Sea Urchin Embryonic Cleavage Rates are Directly Affected by Environmental Copper Concentrations

Jason Huska and Ben Myers

Millersville University

 

Introduction

 

In certain countries of South America, mining is the most important commercial activity. In Chile, for example, 60% of its total national income is produced from mining. However, in past years, there haven’t been any strict measures or regulations for the control of the pollution. As a result, heavy metals such as copper and iron ore were discharged at high levels into the surrounding waters (Vasquez et al, 2000). With the abundance of heavy metals in the water, there may be adverse effects for the native sea urchin population, particularly affecting the growth and development of embryos.

Kobayashi and Okamura (2005) have demonstrated that sea urchin embryo development is negatively effected in an environment where heavy metals are in high concentrations. They also suggest that copper and zinc are the most detrimental to development. However, because they used ASW that contained a combination of 5 different heavy metals, the effects cannot be attributed to a single pollutant. It may have been that a combination of the metals intensified the toxicity as compared to the damage caused by a single heavy metal ion. For this reason, we plan to study how excess copper in the water affects fertilization as well as growth and development of sea urchins embryos.

 

Methods

 

Preparation of Gametes

 

Adult female urchins were stimulated to ovulate as described previously (Cebra-Thomas, 2006). The oocytes were collected and separated equally in four sterile test tubes. The gametes were washed three times with their designated copper chloride ASW solutions: 0 mM, 5 mM, 10 mM, and 20 mM. Male gametes and copper chloride solutions were graciously prepared and provided by Dr. Cebra-Thomas.

 

Induction of Fertilization and Analysis of Develpoment

 

Two drops of sea urchin sperm were added to each of the 4 oocyte containing copper chloride solutions and incubated at room temperature. At times 15, 55, and 80 minutes, aliquots from each test tube were removed, placed onto depression slides, and development was observed by phase-contrast microscopy.

 

Results

 

Phase contrast microscopy was utilized to examine the development of sea urchin embryos from fertilization envelope formation to the 8-cell zygote stage from each of the 4 copper chloride solutions. At time 15 minutes, formation of the fertilization envelope was observed in all concentrations of copper chloride ASW (Table 1). After 55 minutes elapsed, both the fertilization envelope and 2-cell stage embryos were also observed in all solutions. However, Table 2 indicates that the majority of the embryos developing in the 0 mM and 5 mM copper chloride solutions were in the 2 cell stage (>60%), while the majority in the 10 mM and 20 mM copper chloride solutions only exhibited the fertilization envelope (>50%). Lack of fertilization was ~10% in each of the various copper chloride solutions at this time point.

At 80 minutes, cells in the 0 mM copper chloride water were predominately in the 2-cell stage (64%) with a portion developing into 4-cell embryos (15%). In the 5 mM copper solution, 43% of the embryos exhibited 4-cell stage and 39% had developed to the 2-cell stage. Three embryos (7%) had progressed to the 8-cell stage. At 10 mM copper chloride, progression to the 8-cell embryo was observed, however, the cells were distorted in appearance. Both 4-cell and 2-cell embryos still remained and appeared normal at this concentration. At a concentration of 20 mM copper chloride, few embryos progressed beyond the fertilization envelope (16% total development), and those that did exhibited abnormalities (Table 3).

 

Table 1 Sea urchin development in 5, 10, and 20 mM copper chloride solutions after 15 minutes.

 

[Copper Chloride]

Fertilized

% Fertilization

0 mM

21/36

58

5 mM

31/36

86

10 mM

25/36

69

20 mM

33/46

72

Table 2 Sea urchin development in 5, 10, and 20 mM copper chloride solutions after 55 minutes.

 

[CopperChloride]

2-Cell

% 2-Cell

Fertilized

%Fertilization

0 mM

39/58

67

13/58

22

5 mM

40/67

60

18/67

27

10 mM

20/58

34

33/58

57

20 mM

19/55

35

29/55

53

 

Table 3 Sea urchin development in 5, 10, and 20 mM copper chloride solutions after 80 minutes.

 

[Copper Chloride]

8-Cell

4-Cell

2-Cell

Fertilized

0 mM

0/53 (0%)

8/53 (15%)

34/53 (64%)

6/53 (11%)

5 mM

3/46 (7%)

20/46 (43%)

18/46 (39%)

2/46 (4%)

10 mM

17/55(31%)a

15/55 (27%)

15/55 (27%)

8/55 (15%)

20 mM

1/53 (2%)b

3/53 (6%)

4/53 (8%)

45/53 (85%)

a.      Cells appeared abnormal and improperly organized.

b.      Cells appeared abnormal and improperly organized.

