• Clinical and Experimental Reproductive Medicine
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• v.48 no.4
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• pp.352-361
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• 2021

Objective: The study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. Methods: In total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5. Results: In total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). Conclusion: Cilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

• Development and Reproduction
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• v.12 no.2
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• pp.125-132
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• 2008

Melatonin (N-acetyl-5-methoxytryptamine), a major hormone of pineal gland in vertebrates, is known to be associated with regulation of the dynamic physiological functions in general and has some functions on reproduction in the ovarian follicles in particular. And its antioxidant properties as a scavenger are also reported. The aim of this study was to investigate the effect of melatonin on the in vitro maturation of mouse germinal vesicle (GV)-stage oocytes. Oocyte maturation, apoptosis, and mRNA expression of melatonin receptor were analyzed in the cumulus cell-enclosed oocytes (CEOs) cultured with melatonin for 18 h. The CEOs were obtained from 3 wk-old ICR female mice cultured in media with 0, 0.1 nM, 10 nM, or 1,000 nM melatonin for 18 h. And then the extrusion of the first polar body was assessed to evaluate the maturation rate. The apoptosis and mRNA expression of melatonin receptor (Mtnr1-a and Mtnr1-b) in cumulus cells of each group were measured by TUNEL assay, ELISA, and real time RT-PCR after in vitro maturation(IVM). The addition of melatonin in the IVM medium significantly improved nuclear maturation of the mouse GV oocytes and the highest maturation rate were obtained from the group treated with 1,000 nM melatonin. Apoptosis was not detected in IVM oocytes, but detected in cumulus cells. And cumulus cells treated with 1,000 nM melatonin exhibited significantly lower apoptosis. In the group treated with 1,000 nM melatonin, the expression of melatonin receptor mRNA was decreased in CEOs. In conclusion, melatonin has a potentially important role for regulating oocyte maturation and reduces the apoptosis of cumulus cells in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.299-307
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• 2003

Objectives: The present study was conducted to evaluate the mobile transposon-like element, clone MTi7 (MTi7) expression in the mouse ovary and to determine its role(s) in the mouse oocytes by RNA interference (RNAi). Methods: MTi7 mRNA expression was localized by in situ hybridization in day5 and adult ovaries. Double stranded RNA (dsRNA) was prepared for c-mos, a gene with known function as control, and the MTi7. Each dsRNA was microinjected into the germinal vesicle (GV) stage oocytes then oocyte maturation and intracellular changes were evaluated. Results: In situ hybridization analysis revealed that MTi7 mRNA localized to the oocyte cytoplasm from primordial to preovulatory follicles. After dsRNA injection, we found 43-54% GV arrest of microinjected GV oocytes with 68%-90% decrease in targeted c-mos or MTi7 mRNA. Conclusions: This is the first report of the oocyte-specific expression of the MTi7 mRNA. From results of RNAi for MTi7, we concluded that the MTi7 is involved in the germinal vesicle breakdown in GV oocytes, and MTi7 may be implicated with c-mos for its function. We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions.

• Parasites, Hosts and Diseases
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• v.54 no.5
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• pp.653-658
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• 2016

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.183-192
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• 1993

A transmission electron microscopic study was performed to observe the ultrastructures of the male germinal cells and spermatozoa of Fibricola seoulensis. Spermatogonia were found in the periphery of the testis and characterized by large nuclei and comparatively little cytoplasms. Spermatocytes contained an oval to spherical nucleus. Their nuclear volume was little larger in comparative to that of cytoplasm, and the chromatin was comparatively little. The early spermatids were characterized by a great amount of cytoplasm, and numerous mitochondria encircled the nucleus. In a more advanced spermatids the electron-dense strands of chromatin appeared in the nucleus, and a pair of rootlet of the axoneme and a microtubule-organizing center (MTOC) were observed near the nucleus. The sectioned spermatozoa were found in the testis and the seminal vesicle. Their cross sectional views were divided into 6 types when they were distinguished on the basis of the morphology and components. The spermatozoa of F. seoulensis showed two flagella of 9+1 type axoneme.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.26-31
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• 2001

