Effects of Di(2-ethylhexyl) Phthalate on StrongylocentrotusPurpuratus Sperm Motility, Fertility and Viability

Anthony Gizzi, Department of Biological Sciences, York College of Pennsylvania

Introduction

Over the last 50 years, large quantities of estrogenic man-made chemicals (xenoestrogens) have been released into the environment. Consequently, there is mounting concern that these estrogenic chemicals may have harmful influences on the male reproductive system in many animal species, including humans. To date, there is an abundance of research evaluating the effects of xenoestrogens on rats and other internal reproducers. However, many aquatic and marine organisms reproduce via an external pathway wherein gametes are directly exposed to the surrounding water. Unfortunately, little is known regarding how the gametes of aquatic and marine organisms will react when exposed to these xenoestrogenic chemicals, specifically, Di(2-ethylhexyl) phthalate (DEHP), during spawning (Figure 1).

Research Objective

Using a sperm/fertilization bioassay this study will evaluate the effects of DEHP exposure on the sperm of Strongylocentrotus purpuratus.

Review of literature

Mean sperm counts in some men have declined by 40-50% over the past 50 years (Carlson et., al 1992)

Evidence suggests that the presence of environmental xenoestrogens may have deleterious effects on wildlife populations by decreasing fertility (Colbern & Clement, 1992).

Ghirardini et al., (2001) found toxic effects on sea urchin fertility after exposure to 50 µg/kg of polychlorinated biphenyl (PCB), a known xenoestrogen.

In some areas of the U.S, PCB levels are as high as 100 µg/kg in dry sediment (Wong et al., 1992-1995).

Exposure to DEHP (Figure 1), a commonly used plasticizer, causes testicular atrophy accompanied by selective depletion of testicular zinc in mice (Agarwal, et al., 1986).

Decrease in testes weight (Figure 2) results after exposure to OPP, a xenoestrogen, in rats (Sharpe et al., 1995).

Human sperm exposed to 17b-estradiol, a synthetic estrogen, show reduced motility and fertility (Luconi et al., 1999).

Hypothesized Result

Sperm motility, fertility and viability will be significantly reduced, in a dose-dependent manner, following DEHP treatment (Figure 6).

Figure 6. The dose dependant response of S. purpuratus to DEHP exposure. The percentage of viable (gray line) and motile (red line) sperm cells is expected to decline with increasing concentrations of DEHP. Similarly, sperm fertility, measured by the appearance of a fertilization membrane (blue line), is also expected to decline with increasing concentrations of DEHP.

Acknowledgements I would like to thank Dr. Ricker for the time she spent helping to devise a synthesis for which my thesis was based upon. I would also like to thank Dr. Nolan for her support and criticism.

Literature Cited:Carlsen E, Giwercman A, Keiding N,Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. Br Med J305:609-613, 1992Colborn T, Clement C, eds. Chemically induced alterations in sexual and functional development: the wildlife/human connection. Princeton, NJ:Princeton Scientific Publishing,1992Agarwal D, Eustis S, Lamb J. C., Reel R., Kluwe, W. Effects of Di(2-Ethylhexly) Phthalate on the Gonadal Pathophysiology, Sperm Morphology, and Reproductive Performance of Male Rats. Environmental Health Perspectives, Vol. 65, pp. 343-350, 1986Luconi M, Muratori M, Forti G, and Baldi E. Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects. J Clin Endocrinol Metab 84: 1670–1678, 1999Thomas J. A., and Thomas M.J. Biological effects of di-(2-ethyl-hexyl) phthalate and other phthalic acid esters. CRC Crit. Rev Toxicol. 13: 283-317, 1984Sharpe RM, Fisher JS, Millar MM, Jobling S & Sumpter JP. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environmental Health Perspectives. 103 1136–1143, 1995 Wong, C.S., P.D. Capel, and L.H. Nowell. Organochlorine pesticides and PCBs in streamsediment and aquatic biota – initial results from the National Water Quality Assessment Program, 1992-1995. Water-Resources Investigations Report 00-4053. United States Geological Survey. Sacramento, CA, 1995

Fig 2. Scatter plot of testicular size versus body weight for control rats (blue) and those treated with octylphenol poly-ethoxylate (OPP), a xenoestrogen (black) (Sharpe et al., 1995).

Statistical Analysis: For each parameter of this study, an Analysis of Variance will be performed to determine whether there are significant effects among the treatment groups. If significant differences exist (p<0.05), pairwise comparisons will be performed using a Tukey test.

Figure 1. Molecular structure of xenoestrogen, Di(2-ethylhexyl) phthalate (DEHP). Its estrogen mimicking properties allow it to bind to estrogen receptors in cells (www.inchem.org) .

Figure 3. Sea urchin sperm on the surface ofan egg (www.luc.edu).

Figure 4. Fertilization membrane (arrow) surrounding seaurchin egg immediately following fertilization (www.swarthmore.edu).

CollectSperm

CollectEggs

Expose sperm for 10 min. to sediment* containing DEHP(0-20,000ppm)

Maintain eggs in artificial sea water

Mix treated sperm and eggs in a beaker for 20 minutes. Fertilization success, as indicated by the presence or absence of the fertilization membrane (Figures 3 & 4), will be measured.

Overall percent motility will be measured using a warm hemocytometer. Sperm viability will be examined using a differential stain (Figure 5).

Inject urchin w/ 1.0 mL of 0.5M KCl (aboral

surface)

(Corbisimages.com)

*commercially available sediment, containing no detectable estrogens, will be spiked with sunflower seed oil containing 0-20,000 µg/kg of DEHP.

Potential SignificanceThere are many possible routes of exposure to xenoestrogenic compounds which can affect the mobility, fertility and viability of sperm cells. One of the potential explanations is that cellular insult is taking place directly at the level of the sperm itself, supporting this study’s hypothesis. The lack of a mechanism indicating how sperm and other cells in the reproductive tract are impacted by DEHP, necessitates further molecular, cellular, and physiological research.

Methods

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(www.colostate.edu)

Figure 5. Differential staining of human sperm. Prewarmed nigrosin-eosin stain was used (100x magnification).