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Characteristics and Practical Application of Spermatogonial Stem Cells
Buom-Yong Ryu 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1
Spermatogenesis is a series of complex processes that produce spermatozoa in male testis and it occurs through consecutive cell divisions and differentiation of germ cells (Russell et al., 1990). This process is initiated by a small number of spermatogonial stem cells (SSCs) that are only two or three per 104 testis cells in mouse case, and finally gives rise to many functional spermatozoa. Similar to the characteristics found in other adult stem cells, SSCs have the capability of self‐renewal and differentiation (Meistrich and van Beek, 1993). SSCs that have these two capabilities are the source of maintaining male postnatal fertility for lifetime. SSCs that exist inside the male testis maintain the numerical equilibrium through self‐renew after birth and they are the only germ‐line stem cell that can transfer the genetic information to the next generation through spermatogenesis. Therefore, when genetic modification is performed at the SSC stage, it can produce transgenic offspring in the next generation as well as germ‐line modification so that it can deliver the transformed character stably to the descendants. Past studies regarding the SSC had been dependent on morphological observations due to the absence of a marker system that can distinguish the SSCs. Brinster et al. (1994) published a groundbreaking turning point in identifying characteristics of SSC by developing SSC transplantation technique (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). Utilizing the SSC transplantation technique, the self‐renew and production capability of differentiated tissue derived from transplanted SSC within the recipient’s seminiferous tubule can be directly analyzed. The biological activity of SSCs can also be investigated objectively by the SSC transplantation technique. Since the advent of the SSC transplantation technique, there have been a lot of progresses in the biological field of SSC. Recently, the enrichment technique of SSCs using FACS and specific surface marker, in vitro culture, and genetic modification techniques of SSCs have been developed in rodents. These techniques have potential to enhance the practical applications of SSCs. Characterization and development of useful technique for SSCs are now extending to livestock species.
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),권재희(Jae Hee Kwon),지병철(Byeong Chul Jee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.12
N/A The proportion of male factor infertility due to quantitative and qualitative sperm disorders is approximately 50-60% in infertile couples. In IVF-ET, lower or failed fertilization of oocytes usually results from subnormal count of total motile sperms, but this may occur in infertile couples even with normal sperm count. It has been suggested that some functional defects in sperms are responsible for lower or failed fertilization. Routine semen analysis based on numerical background has limits for the assessment of fertilization capacity of sperm in infertile males, and the andrologic test for the prediction of fertilization capacity must be objective, repeatable, quick, economic, and easily applicable for the clinical settings. The purposes of this study were to develop the analysis method of strict morphology of sperm using the strict criteria as a simple, inexpensive and useful test of sperm fertilization capacity, to establish the normal fertile range and the cut-off value of strict morphology, and to evaluate the validity of strict morphology as a prognostic indicator of fertilization capacity in IVF-ET. In establishing the effectiveness of strict morphology of sperm, ROC curve was used. Among the various thresholds for the prediction of fertilizing ability, normal morphologic value 10.0 corresponding to the value with higher sensitivity and lesser false positive rates was determined as a cut-off value. Using this cut-off point, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of strict morphology for the prediction of fertilization capacity was 73.9%, 81.0%, 80.6%, and 72.7%, respectively. To evaluate the clinical validity of strict morphology as a prognostic indicator of fertilization capacity, this cut-off point was applied to 133 patients undergoing IVF-ET. For the prediction of fertilization rate >30% in IVF-ET, the sensitivity, specificity, PPV, and NPV was 77.3%, 77.8%, 87.2%, and 63.6%, respectively. In conclusion, the strict morphology of sperm is one of the most simple and useful test for the assessment of fertilization capacity of sperm and the prediction of IVF-ET outcomes in infertile couples.
고식적 체외수정시술시 3 일째 배아이식술의 효용성에 관한 연구
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),김정구(Jung Gu Kim),지병철(Byung Ch 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3
N/A Objective: To investigate the positive or negative effect of delaying embryo transfer(ET) one day in IVF-ET. Methods: From May to July, 1997, a total of 64 patients was emolled in this prospective randomized case-controlled study. When the timing of oocyte retrieval was decided, random allocation of patients was made to one of the two groups: day 2 transfer or day 3 transfer. In day 3 transfer group, embryos were cultured in M3 media(Medi-Cult) for further 24 hours. Results: There were no significant differences in age of patients, infertility factor, basal serum FSH level, and serum E2 level on hCG day between two poups, but number of previous IVF-ET cycles was significantly higher in day 3 transfer group(p 0.042). Number of oocytes retrieved, fertilization rate, cleavage rate, and number of embryos transferred had no significant difference, but cumulative embryo score(CES) was significantly higher in day 3 transfer group(p 0.0001). Clinical pregnancy and implantation rates were bigher in day 3 transfer group, but without significance(34.4% vs. 21.9%; 8.7% vs, 5.4%). There were also no significant differences in spontaneous miscarriage and multiple pregnancy rates. Especially in patients over 35 years of age, clinical pregnancy and implantation rates were more higher in day 3 transfer group, but without significance(41.7% vs, 8.3%; 8.5% vs. 1.6%). Conclusion: Considering the higher number of previous cycles in day 3 transfer group, it is at least likely that delaying ET one day may be clinically beneficial in IVF-ET, especially in patients with old age or repeated failure of previous cycles.
