Seascape Genetics of the Atlantic Spotted Dolphin (Stenella frontalis) Based on Mitochondrial DNA.

The Atlantic spotted dolphin (Stenella frontalis) is endemic to tropical, subtropical, and warm temperate waters of the Atlantic Ocean. Throughout its distribution, both geographic distance and environmental variation may contribute to population structure of the species. In this study, we follow a seascape genetics approach to investigate population differentiation of Atlantic spotted dolphins based on a large worldwide dataset and the relationship with marine environmental variables. The results revealed that the Atlantic spotted dolphin exhibits population genetic structure across its distribution based on mitochondrial DNA control region (mtDNA-CR) data. Analyses based on the contemporary landscape suggested, at both the individual and population level, that the population genetic structure is consistent with the isolation-by-distance model. However, because geography and environmental matrices were correlated, and because in some, but not all analyses, we found a significant effect for the environment, we cannot rule out the addition contribution of environmental factors in structuring genetic variation. Future analyses based on nuclear data are needed to evaluate whether local processes, such as social structure and some level of philopatry within populations, may be contributing to the associations among genetic structure, geographic, and environmental distance.

Open Fetal Microneurosurgery for Intrauterine Spina Bifida Repair.

OBJECTIVES: The aim of the study was to describe the feasibility of open fetal microneurosurgery for intrauterine spina bifida (SB) repair and to compare perinatal outcomes with cases managed using the classic open fetal surgery technique. METHODS: In this study, we selected a cohort of consecutive fetuses with isolated open SB referred to our fetal surgery center in Queretaro, Mexico, during a 3.5-year period (2016-2020). SB repair was performed by either classic open surgery (6- to 8-cm hysterotomy with leakage of amniotic fluid, which was replaced before uterine closure) or open microneurosurgery, which is a novel technique characterized by a 15- to 20-mm hysterotomy diameter, reduced fetal manipulation by fixing the fetal back, and maintenance of normal amniotic fluid and uterine volume during the whole surgery. Perinatal outcomes of cases operated with the classic open fetal surgery technique and open microneurosurgery were compared. RESULTS: Intrauterine SB repair with a complete 3-layer correction was successfully performed in 60 cases either by classic open fetal surgery (n = 13) or open microneurosurgery (n = 47). No significant differences were observed in gestational age (GA) at fetal intervention (25.4 vs. 25.1 weeks, p = 0.38) or surgical times (107 vs. 120 min, p = 0.15) between both groups. The group with open microneurosurgery showed a significantly lower rate of oligohydramnios (0 vs. 15.4%, p = 0.01), preterm rupture of the membranes (19.0 vs. 53.8%, p = 0.01), higher GA at birth (35.1 vs. 32.7 weeks, p = 0.03), lower rate of preterm delivery <34 weeks (21.4 vs. 61.5%, p = 0.01), and lower rate of perinatal death (4.8 vs. 23.1%, p = 0.04) than the group with classic open surgery. During infant follow-up, the rate of hydrocephalus requiring ventriculoperitoneal shunting was similar between both groups (7.5 vs. 20%, p = 0.24). All patients showed an intact hysterotomy site at delivery. CONCLUSION: Intrauterine spina repair by open fetal microneurosurgery is feasible and was associated with better perinatal outcomes than classic open fetal surgery.

Advanced puberty triggered by bi-weekly changes in reproductive factors during the photolabile period in a male teleost fish, Dicentrarchus labrax L.

This study evaluated the impact of continuous light (LL) within the photolabile period on advanced puberty in juvenile male European sea bass. The exposure to an LL regime for 1 month, from August 15 to September 15 (LLa/s), was compared to a constant simulated natural photoperiod (NP) and constant continuous light conditions year-round (LLy). Somatic growth, hormone plasma levels, rates of testicular maturation and spermiation, as well as the mRNA levels of some reproductive genes were analyzed. Our results demonstrated that both LLa/s and LLy treatments, which include LL exposure during the photolabile period, were highly effective in inhibiting the gametogenesis process that affects testicular development, and clearly reduced the early sexual maturation of males. Exposure to an LL photoperiod affected body weight and length of juvenile fish during early gametogenesis and throughout the first year of life. Interestingly, LL induced bi-weekly changes in some reproductive factors affecting Gnrh1 and Gnrh2 content in the brain, and also reduced pituitary fshß expression and plasmatic levels of 11-KT, E2, Fsh throughout early gametogenesis. We suggest that low levels of E2 in early September in the LL groups, which would be concomitant with the reduced number of spermatogonial mitoses in these groups, might indicate a putative role for estrogens in spermatogonial proliferation during the early gonadal development of this species. Furthermore, a significant decrease in amh expression was observed, coinciding with low plasma levels of 11-KT under LL regimes, which is consistent with the idea that this growth factor may be crucial for the progress of spermatogenesis in male sea bass.

Leptin receptor gene in the European sea bass (Dicentrarchus labrax): Cloning, phylogeny, tissue distribution and neuroanatomical organization.

