Materials and Reagents

Scanning Electron Microscopy (SEM) and the motile sperm cell architecture

Pipette tips 1-200 µl (Carolina Biological Supply, catalog number: 215055 )
Pasteur pipettes (Fisher Scientific, catalog number: 13-678-4)
Manufacturer: Corning, catalog number: C7095B5X .
1.5-ml centrifuge tubes (USA Scientific, catalog number: 1615-5500 )
Glass coverslips 22 x 22 mm (Fisher Scientific, catalog number: 12-542B )
Male plants with mature sperm cells
1. Pellia epiphylla
2. Conocephalum conicum
3. Equisetum arvense
4. Ceratopteris richardii
Sorensens phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences, catalog number: 11600-10 )
Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16120 )
Sodium cacodylate buffer, 0.2 M, pH 7.4 (Electron Microscopy Sciences, catalog number: 11652 )
Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
Ethanol (Decan Laboritories, catalog number: 2705HC )
Hexamethyl disilizane (HMDS) (Electron Microscopy Sciences, catalog number: 16700 )
0.01 M phosphate buffer (pH 7.2) (see Recipes)
0.05 M phosphate buffer (pH 7.2) (see Recipes)
0.02 M phosphate buffer (pH 7.2) (see Recipes)
2.5% glutaraldehyde (see Recipes)
0.05 M sodium cacodylate buffer (pH 7.2) (see Recipes)
2% aqueous osmium tetroxide (see Recipes)

Equipment

Scanning electron microscope (SEM) (FEI, model: Quanta^TM 450 FEG )
Shaker (Thermolyne, model: M-16715 )
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: mySPIN^TM 6 , catalog number: 75004061)
Oven (General Signal, model: Gravity convection )
SEM Specimen Mount Stubs, Aluminum Slotted Head (Electron Microscopy Sciences, catalog number: 75220 )
Samdri 790 Critical Point Dryer (Tousumis)
Sputter coater (Denton Vacuum, model: Desk V )

Procedure

Scanning Electron Microscopy (SEM) and the motile sperm cell architecture:
SEM examination of mature sperm cells has enabled rapid visualization and comparisons of whole gamete architecture compared with laborious TEM observations and cell reconstructions. The diversity of gamete architecture and organization of flagella in mature cells is readily seen in the SEM (Figure 1).

Figure 1. Scanning electron micrographs of plant sperm cells. A. Pellia epiphylla, a simple thalloid liverwort. This is one of the longest sperm cells produced by bryophytes. The two flagella (f) are staggered in their insertion near the cell anterior (a) and the cell body consists of a long cylindrical nucleus (n) with a plastid and mitochondrion at the posterior (arrow). B. Conocephalum conicum, a complex thalloid liverwort. This sperm cell is much smaller than that of Pellia. The two flagella (f) are inserted anteriorly (a) and are much longer than the cell body, which consists of a cylindrical nucleus (n) and posterior cytoplasm that in this case is not fully extended. C. Equisetum arvense, a eusporangiate fern. The approximately 36 basal bodies (bb) attach flagella (f) along the anterior coils of the cell. The nucleus (n) extends to the cell posterior (not visible). D. Ceratopteris richardii, a leptosporangiate fern. This cell coils four times and has about 80 flagella (f) inserted anteriorly (a). The cylindrical nucleus (n) extends over three coils and tapers at the cell posterior. Bars = 5.0 µm (A), 1.0 µm (B, C, D).