폐쇄성 무정자증 환자에서 정자 채취 후 난자 세포질내 정자 주입술의 결과 : 결핵성 부고환염의 영향
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),김정구(Jung Gu Kim),지병철(Byung Ch 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3
N/A Objective: To investigate the intluence of previous tuberculous epididymitis in infertile males with obstructive azoospermia on the outcome of sperm retrieval and intracytoplasmic sperm injection(ICSI) in IVF-ET propam. Methods: Retrospective analysis was paformed in 26 patients with obstructive azoospermia undergoing sperm retrieval and ICSI at Seoul National University Hospital from January, 1996 to August, 1997; 12 cycles in 5 patients with tuberculous obstructive azoospermia(Group A), and 40 cycles in 21 patients with non-tuberculous obstructive azoospermia(Group B). Results: There was no significant difference in the clinical pregnancy rate(PR) per fresh transfer between Groups A and B(20.0%[2/10] vs. 26.8%(11/41)). The rates of embryo implantation and clinical miscarriage were also comparable in both groups(6.3% vs. 11.1%, 50.0% vs. 9.1%). This tendencies were also similar after including five cryopreserved-thawed transfer cycles. Conclusion: Embryo quality and pregnancy outcome in sperm retrieval and ICSI were comparable in both the tuberculous and non-tuberculous obstructive azoospermia patients. Our results suggest that previous history of tuberculous epididymitis in patients with obstructive azoospermia does not affect the outcome of sperm retrieval and ICSI.
미수정 및 저수정율의 기왕력이 있는 체외수정시술 환자에서의 난자 세포질내 정자 주입술 : 중증 남성인자 환자와의 비교
문신용(Shin Yong Moon),최영민(Young Min Choi),김석현(Seok Hyun Kim),오선경(Sun Kyung Oh),서창석(Chang Suk Suh),이진용(Jin Yong Lee),정병준(Byeong Jun Jung),김희선(Hee Sun Kim),류범용(Buom Yong Ryu),방명걸(Myung Geol Pang),김정구(Jung 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.2
N/A Objective: The purpose of this study was to determine whether intracytoplasmic sperm injection(ICSI) could overcome the defects of oocytes in IVF-ET patients with previous fertilization failure by conventional fertilization technique. Design: Retrospective study Materials and Methods: A total of 119 ICSI cycles in 57 IVF-ET patients performed from May, 1995 to December, 1997 was enrolled. Subjects were divided into two groups: FR group included 66 ICSI cycles in 35 patients with normal sperm who underwent ICSI due to past history of failed or poor fertilization in the previous IVF-ET cycles, and OAT group included 53 ICSI cycles in 22 patients with severe oligoasthenoterato- zoospermia(OAT) which was defined as sperm concentration < 20 million/ml, mo#dlity < 30% and normal morphology < 4% by strict morphologic criteria. The outcomes of ICSI were analyzed and compared in both groups. Results: The age of female patients, basal serum FSH level, and the numbers of oocytes retrieved and metaphase II oocytes were all comparable in both groups. The fertilization rate after ICSI was similar in both groups(68.7+-25.3% vs. 67.7+-24.5%), as were the cleavage rate of normally fertilized oocytes(93.1+-21.4% vs. 89.3+-21.6%), the number of embryos transferred(4,00+-1.98 vs. 4.64+-2.10), and cumulative embryo score(CES) indicating the quality of embryos(47.3+-33.2 vs. 54.1+-33.2). The implantation rate(4.3+-10.5% vs. 3.8+-11.0%) and the clinical pregnancy rate per cycle(15.2% vs. 13.2%) were also comparable in both groups. Conclusions: Although it has been shown that there is a higher risk of chromosomal abnormalities in oocytes from IVF-ET patients with pevious failed or poor fertilization, higher implantation and clinical pregnancy rates wer#e not observed in patients with OAT following ICSL Therefore, the functional defect of sperm such as loss of capacitation, defect of aaasome reaction, and abnormality of nucleus decondensation should be also considered in patients with previous failed or poor fertilization.
Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing
Kim, Ki-Jung,Lee, Yong-An,Kim, Bang-Jin,Kim, Yong-Hee,Kim, Byung-Gak,Kang, Hyun-Gu,Jung, Sang-Eun,Choi, Sun-Ho,Schmidt, Jonathan A.,Ryu, Buom-Yong Elsevier 2015 Cryobiology Vol.70 No.2
<P><B>Abstract</B></P> <P>Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200mM trehalose is an efficient cryopreservation protocol for bovine SSCs.</P>
Kim, Bang-Jin,Lee, Yong-An,Kim, Ki-Jung,Kim, Yong-Hee,Jung, Mi-Seon,Ha, Seung-Jung,Kang, Hyun-Gu,Jung, Sang-Eun,Kim, Byung-Gak,Choi, Yu-Ri,Do, Jeong Tae,Ryu, Buom-Yong D.A. Spandidos 2015 International journal of molecular medicine Vol.36 No.1
<P>Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.</P>