Accumulation of the Toxic Metal Mercury in Multiple Tissues of Marine-Associated Birds from South Florida.

One of the best studied global "hot spots" for ecological mercury (Hg) contamination is south Florida (USA), where elevated Hg concentrations in environmental media and regional wildlife were first described over thirty years ago. While Hg contamination has lessened in this region, it is still critical to monitor Hg uptake and potential risks in south Florida wildlife, especially in marine-associated birds, which are known to accumulate potentially toxic Hg levels. In this study, total Hg (THg) concentrations were measured in liver, kidney, muscle, and feathers of 101 individuals from seven species of south Florida birds: brown pelican Pelecanus occidentalis, double-crested cormorant Phalacrocorax auratus, herring gull Larus argentatus, laughing gull Leucophaeus atricilla, northern gannet Morus bassanus, royal tern Thalasseus maximus, and osprey Pandion halietus. A sizeable proportion of individuals (> 40%) were found to contain THg concentrations in internal tissues that exceeded estimated toxicity thresholds for Hg-related effects. Certain species, especially osprey, were found to exhibit a higher rate of threshold exceedances than others and should continue to be monitored for Hg-related effects in future studies. Feather THg concentrations exhibited a lower rate of toxicity threshold exceedances (12%) and were not significantly correlated with those in internal tissues in most cases, suggesting that they may not be well suited for monitoring Hg exposure in these species unless sources of data variation can be better understood. The results of the present study contribute significantly to our understanding of trends in Hg accumulation and Hg-related health risks in south Florida marine-associated birds.

Shark tooth collagen stable isotopes (δ15 N and δ13 C) as ecological proxies.

The isotopic composition of tooth-bound collagen has long been used to reconstruct dietary patterns of animals in extant and palaeoecological systems. For sharks that replace teeth rapidly in a conveyor-like system, stable isotopes of tooth collagen (δ13 CTeeth & δ15 NTeeth ) are poorly understood and lacking in ecological context relative to other non-lethally sampled tissues. This tissue holds promise, because shark jaws may preserve isotopic chronologies from which to infer individual-level ecological patterns across a range of temporal resolutions. Carbon and nitrogen stable isotope values were measured and compared between extracted tooth collagen and four other non-lethally sampled tissues of varying isotopic turnover rates: blood plasma, red blood cells, fin and muscle, from eight species of sharks. Individual-level isotopic variability of shark tooth collagen was evaluated by profiling teeth of different ages across whole jaws for the shortfin mako shark Isurus oxyrinchus and sandbar shark Carcharhinus plumbeus. Measurements of δ13 CTeeth and δ15 NTeeth were positively correlated with isotopic values from the four other tissues. Collagen δ13 C was consistently 13 C-enriched relative to all other tissues. Patterns for δ15 N were slightly less uniform; tooth collagen was generally 15 N-enriched relative to muscle and red blood cells, but congruent with fin and blood plasma (values clustered around a 1:1 relationship). Significant within-individual variability was observed across whole shortfin mako shark (δ13 C range = 1.4‰, δ15 N range = 3.6‰) and sandbar shark (δ13 C range = 1.2‰-2.4‰, δ15 N range = 1.7‰-2.4‰) jaws, which trended with tooth age. We conclude that amino acid composition and associated patterns of isotopic fractionation result in predictable isotopic offsets between tissues. Within-individual variability of tooth collagen stable isotope values suggests teeth of different ages may serve as ecological chronologies, that could be applied to studies on migration and individual-level diet variation across diverse time-scales. Greater understanding of tooth replacement rates, isotopic turnover and associated fractionation of tooth collagen will help refine potential ecological inferences, outlining clear goals for future scientific inquiry.

Female sperm storage in the bonnethead Sphyrna tiburo oviducal gland: Immunolocalization of steroid hormone receptors in sperm storage tubules.