 

 

 

 

Discussion

 

We have shown that the development of sea urchin embryos is negatively affected in an environment containing excess copper. Fertilization occurred in each of the copper chloride concentrations, suggesting that the excess ion does not affect this process. However, the rate of cleavage appeared to slow in the embryos exposed to 10 mM and 20 mM copper chloride. At time point 55 minutes, >60% of the embryos in the 0 and 5 mM copper solutions progressed to the 2-cell stage as expected. At the same time point, only 34% of the embryos developing in the 10 mM and 20 mM copper chloride solutions reached this stage. Normal embryonic development to the 4-cell stage was observed after 80 minutes in both the 0 mM and 5 mM copper chloride solutions. However, contradictory to our hypothesis, the embryos in the 5 mM solution appeared to divide faster, indicated by the presence of 8-cell embryos. The 8-cell embryo was also observed in the 10 mM copper chloride environment; however, the cells appeared distorted and out of place. We also noted similar abnormalities in the only 8-cell embryo in the 20 mM solution. Development also appeared to halt in the 20 mM solution; only 16% of the total cells progressed beyond first cleavage. The abnormalities and slow growth were only observed in higher concentrations of copper chloride, suggests that the heavy metal has adverse effects on early cleavage events within the embryo.

Kobayashi and Okamura (2005) indicate a dose dependent inhibition of embryonic development when using a variety of heavy metals in ASW. Our data also suggests a dosage dependent developmental inhibition of embryonic cleavage events. At the 80 minute mark, only 11% of the 0 mM copper chloride group had not progessed further than the fertilization envelope compared to 85% of observed embryos arresting at this stage in the 20 mM copper chloride solution. Those that did develop further in the 20 mM solution, however, appeared abnormal. Based upon our observations, the embryos in higher concentrations of copper would not develop into viable pluteus larvae considering that the majority of the cells did not progress beyond the fertilization envelope.

At high concentrations of copper chloride, cleavage was inhibited, thereby stopping development of the embryo. In lower concentrations, however, cell division proceeded more rapidly than in the absence of copper. At 80 minutes, 64% of the control group divided once, resulting in the majority of 2-cell embryos as expected. The majority of the embryos in the 5 mM copper chloride solution, however, had developed beyond this stage with 43% exhibiting 4-cell embryos and 7% progressing to the 8 cell-stage. This pattern was also observed in the 10 mM copper solution with 27% of the total embryos in the 4-cell stage and 31% in the 8-cell stage. While the majority did progress much faster, those in the 8-cell stage at 10 mM appeared abnormal and had blastomeres out of place. This trend suggests that a limited amount of copper may help in the development of the embryo, however there is a threshold that when crossed, inhibits development. Our findings confirm the hypothesis that excess copper in the water leads to adverse effects in sea urchin embryonic development. This confirms suspicions that environmental pollutants resulting from mining operations may have detrimental effects to native marine organisms. We also describe that small amounts of copper may help to speed embryonic development which could be potentially beneficial for the species. We plan to test this theory further by exposing developing urchin embryos to concentrations of copper chloride ranging from 1-7 mM and allowing development to proceed to the pluteus larvae. This would allow us to observe if small levels of copper metal are adverse in the later stages of development.

 

References

 

Cebra-Thomas, J. (2006). Isolatin of Gametes of Sea Urchins [Protocol for the isolation of gametes of Sea Urchins]. Retrieved from http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Urchin/Urchin_gamete.html

 

Kobayashi, N. and Okamura, H. (2004). Effects of Heavy Metals on Sea Urchin Embryo Development. Part 1. Tracing the cause by effects. Chemosphere. 55(10): 1403-1412.

 

Kobayashi, N. and Okamura, H. (2005). Effects of Heavy Metals on Sea Urchin Development. Part 2. Interactive Toxic Effects of Heavy Metals in Synthetic Mine Effluents. Chemosphere. 61(8): 1198-1203.


Vasquez, J., Matsuhiru, B., Vega, M., Pardo L., and Veliz, D. (2000). The Effects of Mining Pollution on Subtidal Habitats of Northen Chile. Internation Journal of Environment and Pollution. 13(1-6):2-25.