Background : Follicular dendritic cells (FDCs) play key roles during T cell-dependent humoral immune responses by allowing antigen-specific B cells to survive, proliferate, and differentiate within the FDC networks of secondary follicles, i.e., germinal centers (GC). Methods: A novel monoclonal antibody, 3C8, was generated by immunizing with an FDC line HK, in order to understand the molecular signals involved in the FDC-B cell interactions in the microenvironment of the GC. Results: The 3C8 antibody did not bind to mononuclear cells, including T cells, B cells, and monocytes. Murine L929 and human skin fibroblasts exhibited no or little reactivity to 3C8. However, 3C8 specifically recognized HK cells by flowcytometry. Furthermore, the antigen recognized by 3C8 was restricted to the GC of the human tonsil. Dendritic networks of the GC were intensely stained by 3C8, but cells outside the GC were not. Conclusion: Our results suggest that the antigen 3C8 may play some unique role on FDCs during the GC reactions.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.2
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• pp.58-67
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• 2012

Objective: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). Methods: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). Results: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. Conclusion: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3037-3046
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• 2012

The diffuse large B-cell lymphoma (DLBCL) encompasses two major groups of tumors with uneven survival outcomes - germinal center B-cell (GCB) and non-germinal center B-cell (non-GCB). In the present study, we investigated the expression of GCB markers (BCL-6 and CD10) and non-GCB markers (CD138 and MUM-1) in an effort to evaluate their prognostic value. Paraffin-embedded tumor biopsies of 46 Jordanian DLBCL patients were analyzed, retrospectively, by immunohistochemistry to investigate the expression of BCL-6, CD10, CD138 and MUM-1. In addition, survival curves were calculated with reference to marker expression, age, sex and nodal involvement. Positive expression of BCL-6, CD10, CD138 and MUM-1 was shown in 78%, 61%, 39% and 91% of the cases, respectively, that of BCL-6 being associated with better overall survival (p = 0.02), whereas positive CD138 was linked with poor overall survival (p = 0.01). The expression of CD10 and MUM-1 had no impact on the overall survival. Among the clinical characteristics studied, diagnosis at an early age, nodal involvement and maleness were associated with a higher overall survival for DLBCL patients. Our results underline the importance of BCL-6 as a marker of better prognosis and CD138 as a marker of poor prognosis for DLBCL patients.

• Biomolecules & Therapeutics
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• v.30 no.6
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• pp.546-552
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• 2022

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcK^P) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcK^P showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-Fc^M). The animal group injected with EpCAM-FcK^P (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-Fc^M (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

• IMMUNE NETWORK
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• v.5 no.2
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• pp.124-129
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• 2005

Background: CM1 (centrocyte/-blast marker 1) is originally defined as a germinal center B cell marker. It is known that CM1 plays a critical role on B cell development in germinal center. In addition, we have found that CM1 is expressed on lymphoma cell lines, such as Raji, Ramos and IM-9. This means that CM1 might be served as a tumor marker as well. In the present study, we examined the expression of CM1 on the surface of the other tumors and the possibility of the development of tumor screening ELISA kit by using CM1. Methods: First, we have examined the expression of CM1 on stomach cancer and hepatoma, which are predominantly (discovered) occurred in Korean, by flow cytometry analysis. After purifying of CM1 antigen from Raji and Ramos, the optimal ELISA condition was determined. And then we compared the level of CM1 between normal individuals and cancer patients by ELISA. To decrease the non-specific binding of anti-CM1 mAb with serum components except CM1 and to enhance the diagnostic accuracy, albumin depletion spin column was used. Results: CM1 was highly expressed on stomach cancer and hepatoma cell lines. In addition, we have also confirmed the increased CM1 expression on cancer patients. The difference of CM1 expression between normal individuals and cancer patients were more clearly observed, after deletion of serum albumin by using albumin depletion spin column. Conclusion: Based on the results from this study, CM1 might be a useful molecule for the early diagnosis of cancer. In addition, further studies for the increase of ELISA sensitivity and appropriate albumin depletion methods should be needed.