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Spermatozoa mitochondrial ferritin (MtF) content is related to sperm motility
otherwise normozoospermic patients may provide a diagnostic marker for sperm quality. Supported by: the Jeffress Trust and the Virginia Commonwealth Health Research Board.
Monday, October 13, 2003 4:15 P.M. O-80 Spermatozoa mitochondrial ferritin (MtF) content is related to sperm motility. Federico Calzi, Sonia Levi, Paolo Santambrogio, Lucia De Santis, Elisa Rabellotti, Viviana Bonzi. IRCCS Hosp San Raffaele, Milan, Italy; Protein Engineering Unit, DIBIT, IRCCS Hosp San Raffaele, Milan, Italy. Objective: To evaluate the mitochondrial ferritin (MtF) (1) content in spermatozoa and its relationship with sperm mobility. Design: Our study was divided into two steps: First we wanted to determine the presence of the mitochondrial type of ferritin in human sperm (previous data had shown its presence in mouse testis). Then human semen samples were divided into three groups according to spermatozoa concentration and motility and MtF content was evaluated. Materials and Methods: The first part of our study included 10 human semen samples randomly chosen from those obtained in our assisted reproductive program. We used an ELISA method, based on polyclonal anti-MtF antibodies, to determine the presence of the mitochondrial ferritin. The method is specific for the MtF and does not recognize the citosolic ferritins. In all of the samples we found the presence of the mitochondrial type of ferritin. After this statement, we started the second part of the study. Between November 2002 and February 2003, 104 semen samples were collected in our experimental program and divided into three groups; group A: normospermic samples (n°3 20X106/ml and progressive motility3 40%); group B: oligo-asthenospermic samples (n°3 ⬍ 20X106/ml and progressive motility ⬍ 40%); and group C: normo-asthenospermic samples (n°3 20X106/ml and progressive motility ⬍ 40%). MtF content was quantified on cells homogenated by 1% Triton-⫻100 treatment, using ELISA method. All results were expressed as ng of MtF/ mg of total protein and statistical analysis were performed using Student’s t test. Results: The study revealed the presence of the mitochondrial type of ferritin in all samples and the value varied from 31.4 ng/mg of total protein to 1017.0 ng/mg protein. The MtF content in group A (n° ⫽ 35) was 609.5 ⫾ 216.5 ng of MtF/ mg protein; in group B (n° ⫽ 41) the content of MtF was 225.2 ⫾ 171.7 ng/mg of protein (p ⬍ 0.0001 vs. group A) and in group C (n° ⫽ 28) we found 438.1 ⫾ 174.2 ng of MtF/mg of protein (p ⬍ 0.001 vs. group A). Conclusion: Our preliminary data demonstrated, for the first time, the presence of mitochondrial ferritin in human spermatozoa. We also found that there is a statistically significative difference in Mitochondrial Ferritin content in the three groups in which we divided our samples. In previous work it has been proposed that MtF has a cito-protective role related to its property to defend mitochondria against oxidative damage. Because of the putative protective role of mitochondrial ferritin, the less content of this protein in spermatozoa mitochondria, in group B and C, can be related to the poor quality of semen samples, especially for the reduced motility in group C. Further research is required in order to confirm our preliminary data. 1. Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, Arosio P, Drysdale J. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem. 2001 Jul 6;276(27):24437-40.
Monday, October 13, 2003 4:30 P.M. O-81 Gene expression profiles during mouse spermatogenesis by 8.0K cDNA microarray experiment. Suman Lee, Jung Hoon Park, Kye-Sung Kim, Hyun-Joo Kim, Kwang Yul Cha, Sook-Hwan Lee. Pochon CHA Univ, Seoul, Republic of Korea. Objective: To gain a comprehensive view of gene expression and regulation mechanism involved in germ cell development, especially pachytene spermatocytes and round spermatids.
FERTILITY & STERILITY威
Design: Expression profiling by cDNA microarray analysis. Main Outcome Measure(s): Gene expression profiles of spermatogenic cells obtained by 8.0K DNA microarray analysis. Materials and Methods: Mouse spermatogenic cells, pachytene spermatocytes and round spermatids, were isolated by sedimentation velocity at unit gravity. Total RNA was extracted cell using the Qiagen RNeasy kit. Probe was prepared with Cy3 and Cy5 labeled dUTP, and then hybridized mouse 8K cDNA chip. The slide was scanned on GenePix 4000B and analyzed with GenePix pro 4.1. The data generated by image processing were classified with category by GeneSpring program. To verify the microarray data, we examined the mRNA level by RT-PCR. Finally, the data were confirmed with in situ hybridization of mouse testis. Results(s): We found that 170 genes whose expression was higher in pachytene spermatocytes and 350 genes were higher in round spermatids. The nearly half of 170 upregulated genes in pachytene spermatocytes increased their gene expression less than 3 times; 86 increased 3–5 times; 14 increased ⬎ 5 times. Most of the 350 upregulated genes in round spermatids increased expression less than 3 times; 105 increased 3–5 times; 14 increased ⬎ 5 times. In pachytene spermatocytes, the expression of cell cycle and proliferation-related genes, cyclin-dependent kinase 2a and PCNA was increased over two times. In round spermatids, the expression levels of ubiqutin-related genes, ubiquitous specific protease 9 (USP9Y) and ubiquitin specific protease 9 are upregulated. The expression of the other RBM protein was increased over twofold in pachytene spermatocytes and round spermatids, RNA binding motif protein and RNA binding motif protein 4, respectively. Conclusion(s): Our data showed that the expression of more than 500 genes is changed between pachytene spermatocytes and round spermatids. The application of DNA chip technology for the study of gene expression in pachytene spermatocytes and round spermatids will allow a greater understanding of the control of germ cell development. Keywords: DNA microarray, expression profile, pachytene spermatocyte, round spermatid Supported by: a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002).
