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1.
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
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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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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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旨在揭示白消安处理对睾丸的损伤机理,提高精原干细胞（SSCs）移植受体制备效率及安全性,缓解或避免因白消安毒性导致的雄性不育。对白消安处理小鼠进行睾丸生物素示踪试验,以验证血睾屏障（BTB）完整性;采用液质联用靶向测定其附睾尾精液中白消安的含量,以验证白消安是否可以直接穿过睾丸BTB进入曲细精管;并对小鼠血清进行炎性因子ELISA测定,以验证其浓度是否发生变化。结果发现,小鼠睾丸注射白消安后,曲细精管内最早24 h可检测到生物素示踪红色荧光,表明BTB已被破坏;注射白消安30 min后,附睾精液中检测到最高水平白消安,随后,其水平逐渐降低;小鼠睾丸注射白消安后,血清中TNF-α、IL-1β和IL-6浓度逐渐增加,36 h后均显著高于对照组（P<0.05）,21 d后达到峰值时,TNF-α浓度为（1 855.51±10.32） pg·mL-1,IL-1β浓度为（293.59±3.34） pg·mL-1,IL-6浓度为（340.30±12.55） pg·mL-1,随后逐渐下降。综上表明,白消安可自由穿越BTB,且于24 h生物示踪素可突破BTB,血清炎性因子TNF-α、IL-1β和IL-6浓度逐渐升高并达显著水平,在21 d达到最高后逐渐下降。 相似文献
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精原干细胞移植技术是一种新兴的动物繁殖技术,可以提高雄性动物的生殖能力.该技术是从适龄雄性供体动物采集精原干细胞,注射入适龄受体动物的生精小管中使其产生精子的技术.精原干细胞移植首先在小鼠试验中获得成功,接着人们将这项技术应用到家畜等大中型动物中并获得成功.随着这项技术的不断深入研究,精原干细胞不但可以在同种间进行移植,而且在异种间的移植也获得成功.通过对培养体系的不断完善,筛选、移植方法的不断改进,可以获得更高的移植成功率.精原干细胞移植为提高优良品种家畜的生产效率、保护野生动物资源、转基因动物的生产及不育症的治疗提供了一种新的方法. 相似文献
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Takashi TANAKA Mito KANATSU-SHINOHARA Takashi SHINOHARA 《The Journal of reproduction and development》2015,61(4):305-316
Spermatogonial stem cells (SSCs) undergo self-renewal divisions to provide the foundation for spermatogenesis. Although Rb1 deficiency is reportedly essential for SSC self-renewal, its mechanism has remained unknown. Here we report that Rb1 is critical for cell cycle progression and protection of SSCs from DNA double-strand breaks (DSBs). Cultured SSCs depleted of Cdkn1b proliferated poorly and showed diminished expression of CDK4 and RB1, thereby leading to hypophosphorylation of RB1. Rb1 deficiency induced cell cycle arrest and apoptosis in cultured SSCs, which expressed markers for DNA DSBs. This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency. Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell
cycle arrest and apoptosis. These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage. 相似文献
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Xinpeng Yang Yue Feng Yang Li Dake Chen Xuanyan Xia Jialian Li Fenge Li 《Reproduction in domestic animals》2021,56(3):416-426
Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis. 相似文献
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精原干细胞（spermatogonial stem cells,SSCs）位于睾丸曲细精管基底小室内,因其具有持续的自我更新能力和分化为精子的能力,被认为是雄性哺乳动物体内唯一能将自身遗传物质传递给后代的成体干细胞。对SSCs的研究已成为干细胞学科研究的热点之一,但目前SSCs的研究多集中于啮齿类动物,而猪SSCs（porcine SSCs,pSSCs）的研究进展相对落后,主要是由于存在以下几个问题：睾丸中pSSCs本身数量极少,且缺乏特异的分子标记;pSSCs的分离纯化技术仍不成熟;pSSCs体外培养体系仍不够完善。这些问题的存在导致pSSCs分离纯化效率低,并且难以在体外长期稳定培养及传代,以至于pSSCs的相关机理研究缺乏稳定的材料,为其体外研究及应用带来了不便。基于以上现状,文章总结了pSSCs体外分离培养及移植的研究进展,详细介绍了pSSCs的分子标记、分离纯化和体外培养的相关方法,以及pSSCs移植技术的研究现状,旨在为pSSCs研究提供参考,以期加快pSSCs在猪的遗传育种与繁殖领域以及男性生殖医学领域的研究和应用。 相似文献
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Apoptosis of male germ cells is a complex phenomenon in many animal species. Understanding its mechanisms could be useful in the diagnosis and therapy of male infertility. To examine the differences of distribution of apoptosis among mouse strains, the terminal transferase-mediated nick end labelling (TUNEL) method was employed. In the testes of MRL mice, many TUNEL-positive cells were identified at the metaphases of meiotic spermatocytes. Morphometrical analysis revealed that metaphase-specific apoptosis occurred at the region between secondary spermatocytes and step 1 spermatids in stage XII seminiferous tubules. In the investigation of the developing first-wave of seminiferous tubules, there were some metaphases showing apoptotic morphology prior to becoming secondary spermatocytes. Details of the apoptotic structure revealed by electron microscopy showed that cellular arrest occurred after the beginning of the M phase of the cell cycle. These results suggested that metaphase-specific apoptosis in the testis of the MRL mouse strain took place at least at the first meiotic division, perhaps showing the spindle assembly checkpoint of the cell cycle. 相似文献
