formation of sertoli cells leading to impairment of the integrity of the blood-testis barrier and alterations in endocrine and spermatogenic function. Supported by: None.
P-793 ANALYSIS OF ANDROGENIC EFFECT DURING SPERMATOGENESIS IN TESTIS. Y. Nakamura, S. Komori, H. Kasumi, H. Tanaka, K. Koyama. Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Laboratory of Developmental Biology and Reproduction, Insititute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan. OBJECTIVE: The identification of proteins induced by androgen in Sertoli cells is a major issue in spermatogenesis. DESIGN: Laboratory study. MATERIALS AND METHODS: In this study, we analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone using SELDITOF mass spectrometry. RESULTS: We found increases in expression of a 5.0 kDa protein at 15 min, a 11.3 kDa protein at 24 hrs and 4.3, 5.7, 5.8, 9.95 and 9.98 kDa proteins at 48 hrs. On the other hand, expression of 6.3 and 8.6 kDa proteins were decreased at 30 min, and 4.9, 5.0, 12.4 and 19.8 kDa proteins at 48 hrs. The 11.3 kDa increased molecule was identified as macrophage migration inhibitory factor. The 9.98 kDa increased molecule was identified as calgizzarin known to bind to Ca2þ. The 19.8 kDa molecule was identified as translationally controlled tumor protein (TCTP) known to bind to tublin and Ca2þ and regulates cell proliferation. The timing of their expression suggests that these proteins are involved late in androgen regulation of spermatogenesis in Sertoli cells. CONCLUSIONS: The late change in the expression of these proteins suggests that androgenic effects on spermatogenesis are exerted eventually through these proteins. Supported by: This work was supported in part by a Grant-in aid for Scientific Research from the Ministry of education, Science and Culture (No 16591693 for S Komori and No14571594 for H Kasumi).
S369
P-792
ULTRASONOGRAPHICALLY MEASURED TESTICULAR VOLUMES IN 0 TO 6 YEAR OLD BOYS. E. A. Kuijper, J. van Kooten, J. I. Verbeke, M. van Rooijen, C. B. Lambalk. Reproductive Medicine, VUmc, Amsterdam, Noord-Holland, Netherlands; Radiology, VUmc, Amsterdam, Noord-Holland, Netherlands; Infant Welfare Center, GG&GD, Amsterdam, Noord-Holland, Netherlands.
MONO-(2-ETHYLHEXYL) PHTHALATE RAPIDLY DECREASES CLAUDIN-11 IN SERTOLI CELLS. Y. Kondo, T. Ishikawa, K. Yamaguchi, T. Haraguchi, Y. Sakamoto, M. Fujisawa. Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
OBJECTIVE: Aside from converted orchidometer measurements, there are no referential values for testicular ultrasound measurements in children available. Therefore, the aim of the current study was to obtain ultrasonographically measured normative data for testicular volumes in 0 to 6 year old boys. DESIGN: Cohort Study. MATERIALS AND METHODS: A total of 344 healthy, term born, singleton boys of different ethnical origin (Caucasian, Mediterranean, African, Asian) ranging in age from 1 to 69 months, were included in the study. RESULTS: No significant differences were found neither between the various ethnical groups, nor between the left and right testicle. Therefore, data were pooled and the mean testicular volume was used as a unit of analysis per child. Mean testicular volume was compared between the different age categories. Testicular volume increases significantly in the first 5 months after birth from 0.27 (0.02) to 0.44 (0.03) after which the volume decreases to 0.31 (0.02) at 9 months. Over the following years testicular volume remains stable. CONCLUSIONS: This study provides normal values for ultrasonographically measured testicular volumes in 0 to 6 year old boys. Ultrasound is a valid method for measuring small pre-pubertal testicles as it is able to detect minor changes in volume in relation to established physiological changes in the first year of life. Supported by: None.