Female sperm storage (FSS) has been demonstrated to occur in representatives from all major vertebrate groups and has been hypothesized to have several possible adaptive benefits that may maximize reproductive success of its practitioners. However, while the range of taxa that exhibit FSS and its possible evolutionary benefits have received significant attention in past years, the physiological mechanisms by which FSS occurs in vertebrates have only recently been explored. In this study, we examined the potential role of gonadal steroid hormones in regulating FSS in the bonnethead Sphyrna tiburo, a small hammerhead species in which females have been shown to be capable of storing male spermatozoa for up to 6 - 7 months following copulation. Like past studies on this species, we observed associations between plasma concentrations of the gonadal steroids 17ß-estradiol, testosterone, and progesterone with FSS in female bonnetheads, suggesting roles for these hormones in regulating this process. Using immunohistochemistry, we also observed presence of androgen receptor, estrogen receptor alpha (ERα), and progesterone receptor in epithelial cells of sperm storage tubules in the bonnethead oviducal gland, as well as occurrence of ERα in stored spermatozoa, specifically during the sperm storage period. These results suggest that E2, T, and P4 may regulate certain aspects of FSS in bonnethead indirectly through actions on the female reproductive tract, whereas E2 may also have direct effects on sperm function. This is the first study on the regulation of FSS in sharks and has formed a basis for future work geared towards improving our understanding of this process in chondrichthyans.

Analysis of Trace Element Concentrations and Antioxidant Enzyme Activity in Muscle Tissue of the Atlantic Sharpnose Shark, Rhizoprionodon terraenovae.

Metals occur naturally in the environment; however, anthropogenic practices have greatly increased metal concentrations in waterways, sediments, and biota. Metals pose health risks to marine organisms and have been associated with oxidative stress, which can lead to protein denaturation, DNA mutations, and cellular apoptosis. Sharks are important species ecologically, recreationally, and commercially. Because they occupy a high trophic level, assessing muscle tissue metal concentrations in sharks may reflect metal transfer in marine food webs. In this study, concentrations of cadmium, copper, lead, nickel, selenium, silver, and zinc were measured in the muscle of Rhizoprionodon terraenovae (Atlantic sharpnose shark) from 12 sites along the coast of the southeastern United States. Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) also were examined in the muscle tissue of R. terraenovae. A total of 165 samples were analyzed, and differences in trace element bioaccumulation and enzyme activity were observed across sites. R. terraenovae samples collected from South Florida and South Carolina had the highest cumulative trace element concentrations whereas those collected from North Carolina and Alabama had the lowest cumulative concentrations. Trace element concentrations in shark muscle tissue were significantly correlated to antioxidant enzyme activity, particularly with glutathione peroxidase, suggesting that this enzyme may serve as a non-lethal, biomarker of metal exposure in R. terraenovae. This is one of the most extensive studies providing reference levels of trace elements and oxidative stress enzymes in a single elasmobranch species within the U.S.

Resource-use dynamics of co-occurring chondrichthyans from the First Coast, North Florida, USA.

Recent studies on shark assemblages on the northeast Florida and southeast Georgia coast (hereafter referred to collectively as the "First Coast") have demonstrated differences in species and age-class composition of catch from previously characterized estuaries and newly surveyed area beaches, demonstrating that these regions may provide a critical habitat to different segments (i.e., life stages) of local shark populations. In this study, carbon and nitrogen stable isotopes (δ13 C and δ15 N) from muscle tissue and blood plasma were used to examine trophic dynamics (and temporal variability thereof) of the three dominant co-occurring species found along First Coast beaches (the Atlantic Sharpnose shark Rhizoprionodon terraenovae, Blacknose shark Carcharhinus acronotus and Blacktip shark Carcharhinus limbatus) to determine if they exhibit overlap in resource use along with spatial and temporal habitat use. Although considered spatially segregated from the beach species, a dominant, age-class species found in First Coast estuaries (juvenile Sandbar sharks Carcharhinus plumbeus) was also included in this analysis for comparison. Temporal variability of resource-use characteristics was detected at the species level. Resource-use overlap among species varied by tissue type and was generally higher for blood plasma, suggesting greater resource sharing over more recent time periods. Over longer time periods Atlantic Sharpnose and Blacktip sharks exhibited resource-use expansion, whereas Blacknose sharks exhibited a narrowing in resource use, suggesting a more specialized foraging strategy compared to the other species. The resource-use breadth of Sandbar sharks also expanded between blood plasma and muscle tissue. Significant size relationships were detected in Blacktip and Sandbar sharks, indicating ontogenetic resource shifts for both species. A diversity of highly productive resource pools likely support shark populations along the First Coast such that resource-use differentiation is not required to facilitate species co-occurrence. This work may shed light on understanding patterns of species co-occurrence as well as aid in future conservation efforts.

Reproductive cycle and fecundity of the bonnethead Sphyrna tiburo L. from the northwest Atlantic Ocean.

