The endocrine disruptive effects of the Deep Horizon crude oil contamination on
the reproductive biology of coastal sharks in the northern Gulf of Mexico.
Principal Investigators:
Eric R. Hoffmayer, Center for Fisheries Research and Development, University of Southern
Mississippi, Ocean Springs, MS 39564. 228-872-4257, eric.hoffmayer@usm.edu
James A. Sulikowski, Marine Science Center, University of New England, Biddeford, ME 04005.
William B. Driggers III, NOAA Fisheries, Mississippi Laboratories, Pascagoula, MS 39568.
R. Joe Griffitt, Department of Coastal Sciences, University of Southern Mississippi, Ocean
Springs, MS 39564
Paul C.W. Tsang, Molecular, Cellular and Biomedical Sciences, University of New Hampshire,
Durham, NH 03824.
Cluster: Observe and Understand
Rationale: Endocrine disruptors (EDs) are a structurally diverse group of compounds that have been
shown to adversely affect all forms of life, and their progenies, by perturbing sensitive hormone
pathways that regulate reproductive functions (e.g. Gillesby and Zacharewski, 1998, Porte et al.
2006). Because of the lipophilic and persistent nature of most EDs and their metabolites, many
bioaccumulate and biomagnify especially in apex type predators. While endocrine disruption in
marine organisms is still poorly understood (Porte et al., 2006) some information regarding their
affects on bony fishes are beginning to surface. For example, exposure to EDs may result in
decreased fertility and egg production in females, or lead to reduced gonad size or feminization of
genetic male fish (e.g. Arcand-Hoy and Benson, 1997). However, few studies have described the
affects of EDs on cartilaginous fish. Although polycynic aromatic hydrocarbons (PAHs) and
aliphatic hydrocarbons (AHs) have been shown to accumulate within elasmobranchs exposed to oil
spills (Al-Hassan et al., 2000), the long term effects of this exposure are unknown. This is
unfortunate as this group is extremely susceptible to the deleterious bioaccumulation effects of these
and other EDs due their relative longevity, moderate to large size, slow metabolism, and high
trophic position (e.g. Storelli et al, 2005). Since most sharks are predators at, or near, the top of
marine food chains, their inability to fill this role, due to effects of EDs, can negatively affect the
structure and function of marine ecosystems in many ways.
Objectives: We will test three broad hypotheses, including 1) hydrocarbon and polycyclic aromatic
hydrocarbon exposure in coastal shark species will have endocrine disruptive effects which will
influence their gross reproductive biology and endocrinology, 2) exposure to EDs will cause
perturbations in the cellular steroidogenic biosynthetic pathways of the ovary, testis and placenta,
and 3) perturbations in steroidogenesis by the ovary as a result of exposure to EDs will increase
embryonic mortality and therefore lower fecundity due to an inadequate uterine environment.
Model Species. The Atlantic sharpnose shark, Rhizoprionodon terraenovae, will be used a model
species to evaluate the deleterious long-term effects of the crude oil exposure on coastal shark
species. This species was chosen due to their high abundance in the region, their relatively short
generation time (2 years), and their limited range, which includes the estuaries and deeper near
shore waters that are currently being exposed to the oil spill. Since mature female Atlantic
sharpnose sharks exhibit extreme sexual segregation and remain in deeper waters once mature
(Parsons and Hoffmayer, 2005) they are currently being exposed to the Deep Horizon oil spill.
Additionally, we have long-term (12-16 years) abundance data for Atlantic sharpnose sharks from
northcentral Gulf of Mexico (ncGOM) waters, as well as baseline reproductive data, including
hormones, histology, and morphological data, from the last two years (Hoffmayer, unpub. data).