ENDOMETRIOSIS Monday, October 13, 2003 2:00 P.M. O-82 The broad-spectrum chemokine inhibitor NR58-3.14.3 suppresses the implantation and progression of human endometrial implants in the nude mice endometriosis model. Murat Berkkanoglu, Lufang Zhang, Umit A. Kayisli, Levent Senturk, Janelle Luk, Aydin Arici. Yale Univ Sch of Medicine, New Haven, CT. Objective: Endometriosis is a common gynecologic disorder, affecting 3-10% of reproductive-aged women and is characterized by the growth of endometrial tissue outside the uterine cavity. Although the precise pathogenesis of endometriosis is still not known, there is substantial evidence to support that changes in the cytokine network including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and several other proinflammatory chemoattractant cytokines play a role in the pathogenesis of this disease. NR58-3.14.3 is a novel broad-spectrum inhibitor of chemokines including MCP-1, IL-8, macrophage inflammatory protein-1␣ (MIP-1␣), and RANTES (regulated on activation, normal T-cell expressed and secreted). The aim of the present study was to evaluate the efficacy of this broad-spectrum chemokine inhibitor (BSCI) to prevent human endometrium to implant and grow in ectopic sites in the nude mice endometriosis model. Design: Prospective study. Materials and Methods: A total of 31 eight-week-old ovariectomized athymic nude mice (weighing 25-30 g) were used. They all underwent laparotomy through 0.2 cm vertical midline incision. Equal number of 1mm3 fragments of human proliferative phase endometrium were given via tuberculin syringes without attached needles into the peritoneal cavity of mice. After the closure of the abdominal wall, animals were assigned randomly to receive daily intraperitoneal injections of either PBS (control) or the BSCI (1 mg/animal in 0.1 ml PBS). In addition, each mouse was given intramuscular estradiol valerate (100 g/kg im) once a week. Fourteen days later, all animals were sacrificed and the number, extent, and
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Monday, October 13, 2003 4:15 P.M. O-80 Spermatozoa mitochondrial ferritin (MtF) content is related to sperm motility. Federico Calzi, Sonia Levi, Paolo Santambrogio, Lucia De Santis, Elisa Rabellotti, Viviana Bonzi. IRCCS Hosp San Raffaele, Milan, Italy; Protein Engineering Unit, DIBIT, IRCCS Hosp San Raffaele, Milan, Italy. Objective: To evaluate the mitochondrial ferritin (MtF) (1) content in spermatozoa and its relationship with sperm mobility. Design: Our study was divided into two steps: First we wanted to determine the presence of the mitochondrial type of ferritin in human sperm (previous data had shown its presence in mouse testis). Then human semen samples were divided into three groups according to spermatozoa concentration and motility and MtF content was evaluated. Materials and Methods: The first part of our study included 10 human semen samples randomly chosen from those obtained in our assisted reproductive program. We used an ELISA method, based on polyclonal anti-MtF antibodies, to determine the presence of the mitochondrial ferritin. The method is specific for the MtF and does not recognize the citosolic ferritins. In all of the samples we found the presence of the mitochondrial type of ferritin. After this statement, we started the second part of the study. Between November 2002 and February 2003, 104 semen samples were collected in our experimental program and divided into three groups; group A: normospermic samples (n°3 20X106/ml and progressive motility3 40%); group B: oligo-asthenospermic samples (n°3 ⬍ 20X106/ml and progressive motility ⬍ 40%); and group C: normo-asthenospermic samples (n°3 20X106/ml and progressive motility ⬍ 40%). MtF content was quantified on cells homogenated by 1% Triton-⫻100 treatment, using ELISA method. All results were expressed as ng of MtF/ mg of total protein and statistical analysis were performed using Student’s t test. Results: The study revealed the presence of the mitochondrial type of ferritin in all samples and the value varied from 31.4 ng/mg of total protein to 1017.0 ng/mg protein. The MtF content in group A (n° ⫽ 35) was 609.5 ⫾ 216.5 ng of MtF/ mg protein; in group B (n° ⫽ 41) the content of MtF was 225.2 ⫾ 171.7 ng/mg of protein (p ⬍ 0.0001 vs. group A) and in group C (n° ⫽ 28) we found 438.1 ⫾ 174.2 ng of MtF/mg of protein (p ⬍ 0.001 vs. group A). Conclusion: Our preliminary data demonstrated, for the first time, the presence of mitochondrial ferritin in human spermatozoa. We also found that there is a statistically significative difference in Mitochondrial Ferritin content in the three groups in which we divided our samples. In previous work it has been proposed that MtF has a cito-protective role related to its property to defend mitochondria against oxidative damage. Because of the putative protective role of mitochondrial ferritin, the less content of this protein in spermatozoa mitochondria, in group B and C, can be related to the poor quality of semen samples, especially for the reduced motility in group C. Further research is required in order to confirm our preliminary data. 1. Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, Arosio P, Drysdale J. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem. 2001 Jul 6;276(27):24437-40.
