Drosophila melanogaster and Eucypris virens giant spermatozoa as visualized by cell inclusion in microgels

A new technique, based on live Sperm Inclusion in Microgels (SIM), allows quick and easy analysis of giant spermatozoa under bright field or fluorescence microscopy. The technique has been assayed on Drosophila melanogaster (Diptera) and Eucypris virens (Ostracoda) spermatozoa and based on the inclusion of freshly obtained male gametes in low melting agarose microgels at 37 degrees C to prevent cell damage. Gametes spread onto pretreated slides are dehydrated and directly observed under phase contrast microscopy or stained with specific fluorochromes for DNA or proteins. Results show that the morphology of whole sperm is highly preserved allowing identification of sperm characteristics difficult to visualize after using standard fixation procedures. In Drosophila melanogaster, SIM allows the simultaneous visualization of the complete flagellum plus a clear delineation of the DNA. It also allows observation of morphological changes in the perforatorium as sperm elongation takes place. In E. virens, SIM slides visualized under phase contrast and fluorescence microscopy show three main morphological regions on the entire spermatozoa. Spermatozoa of this species are auto-fluorescent under wavelength excitation from 387 to 562 nm. In spite of this, clear localization of the DNA molecule at the posterior 1/3 part of the whole spermatozoa can also be achieved after single DAPI staining or in combination with Mercuri-diBrom-Fluorescein, a fluorochrome for protein targeting.