Elemental analysis of vertebrae discerns diadromous movements of threatened non-marine elasmobranchs.

River sharks (Glyphis spp.) and some sawfishes (Pristidae) inhabit riverine environments, although their long-term habitat use patterns are poorly known. We investigated the diadromous movements of the northern river shark (Glyphis garricki), speartooth shark (Glyphis glyphis), narrow sawfish (Anoxypristis cuspidata), and largetooth sawfish (Pristis pristis) using in situ laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) on vertebrae to recover elemental ratios over each individual's lifetime. We also measured elemental ratios for the bull shark (Carcharhinus leucas) and a range of inshore and offshore stenohaline marine species to assist in interpretation of results. Barium (Ba) was found to be an effective indicator of freshwater use, whereas lithium (Li) and strontium (Sr) were effective indicators of marine water use. The relationships between Ba and Li and Ba and Sr were negatively correlated, whereas the relationship between Li and Sr was positively correlated. Both river shark species had elemental signatures indicative of prolonged use of upper-estuarine environments, whereas adults appear to mainly use lower-estuarine environments rather than marine environments. Decreases in Li:Ba and Sr:Ba at the end of the prenatal growth zone of P. pristis samples indicated that parturition likely occurs in fresh water. There was limited evidence of prolonged riverine habitat use for A. cuspidata. The results of this study support elemental-environment relationships observed in teleost otoliths and indicate that in situ LA-ICP-MS elemental characterization is applicable to a wide range of elasmobranch species as a discriminator for use and movement across salinity gradients. A greater understanding of processes that lead to element incorporation in vertebrae, and relative concentrations in vertebrae with respect to the ambient environment, will improve the applicability of elemental analysis to understand movements across the life history of elasmobranchs into the future.

Arginine site 264 in murine estrogen receptor-α is dispensable for the regulation of the skeleton.

Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα. Estrogen protects against bone loss but is not a suitable treatment due to adverse effects in other tissues. Therefore, increased knowledge regarding estrogen signaling in estrogen-responsive tissues is warranted to aid the development of bone-specific estrogen treatments. Estrogen receptor-α (ERα), the main mediator of estrogenic effects in bone, is widely subjected to posttranslational modifications (PTMs). In vitro studies have shown that methylation at site R260 in the human ERα affects receptor localization and intracellular signaling. The corresponding amino acid R264 in murine ERα has been shown to have a functional role in endothelium in vivo, although the methylation of R264 in the murine gene is yet to be empirically demonstrated. The aim of this study was to investigate whether R264 in ERα is involved in the regulation of the skeleton in vivo. Dual-energy X-ray absorptiometry (DEXA) analysis at 3, 6, 9, and 12 mo of age showed no differences in total body areal bone mineral density (BMD) between R264A and wild type (WT) in either female or male mice. Furthermore, analyses using computed tomography (CT) demonstrated that trabecular bone mass in tibia and vertebra and cortical thickness in tibia were similar between R264A and WT mice. In addition, R264A females displayed a normal estrogen treatment response in trabecular bone mass as well as in cortical thickness. Furthermore, uterus, thymus, and adipose tissue responded similarly in R264A and WT female mice after estrogen treatment. In conclusion, our novel finding that mutation of R264 in ERα does not affect the regulation of the skeleton, together with the known role of R264 for ERα-mediated endothelial effects, supports the concept that R264 determines tissue specificity of ERα.NEW & NOTEWORTHY Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα.

A comparison of the structure, composition and mechanical properties of anosteocytic vertebrae of medaka (O. latipes) and osteocytic vertebrae of zebrafish (D. rerio).

Medaka (O. latipes) and zebrafish (D. rerio) are two teleost fish increasingly used as models to study human skeletal diseases. Although they are similar in size, swimming pattern and many other characteristics, these two species are very distant from an evolutionary point of view (by at least 100 million years). A prominent difference between the skeletons of medaka and zebrafish is the total absence of osteocytes in medaka (anosteocytic), while zebrafish bone contains numerous osteocytes (osteocytic). This fundamental difference suggests the possibility that the bony elements of their skeleton may be different in a variety of other aspects, structural, mechanical or both, particularly in heavily loaded bones like the vertebrae. Here we report on the results of a comparative study that aimed to determine the similarities and differences in medaka and zebrafish vertebrae in terms of their macro- to nanostructure, composition and mechanical properties. Our results reveal many similarities between medaka and zebrafish vertebrae, making the lack or presence of osteocytes the only major difference between the bones of these two species.