Approach. Each month, mature and immature Atlantic sharpnose sharks will be collected from
inshore and offshore waters to evaluate the contaminant level in their tissues and document the
deleterious effect of their exposure for a period of one year. All sharks collected will be brought
back to the Gulf Coast Research Laboratory for gross reproductive examination. Males and females
will be examined for stage of maturity and several reproductive measurements will be taken,
including clasper length, testis mass, and epididymis width for males, and ovary weight, follicle
diameter, and oviducal width for females (Driggers et al. 2004; Sulikowski et al., 2007). All eggs
will be assessed for viability and all pups will be examined for any abnormalities. In addition, males
will be assessed by microscopic analyses for anomalies in spermatogenic development (Sulikowski
et al. 2009) and radioimmunoassay (Sulikowski et al. 2007) for anomalies in circulating levels of
testosterone (T), dihydrotestosterone (DHT), progesterone (P4), estradiol (E2) and triiodothyronine
(T3) and thyroxine (T4). Likewise, females will be assessed the same techniques listed above for
anomalies in oocyte development and in circulating levels of testosterone (T), dihydrotestosterone
(DHT), progesterone (P4), estradiol (E2) and triiodothyronine (T3) and thyroxine (T4). In addition,
the concentrations of the aforementioned hormones will also be assayed in the egg yolk of
developing embryo’s to investigate hormonal anomalies in this tissue.
For in vitro studies, we will culture follicle, testicular, and placental cells, with an androgen, such as
testosterone, and use specific radioimmunoassay to determine their ability to synthesize estradiol,
estriol and estrone. Similarly, by providing cultured testicular cells with progesterone or
pregnenolone, we will use specific radioimmunoassay to determine their ability to synthesize
androgens such as androstenedione, testosterone and dihydrotestosterone (Tsang and Callard 1987).
At the same time, we will test the responsiveness of these cells to homologous gonadotropins using
a ventral lobe extract (Tsang and Callard 1988). Finally, each cell type will be incubated in the
presence of a tritiated steroid precursor, for example, tritiated pregnenolone or tritiated
progesterone.
Uterine tissues from adult and juvenile sharks will be extracted for total proteins in preparation for
substrate gel electrophoresis or zymography, a method we previously used for bovine luteal tissue
(Goldberg et al. 1995). Zymography is a modified form of sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) whereby substrates for specific matrix metalloproteinases (MMP)
are co-polymerized with the polyacrylamide. This method is ideal since enzyme identification and
enzyme activity are based on substrate preferences, bypassing the need for antibodies (which are
unavailable for elasmobranchs). We have unpublished data on the identification of MMPs in the
plasma of spiny dogfish as well as from uterine extracts of skates (Tsang, unpublished data).
We will use Quantitative PCR (QPCR) to examine induction of four genes known to be highly
responsive to and indicative of, reproductive success and hydrocarbon exposure. Genes to be
examined indicative of endocrine disrupting effects include Estrogen receptor alpa (ER),
vitellogenin (VTG), and aromatase (AROM). To assess molecular responses to crude or dispersed
oil contamination, we will examine expression levels of the Aryl Hydrocarbon Receptor (AhR),
Cytochrome p450 1A (CYP1A), Glutathione S-transferase (GST), and acetylcholinesterase (AChE).
All genes will be quantified in brain, liver, and gonad, as appropriate. Gene expression will be
calculated as fold-change from “Not Impacted” and “Baseline” samples, and will be normalized to
expression of 18S. All necessary expertise and equipment is present at GCRL for QPCR.
Expected Outcomes: If coastal and pelagic shark species were exposed to oil, dispersant, or
dispersed oil, then depending on the level of exposure there may be a high likelihood that ED
effects will manifest themselves.
We expect to see lower circulating steroid hormone
concentrations in adult sharks, which will be due to the adverse effects of EDs on the enzymes of
the steroidogenic pathways in ovarian, testicular and placental cells. We also expect that alterations
in morphological parameters (i.e. reduced testes size) and sex ratios of pups will occur. In addition,
culturing ovarian and testicular cells from immature and juvenile sharks may reveal precocious
abilities of these cells to respond to gonadotropins and start synthesis of steroid hormones, which is
reminiscent of the reported cases of precocious puberty in humans. We expect to find that the lower
fecundity due to exposure to EDs is due in part to their effects on MMPs, perhaps by preventing
their activation and therefore crippling their actions on the uterus to create an optimal environment
for implantation. Alternatively, the EDs could have an effect on the tissue inhibitors of
metalloproteinases (TIMPs), which are endogenous regulators of MMP activity.
Approximate Cost: $198,376
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