A Method for Observing Ceratopteris richardii Sperm
Heather Cousino and Jun Tsuji, Siena Heights University, Department of Biology, 1247 E. Siena Heights Drive, Adrian, Michigan 49221-1796, USA

ABSTRACT

The fern Ceratopteris richardii produces motile sperm that are challenging to observe and study due to their rapid speed. To assess the effectiveness of Protoslo in restricting the movement of fern sperm, we measured the speeds of wild type and slo1 mutant sperm in water, Protoslo, methyl cellulose, and hydroxyethyl cellulose. We found that diluted Protoslo restricted the movement of the fern sperm as well as 1% methyl cellulose, while having an optical clarity similar to that of water. For these reasons, Protoslo functioned better than the other test solutions.

INTRODUCTION

Ceratopteris richardii is a small, homosporous fern that has been used as a model plant for research and educational studies. C. richardii has a rapid life cycle consisting of separate sporophytic and gametophytic generations. In only 5 to 10 days, for example, mature gametophytes can develop from germinated spores (Hoffman and Vaughn 1995). The male gametophytes and hermaphrodites of C. richardii produce multiflagellated, motile sperm (spermatozoids) that are difficult to observe and study because of their rapid movements (Hickok and Warne 1998). C. richardii sperm can be studied using mutants that produce slow moving sperm (Duckett and others 1979), like the slo1 mutant; however, this precludes conducting experiments directly with the wild type fern and requires the extra time and expense of growing a second culture.

Scientists have used various liquids to restrict the movement of motile organisms for microscopic examination. Protoslo, a clear, viscous, non-lethal, aqueous solution composed of 10% methyl cellulose has been used to reduce the movement of many organisms including Paramecium (Lytle 2000), Euglena (Hickman and Hickman 1993), rotifers (Fox 2001), Amoeba (Fox 2001), and some ciliates (NSTA 1996). By slowing down the movement of these microorganisms, scientists have been able to study these organisms and their organelles in greater detail (Fox 2001). To our knowledge, there has not been a report of the use of Protoslo with C. richardii sperm. The purpose of this study was to assess the use of Protoslo in observing C. richardii sperm.

MATERIALS AND METHODS

To grow the ferns, we obtained wild type and slo1 mutant C. richardii spores from the Carolina Biological Supply Company (Burlington, NC). We then put the spores in petri dishes containing C. richardii Basic Medium (Carolina Biological Supply Co.) and placed the dishes under fluorescent illumination with a light intensity of 1463 lux to allow the spores to germinate and develop into mature gametophytes (approximately 3 weeks).

For microscopic examination, we put one or two of the wild type or mutant gametophytes on a glass concavity microscope slide (15mm x 0.2mm x 0.8mm). We then added 0.1 mL of either water, 67% Protoslo, (6.7% methyl cellulose, Carolina Biological Supply Co.), 0.1% hydroxyethyl cellulose (Aldrich Chemical Company), 1% hydroxyethyl cellulose, 0.1% methyl cellulose (The Matheson Company), or 1% methyl cellulose, where all of the solutions were prepared in water. Afterwards, we viewed and recorded the C. richardii sperm using a Wolfe light microscope of 100x total magnification attached to a Hitachi video camera, which was connected to a 20-inch screen TV/VCR combination. Upon replaying the video tape, we measured the distance that each sperm traveled on the television screen over a 5 second interval. Since a distance of 38 cm on the television screen corresponded to 1 mm on the microscope slide, we then converted the monitor measurements into the actual distances that the sperm traveled. We collected the data for 50 different sperm for each test solution. Each experiment was then repeated for a total of three times using different fern cultures. Since the averages of the three trials for each of the 12 experimental groups fit the assumptions of distribution and homogeneity of variance for parametric analysis, we used an analysis of variance (ANOVA) to test for significant differences. All ANOVA tests were 2-tailed and a probability level ≤0.05 was considered significant.

RESULTS AND DISCUSSION

The C. richardii sperm were initially observed in water. A few minutes after the addition of water, large amounts of sperm were released from the antheridia. Each sperm was spiral-shaped, between 13 to 25 micrometers in length, and moved in a circular motion with the aid of its numerous flagella. The sperm of both the wild type and the slo1 mutant were indistinguishable from each other. The C. richardii sperm were similar in size and appearance to those reported previously (Hickok and Warne 1998; Javalgekar 1960).