Resource use of great hammerhead sharks (Sphyrna mokarran) off eastern Australia.

Great hammerhead sharks Sphyrna mokarran are the largest member of Sphyrnidae, yet the roles of these large sharks in the food webs of coastal ecosystems are still poorly understood. Here we obtained samples of muscle, liver and vertebrae from large S. mokarran (234-383 cm total length; LT ) caught as by-catch off eastern Australia and used stable-isotope analyses of δ15 N, δ13 C and δ34 S to infer their resource use and any associated ontogenetic patterns. The results indicated large S. mokarran are apex predators primarily relying on other sharks and rays for their diet, with a preference for benthic resources such as Australian cownose rays Rhinoperon neglecta during the austral summer. Teleosts, cephalopods and crustaceans were not significant components of S. mokarran diets, though some conspecifics appeared to rely on more diverse resources over the austral summer. Ontogenetic shifts in resource use were detected but trajectories of the increases in trophic level varied among individuals. Most S. mokarran had non-linear trajectories in ontogenetic resource-use shifts implying size was not the main explanatory factor. Stable isotope values of δ13 C and δ34 S in muscle suggest S. mokarran span coastal, pelagic and benthic food webs in eastern Australia.

Elements of time and place: manganese and barium in shark vertebrae reflect age and upwelling histories.

As upper-level predators, sharks are important for maintaining marine food web structure, but populations are threatened by fishery exploitation. Sustainable management of shark populations requires improved understanding of migration patterns and population demographics, which has traditionally been sought through physical and/or electronic tagging studies. The application of natural tags such as elemental variations in mineralized band pairs of elasmobranch vertebrae cartilage could also reveal endogenous and exogenous processes experienced by sharks throughout their life histories. Here, elemental profiles were characterized in vertebrae encompassing complete life histories (birth-to-death) of shortfin mako (Isurus oxyrinchus), common thresher (Alopias vulpinus) and blue shark (Prionace glauca) of known tag and recapture locations in the eastern North Pacific Ocean. All sharks were injected with oxytetracycline at initial capture, released and subsequently recaptured, with individual liberty times ranging from 215 days to 6 years. Vertebral band pairs forming over the liberty intervals were verified by counting the number of band pairs deposited since the oxytetracycline band. Regular oscillations in vertebrae manganese (Mn) content corresponded well with the number of validated band pairs, suggesting that Mn variation could be used to age sharks. Increases in vertebrae barium concentration were correlated with times when individuals occupied areas with high coastal upwelling indices, the timing and spatial intensity of which varied from year to year. Interspecific relationships were probably influenced by behavioural differences in horizontal and vertical habitat use, feeding habits and thermoregulatory physiology. These results indicate that vertebral sclerochronology has the potential to advance our knowledge of elasmobranch life history including age and growth estimation and environmental reconstruction.

Collagen fibril organization within rat vertebral bone modified with metastatic involvement.

Metastatic involvement diminishes the mechanical integrity of vertebral bone, however its specific impact on the structural characteristics of a primary constituent of bone tissue, the collagen-I fibril matrix, has not been adequately characterized. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively. A maximum of 21days was allowed between inoculation and rat sacrifice for vertebrae extraction. Linear polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and transmission electron microscopy (TEM) imaging was utilized to assess the impact of metastatic involvement on collagen fibril organization. Increased observations of deviations in the typical plywood motif or a parallel packing structure and an increased average measured susceptibility ratio (related to relative degree of in-plane vs. out-plane fibrils in the analyzed tissue area) in bone adjacent to metastatic involvement was indicative of change in fibrilar organization compared to healthy controls. In particular, collagen-I fibrils in tumour-induced osteoblastic bone growth showed no adherence to the plywood motif or parallel packing structure seen in healthy lamellar bone, exhibiting a much higher susceptibility ratio and degree of fibril disorder. Negative correlations were established between measured susceptibility ratios and the hardness and modulus of metastatic bone tissue assessed in a previous study. Characterizing modifications in tissue level properties is key in defining bone quality in the presence of metastatic disease and their potential impact on material behaviour.

Effects of sample cleaning and storage on the elemental composition of shark vertebrae.

