Differentiation of murine male germ cells to spermatozoa in a soft agar culture system
Differentiation of murine male germ cells to spermatozoa in a soft agar culture system
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DOI：
10.1038/aja.2011.112
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发表时间：
2012-03-01
影响因子：
2.9
通讯作者：
Huleihel, Mahmoud
中科院分区：
文献类型：
--
作者：
Abu Elhija, Mahmoud;Lunenfeld, Eitan;Huleihel, Mahmoud
建立一个允许睾丸生殖细胞发育为精子的体外系统将对精子发生的研究和未来男性不育的治疗具有重要价值。在本研究中，我们利用三维琼脂培养系统(SACS)建立了体外培养条件，该系统能够诱导睾丸生殖细胞达到精子发生的最后阶段，包括精子的产生。用酶法分离7日龄小鼠睾丸曲细精管，在添加胎牛血清(FCS)的RPMI培养液中，在小管囊的上层培养曲细精管内细胞。囊的下层仅含有RPMI附加FCS的培养液。在培养14天和28天后分离上层的菌落，并根据其大小进行分类。免疫荧光和实时定量聚合酶链式反应分析未分化和分化的精原细胞(Vasa、DAZL、Oct-4、C-Kit、GFR-α-1、CD9和α-6-整合素)、减数分裂细胞(LDH、Crem-1和Boule)和减数分裂后细胞(鱼精蛋白-1、顶体蛋白和SP-10)表达的特异性标志物。我们的结果表明，它可以诱导小鼠睾丸减数分裂前生殖细胞的扩增，并诱导它们分化为囊内的精子。精子形态正常，含有顶体。因此，我们的结果表明，SACS可以作为一种新的体外系统用于小鼠减数分裂前生殖细胞成熟到减数分裂后阶段和形态正常的精子。《亚洲男科学杂志》(2012年)14285-293；DOI：10.1038/aja.2011.112；2011年11月7日在线发布
Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-alpha-1, CD9 and alpha-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa. Asian Journal of Andrology (2012) 14, 285-293; doi:10.1038/aja.2011.112; published online 7 November 2011