The present study examined temporal changes in plasma sex hormone concentrations and the morphology and histology of reproductive organs in mature northwest Atlantic (NWA) bonnetheads Sphyrna tiburo L. to characterize reproductive cycle, breeding periodicity and fertility in this still poorly studied population. Progressive increases in testis width, epididymis head width, plasma testosterone (T) concentrations, and occurrence of mature spermatozoa were observed in male S. tiburo from June to September, demonstrating that spermatogenesis occurs during the summer. Nonetheless, increases in maximum follicle diameter, oviducal gland width, plasma 17ß-estradiol and T concentrations, and occurrence of vitellogenic follicles were not observed in mature females until between October and April, demonstrating non-synchronous patterns of gametogenesis in males and females. Fresh copulatory wounds were observed in females collected during late September along with histological evidence for sperm presence in the oviducal gland between September and April, confirming a 6- to 7 month period of female sperm storage. Ovulation occurred between mid-April and early May in concert with increases in female plasma progesterone concentrations. Gestation occurred during a 4.5- to 5 month period between May and early September, and 97% of mature females collected during this period were gravid, indicating a highly synchronized, annual reproductive periodicity. Brood size was significantly correlated with maternal size and ranged from 1 to 13 pups with a mean ± S.D. of 8.1 ± 2.2, which was significantly lower than reported in Gulf of Mexico (GOM) populations. The occurrence of non-fertile offspring was observed in 17% of broods with a range of 1-7 non-fertile eggs present in individual females. Thus, as previously reported in GOM S. tiburo, this unusual form of infertility also appears to be prevalent in the NWA population and requires further study. This study has demonstrated meaningful differences in reproductive biology of these populations, emphasizing the need for region-specific approaches for population management.

Androgen receptors in the bonnethead, Sphyrna tiburo: cDNA cloning and tissue-specific expression in the male reproductive tract.

As demonstrated in past studies, androgens appear to play critical roles in regulating reproduction in male sharks. However, little is known about the cell-specific actions of androgens in these fishes. To address this, this study examined androgen targets in reproductive organs of a seasonally reproducing shark, the bonnethead (Sphyrna tiburo). A partial bonnethead AR cDNA clone was isolated and found to exhibit strong homology with known vertebrate ARs. Using RT-PCR and in situ hybridization, AR was found to be expressed in multiple cell types in the male bonnethead testis (premeiotic germ cells, Leydig-like interstitial cells, Sertoli cells, peritubular myoid cells, and mature spermatozoa) and gonadal ducts (stromal cells, luminal epithelial cells, mature spermatozoa). Furthermore, AR expression in these organs was found to vary temporally in relation to the seasonal reproductive cycle. Based on immunocytochemistry, the presence of AR protein in male bonnethead reproductive organs was largely consistent with patterns of AR gene expression with the single exception of mature spermatozoa, which exhibited consistently strong mRNA expression but only inconsistent and weak AR protein immunoreactivity. These results suggest important roles for androgens in regulating germ cell proliferation, hormone production, spermatid elongation, spermiation, and gonadal duct function in male bonnetheads. In addition, high abundance of AR mRNA in bonnethead spermatozoa suggest the potential for de novo protein synthesis following spermiation/copulation and/or a role for AR mRNA in early embryonic development, both of which have been proposed to explain the occurrence of mRNA transcripts in spermatozoa from various vertebrates.

Evaluation of the use of metallothionein as a biomarker for detecting physiological responses to mercury exposure in the bonnethead, Sphyrna tiburo.

Previous studies have demonstrated that sharks, perhaps more so than any other fishes, are capable of bioaccumulating the non-essential toxic metal mercury (Hg) to levels that threaten the health of human seafood consumers. However, few studies have explored the potential effects of Hg accumulation in sharks themselves. Therefore, the goal of this study was to examine if physiological effects occur in sharks in response to environmentally relevant levels of Hg exposure. To address this goal, the relationship between muscle Hg concentrations and muscle/hepatic levels of metallothionein (MT), a widely used protein biomarker of toxic metal exposure in fish, was examined in bonnetheads, Sphyrna tiburo, from three Florida estuaries. Total Hg concentrations in bonnethead muscle, as determined using thermal decomposition and atomic absorption spectrometry, ranged from 0.22 to 1.78 µg/g wet weight and were correlated with animal size. These observations were consistent with earlier studies on Florida bonnetheads, illustrating that they experience bioaccumulation of Hg, often to levels that threaten the health of these animals or consumers of their meat. However, despite this, MT concentrations measured using Western blot analysis were not correlated with muscle Hg concentrations. These results suggest that either environmentally relevant levels of Hg exposure and uptake are below the physiological threshold for inducing effects in sharks or MT is a poor biomarker of Hg exposure in these fishes. Of these two explanations, the latter is favored based on a growing body of evidence that questions the use of MTs as specific indicators of Hg exposure and effects in fish.