RATIONALE: Application of vertebral chemistry in elasmobranchs has the potential to progress our understanding of individual migration patterns and population dynamics. However, the influence of handling artifacts such as sample cleaning and storage on vertebral chemistry is unclear and requires experimental investigation. METHODS: Vertebrae centra from blacktip sharks (Carcharhinus limbatus) were cleaned with bleach (NaOCl) for 5 minutes (min), 1 hour (h) and 24 (h) in a cleaning experiment and stored frozen, in 70% ethanol, and 10% formalin treatments for 20 days in a storage experiment. Element concentrations (Li, Na, Mg, Mn, Cu, Zn, Sr, Ba, Pb) were quantified in the outer edges of vertebrae centra using laser ablation inductively coupled plasma mass spectrometry and the [element:Ca] molar ratios were compared among treatments and individual sharks. RESULTS: Bleach cleaning significantly increased [Na:Ca] and formalin storage decreased [Na:Ca] and [Mg:Ca], but ethanol storage did not affect any [element:Ca] ratios. Vertebrae edge [Sr:Ca], [Ba:Ca] and [Mn:Ca] varied among individual sharks, potentially reflecting different environments that they had previously inhabited. CONCLUSIONS: This study shows how archiving methods for vertebrae cartilage can affect primary element:Ca compositions. We demonstrate greatest element:Ca stabilities for vertebrae with limited bleach exposure that are either stored in ethanol or frozen, supporting the use of comparably archived sample sets in future elemental studies.

Age and Growth of Elasmobranchs and Applications to Fisheries Management and Conservation in the Northeast Pacific Ocean.

In addition to being an academic endeavour, the practical purpose of conducting age and growth studies on fishes is to provide biological data to stock assessment scientists and fisheries managers so they may better understand population demographics and manage exploitation rates. Age and size data are used to build growth models, which are a critical component of stock assessments. Though age determination of elasmobranchs in the northeast Pacific Ocean (NEP) began in the 1930s, the field has evolved substantially in recent years, allowing scientists to incorporate age data into assessments for more species than ever before. Owing to the highly diverse biology of this group of fishes, each species has its own set of challenges with regard to age determination. Age determination methods typically rely on semicalcified hard structures that form regular growth patterns; however, the structure selected and preparation method used is often species specific. New staining techniques have improved the ability to assess age and improve ageing precision for some species, and advances in microchemical methods have allowed for independent means of estimating age and validating age determination accuracy. Here we describe current age determination methods for NEP elasmobranchs. While the library of available techniques is increasing, there are still some NEP species for which reliable ageing methods have yet to be defined; we discuss these challenges and potential avenues of future research. Finally, we conclude by describing how age estimates are used in growth models and subsequently in stock assessments of selected NEP elasmobranchs.

Natural variability and effects of cleaning and storage procedures on vertebral chemistry of the blacktip shark Carcharhinus limbatus.

Key methodological assumptions regarding the degree of natural variability and influence of sample handling and storage of elasmobranch vertebral chemistry were assessed using laser-ablation inductively coupled plasma mass spectrometry. Vertebral chemistry of juvenile blacktip sharks Carcharhinus limbatus was examined to identify whether differences existed among different regions of the vertebral column, between thoracic vertebrae of individual fish or within individual vertebrae. Additionally, the effects of bleach exposure and storage in ethanol on vertebral chemistry were compared. No significant variation in vertebral chemistry was found among different regions of the vertebral column or between thoracic vertebrae, but significant differences among transect locations within individual vertebrae were observed. The variation at all three levels appears comparable with published data on sagittal otoliths of bilaterally symmetrical teleost fishes. The experimental assessment of potential treatment effects indicated vertebral chemistry was not significantly affected by bleach or exposure to ethanol. Taken together, these results support the idea that vertebrae taken from the same region of the vertebral column can be treated as equivalent and at least certain elements remain robust to exposure to bleach and ethanol.

Elements and elasmobranchs: hypotheses, assumptions and limitations of elemental analysis.

Quantifying the elemental composition of elasmobranch calcified cartilage (hard parts) has the potential to answer a range of ecological and biological questions, at both the individual and population level. Few studies, however, have employed elemental analyses of elasmobranch hard parts. This paper provides an overview of the range of applications of elemental analysis in elasmobranchs, discussing the assumptions and potential limitations in cartilaginous fishes. It also reviews the available information on biotic and abiotic factors influencing patterns of elemental incorporation into hard parts of elasmobranchs and provides some comparative elemental assays and mapping in an attempt to fill knowledge gaps. Directions for future experimental research are highlighted to better understand fundamental elemental dynamics in elasmobranch hard parts.

