अलगाव और इन विट्रो में सक्रियकरण Caenorhabditis एलिगेंस शुक्राणु
Summary
अलग और पुरुष से spermatids सक्रिय करने के लिए एक प्रोटोकॉल<em> सी. एलिगेंस</em> यहाँ वर्णित है. पुरुष विज्ञप्ति spermatids के पीछे के अंत काटना. spermatids protease के अलावा द्वारा सक्रिय किया जा सकता है है.
Abstract
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 oocytes1. 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 worm2. 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 chromosome3. 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 population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. 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 spermatids2.
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. Shakes5, and Nelson6 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 process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.
Protocol
Discussion
विशेष म्यूटेंट का विश्लेषण करने के अलावा, इस प्रोटोकॉल अन्य महत्वपूर्ण अनुप्रयोगों, जैसे उम्र बढ़ने के साथ शुक्राणु morphology का विश्लेषण है. Spermatids और अलग शुक्राणु इस प्रोटोकॉल का उपयोग जैसे अन्य बहाव के प्र…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Diane सी., हम शमूएल वार्ड धन्यवाद हिलाता है और इस तकनीक में अग्रणी करने के लिए ग्रेगरी नेल्सन. हम सक्रिय शुक्राणु दिखा वीडियो के लिए धन्यवाद पवन Kadandale और ब्रायन Geldziler, Singson उपयोगी विचार – विमर्श के लिए प्रयोगशाला के सदस्यों, CGC उपभेदों और NIH के लिए अनुदान (R01HD054681) के माध्यम से हमें समर्थन करने के लिए.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Tuberculin syringe | Becton Dickinson | 309623 | Syringe: 1 ml, needle: 27G X ½ in |
|
| Pap pen | Zymed | 00-8888 | ||
| VWR VistaVision HistoBond Microscope Slides |
VWR International | 16004-406 | Dimension: 75X25X1mm |
|
| Cover glass | VWR International | 48366045 | ||
| Protease | Sigma | P-6911 |
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