Histological comparison of shark dermis across various ecomorphologies.

The integument plays essential roles in the structural support, protection, and hydrodynamic capability among fishes. Most research on shark skin has focused on the external epidermal layer, while the larger dermis anchoring the dermal denticles has been mostly ignored. Shark dermis is composed of two layers, the upper stratum laxum and the lower stratum compactum, holding supportive collagen and elastic fibers. There may be morphological and compositional differences in the dermis across various species of sharks that could relate to their different swimming modes and ecologies. The goal of this study was to characterize and describe the dermis among three shark species, Ginglymostoma cirratum, Sphyrna mokarran, and Isurus oxyrinchus, each representing a different swimming mode. Histological characterizations were performed at 16 locations along the body of each shark; variables such as dermal thickness, abundance of collagen and elastic fibers, and fiber size were quantified. Results showed G. cirratum has the thickest skin overall, and the largest fiber size for both collagen and elastic fibers, with overall patterns of increased amounts of collagen fibers and decreased amount of elastic fibers. At the opposite end of the spectrum, I. oxyrinchus showed the thinnest dermis along the flank region, with overall patterns of increased elastic fibers and decreased collagen fibers. These findings may challenge our original assumptions of a rigid body in fast moving sharks and a more flexible body in slower moving sharks and highlight the diversity of the shark integument.

Associations between total mercury, trace minerals, and blood health markers in Northwest Atlantic white sharks (Carcharodon carcharias).

The ecology and life-histories of white sharks make this species susceptible to mercury bioaccumulation; however, the health consequences of mercury exposure are understudied. We measured muscle and plasma total mercury (THg), health markers, and trace minerals in Northwest Atlantic white sharks. THg in muscle tissue averaged 10.0 mg/kg dry weight, while THg in blood plasma averaged 533 µg/L. THg levels in plasma and muscle were positively correlated with shark precaudal length (153-419 cm), and THg was bioaccumulated proportionally in muscle and plasma. Nine sharks had selenium:mercury molar ratios in blood plasma >1.0, indicating that for certain individuals the potential protective effects of the trace mineral were diminished, whereas excess selenium may have protected other individuals. No relationships between plasma THg and any trace minerals or health markers were identified. Thus, we found no evidence of negative effects of Hg bioaccumulation, even in sharks with very high THg.

Total mercury concentrations in invasive lionfish (Pterois volitans/miles) from the Atlantic coast of Florida.

Invasive lionfish (Pterois volitans/miles) pose a serious threat to marine ecosystems throughout the western Atlantic Ocean and Caribbean Sea. The development of a fishery for lionfish has been proposed as a strategy for controlling populations; however, there is concern about consumption of this species by humans due to its high trophic position and potential for bioaccumulation of mercury. We analyzed total mercury (THg) in tissues of lionfish from two locations on the east coast of Florida. THg in lionfish increased with size and differed by location and sex. THg was highest in muscle tissue and was strongly positively correlated among tissues. THg in lionfish was lower than other commonly consumed marine fishes, and falls into Florida's least restrictive advisory level. Consumption of lionfish poses a low risk and concerns over mercury bioaccumulation should not present a significant barrier to lionfish harvest.

Organochlorine concentrations, reproductive physiology, and immune function in unique populations of freshwater Atlantic stingrays (Dasyatis sabina) from Florida's St. Johns River.

Within the past decade, reproductive and health disorders have been reported to occur in unique populations of Atlantic stingrays (Dasyatis sabina) inhabiting certain components of Florida's St. Johns River. Since these irregularities are consistent with the alleged effects of organochlorine (OC) contaminant exposure in other Florida wildlife, the goal of this study was to examine possible associations between OC concentrations and reproduction and/or immune function in stingrays from this river system. Liver concentrations of 30 OC pesticides/pesticide metabolites and total polychlorinated biphenyls (PCBs) were measured and compared in D. sabina collected from four central Florida lakes of the St. Johns River: Lake George, Lake Harney, Lake Jesup, and Lake Monroe. Reproductive biology, serum testosterone and 17beta-estradiol concentrations, and circulating white blood cell counts were examined and compared in stingrays from lakes that were determined to contain low (Lake George), intermediate (Lake Monroe), and high (Lake Jesup) levels of pesticide contamination, based on the results of liver OC assessments. Successful breeding occurred in Lake Jesup stingrays, indicating that the degree of OC accumulation in these animals is not high enough to cause reproductive impairment. However, elevated serum steroid concentrations and white blood cell counts were observed in Lake Jesup stingrays, suggesting that endocrine and immune function may be altered in these animals due to OC exposure and/or other, as yet unknown, ecological factors. Inconsistencies in the reproductive success of Lake Monroe stingrays were observed, confirming earlier reports of reproductive complications in this sub-population. Based on these findings, previous occurrences of reproductive failure in St. Johns River stingrays may be due to environmental factors other than OC exposure.