Raloxifene improves skeletal properties in an animal model of cystic chronic kidney disease.

Patients with chronic kidney disease (CKD) have an increased risk of fracture. Raloxifene is a mild antiresorptive agent that reduces fracture risk in the general population. Here we assessed the impact of raloxifene on the skeletal properties of animals with progressive CKD. Male Cy/+ rats that develop autosomal dominant cystic kidney disease were treated with either vehicle or raloxifene for five weeks. They were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole-bone mechanics, and material properties). Their normal littermates served as controls. Animals with CKD had significantly higher parathyroid hormone levels compared with normal controls, as well as inferior structural and mechanical skeletal properties. Raloxifene treatment resulted in lower bone remodeling rates and higher cancellous bone volume in the rats with CKD. Although it had little effect on cortical bone geometry, it resulted in higher energy to fracture and modulus of toughness values than vehicle-treated rats with CKD, achieving levels equivalent to normal controls. Animals treated with raloxifene had superior tissue-level mechanical properties as assessed by nanoindentation, and higher collagen D-periodic spacing as assessed by atomic force microscopy. Thus, raloxifene can positively impact whole-bone mechanical properties in CKD through its impact on skeletal material properties.

Stable isotope analysis of vertebrae reveals ontogenetic changes in habitat in an endothermic pelagic shark.

Ontogenetic changes in habitat are driven by shifting life-history requirements and play an important role in population dynamics. However, large portions of the life history of many pelagic species are still poorly understood or unknown. We used a novel combination of stable isotope analysis of vertebral annuli, Bayesian mixing models, isoscapes and electronic tag data to reconstruct ontogenetic patterns of habitat and resource use in a pelagic apex predator, the salmon shark (Lamna ditropis). Results identified the North Pacific Transition Zone as the major nursery area for salmon sharks and revealed an ontogenetic shift around the age of maturity from oceanic to increased use of neritic habitats. The nursery habitat may reflect trade-offs between prey availability, predation pressure and thermal constraints on juvenile endothermic sharks. The ontogenetic shift in habitat coincided with a reduction of isotopic niche, possibly reflecting specialization upon particular prey or habitats. Using tagging data to inform Bayesian isotopic mixing models revealed that adult sharks primarily use neritic habitats of Alaska yet receive a trophic subsidy from oceanic habitats. Integrating the multiple methods used here provides a powerful approach to retrospectively study the ecology and life history of migratory species throughout their ontogeny.

Historical DNA reveals the demographic history of Atlantic cod (Gadus morhua) in medieval and early modern Iceland.

Atlantic cod (Gadus morhua) vertebrae from archaeological sites were used to study the history of the Icelandic Atlantic cod population in the time period of 1500-1990. Specifically, we used coalescence modelling to estimate population size and fluctuations from the sequence diversity at the cytochrome b (cytb) and Pantophysin I (PanI) loci. The models are consistent with an expanding population during the warm medieval period, large historical effective population size (NE), a marked bottleneck event at 1400-1500 and a decrease in NE in early modern times. The model results are corroborated by the reduction of haplotype and nucleotide variation over time and pairwise population distance as a significant portion of nucleotide variation partitioned across the 1550 time mark. The mean age of the historical fished stock is high in medieval times with a truncation in age in early modern times. The population size crash coincides with a period of known cooling in the North Atlantic, and we conclude that the collapse may be related to climate or climate-induced ecosystem change.

Effect of sample preparation techniques for carbon and nitrogen stable isotope analysis of hydroxyapatite structures in the form of elasmobranch vertebral centra.

