Vertebral cartilage of the clearnose skate, Raja eglanteria: Development, structure, ageing, and hormonal regulation of growth

Incremental marks formed in the vertebral cartilage of most sharks, skates and rays are widely used as indicators of age in elasmobranch growth studies. Such information is essential for fisheries management, thus vertebral ageing has become an invaluable tool for investigating elasmobranch life history. Unfortunately, lack of information on the processes that regulate vertebral growth and mineralization limit efforts to correlate episodic stimuli with increment production. to address this research need, this dissertation investigated these processes through a detailed study on the vertebral cartilage of the clearnose skate, Raja eglanteria. Histologic observations indicated that changes in appositional cartilage growth are the catalyst for increment formation. Observations by scanning electron microscopy supported this conclusion by demonstrating that growth increments differ in the proportion of cells to mineralized matrix. In contrast, elemental analysis of vertebrae using energy-dispersive spectrophotometry demonstrated no change in mineral concentration between seasonal growth layers. This observation was advantageous, because uptake of the radiotracer &\sp{lcub}45{rcub}&Ca was ineffective in estimating the rates of vertebral calcification in captive R. eglanteria. Failure of this standard method appeared to reflect the free exchange of calcium between skeletal and serologic reservoirs. An in vitro method for measuring vertebral growth was developed using incorporation of &\sp{lcub}35{rcub}&S-sulfate as a marker for cartilage matrix synthesis. Certain conserved among elasmobranchs and higher vertebrates. The growth hormone-dependent serum factor insulin-like growth factor-I (IGF-I) increased vertebral matrix synthesis, suggesting an important role for this hormone in regulating elasmobranch skeletal growth. In contrast, corticosterone reduced &\sp{lcub}35{rcub}&S-sulfate uptake in vertebral cartilage, suggesting an inhibitory role for glucocorticosteroids in elasmobranch chondrogenesis. Calcitonin also inhibited vertebral matrix synthesis and, in vivo, may play some role in skeletal development or mineral homeostasis. Finally, nutritional status also appeared to influence vertebral growth in vitro, perhaps indirectly through effects on hormone production. In sum, this dissertation identified changes in cartilage growth as the impetus for vertebral growth zone production in R. eglanteria. Physiological mechanisms that likely regulate vertebral growth are described in this pioneer study on elasmobranch skeletal growth

Age And Growth Of The Smooth Dogfish (Mustelus canis) In The Northwest Atlantic Ocean

The northwest Atlantic population of smooth dogfish (Mustelus canis) ranges from Cape Cod, Massachusetts, to South Carolina. Although M. canis is seasonally abundant in this region, very little is known about important aspects of its biology, such as growth and reproductive rates. In the early 1990s, commercial fishery landings of smooth dogfish dramatically increased on the east coast of the United States. This study investigated growth rates of the east coast M. canis population through analysis of growth patterns in vertebral centra. Marginal increment analysis, estimates of precision, and patterns in seasonal growth supported the use of vertebrae to age these sharks. Growth bands in vertebral samples were used to estimate ages for 894 smooth dogfish. Age-length data were used to determine von BertalanfFy growth parameters for this population: K = 0.292/yr, L-infinity = 123.57 cm, and t(0) = -1.94 years for females, and K = 0.440/yr, L-infinity = 105.17 cm, and t(0) = -1.52 years for males. Males matured at two or three years of age and females matured between four and seven years of age. The oldest age estimate for male and female samples was ten and sixteen years, respectively

Environmental Emission of Pharmaceuticals from Wastewater Treatment Plants in the USA

The residual drugs, drug bioconjugates, and their metabolites, mostly from human and veterinary usage, are routinely flushed down the drain, and enter wastewater treatment plants (WWTP). Increasing population, excessive use of allopathic medicine, continual introduction of novel drugs, and existing inefficient wastewater treatment processes result in the discharge of large volumes of pharmaceuticals and their metabolites from the WWTPs into the environment. The effluent from the WWTPs globally contaminate ~25% of rivers and the lakes. Pharmaceuticals in the environment, as contaminants of emerging concerns, behave as pseudo-persistent despite their relatively short environmental half-lives in the environment. Therefore, residual levels of pharmaceuticals in the environment not only pose a threat to the wildlife but also affect human health through contaminated food and drinking water. This chapter highlights WWTPs as point-sources of their environmental emissions and various effects on the aquatic and terrestrial ecosystem

p21WAF1/CIP1expression in stage I cutaneous malignant melanoma: its relationship with p53, cell proliferation and survival

The expression of p21, p53 and proliferating cell nuclear antigen (PCNA) was analysed by immunohistochemistry in a consecutive series of 369 clinical stage I cutaneous malignant melanoma patients. Correlation of the detected expression levels with each other, with clinicopathological data and with melanoma survival were statistically evaluated. p21 expression was significantly associated with p53 and PCNA expression levels. In addition, high levels of p53 and PCNA were significantly interrelated. Tumour thickness, recurrent disease, high TNM category and older (≥ 55 years) age at diagnosis were inversely associated with p21 expression. Gender, bleeding, tumour thickness, Clark's level of invasion, TNM category and p53 index were all important predictors of both recurrence-free and overall survival of melanoma. In Cox's multivariate analysis including 164 patients with a complete set of data, only high tumour thickness and bleeding predicted poor recurrence-free survival (P= 0.0042 and 0.0087 respectively) or overall survival (P= 0.0147 and 0.0033 respectively). Even though elevated p21 expression may be associated with more favourable prognosis in clinical stage I cutaneous melanoma, our results suggest that cell cycle regulatory effects of p21 can be overcome by some other and stronger, partly yet unknown, mechanisms. 1999 Cancer Research Campaig

