Isolation and in vitro activation of Caenorhabditis elegans sperm.
Isolation and in vitro activation of Caenorhabditis elegans sperm.
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DOI：
10.3791/2336
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发表时间：
2011-01-31
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影响因子：
--
通讯作者：
Singson, Andrew
中科院分区：
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作者：
Singaravelu, Gunasekaran;Chatterjee, Indrani;Singson, Andrew
雄性和雌雄同体是C.优雅的变形虫精子由雄性和雌雄同体产生。在配子发生的早期阶段，雌雄同体的生殖细胞分化成有限数量的精子（大约300个），并储存在一个叫做受精囊的小“袋子”中。后来，雌雄同体继续产生卵母细胞。相反，男性在整个成年期只产生精子。雄性产生如此多的精子，以至于它占典型成年蠕虫总细胞的50%以上。因此，从雄性中分离精子比从雌雄同体中分离精子容易。由于X染色体的自发不分离，只有一小部分雄性是自然产生的。将雌雄同体与雄性杂交，或者更方便地，引入突变以产生Him（雄性高发病率）表型，是可以丰富雄性群体的一些策略。通过观察尾巴的形态，可以很容易地将雄性与雌雄同体区分开来。雌雄同体的尾巴是尖的，而男性的尾巴是圆形的交配结构。切断尾巴会释放出大量储存在雄性生殖道内的精子细胞。在体视显微镜下使用27号针进行解剖。由于精子细胞不与任何其他细胞物理连接，液压排出男性身体的内部内容物，包括精子细胞。在一小滴“精子培养基”上直接解剖雄性。精子细胞对pH值的变化敏感。因此，在精子培养基中使用HEPES，一种具有良好缓冲能力的化合物。精子培养基中的葡萄糖和其他盐有助于维持渗透压，以保持精子的完整性。精子细胞减数分裂后分化为精子称为精子发生或精子激活。Shakes和纳尔逊先前表明，圆形精子细胞可以通过加入各种活化化合物（包括链霉蛋白酶E）诱导分化为精子。在这里，我们展示了体外精子发生的C。用Pronase E.成功的精子发生是育性的先决条件，因此精子发生缺陷的突变体是不育的。近年来，一些突变体已被证明在精子发生过程中有特异性缺陷。在体外激活新型Spe（精子发生缺陷）突变体过程中发现的异常将有助于我们发现参与这一事件的其他参与者。
Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm--around 300--and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for > 50% of the total cells in a typical adult worm. Therefore, isolating sperm from males is easier than from that of hermaphrodites. Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population. Males can be easily distinguished from hermaphrodites by observing the tail morphology. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures. Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids. Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm. Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes, and Nelson previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E. Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.