RATIONALE: Bulk stable isotope analysis (SIA) provides an important tool for the study of animal ecology. Elasmobranch vertebral centra can be serially sampled to obtain an isotopic history of an individual over ontogeny. The measured total δ(13)C value, however, may be misinterpreted due to the inclusion of the (13)C-rich inorganic portion. Hydrochloric acid (HCl) is commonly used to remove the inorganic portion of hydroxyapatite structures before undertaking SIA, but more recently ethylenediaminetetraacetic acid (EDTA) has been recommended for elasmobranch vertebrae. These acid treatments may introduce uncertainty on measured δ(13)C and δ(15)N values above instrument precision and the effect of small sample size remains untested for elasmobranch vertebrae. METHODS: Using a non-dilution program on an isotope ratio mass spectrometer the minimum sample weight of vertebrae required to obtain accurate isotopic values was determined for three shark species: white (Carcharodon carcharias), tiger (Galeocerdo cuvier), and sand tiger (Carcharias taurus). To examine if acid treatment completely removes the inorganic component of the vertebrae or whether the technique introduces its own uncertainty on measured δ(13)C and δ(15)N values, vertebrae samples were analyzed untreated and following EDTA treatment. RESULTS: The minimum sample weight required for accurate stable isotope values and the percentage sample yield following EDTA treatment varied within and among species. After EDTA treatment, white shark vertebrae were all enriched in (13)C and depleted in (15) N, tiger shark vertebrae showed both enrichment and depletion of (13)C and (15)N, and sand tiger shark vertebrae were all depleted in (13)C and (15)N. CONCLUSIONS: EDTA treatment of elasmobranch vertebrae produces unpredictable effects (i.e. non-linear and non-correctable) among species in both the percentage sample yield and the measured δ(13)C and δ(15)N values. Prior to initiating a large-scale study, we strongly recommend investigating (i) the minimum weight of vertebral material required to obtain consistent isotopic values and (ii) the effects of EDTA treatment, specific to the study species and the isotope ratio mass spectrometer employed.

Immunolocalisation of fibrillin microfibrils in the calf metacarpal and vertebral growth plate.

Overgrowth of limbs and spinal deformities are typical clinical manifestations of Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), caused by mutations of the genes encoding fibrillin-1 (FBN1) and fibrillin-2 (FBN2), respectively. FBN1 mutations are also associated with acromicric (AD) and geleophysic dysplasias (GD), and with Weill-Marchesani syndrome (WMS), which is characterised by short stature. The mechanisms leading to such abnormal skeletal growth and the involvement of the fibrillins are not understood. Postnatal longitudinal bone growth mainly occurs in the epiphyseal growth plate. Here we investigated the organisation of fibrillin microfibrils in the growth plate of the long bone and vertebra immunohistochemically. Fibrillin-1 was dual-immunostained with elastin, with fibrillin-2 or with collagen X. We report that fibrillin microfibrils are distributed throughout all regions of the growth plate, and that fibrillin-1 and fibrillin-2 were differentially organised. Fibrillin-1 was more abundant in the extracellular matrix of the resting and proliferative zones of the growth plate than in the hypertrophic zone. More fibrillin-2 was found in the calcified region than in the other regions. No elastin fibres were observed in either the proliferative or hypertrophic zones. This study indicates that, as fibrillin microfibrils are involved in growth factor binding and may play a mechanical role, they could be directly involved in regulating bone growth. Hence, mutations of the fibrillins could affect their functional role in growth and lead to the growth disorders seen in patients with MFS, CCA, AD, GD and WMS.

Mechanical properties of sand tiger shark (Carcharias taurus) vertebrae in relation to spinal deformity.

Approximately 35% of sand tiger sharks (Carcharias taurus) in public aquaria exhibit spinal deformities ranging from compressed vertebrae and loss of intervertebral space to dislocated spines with vertebral degeneration and massive spondylosis caused by excessive mineralization both within vertebrae and outside the notochordal sheath. To identify the mechanical basis of these deformities, vertebral centra from affected (N=12) and non-affected (N=9) C. taurus were subjected to axial compression tests on an MTS 858 Bionix material testing system, after which mineral content was determined. Vertebral centra from affected sharks had significantly lower mineral content and material behavior in nearly all variables characterizing elasticity, plasticity and failure. These mechanical deficiencies are correlated with size at capture, capture method, vitamin C and zinc deficiency, aquarium size and swimming behavior in public aquaria. Non-affected C. taurus had greater stiffness and toughness even though these properties are generally incompatible in mineralized structures, suggesting that the biphasic (mineralized, unmineralized phases) nature of chondrichthyan vertebrae yields material behavior not otherwise observed in vertebrate skeletons. However, vertebral centra from non-affected sharks had lower mineral content (33%), stiffness (167 MPa), yield strain (14%) and ultimate strength (16 MPa) than other species of sharks and bony vertebrates, indicating that biomechanical precautions must be taken in the husbandry of this species.

Proton-density fat fraction and simultaneous R2* estimation as an MRI tool for assessment of osteoporosis.

