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The in vivo imaging of fertilizing spermatozoa traveling in female reproductive tract
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
production in various tissues to recruit monocytes and neutrophils to the site of inflammation, similar to Interferon gamma. Our objective was to examine the effect of IL-4 on IL-17 production in order to find out how IL-4 can involve in Collagen-Induced Arthritis (CIA) mice. In this study, a chicken collagen-II-induced experimental arthritis (CIA) model was used in DBA/1 mice to investigate the relationship between IL-4 and IL-17 as well as other inflammatory factors. On the 38th day after the mice were induced with CIA, the expression of IL -17 and IL - 4 as well as IFN␥ and IL- 13 in serum of the mice was measured by ELISA and QRT-PCR. The result of QRT-PCR analysis of IL-17 and IL-4 mRNA levels in the spleen showed that IL-17 is increased significantly at the onset of CIA in the spleen (p <0.01). Meanwhile, IL-17 is generally reduced in the peak of CIA but IL-4 is increased significantly at the peak of CIA in the spleen (p <0.05) when it was compared with the weight of the animal.IL-4 can involve on production of IL-17 at especially the peak of CIA. These results imply that the inhibition of IL-17 can decrease the expression of IL-1 and IL-6 production which will result in the aggravation of arthritis doi:10.1016/j.jri.2012.03.430 P 129 The in vivo imaging of fertilizing spermatozoa traveling in female reproductive tract M. Okabe ∗ , Y. Muro, M. Ikawa Osaka University, Research Institute for Microbial Diseases, Suita, Japan BACKGROUND: Millions of spermatozoa are deposited in female reproductive tract but only a few of the spermatozoa are reported to reach the fertilization site. However, the manner of fertilizing spermatozoa reaching to the unfertilized eggs “in vivo” is largely unknown. PURPOSE: To clarify how the fertilizing spermatozoa reach to the eggs, the spermatozoa in vivo were live imaged and the numbers and movement of spermatozoa in various parts of the female reproductive tract were observed using mouse as an experimental model. METHOD: We established transgenic mouse line which produces spermatozoa expressing fluorescent protein in their acrosome (tagged by GFP driven by acrosin promoter) and in their midpiece (tagged by RFP with mitochondria migrating signal, also driven by acrosin promoter). This enabled us to observe the spermatozoa in vivo through the wall of female reproductive tract using a fluorescent microscope (Olympus BX50 and Keyence BZ-8000). Small numbers of spermatozoa were counted by eyes under fluorescent microscope and massive numbers were quantitated by western blotting using anti-sperm antibody. RESULT AND DISCUSSION: The spermatozoa are directly ejaculated in uterus in mouse. The average amount of sperm found in the uterus was examined and it was about 1.8 × 107ˆ right after the coitus. The ejaculated spermatozoa started to migrate into the oviduct and sperm number gradually increased in the oviduct and reached its maximum 9 hours after coitus. The number of oviductal spermatozoa
101
ˆ which is approximately one over one was about 2.9 × 104, thousand of the uterine spermatozoa. Utilizing the fluorescent protein tagged spermatozoa, we observed the spermatozoa in female reproductive tract. The various movies will be shown in this presentation and the process of spermatozoa reaching to the unfertilized eggs will be discussed. doi:10.1016/j.jri.2012.03.431 P 130 Vitamin D3 inhibits inflammatory cascade in endometrial cells through down regulation of TLR signaling A.H. Zarnani 1,2,∗ , N. Rashidi 3 , M. Mirahmadian 3 , M. JeddiTehrani 4 , S. Rezania 5 , A. Kalantari 2 1
Avicenna Research Institute, Academic Center for Education, Culture & Research (ACECR), Reproductive Biotechnology Research Center, Tehran, Iran, Islamic Republic of 2 Tehran University of Medical Sciences, Immunology Research Center, Tehran, Iran, Islamic Republic of 3 Faculty of Medicine, Tehran University of Medical Sciences, Immunology, Tehran, Iran, Islamic Republic of 4 Avicenna Research Institute, ACECR, Monoclonal Antibody Research Center, Tehran, Iran, Islamic Republic of 5 Medical University of Graz, Biophysics Institute, Graz, Austria PROBLEM: Toll-like receptors (TLR) have been appeared as important upstream mediators of inflammation at many human tissues. TLR-mediated inflammatory processes have been suggested to be involved in pathophysiology of such pregnancy-related disorders as spontaneous abortion, preterm labor, preeclampsia, and intrauterine growth restriction. Bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are among the pathogen-associated molecular patterns (PAMP) which induce TLR signaling leading to release of pro-inflammatory cytokines. Over the past decade, clinical evidence has been accumulating that vitamin D3 (VD3) is effective in the treatment of diseases with inflammatory nature. The aim of this study was to investigate the effects of VD3 on production of inflammatory cytokines and expression of TLR2, TLR4 and MyD88, the main adaptor of TLR signaling pathway, in endometrial cells in response to LPS and LTA stimulation. METHODS: Endometrial tissues were obtained from cycling women undergoing operations for benign gynecological conditions. Whole endometrial (WECs) and purified endometrial stromal cells (ESCs) were examined for expression of TLR2, TLR4 and MyD88 genes in LPS- and LTAstimulated or non-stimulated conditions at both gene and protein levels by semi-quantitative RT-PCR, western blotting and flow cytometry, respectively. TLR stimulation was functionally assessed by measurement of TNF-␣, IL-6, and IL-8 production in cell culture supernatants by ELISA. In parallel, immunomodulatory effects of VD3 on the aforesaid parameters were assessed. RESULTS: Isolated ESCs exhibited high expression of CD10 and vimentin, but failed to express cytokeratin indicating their high purity. Flow cytometry analysis demonstrated that ESCs do not express TLR2 and TLR4 at