Using excised male tissue (antheridia of male gametophytes), place several antheridia in each droplet (ca. 100 µl) of 0.01 M phosphate buffer, pH 7.2 (see Recipes), on a dental wax plate, cover to retard evaporation, and leave overnight at room temperature to facilitate release of sperm. Alternatively, excised antheridia can be placed in 1.5 ml Eppendorf centrifuge tubes adding 1 ml of 0.01 M phosphate buffer, pH 7.2 to completely cover tissue and left overnight. Antheridial tissue can be removed and discarded the next day. Swimming sperm will be in the buffer.
Remove tissue and place the droplet in a 1.5 ml Eppendorf centrifuge tube containing 1 ml 2.5% glutaraldehyde (see Recipes) in 0.05 M sodium cacodylate buffer, pH 7.2 (see Recipes).
Shake tube gently and fix the spermatozoid suspension for 1 h at room temperature.
Note: From this point on, spermatozoids are concentrated by centrifugation at 100 x g for 5 min between each solution change. Because the sperm are colorless, you will most likely not see a pellet after centrifugation.
Remove the glutaraldehyde supernatant down to 0.1 ml leaving the pellet at bottom of tube. Rinse fixed sperm cells three times (10 min each) in 1 ml 0.05 M sodium cacodylate buffer.
Post-fix tissue for 20 min in 1 ml 2% aqueous osmium tetroxide (see Recipes).
Rinse specimens twice in 1 ml double distilled autoclaved water (10 min), once each in 25%, 50% and in 75% ethanol (10 min), twice in 100% ethanol (10 min), and then twice in 100% hexamethyl disilizane (HMDS; 10 min).
Pipet sperm cells in 1 ml HMDS on to a glass coverslip affixed to a standard aluminum pin-type mounting stub and dry for 20 min in a 60 °C oven.
Specimens on mounting stubs are sputter-coated with an ultra-thin layer (ca. 390 nm) of a conducting metal (palladium–gold) that prevents charging from non-conducting biological specimens, increases the number of secondary electrons from the specimen surface that improves topographical contrast, and minimizes damage to the specimen.
Basic steps:
1. Vacuum down sputter coater
2. Out-gas with Argon gas
3. Apply voltage
4. Open gas to the specific emissions
5. Coat with ca. 390 nm palladium–gold
View on a high-resolution SEM.

Note: Often cells aggregate around the edges of the coverslip, so it is better to put a very small drop of sperm cell suspension in HMDS in the middle of the coverslip to dry.

Recipes

0.01 M phosphate buffer (pH 7.2)
Add 5 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 95 parts double distilled autoclaved water
0.05 M phosphate buffer (pH 7.2)
Add 25 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 75 parts double distilled autoclaved water
0.02 M phosphate buffer (pH 7.2)
Add 1 part 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 9 parts double distilled autoclaved water
2.5% glutaraldehyde
1. Add 1 part 10% glutaraldehyde to 1 part double distilled autoclaved water
2. Add 1 part 0.20 M Sorensen’s phosphate buffer (pH 7.2) to 1 part double distilled autoclaved water
3. Add 1 part 5% glutaraldehyde to 1 part 0.10 M Sorensen’s phosphate buffer (pH 7.2)
0.05 M sodium cacodylate buffer (pH 7.2)
Add 1 part 0.30 M sodium cacodylate buffer (pH 7.2) to 5 parts double distilled autoclaved water
2% aqueous osmium tetroxide
Add 1 part 4% aqueous osmium tetroxide to 1 part double distilled autoclaved water

Acknowledgments

This research was supported by research grants (DEB-0322664, DEB-0423625, DEB0521177, and DEB-0228679) from the National Science Foundation as part of the Research Experience for Undergraduates and Assembling the Tree of Life Programs.

References

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Lopez-Smith, R. and Renzaglia, K. (2008). Sperm cell architecture, insemination, and fertilization in the model fern, Ceratopteris richardii. Sex Plant Reprod 21:153-167.
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Renzaglia, K. S., Rasch, E. M. and Pike, L. M. (1995). Estimates of nuclear DNA content in bryophyte sperm cells: phylogentic considerations. Am J Bot 82: 18-25.
Southworth, D. and Cresti, M. (1997). Comparison of flagellated and nonflagellated sperm in plants. Am J Bot 84:1301-1311.