Monday, October 13, 2003 4:30 P.M. O-81 Gene expression profiles during mouse spermatogenesis by 8.0K cDNA microarray experiment. Suman Lee, Jung Hoon Park, Kye-Sung Kim, Hyun-Joo Kim, Kwang Yul Cha, Sook-Hwan Lee. Pochon CHA Univ, Seoul, Republic of Korea. Objective: To gain a comprehensive view of gene expression and regulation mechanism involved in germ cell development, especially pachytene spermatocytes and round spermatids.
FERTILITY & STERILITY威
Design: Expression profiling by cDNA microarray analysis. Main Outcome Measure(s): Gene expression profiles of spermatogenic cells obtained by 8.0K DNA microarray analysis. Materials and Methods: Mouse spermatogenic cells, pachytene spermatocytes and round spermatids, were isolated by sedimentation velocity at unit gravity. Total RNA was extracted cell using the Qiagen RNeasy kit. Probe was prepared with Cy3 and Cy5 labeled dUTP, and then hybridized mouse 8K cDNA chip. The slide was scanned on GenePix 4000B and analyzed with GenePix pro 4.1. The data generated by image processing were classified with category by GeneSpring program. To verify the microarray data, we examined the mRNA level by RT-PCR. Finally, the data were confirmed with in situ hybridization of mouse testis. Results(s): We found that 170 genes whose expression was higher in pachytene spermatocytes and 350 genes were higher in round spermatids. The nearly half of 170 upregulated genes in pachytene spermatocytes increased their gene expression less than 3 times; 86 increased 3–5 times; 14 increased ⬎ 5 times. Most of the 350 upregulated genes in round spermatids increased expression less than 3 times; 105 increased 3–5 times; 14 increased ⬎ 5 times. In pachytene spermatocytes, the expression of cell cycle and proliferation-related genes, cyclin-dependent kinase 2a and PCNA was increased over two times. In round spermatids, the expression levels of ubiqutin-related genes, ubiquitous specific protease 9 (USP9Y) and ubiquitin specific protease 9 are upregulated. The expression of the other RBM protein was increased over twofold in pachytene spermatocytes and round spermatids, RNA binding motif protein and RNA binding motif protein 4, respectively. Conclusion(s): Our data showed that the expression of more than 500 genes is changed between pachytene spermatocytes and round spermatids. The application of DNA chip technology for the study of gene expression in pachytene spermatocytes and round spermatids will allow a greater understanding of the control of germ cell development. Keywords: DNA microarray, expression profile, pachytene spermatocyte, round spermatid Supported by: a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002).
ENDOMETRIOSIS Monday, October 13, 2003 2:00 P.M. O-82 The broad-spectrum chemokine inhibitor NR58-3.14.3 suppresses the implantation and progression of human endometrial implants in the nude mice endometriosis model. Murat Berkkanoglu, Lufang Zhang, Umit A. Kayisli, Levent Senturk, Janelle Luk, Aydin Arici. Yale Univ Sch of Medicine, New Haven, CT. Objective: Endometriosis is a common gynecologic disorder, affecting 3-10% of reproductive-aged women and is characterized by the growth of endometrial tissue outside the uterine cavity. Although the precise pathogenesis of endometriosis is still not known, there is substantial evidence to support that changes in the cytokine network including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and several other proinflammatory chemoattractant cytokines play a role in the pathogenesis of this disease. NR58-3.14.3 is a novel broad-spectrum inhibitor of chemokines including MCP-1, IL-8, macrophage inflammatory protein-1␣ (MIP-1␣), and RANTES (regulated on activation, normal T-cell expressed and secreted). The aim of the present study was to evaluate the efficacy of this broad-spectrum chemokine inhibitor (BSCI) to prevent human endometrium to implant and grow in ectopic sites in the nude mice endometriosis model. Design: Prospective study. Materials and Methods: A total of 31 eight-week-old ovariectomized athymic nude mice (weighing 25-30 g) were used. They all underwent laparotomy through 0.2 cm vertical midline incision. Equal number of 1mm3 fragments of human proliferative phase endometrium were given via tuberculin syringes without attached needles into the peritoneal cavity of mice. After the closure of the abdominal wall, animals were assigned randomly to receive daily intraperitoneal injections of either PBS (control) or the BSCI (1 mg/animal in 0.1 ml PBS). In addition, each mouse was given intramuscular estradiol valerate (100 g/kg im) once a week. Fourteen days later, all animals were sacrificed and the number, extent, and
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