production in various tissues to recruit monocytes and neutrophils to the site of inflammation, similar to Interferon gamma. Our objective was to examine the effect of IL-4 on IL-17 production in order to find out how IL-4 can involve in Collagen-Induced Arthritis (CIA) mice. In this study, a chicken collagen-II-induced experimental arthritis (CIA) model was used in DBA/1 mice to investigate the relationship between IL-4 and IL-17 as well as other inflammatory factors. On the 38th day after the mice were induced with CIA, the expression of IL -17 and IL - 4 as well as IFN␥ and IL- 13 in serum of the mice was measured by ELISA and QRT-PCR. The result of QRT-PCR analysis of IL-17 and IL-4 mRNA levels in the spleen showed that IL-17 is increased significantly at the onset of CIA in the spleen (p <0.01). Meanwhile, IL-17 is generally reduced in the peak of CIA but IL-4 is increased significantly at the peak of CIA in the spleen (p <0.05) when it was compared with the weight of the animal.IL-4 can involve on production of IL-17 at especially the peak of CIA. These results imply that the inhibition of IL-17 can decrease the expression of IL-1 and IL-6 production which will result in the aggravation of arthritis doi:10.1016/j.jri.2012.03.430 P 129 The in vivo imaging of fertilizing spermatozoa traveling in female reproductive tract M. Okabe ∗ , Y. Muro, M. Ikawa Osaka University, Research Institute for Microbial Diseases, Suita, Japan BACKGROUND: Millions of spermatozoa are deposited in female reproductive tract but only a few of the spermatozoa are reported to reach the fertilization site. However, the manner of fertilizing spermatozoa reaching to the unfertilized eggs “in vivo” is largely unknown. PURPOSE: To clarify how the fertilizing spermatozoa reach to the eggs, the spermatozoa in vivo were live imaged and the numbers and movement of spermatozoa in various parts of the female reproductive tract were observed using mouse as an experimental model. METHOD: We established transgenic mouse line which produces spermatozoa expressing fluorescent protein in their acrosome (tagged by GFP driven by acrosin promoter) and in their midpiece (tagged by RFP with mitochondria migrating signal, also driven by acrosin promoter). This enabled us to observe the spermatozoa in vivo through the wall of female reproductive tract using a fluorescent microscope (Olympus BX50 and Keyence BZ-8000). Small numbers of spermatozoa were counted by eyes under fluorescent microscope and massive numbers were quantitated by western blotting using anti-sperm antibody. RESULT AND DISCUSSION: The spermatozoa are directly ejaculated in uterus in mouse. The average amount of sperm found in the uterus was examined and it was about 1.8 × 107ˆ right after the coitus. The ejaculated spermatozoa started to migrate into the oviduct and sperm number gradually increased in the oviduct and reached its maximum 9 hours after coitus. The number of oviductal spermatozoa
101
ˆ which is approximately one over one was about 2.9 × 104, thousand of the uterine spermatozoa. Utilizing the fluorescent protein tagged spermatozoa, we observed the spermatozoa in female reproductive tract. The various movies will be shown in this presentation and the process of spermatozoa reaching to the unfertilized eggs will be discussed. doi:10.1016/j.jri.2012.03.431 P 130 Vitamin D3 inhibits inflammatory cascade in endometrial cells through down regulation of TLR signaling A.H. Zarnani 1,2,∗ , N. Rashidi 3 , M. Mirahmadian 3 , M. JeddiTehrani 4 , S. Rezania 5 , A. Kalantari 2 1
Avicenna Research Institute, Academic Center for Education, Culture & Research (ACECR), Reproductive Biotechnology Research Center, Tehran, Iran, Islamic Republic of 2 Tehran University of Medical Sciences, Immunology Research Center, Tehran, Iran, Islamic Republic of 3 Faculty of Medicine, Tehran University of Medical Sciences, Immunology, Tehran, Iran, Islamic Republic of 4 Avicenna Research Institute, ACECR, Monoclonal Antibody Research Center, Tehran, Iran, Islamic Republic of 5 Medical University of Graz, Biophysics Institute, Graz, Austria PROBLEM: Toll-like receptors (TLR) have been appeared as important upstream mediators of inflammation at many human tissues. TLR-mediated inflammatory processes have been suggested to be involved in pathophysiology of such pregnancy-related disorders as spontaneous abortion, preterm labor, preeclampsia, and intrauterine growth restriction. Bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are among the pathogen-associated molecular patterns (PAMP) which induce TLR signaling leading to release of pro-inflammatory cytokines. Over the past decade, clinical evidence has been accumulating that vitamin D3 (VD3) is effective in the treatment of diseases with inflammatory nature. The aim of this study was to investigate the effects of VD3 on production of inflammatory cytokines and expression of TLR2, TLR4 and MyD88, the main adaptor of TLR signaling pathway, in endometrial cells in response to LPS and LTA stimulation. METHODS: Endometrial tissues were obtained from cycling women undergoing operations for benign gynecological conditions. Whole endometrial (WECs) and purified endometrial stromal cells (ESCs) were examined for expression of TLR2, TLR4 and MyD88 genes in LPS- and LTAstimulated or non-stimulated conditions at both gene and protein levels by semi-quantitative RT-PCR, western blotting and flow cytometry, respectively. TLR stimulation was functionally assessed by measurement of TNF-␣, IL-6, and IL-8 production in cell culture supernatants by ELISA. In parallel, immunomodulatory effects of VD3 on the aforesaid parameters were assessed. RESULTS: Isolated ESCs exhibited high expression of CD10 and vimentin, but failed to express cytokeratin indicating their high purity. Flow cytometry analysis demonstrated that ESCs do not express TLR2 and TLR4 at