共查询到20条相似文献,搜索用时 15 毫秒
1.
Min Hee Park Jung Im Yun Eunsong Lee Seung Tae Lee 《Reproduction in domestic animals》2019,54(11):1497-1500
A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α9 and β1 subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α9β1 actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α9β1 on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α9β1 significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α9 and β1 subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state. 相似文献
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Narong TIPTANAVATTANA Mongkol TECHAKUMPHU Theerawat THARASANIT 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(11):1347-1353
The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal
donors is currently poor primarily, because of contamination with other testicular cells.
This study aimed to purify SSC-like cells using different extracellular matrixes and a
discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology
and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic
digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish.
The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment
2). Experiment 3 was performed to test whether or not the additional step of Percoll
gradient density centrifugation could really improve purification of SSC-like cells.
Testicular histology revealed complete spermatogenesis with laminin expression essentially
at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4
in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells
expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest
SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other
incubation times and gelatin treatment (P<0.05). Percoll treatment
prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ±
0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51%
for GFRα-1, P<0.05). These attached cells demonstrated a typical
SSC-like cell morphology and also expressed POU5F1, RET
and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient
density centrifugation and laminin plating highly enriched the SSC-like cells
population. 相似文献
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Xue-Ming Zhang 《Anatomia, histologia, embryologia》2021,50(1):13-14
Identification of the specific biomarkers is of great importance to enrich and expand the gonocytes or spermatogonial stem cells (SSCs) in livestock. The glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFRα-1) is a conserved marker of the gonocytes/SSCs in multiple species including rodents, primates and human; however, its expression in bovine gonocytes/SSCs is debated. Recently, we and other teams clearly demonstrated the expression of GFRα-1 in bovine gonocytes/SSCs. This is useful for bovine gonocytes/SSCs-related research or application. Nonetheless, new methods still need to be developed to identify the undifferentiated spermatogonial subsets in large livestock and elucidate their spermatogenic potency. 相似文献
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为研究如何在体外获得更好的精原干细胞培养效果,以出生后6~8 d的小鼠为对象,应用两步酶消化法和差速贴壁法分离纯化精原干细胞.分别将浓度为100、200、300和400 ng/mL的红景天多糖加入以Sertoli细胞为饲养层的精原干细胞培养液中,以不添加红景天多糖组为对照,通过RT-PCR法和碱性磷酸酶(AP)染色鉴定细胞,MTT法研究红景天多糖对精原干细胞增殖的影响.RT-PCR和染色结果显示,分离得到的细胞为精原干细胞;MTT结果显示试验组比对照组细胞数量有显著增多(P<0.05),增殖率可达152%,且红景天多糖的最适添加量300 ng/mL.说明红景天多糖能显著促进小鼠精原干细胞的体外增殖. 相似文献
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JI Yun HJ Park MH Park MS Kim JH Choi E Lee SP Gong JM Lim ST Lee 《Reproduction in domestic animals》2014,49(5):705-710
Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats. 相似文献
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Wahono Esthi Prasetyaningtyas Ni Wayan Kurniani Karja Srihadi Agungpriyono Mokhamad Fahrudin 《Animal Science Journal》2020,91(1)
The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO2 atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells. 相似文献
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Huimin Zhao Tingting Li Huan Yang Muhammad Usman Mehmood Yangqing Lu Xingwei Liang Xiaogan Yang Huiyan Xu Kehuan Lu Shengsheng Lu 《Reproduction in domestic animals》2019,54(12):1574-1582
The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini‐pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line‐derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha‐1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo‐derived thioguanine‐ and ouabain‐resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT‐PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1‐, DBA‐ and CDH1‐positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1. 相似文献