Molecular identification and functional characteristics of peptide transporters in the bonnethead shark (Sphyrna tiburo).

Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark (Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H(+)-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies.

Uptake of human pharmaceuticals in bull sharks (Carcharhinus leucas) inhabiting a wastewater-impacted river.

The presence of human pharmaceuticals in sewage-impacted ecosystems is a growing concern that poses health risks to aquatic wildlife. Despite this, few studies have investigated the uptake of active pharmaceutical ingredients (APIs) in aquatic organisms. In this study, the uptake of 9 APIs from human drugs was examined and compared in neonate bull sharks (Carcharhinus leucas) residing in pristine (Myakka River) and wastewater-impacted (Caloosahatchee River) tributaries of Florida's Charlotte Harbor estuary. The synthetic estrogen used in human contraceptives (17α-ethynylestradiol) and 6 of the selective serotonin/norepinephrine reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, venlafaxine) used in human antidepressants were observed at detectable and, in some cases, quantifiable levels in plasma of Caloosahatchee River sharks. Comparatively, only venlafaxine was detected in the plasma of a single Myakka River shark at a level below the limit of quantitation. These results suggest that sharks residing in wastewater-impacted habitats accumulate APIs, a factor that may pose special risks to C. leucas since it is one of few shark species to regularly occupy freshwater systems. Further research is needed to determine if the low levels of API uptake observed in Caloosahatchee River bull sharks pose health risks to these animals.

Molecular classification of an elasmobranch angiotensin receptor: quantification of angiotensin receptor and natriuretic peptide receptor mRNAs in saltwater and freshwater populations of the Atlantic stingray.

Among the most conserved osmoregulatory hormone systems in vertebrates are the renin-angiotensin system (RAS) and the natriuretic peptides (NPs). We examined the RAS and NP system in the euryhaline Atlantic stingray, Dasyatis sabina (Lesueur). To determine the relative sensitivity of target organs to these hormonal systems, we isolated cDNA sequences encoding the D. sabina angiotensin receptor (AT) and natriuretic peptide type-B receptor (NPR-B). We then determined the tissue-specific expression of their mRNAs in saltwater D. sabina from local Texas waters and an isolated freshwater population in Lake Monroe, Florida. AT mRNA was most abundant in interrenal tissue from both populations. NPR-B mRNA was most abundant in rectal gland tissue from both populations, and also highly abundant in the kidney of saltwater D. sabina. This study is the first to report the sequence of an elasmobranch angiotensin receptor, and phylogenetic analysis indicates that the D. sabina receptor is more similar to AT(1) vs. AT(2) proteins. This classification is further supported by molecular analysis of AT(1) and AT(2) proteins demonstrating conservation of AT(1)-specific amino acid residues and motifs in D. sabina AT. Molecular classification of the elasmobranch angiotensin receptor as an AT(1)-like protein provides fundamental insight into the evolution of the vertebrate RAS.

Comparative thyroid hormone concentration in maternal serum and yolk of the bonnethead shark (Sphyrna tiburo) from two sites along the coast of Florida.

Maternally provisioned yolk hormones have been determined to play critical roles in development across vertebrate taxa. This study ascertained the presence and concentration of thyroid hormones triiodothyronine (T(3)) and thyroxine (T(4)) in the maternal serum and yolk of the developing placental viviparous shark Sphyrna tiburo from one site adjacent to Tampa Bay and another within Florida Bay, Florida, USA. The developmental profile of T(3) in yolk showed a steady increase from pre-ovulation to post-ovulation and peaked to its highest concentration during the pregnancy stage. There was an increase in the T(3)/T(4) ratio in yolk during the pregnancy stage which suggests a possible increase in the conversion of T(4) to T(3) within yolk, possible embryonic endogenous production, or passive uptake of T(3) from uterine fluids. Similar to the pattern seen in yolk, maternal serum T(3) concentrations tended to increase as development progressed. The concentration of T(3) and T(4) in yolk from Tampa Bay was consistently higher than in yolk from Florida Bay. The differences in the patterns of thyroid hormones from these two locations may explain previously reported differences in the rate of embryonic development in the two locations.