P-791 SCREENING OF AN ADULT, HUMAN, TESTIS-SPECIFIC cDNA LIBRARY FOR PUTATIVE BINDING PARTNERS OF THE PROTAMINE 1 PROTEIN. B. R. Emery, D. T. Carrell. Andrology And IVF Laboratories, Univ. of Utah School of Medicine, Salt Lake City, UT. OBJECTIVE: The human protamines are rapidly emerging as a significant indication of malefactor infertility. These proteins are incorporated into the sperm nucleus in a multi-step process as the majority of nuclear histones are removed. It is also clear that substantial phosphorylation is required for this step of spermiogenesis. There has been much attention given to the coding and translation of protamine 1 and 2 with little focus on the post-translational modifications necessary for their proper integration into the chromatin. The purpose of this study is to elucidate novel interacting partners of protamine 1. DESIGN: A yeast two hybrid library screen was used to determine potential protein – protein interactions present between the human protamie 1 and the human testis proteome, represented by a commercial pre-transformed adult human testis cDNA library. MATERIALS AND METHODS: A clone containing the full-length cDNA of human protamine 1 was obtained from Open Biosystems. The cDNA was inserted into the PGBKT7 (Clontech) vector, next to the GAL4 binding domain, and transformed into AH109 yeast. The AH109 yeast strain was mated to Y187 yeast pretransformed with the testis cDNA library, which had been inserted into the PACT2 vector (Clontech) adjacent to the GAL4 activation domain. The mated cell culture was plated onto quadruple dropout agar containing the substrate X a GAL for 14–21 days. Blue colonies were picked and replicated twice. The PACT2 vector was isolated from each colony and the cDNA library insert sequenced. RESULTS: A total of 48 colonies, representing 32 unique cDNA sequences were obtained from two separate screens of the testis library. The 32 cDNA sequences represent mostly previously identified proteins with a known or putative function. Approximately a third of these proteins are in frame with the GAL4 AD, suggesting they may be real binding partners of protamine 1. CONCLUSIONS: Our results suggest that protamine 1 may interact with several proteins. These proteins may play a role in post-translational modification of protamine 1 or the incorporation of the protamines into the sperm chromatin. Co-immunoprecipitation assays are underway to help determine which of the identified proteins are true interacting partners with protamine 1. Supported by: None.
FERTILITY & STERILITYÒ
OBJECTIVE: Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Claudin-11 is involoved in membrane interactions at Sertoli cells tight-junction and with the extracellular matrix. The function of tight-junction is under the control of paracrine and endocrine as well as physiochemical factors in various tissues. So, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and claudin-11 in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP by activating mitogen-activated protein kinases (MAPK) signaling pathways. DESIGN: Controlled animal experiment in vitro. MATERIALS AND METHODS: Sertoli cells were isolated and purified from the testes of 18-d-old Sprague Dawley rats and incubated at 34 C. On day 3 ex vivo, Sertoli cells were treated with MEHP (1 mM, 10 mM, or 100 mM). At 0.5, 1, 3, 6, and 24 h after either vehicle (control) or MEHP, whole cell lysates for protein and total RNA were isolated from each replicate. Claudin-11 mRNAs were evaluated by quantitative real-time RT-PCR analysis and phosphorylated proteins (p38, p44/42, and SAPK/JNK) were assayed by Western blot analysis. We also examined nitric oxide synthases (NOSs), the members of the tumor necrosis factor (TNF) ligands (Fas L and TRAIL) and its receptor (Fas) expressions by RT-PCR and Western blot analysis. RESULTS: MEHP treatment led to a significant time- and dose-dependent decrease in Claudin-11 mRNA. Fas, Fas L, TRAIL, and NOSs protein and mRNA were not changed. The MEHP exposure induced the phosphorylation of p44/42, not p38 and SAPK/JNK. When p44/42 MAPK activity inhibitors are used, decrease of claudin-11 by MEHP is prevented, indicating that these effects depend on activation of enzymes. CONCLUSIONS: These data show that MEHP exposure rapidly alters claudin-11 in Sertoli cells, which might contribute to defects in intact junctional complex formation of sertoli cells leading to impairment of the integrity of the blood-testis barrier and alterations in endocrine and spermatogenic function. Supported by: None.
P-793 ANALYSIS OF ANDROGENIC EFFECT DURING SPERMATOGENESIS IN TESTIS. Y. Nakamura, S. Komori, H. Kasumi, H. Tanaka, K. Koyama. Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Laboratory of Developmental Biology and Reproduction, Insititute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan. OBJECTIVE: The identification of proteins induced by androgen in Sertoli cells is a major issue in spermatogenesis. DESIGN: Laboratory study. MATERIALS AND METHODS: In this study, we analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone using SELDITOF mass spectrometry. RESULTS: We found increases in expression of a 5.0 kDa protein at 15 min, a 11.3 kDa protein at 24 hrs and 4.3, 5.7, 5.8, 9.95 and 9.98 kDa proteins at 48 hrs. On the other hand, expression of 6.3 and 8.6 kDa proteins were decreased at 30 min, and 4.9, 5.0, 12.4 and 19.8 kDa proteins at 48 hrs. The 11.3 kDa increased molecule was identified as macrophage migration inhibitory factor. The 9.98 kDa increased molecule was identified as calgizzarin known to bind to Ca2þ. The 19.8 kDa molecule was identified as translationally controlled tumor protein (TCTP) known to bind to tublin and Ca2þ and regulates cell proliferation. The timing of their expression suggests that these proteins are involved late in androgen regulation of spermatogenesis in Sertoli cells. CONCLUSIONS: The late change in the expression of these proteins suggests that androgenic effects on spermatogenesis are exerted eventually through these proteins. Supported by: This work was supported in part by a Grant-in aid for Scientific Research from the Ministry of education, Science and Culture (No 16591693 for S Komori and No14571594 for H Kasumi).
S369