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旨在研究褪黑素(melatonin,MT)对体外培养猪精原干细胞(spermatogonial stem cells,SSCs)的作用机制。本研究采集3头7日龄健康大白公猪睾丸,利用差速贴壁法获得SSCs。后经形态学观察、碱性磷酸酶染色、标记基因检测及免疫荧光染色鉴定后以SSCs作为试验材料,设置MT浓度梯度(0、50、250、500、1 000 μmol·mL-1)组处理SSCs,每组设3个重复(n=3),空白对照组加入0.1% DMSO处理,分别检测添加MT后猪SSCs的细胞活力、活性氧(reactive oxygen species,ROS)水平、谷胱甘肽(glutathione,GSH)含量及凋亡基因表达变化。结果显示:1)分离的克隆团细胞具有SSCs的生长特性,可被碱性磷酸酶染色并表达干细胞标志基因OCT4、SOX2和SSCs标志基因NANOG、PLZF、UCHL1;2)50 μmol·mL-1以上的MT在处理48 h后可显著提高SSCs的细胞活力(P<0.05);3) MT可显著降低猪SSCs内ROS水平(P<0.05),极显著增加细胞内GSH含量(P<0.01);4) MT可显著抑制猪SSCs内凋亡蛋白Bax和Caspase3的表达(P<0.05)。MT具有清除猪SSCs中的ROS,提高总GSH含量,抑制凋亡基因表达进而提高细胞活力的作用,可为养殖过程中提高公猪繁殖性能提供参考。 相似文献
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Kei ISHII Mito KANATSU-SHINOHARA Takashi SHINOHARA 《The Journal of reproduction and development》2014,60(1):37-46
Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the
seminiferous tubules of the testis of infertile animals. However, only 5–10% of the
transplanted cells colonize recipient testes. In this study, we analyzed the impact of
cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based
cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC
activity of mouse germline stem (GS) cells, a population of cultured spermatogonia
enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M
phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only
showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor
receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this
cell cycle-dependency was not observed when cells were transplanted into immature pup
recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells,
suggesting that cells in the G1 phase may passage through the BTB more readily than cells
in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC
migration to the niche. 相似文献
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《中国畜牧杂志》2019,(12)
精原干细胞(SSCs)是具备自我更新与分化潜能的生殖干细胞,其通过协调自我更新与分化之间的动态平衡来维持SSCs群体数量的稳定并保证精子发生的持续进行。SSCs的自我更新和分化受细胞因子、转录因子和其他增殖分化相关蛋白等多种因素调控。MicroRNAs(miRNAs)是一类很小的非编码内源性RNA,广泛存在于机体各组织器官,且差异性表达于精子发生的各个时期,在精子发生过程中发挥重要调控作用。本文主要综述了miRNAs对SSCs自我更新和分化相关转录因子及其他蛋白等表达的调控,并简要论述了miRNAs调控SSCs增殖与分化的其他方式,以期为后续SSCs的研究提供科学参考。 相似文献
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Haiying CHI Masahiro SATO Mitsutoshi YOSHIDA Kazuchika MIYOSHI 《Animal Science Journal》2012,83(1):88-93
α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B4 (BS‐I‐B4). Staining with BS‐I‐B4 demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage. 相似文献
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Narong TIPTANAVATTANA Araya RADTANAKATIKANON Poul HYTTEL Hanne HOLM Supranee BURANAPRADITKUN Piyathip SETTHAWONG Mongkol TECHAKUMPHU Theerawat THARASANIT 《The Journal of reproduction and development》2015,61(6):581-588
The development of germ cells has not been entirely documented in the cat especially the transition phase of
the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development
and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes
were divided into 3 groups according to donor age (I, < 4 months; II, 4–6 months; and III, > 6 months).
In Exp. 1, we studied testicular development by histology, transmission electron microscopy and
immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow
cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3).
Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of
SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the
highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1+ cells (14.89 ±
5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation
in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed
mRNA for GFRA1, ZBTB16, RET and POU5F1.
Our study found that the G/SSC transition occurs at 4–6 months of age. This period is useful for isolation and
improves the establishment efficiency of cat SSCs in vitro. 相似文献
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本研究首先将水牛睾丸经二步酶消化法制成单细胞悬液,依次采用差速贴壁法和Percoll不连续密度梯度离心法分离和纯化精原细胞。在制备好的小鼠胎儿成纤维细胞饲养层上,采用含2.5%血清的培养液对精原细胞进行体外培养,观察血清和成纤维细胞对精原细胞生长的影响,并在体外成功培养了2周通过提取体外培养精原干细胞总FZNA,设计引物并对其进行基因鉴定和免疫细胞化学鉴定,证实体外培养所得的细胞仍保持有精原干细胞的特性,并且该克隆是处在未分化状态的精原干细胞形成的。上述研究可为体外建立水牛精原干细胞长期培养体系提供技术支撑。 相似文献
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Spermatogonial stem cells (SSCs) reside within specialized microenvironments called 'niches', which are essential for their maintenance and self-renewal. In the mammalian testis, the main components of the niche include the Sertoli cell, the growth factors that this nursing cell produces, the basement membrane, and stimuli from the vascular network between the seminiferous tubules. This review focuses on signalling pathways maintaining SSCs self-renewal and differentiation and describes potential mechanisms of regulation of the spermatogonial stem cell niche. 相似文献