Calcitonin-like immunoreactivity in serum and tissues of the bonnethead shark, Sphyrna tiburo.

Calcitonin is a 32-amino acid peptide hormone that is best known for its actions in maintaining skeletal integrity and calcium homeostasis in mammals. Calcitonin also appears to function in regulating certain aspects of animal reproduction, but the nature of this role remains unclear, particularly in nonmammalian vertebrates. The present study investigated the relationship between calcitonin and reproduction in the bonnethead shark (Sphyrna tiburo), a well-studied member of the oldest living vertebrate group (i.e. elasmobranchs) known to possess a calcitonin-producing organ. Serum calcitonin concentrations were measured in 28 reproductively mature female S. tiburo using a heterologous enzyme-linked immunosorbent assay (ELISA) system. Sites of calcitonin immunoreactivity were detected in tissues of mature female and embryonic S. tiburo using immunocytochemistry. Significant increases in serum calcitonin concentrations of mature female S. tiburo occurred during early stages of gestation, a period characterized by yolk-dependency of developing embryos. Immunoreactive calcitonin was detected in the duodenum and pancreas of embryonic S. tiburo sampled during the same period. The results from this study suggest that calcitonin obtained from endogenous and/or maternal sources may function in regulating yolk digestion in embryonic S. tiburo. Therefore, the association between calcitonin and reproduction in elasmobranchs may reflect an important role for this hormone in embryonic development.

Maternal serum and yolk hormone concentrations in the placental viviparous bonnethead shark, Sphyrna tiburo.

Among vertebrates, maternal transfer of hormones to offspring has been studied extensively in mammals (placental transfer) and more recently in oviparous birds and reptiles (yolk transfer). The placental viviparous bonnethead shark, Sphyrna tiburo, allows the investigation of both yolk and placental hormone transfers in a single organism. In this species, yolk provides nutrition for the first half of embryonic development and placental transfer provides the second half. As sex determination is complete prior to development of placental connections, it was postulated that yolk hormones would have a prominent role in embryonic regulation. The goal of the current study was to determine serum and yolk hormone concentrations during five reproductive stages, from pre-ovulatory through pre-implantation (pre-placental) stages. Radioimmunoassay was used to determine 17beta-estradiol, progesterone, and testosterone concentrations in both serum and yolk. When yolk and serum concentrations were compared, the yolk had significantly higher concentrations of both estradiol and progesterone during post-ovulation and early pregnancy. Yolk concentrations of testosterone were significantly less than serum at pre-ovulation, but there were no differences after that stage. When yolk concentrations were compared between stages, significantly higher concentrations of estradiol were present in ovulatory, post-ovulatory, and pre-implantation stages, while progesterone was significantly higher in post-ovulatory, early pregnancy, and pre-implantation stages and testosterone was higher in pre-ovulation. Most of these results are consistent with the published findings in birds and reptiles. Further, in the bonnethead shark, they suggest that yolk transfer of hormones is adequate for sexual differentiation in embryonic development and that estradiol probably has a significant developmental role.

Serum relaxin concentrations and reproduction in male bonnethead sharks, Sphyrna tiburo.

Relaxin is a 6-kd polypeptide hormone that is responsible for regulating several reproductive processes in female vertebrates, but its role in male reproduction remains unclear. To aid in clarifying this role, the objective of the present study was to investigate changes in endogenous relaxin levels associated with reproductive events in male elasmobranchs, which represent one of only three vertebrate groups known to possess this hormone. Serum relaxin concentrations were measured in 27 immature and 66 mature male bonnethead sharks (Sphyrna tiburo), a species with a well-characterized, seasonal reproductive cycle. Temporal changes in serum relaxin concentrations of immature male S. tiburo were not observed. In contrast, a temporal cycle in serum relaxin concentrations of mature male S. tiburo was observed in individuals from two sampling locations. Significant increases (P<0.05) in serum relaxin concentrations of mature male S. tiburo from both collection sites occurred during late spermatogenesis and the mating period, two critical stages of the reproductive cycle. The results from this study suggest that relaxin may play an important role in regulating semen quality, or other aspects of reproduction in male sharks. This is the first study to demonstrate a temporal pattern in endogenous serum Rlx concentrations associated with reproductive events in feral vertebrates. As such, it strengthens earlier hypotheses that suggested a role for this hormone in regulating male vertebrate fertility and copulatory success.