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

Morphological Changes In The Clasper Gland Of The Atlantic Stingray, Dasyatis Sabina, Associated With The Seasonal Reproductive Cycle

The clasper gland of the Atlantic stingray, Dasyatis sabina, was examined over a 1-year period, covering an entire reproductive cycle. Changes in clasper gland tissue architecture, fluid production, and cell proliferation were assessed. No changes in tissue architecture were observed. Evidence of cell proliferation in the gland epithelium was not detected using immunocytochemistry for proliferating cell nuclear antigen, a cellular marker of mitosis. Epithelial cells were not observed to undergo mitosis, and cell membranes remained intact. The lack of structural changes and epithelial cell proliferation supports the proposed merocrinal mode of fluid secretion. Rays captured in nonbreeding months had clasper glands that exhibited tubules with reduced lumens. In contrast, rays caught during the breeding season had clasper gland tubules with enlarged lumens. Clasper gland fluid production was quantified through measurements of the fluid area and tubule area calculated from digital images. Clasper gland fluid production was significantly higher during the mating period than during months not associated with copulatory activity. These data support the notion that the clasper gland is involved in stingray copulatory activity. This study adds to the limited amount of literature focused on this poorly understood component of reproduction in skates and rays. © 2005 Wiley-Liss, Inc

Mercury Accumulation and Effects in the Brain of the Atlantic Sharpnose Shark (Rhizoprionodon terraenovae)

Few published studies have examined whether the elevated concentrations of the nonessential toxic metal mercury (Hg) often observed in shark muscle also occur in the shark brain or whether Hg accumulation affects shark neurophysiology. Therefore, this study examined accumulation and distribution of Hg in the shark brain, as well as effects of Hg on oxidative stress in the shark central nervous system, with particular focus on the Atlantic sharpnose shark (Rhizoprionodon terraenovae). Sharks were collected along the southeastern U.S. coast throughout most of this species’ U.S. geographical range. Total Hg (THg) concentrations were measured in and compared between shark muscle and brain, whereas known biomarkers of Hg-induced neurological effects, including glutathione depletion, lipid peroxidation, and concentrations of a protein marker of glial cell damage (S100b), were measured in shark cerebrospinal fluid. Brain THg concentrations were correlated with muscle THg levels but were significantly lower and did not exceed most published thresholds for neurological effects, suggesting limited potential for detrimental responses. Biomarker concentrations supported this premise, because these data were not correlated with brain THg levels. Hg speciation also was examined. Unlike muscle, methylmercury (MeHg) did not comprise a high percentage of THg in the brain, suggesting that differential uptake or loss of organic and inorganic Hg and/or demethylation of MeHg may occur in this organ. Although Hg accumulation in the shark brain generally fell below toxicity thresholds, higher THg levels were measured in the shark forebrain compared with the midbrain and hindbrain. Therefore, there is potential for selective effects on certain aspects of shark neurophysiology if brain Hg accumulation is increased

Histological and morphological aspects of reproduction in the sandbar shark Carcharhinus plumbeus in the U.S. south-eastern Atlantic Ocean and Gulf of Mexico

The reproduction of the sandbar shark Carcharhinus plumbeus in the U.S. south-eastern Atlantic Ocean including the Gulf of Mexico was examined using a combination of histological and morphological characteristics of C. plumbeus collected through fishery-dependent and -independent sampling programmes (n = 1,567). Indices of maturity were constructed using measurements of gonads, reproductive tracts and claspers, and sandbar sharks exhibited 50% maturity sizes of 140 and 148cm fork length for males and females respectively. Gonado-somatic indices and variation in reproductive tract condition were used to determine seasonal trends in reproduction of mature C. plumbeus. Sandbar sharks have discrete seasonal reproductive cycles in which males produce sperm from January to May with a peak in May and females develop eggs from January to May with ovulation occurring in June. Females were shown to exhibit a \u3e2year reproductive cycle. Embryonic development was assessed through measurements of masses and lengths of uterine contents. Gestation was 12months, from July to the following June, with parturition in late June. This research highlights a difference from previously reported data on the periodicity of female reproduction in C. plumbeus in the U.S. south-eastern Atlantic Ocean and Gulf of Mexico, which may have major effects on future C. plumbeus stock management

Protein expression of hypoxia-inducible factor 1-alpha (HIF-1α) in spot (Leiostomus xanthurus) exposed to constant and diel-cycling hypoxia

Fish kills often occur overnight from low dissolved oxygen (DO) events, however many mortality events are of unknown cause, since little water quality monitoring occurs overnight. The hypoxia-inducible factor 1α (HIF-1α) protein is an excellent candidate as a biomarker for deciphering idiopathic fish kills. In this study, spot (Leiostomus xanthurus) were exposed to either constant or diel-cycling hypoxia, and HIF-1α expression was compared to normoxic control over three days. The results indicated that HIF-1α protein increased (p \u3c 0.005) in muscle tissue after three days exposure to both constant and a simulated diel-cycling hypoxic event in a laboratory setting when compared to normoxic control animals. In comparison, diel-cycling hypoxia produced higher HIF-1α protein concentrations than constant hypoxia in all treatments, suggesting that the fluctuations of DO induced the expression of protein. The results from this study pose implications for using HIF-1α as a biomarker in wild populations, as overnight hypoxic cycles may produce higher protein concentrations than a constant hypoxic event, which would enable the detection of otherwise unnoticed hypoxic stress. © 2012 Elsevier B.V

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