• Title/Summary/Keyword: Spermatogonial stem cells

Search Result 48, Processing Time 0.022 seconds

Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro

  • Li, Ting-Ting;Geng, Shuang-Shuang;Xu, Hui-Yan;Luo, Ao-Lin;Zhao, Peng-Wei;Yang, Huan;Liang, Xing-Wei;Lu, Yang-Qing;Yang, Xiao-Gan;Lu, Ke-Huan
    • Journal of Veterinary Science
    • /
    • v.21 no.1
    • /
    • pp.13.1-13.14
    • /
    • 2020
  • Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3+ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

Study on germline transmission by transplantation of spermatogonial stem cells in chicken

  • Lee, Young-Mok;Han, Jae-Yong
    • Proceedings of the Korea Society of Poultry Science Conference
    • /
    • 2006.11a
    • /
    • pp.43-58
    • /
    • 2006
  • As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

  • PDF

Development of In Vitro Culture System for Male Germline Stem Cells in Porcine (돼지 웅성 생식선 줄기세포의 체외배양기법 개발)

  • Kim, Yong-Hee;Kim, Byung-Gak;Lee, Yong-An;Kim, Bang-Jin;Kim, Ki-Jung;Lee, Myeung-Sik;Im, Gi-Sun;Ryu, Buom-Yong
    • Reproductive and Developmental Biology
    • /
    • v.33 no.3
    • /
    • pp.171-177
    • /
    • 2009
  • Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

Methylation Status of H19 Gene in Embryos Produced by Nuclear Transfer of Spermatogonial Stem Cells in Pig

  • Lee, Hyun-Seung;Lee, Sung-Ho;Gupta, Mukesh Kumar;Uhm, Sang-Jun;Lee, Hoon-Taek
    • Reproductive and Developmental Biology
    • /
    • v.35 no.1
    • /
    • pp.67-75
    • /
    • 2011
  • The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

YY1 and CP2c in Unidirectional Spermatogenesis and Stemness

  • Cheon, Yong-Pil;Choi, Donchan;Lee, Sung-Ho;Kim, Chul Geun
    • Development and Reproduction
    • /
    • v.24 no.4
    • /
    • pp.249-261
    • /
    • 2020
  • Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

Development of a Three-dimensional Hydrogel System for the Maintenance of Porcine Spermatogonial Stem Cell Self-renewal

  • Park, Ji Eun;Park, Min Hee;Kim, Min Seong;Yun, Jung Im;Choi, Jung Hoon;Lee, Eunsong;Lee, Seung Tae
    • Journal of Embryo Transfer
    • /
    • v.32 no.4
    • /
    • pp.343-351
    • /
    • 2017
  • Porcine spermatogonial stem cells (SSCs) prefer three-dimensional (3D) culture systems to 2D ones for the maintenance of self-renewal. Of the many 3D culture systems, agar-based hydrogels are candidates for supporting porcine SSC self-renewal, and there are various types of agar powder that can be used. In this study, we sought to identify an agar-based 3D hydrogel system that exhibited strong efficacy in the maintenance of porcine SSC self-renewal. First, 3D hydrogels with different mechanics were prepared with various concentrations of Bacto agar, lysogeny broth (LB) agar, and agarose powder, and the 3D hydrogel with the strongest alkaline phosphatase (AP) activity and greatest increase in colony size was identified for the different types of agar powder. Second, among the porcine SSCs cultured in the different 3D hydrogels, we analyzed the colony formation, morphology, and size; AP activity; and transcription and translation of porcine SSC-related genes, and these were compared to determine the optimal 3D hydrogel system for the maintenance of porcine SSC self-renewal. We found that 0.6% (w/v) Bacto agar-, 1% (w/v) LB agar-, and 0.2% (w/v) agarose-based 3D hydrogels showed the strongest maintenance of AP activity and the most pronounced increase in colony size in the culture of porcine SSCs. Moreover, among these hydrogels, the strongest transcription and translation of porcine SSC-related genes and largest colony size were detected in porcine SSCs cultured in the 0.2% (w/v) agarose-based 3D hydrogel, whereas there were no significant differences in colony formation and morphology. These results demonstrate that the 0.2% (w/v) agarose-based 3D hydrogel can be effectively used for the maintenance of porcine SSC self-renewal.

C-kit Expressing Male Germ Cells were Highly Sensitive to Busulfan Treatment and Apoptosis of Male Germ Cells Induced by Busulfan Treatment was not Caused by Fas/FalsL or p53

  • Ok Do-Won;Lee Mi-Suk;Gwon Deuk-Nam;Kim Jin-Hoe
    • Proceedings of the KSAR Conference
    • /
    • 2002.06a
    • /
    • pp.4-4
    • /
    • 2002
  • Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. Spontaneous apoptosis of germ cells was observed in the testis of adult mice and experimentally induced busulfan treated mice increased this apoptosis to such an extent that there was a decrease in the weight of the testis. (omitted)

  • PDF

Etv5, a transcription factor with versatile functions in male reproduction

  • Eo, Jinwon;Song, Haengseok;Lim, Hyunjung Jade
    • Clinical and Experimental Reproductive Medicine
    • /
    • v.39 no.2
    • /
    • pp.41-45
    • /
    • 2012
  • Transcription factors govern diverse aspects of cell growth and differentiation as major switches of gene expression. Etv5, a member of the E26 transformation-specific family of transcription factors, has many stories to share when it comes to reproduction. Etv5 deficient mice show complex infertility phenotypes both in males and females. In males, the infertility phenotype exhibited by Etv5 deficiency is sexually dimorphic, and it involves both somatic cells and germ cells. In $Etv5^{-/-}$ female mice, the problem is more complicated by hormonal involvement. This review synthesizes old and new information on this versatile transcription factor-from the inadvertent discovery of its role in the testes to its newly discovered role in maintaining spermatogonial stem cells.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

  • Jeon, Hye Lyun;Yi, Jung-Sun;Kim, Tae Sung;Oh, Youkyung;Lee, Hye Jeong;Lee, Minseong;Bang, Jin Seok;Ko, Kinarm;Ahn, Il Young;Ko, Kyungyuk;Kim, Joohwan;Park, Hye-Kyung;Lee, Jong Kwon;Sohn, Soo Jung
    • Toxicological Research
    • /
    • v.33 no.2
    • /
    • pp.107-118
    • /
    • 2017
  • Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

New strategies for germ cell cryopreservation: Cryoinjury modulation

  • Sang-Eun Jung;Buom-Yong Ryu
    • Clinical and Experimental Reproductive Medicine
    • /
    • v.50 no.4
    • /
    • pp.213-222
    • /
    • 2023
  • Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.