Protocol - Phase contrast microscopy of Drosophila live testes
It allows to preliminarily classify mutant sterile males based on the morphologies observed on the key stages of spermatogenesis.
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Dissect testes from a newly eclosed male in fresh Testis Buffer (0–1 d old)
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RT
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1'
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Newly eclosed males are used because they show the best morphology.
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|
Transfer the testes to a drop of Testis Buffer on a clean microscope slide
|
RT
|
10''
|
|
|
Open up the testes by cutting halfway along their straight portion.
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RT
|
10''
|
The diameter of the drop of buffer should be approx 7 mm to get good preparations under a 22 × 22-mm2 cover slip. Cysts of spermatocytes or spermatids should stay intact.
|
|
Place a clean cover slip over the testes; this will gently squash the cells.
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RT
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1''
|
|
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Take pictures under a phase-contrast microscope.
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RT
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to 20'
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Squash preparations are only good for approx 20 min. After that, the cells are usually too flat and dead to observe.
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Other informations
Nuclear morphology can be examined in live squashes by staining the DNA with the vital dye Hoechst 33342. This is included in the Testis Buffer at 2–5 μg/mL during dissection. Proceed as described abobe, but allow the testes to sit in the buffer for 5 min after dissection and before adding the cover slip.
If scoring for sperm motility, it is important to note that many males have no mature sperm for about 12 h after eclosion. To be sure of the absence of motile sperm from a mutant, keep males isolated from females for 3 days, then dissect their seminal vesicles. The seminal vesicle from males that produce normal motile sperm will normally be slightly opaque. When it is opened, the sperm spill out. Motile sperm will show a shimmering effect visible even under the dissecting microscope.
Validation info
The protocol has been both published and extensively used to obtain data used in peer-reviewed journals
| Citations |
|---|
| White-Cooper H. Spermatogenesis. In "Drosophila Cytogenetics protocols". Henderson, Daryl S. (Ed.) Humana Press Totowa, NJ. 2004);45-76 |
| Di Cara F, Morra R, Cavaliere D, Sorrentino A, De Simone A, Polito CL, Digilio AF. Structure and expression of a novel gene family showing male germline specific expression in Drosophila melanogaster. Insect Mol Biol. 2006;15:813-22 |
| Di Cara F, Cavaliere D, Galliero V, Polito LC, Digilio FA. Expressional and functional analysis of the male-specific cluster mst36F during Drosophila spermatogenesis. Insect Mol Biol. 2010;19:807-813 |
Funded by: Grants from the Regione Campania (Law n. 5)
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Preparation of growth medium for D. melanogaster.pdf
(application/pdf
54Kb)
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Anesthetizing D. melanogaster.pdf
(application/pdf
25Kb)