OBJECTIVE: To investigate multi-echo chemical shift-encoded MRI-based mapping of proton density fat fraction (PDFF) and fat-corrected R2* in bone marrow as biomarkers for osteoporosis assessment. METHODS: Fifty-one patients (28 female; mean age 69.7 ± 9.0 years) underwent dual energy X-ray absorptiometry (DXA). On the basis of the t score, 173 valid vertebrae bodies were divided into three groups (healthy, osteopenic and osteoporotic). Three echo chemical shift-encoded MRI sequences were acquired at 3 T. PDFF and R2* with correction for multiple-peak fat (R2*MP) were measured for each vertebral body. Kruskal-Wallis test and post hoc analysis were performed to evaluate differences between groups. Further, the area under the curve (AUC) for each technique was calculated using logistic regression analysis. RESULTS: On the basis of DXA, 92 samples were normal (53 %), 47 osteopenic (27 %) and 34 osteoporotic (20 %). PDFF was increased in osteoporosis compared with healthy (P = 0.007). R2*MP showed significant differences between normal and osteopenia (P = 0.004), and between normal and osteoporosis (P < 0.001). AUC to differentiate between normal and osteoporosis was 0.698 for R2*MP, 0.656 for PDFF and 0.74 for both combined. CONCLUSION: PDFF and R2*MP are moderate biomarkers for osteoporosis. PDFF and R2*MP combination might improve the prediction in differentiating healthy subjects from those with osteoporosis.

Determination of isoniazid concentration in rabbit vertebrae by isotope tracing technique in conjunction with HPLC.

Medications compounded with isoniazid (INH) are usually applied to surgical sites at the completion of surgery to locally kill postoperative residual tubercle bacilli. However, the distribution and elimination of INH in the vertebrae in vivo are not known. In this study, isotope tracing was used in conjunction with high-pressure liquid chromatography (HPLC) to address this. INH and technetium-99 m-labeled INH were applied to the vertebrae of rabbits. After 2 and 6 h, osseous tissues containing INH, as determined by radionuclide imaging, were collected for detection with HPLC. The results showed that INH mainly stayed around the vertebrae 6 h after its application and did not permeate widely into the blood or other organs, except for the kidneys. The standard deviations of INH concentrations in the technetium-99 m-INH group were approximately four-fold smaller than those in the INH group. This method of coupling isotope tracing and HPLC can effectively limit experimental error during sample collection, allowing accurate and reliable identification of the concentration levels of INH in osseous tissues in vivo.

Gender- and region-specific variations of estrogen receptor α and ß expression in the growth plate of spine and limb during development and adulthood.

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and ß in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and ß expression were shown in the growth plates. Mainly nuclear staining of ERα and ß immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and ß expression and of region on temporal ERα/ERß expression ratio. However, spatial differences of region-related ERα and ß expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and ß immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and ß gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated.

Effects of long-acting testosterone undecanoate on bone mineral density in middle-aged men with late-onset hypogonadism and metabolic syndrome: results from a 36 months controlled study.

We evaluated the effects of long-term testosterone replacement therapy (TRT) on the bone mineral density (BMD) in obese patients with metabolic syndrome (MS) and late-onset hypogonadism (LOH). Sixty men (mean age 57 ± 10) with low serum testosterone (T < 320 ng/dL) and MS regardless the presence of osteoporosis were enrolled. Forty men received intramuscular T-undecanoate (TU) four times/year for 36 months and 20 age-matched hypogonadal men with MS in whom T treatment was contraindicated were used as controls. Hormonal, biochemical markers, vertebral and femoral BMD by dual-energy x-ray absorptiometry were measured. At baseline, overall patients had mild osteopenia (lumbar BMD= 0.891 ± 0.097 g/cm(2); femoral BMD= 0.847 ± 0.117 g/cm(2)). TU induced a significant improvement of bone mass after 36 months (lumbar BMD=1.053 ± 0.145 g/cm(2); p < 0.002; femoral BMD=0.989 ± 0.109; p < 0.003 g/cm(2)) with a 5%/year increase and a significant reduction in hs-CRP without changes in body mass index. A direct relationship between serum T and BMD increments at the lumbar (r(2) = 0.66, p < 0.0001) and femoral (r(2) =0.52, p < 0.0001) sites was demonstrated. Study adherence was 50% without serious side effects. Long-term TRT in middle-aged men with LOH and MS determines a significant increase in both vertebral and femoral BMD related to increased serum T levels, probably independently from estradiol modifications.