With the microscope, the C. richardii sperm were easier to see in some solutions than others, none of which appeared to affect sperm viability. The sperm were easily visible in water, 67% Protoslo (6.7% methyl cellulose), 0.1% hydroxyethyl cellulose, and 0.1% methyl cellulose. However, in 1% hydroxyethyl cellulose and 1% methyl cellulose, the depth of view was reduced and the sperm appeared a little blurry. Surprisingly, the sperm in 1% methyl cellulose were also harder to see, which may be due to differences in the molecular weights between the methyl cellulose we used and that in Protoslo. In 100% Protoslo, the sperm appeared so blurry that they were nearly impossible to see, which necessitated the dilution of Protoslo with water. Our experience with Protoslo differs from the studies conducted with Paramecium, Euglena, and Amoeba, which did not need to dilute Protoslo. This difference may be due to the transparency and small size of the C. richardii sperm as compared to Paramecium and the other microorganisms.

We calculated the average speed of the wild type C. richardii sperm for 3 independent trials of each of the test solutions (Table 1). The wild type C. richardii sperm traveled the fastest in water. The sperm moved slower in 0.1% hydroxyethyl cellulose and 0.1% methyl cellulose, which were not significantly different. The sperm were even slower in the 1% hydroxyethyl cellulose and moved the slowest in 1% methyl cellulose and 67% Protoslo.

We also calculated the average speed of the slo1 mutant sperm for 3 trials in each of the 6 solutions (Table 1). The mutant sperm traveled the fastest in water. Their speed was reduced by the 0.1% hydroxyethyl cellulose and 0.1% methyl cellulose solutions. The next slowest sperm speed was in 1% hydroxyethyl cellulose, followed closely by 1% methyl cellulose. Protoslo was once again the solution in which the sperm traveled the slowest.

The results of the slo1 mutant fern trials were similar to those of the wild type fern (Table 1). For example, the sperm of both types of ferns moved faster in water, 0.1% hydroxyethyl cellulose, and 0.1% methyl cellulose than in either 1% hydroxyethyl cellulose, 1% methyl cellulose, or Protoslo. In general, the slo1 mutant sperm moved slower than the wild type in each of the solutions, with the exception of 1% methyl cellulose and Protoslo, which were equivalent. Mutant ferns have been reported to make sperm with irregular flagella that contain deep channels or holes (Duckett and others 1979). Such abnormalities may explain why the mutant sperm traveled slower than the wild type sperm in the various test solutions.


Table 1. The average speed of the wild type and slo1 mutant fern sperm in different solutions. Each value is the average of 150 independent measurements. Values with the same letter are not significantly different from each other.
  Speed (mm x s-1)
  Wild Type slo1 mutant
Water 3.6a 2.8c
67% Protoslo 0.80b 0.70b
0.1% hydroxyethyl cellulose 2.7c 2.2e
1% hydroxyethyl cellulose 1.7d 1.2b
0.1% methyl cellulose 2.9c 2.2e
1% methyl cellulose 1.0b 1.0b

Although we did not study the effect of Protoslo on sperm longevity, we did discover that Protoslo can be effectively used to observe C. richardii sperm immediately after they are released from the antheridia. Diluted Protoslo restricted the movement of the fern sperm as well as 1% methyl cellulose, while having an optical clarity similar to that of water. For these reasons, Protoslo functioned better than the other test solutions. Also, since Protoslo can be diluted to different percentages, one can easily reduce the sperm speed to any desirable level. Using Protoslo, rather than a sperm mutant, to study fern sperm also saves the time and expense of having to grow a second culture. By using Protoslo, experimental studies of sperm motility can be conducted directly with the wild type fern, especially for educational purposes.


LITERATURE CITED

Duckett JG, Klekowski EJ, Hickok LG. 1979. Ultrastructural studies of mutant spermatozoids in ferns. I. The mature nonmotile spermatozoid of mutation 230X in Ceratopteris thalictroides (L.)Brogn. Gamete Research 2:317-343.

Fox R. 2001. Invertebrate zoology: Laboratory techniques. http://www.lander.edu/rsfox/310TechniquesLab.html.

Hickman CP Jr, Hickman FM. 1993. Laboratory studies in integrated principles of zoology. St. Louis, Missouri: Mosby-year Book. 420p.

Hickok LG, Warne TR. 1998. C-Fern manual. Burlington, North Carolina: Carolina Biological Supply Company.

Hoffman JC, Vaughn KC. 1995. Using the developing spermatogenous cells of Ceratopteris to unlock the mysteries of the plant cytoskeleton. Int. J. Plant Sci. 156(3):346-358.

Javalgekar SR. 1960. Sporogenesis and prothallial development in Ceratopteris thalictroides. Botanical Gazette 122(1):45-50.

Lytle CF. 2000. General zoology: Laboratory guide. Dubuque, Iowa: McGraw-Hill. 371p.

National Science Teachers Association. 1996. Scope, sequence, and coordination. http://dev.nsta.org/ssc/pd