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FRO de Barros RA Worst GCP Saurin CM Mendes MEOA Assumpção JA Visintin 《Reproduction in domestic animals》2012,47(6):887-890
The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 105 viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression. 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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Shinohara T Ishii K Kanatsu-Shinohara M 《The Journal of reproduction and development》2011,57(2):288-295
Stem cells of the side population (SP) phenotype are found in many self-renewing tissues and can be identified by their unique ability to effectively exclude the dye Hoechst 33342. We previously established a method for expanding spermatogonial stem cells (SSCs) in vitro, but the frequency of SSCs is only about 1 to 2%, limiting detailed SSC analyses. In this study, we sought to isolate SSCs from in vitro cultures by exploiting their ability to exclude Hoechst 33342. In contrast to the findings of previous in vivo studies, we found that SP cells developed in a stochastic manner in vitro. Moreover, SP cells in culture were not enriched in SSCs, but they were interconvertible with non-SP cells. Although SP cells were consistently found in testes after transplantation of cultured cells, they were not enriched in SSCs. These results show that SSCs have an unstable SP phenotype and provide evidence that SSCs change their phenotype characteristics in response to their microenvironment. 相似文献
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Manipulation of spermatogonial stem cells in livestock species 总被引：1，自引：0，他引：1
《畜牧与生物技术杂志(英文版)》2019,(4)
We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells(SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology; 2) the approaches for SSC isolation and purification; 3) the available in vitro systems for the stable expansion of isolated SSCs; 4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis; 5) a thorough overview of the techniques of SSC transplantation in livestock species(including the preparation of recipients for SSC transplantation,the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we wil draw special attention to situations where such translation is not possible. 相似文献
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High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5-4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E(2)) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E(2) and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E(2) and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E(2) but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E(2) production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins. 相似文献
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以新生犊牛睾丸为实验对象,应用组合酶法进行支持细胞分离培养,并研究了冷冻保存后支持细胞的生长特性。结果表明:在细胞分离时,消化睾丸组织,分离曲细精管法所获得的细胞悬液中的有效细胞数高于组织剪碎法。支持细胞体外培养,4 h后开始贴壁,3~4 d铺满培养皿底壁,传代后细胞生长较快,2 d即可增殖一代。HE染色,胞质染色较淡,而细胞核染色较深,呈圆形或椭圆形位于细胞质中央或偏位,核仁明显。采用10%FBS+10%DMSO的DMEM液做冷冻液,对细胞进行冷冻保存时,支持细胞的复苏率在65%以上。解冻后的支持细胞体外培养,4h开始有细胞贴壁,24h后大部分细胞贴壁,3~4d铺满培养皿底壁。 相似文献
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白消安（busulfan）对精子发生具有较强毒害作用,可导致雄性不育,是制备精原干细胞（spermatogonial stem cells,SSCs）移植受体的理想药剂。睾丸炎对生精机能也有重要影响,甚至能导致雄性不育。然而,白消安损害血睾屏障（blood-testis barrier,BTB）,影响精子发生的机制尚不清楚,尚不知其是否会导致非传染性睾丸炎症,并影响细胞因子分泌,损害生育能力。因此,本文综述了白消安对BTB的破坏作用、对睾丸细胞相关功能蛋白的影响,以及白消安损伤睾丸的缓解方法,以深入揭示白消安对睾丸细胞和BTB功能与结构的毒害作用,为研发高效安全的SSCs移植受体制备技术,以及化疗药物治疗和环境毒素作用下生精机能科学防护与恢复提供科学参考。 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