In this study, we report the cloning of three transcripts for leptin receptor in the European sea bass, a marine teleost of economic interest. The two shortest variants, generated by different splice sites, encode all functional extracellular and intracellular domains but missed the transmembrane domain. The resulting proteins are therefore potential soluble binding proteins for leptin. The longest transcript (3605bp), termed sblepr, includes all the essential domains for binding and transduction of the signal. Thus, it is proposed as the ortholog for the human LEPR gene, the main responsible for leptin signaling. Phylogenetic analysis shows the sblepr clustered within the teleost leptin receptor group in 100% of the bootstrap replicates. The neuroanatomical localization of sblepr expressing cells has been assessed by in situ hybridization in brains of sea bass of both sexes during their first sexual maturation. At histological level, the distribution pattern of sblepr expressing cells in the brain shows no clear differences regarding sex or reproductive season. Transcripts of the sblepr have a widespread distribution throughout the forebrain and midbrain until the caudal portion of the hypothalamus. A high hybridization signal is detected in the telencephalon, preoptic area, medial basal and caudal hypothalamus and in the pituitary gland. In a more caudal region, sblepr expressing cells are identified in the longitudinal torus. The expression pattern observed for sblepr suggests that in sea bass, leptin is very likely to be involved in the control of food intake, energy reserves and reproduction.

Is the kisspeptin system involved in responses to food restriction in order to preserve reproduction in pubertal male sea bass (Dicentrarchus labrax)?

Previous works on European sea bass have determined that long-term exposure to restrictive feeding diets alters the rhythms of some reproductive/metabolic hormones, delaying maturation and increasing apoptosis during gametogenesis. However, exactly how these diets affect key genes and hormones on the brain-pituitary-gonad (BPG) axis to trigger puberty is still largely unknown. We may hypothesize that all these signals could be integrated, at least in part, by the kisspeptin system. In order to capture a glimpse of these regulatory mechanisms, kiss1 and kiss2 mRNA expression levels and those of their kiss receptors (kiss1r, kiss2r) were analyzed in different areas of the brain and in the pituitary of pubertal male sea bass during gametogenesis. Furthermore, other reproductive hormones and factors as well as the percentage of males showing full spermiation were also analyzed. Treated fish fed maintenance diets provided evidence of overexpression of the kisspeptin system in the main hypophysiotropic regions of the brain throughout the entire sexual cycle. Conversely, Gnrh1 and gonadotropin pituitary content and plasma sexual steroid levels were downregulated, except for Fsh levels, which were shown to increase during spermiation. Treated fish exhibited lower rates of spermiation as compared to control group and a delay in its accomplishment. These results demonstrate how the kisspeptin system and plasma Fsh levels are differentially affected by maintenance diets, causing a retardation, but not a full blockage of the reproductive process in the teleost fish European sea bass. This suggests that a hormonal adaptive strategy may be operating in order to preserve reproductive function in this species.

The forebrain-midbrain acts as functional endocrine signaling pathway of Kiss2/Gnrh1 system controlling the gonadotroph activity in the teleost fish European sea bass (Dicentrarchus labrax).

Some teleost species, including European sea bass, harbor two different kisspeptin coding genes: kiss1 and kiss2. Both genes are expressed in the brain, but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, we have examined the effects of intracerebroventricular injections of the highly active sea bass peptides Kiss1-15 and Kiss2-12 on spermiating male sea bass. Physiological saline, Kiss1-15, or Kiss2-12 was injected into the third ventricle. To establish the gene expression cascade involved in the action of kisspeptins, the expression of the two sea bass kisspeptin receptor genes (kiss1r and kiss2r) and the three sea bass Gnrh genes (gnrh1, gnrh2, and gnrh3) were analyzed in the forebrain-midbrain and the hypothalamus. In addition, the protein levels of hypothalamic and pituitary Gnrh1 were measured. Blood samples were collected at different times after injection to analyze the effects of kisspeptins on the release of gonadotropins (Lh and Fsh) and androgens (testosterone and 11-ketotestosterone). The present results provide the first evidence that the effects of Kiss2 on central regulation of reproductive function involve the neuroendocrine areas of the forebrain-midbrain in teleost fish. The marked effect of Kiss2 on kiss2r and gnrh1 expression in the forebrain-midbrain and on Gnrh1 release suggest that this neuronal system is involved in the neuroendocrine regulation of gonadotroph activity. This hypothesis was confirmed by a surge of plasma Lh in response to Kiss2, which presumably has a strong stimulatory effect on testosterone release, and thus on sperm quality parameters.

Updating control of puberty in male European sea bass: A holistic approach.

Puberty is the process by which an immature animal acquires the ability to reproduce for the first time; its onset occurs soon after sexual differentiation and is characterized by the beginning of gametogenesis in both sexes. Here we present new insights on when and how the onset of puberty occurs in male European sea bass, its dependence on reaching a critical size, and how it can be controlled by photoperiod, revealing the existence of a photolabile period with important applications in aquaculture. Regarding size, apparently only European sea bass above a certain size threshold attain the ability to carry out gametogenesis during their first year of life, while their smaller counterparts fail to do so. This could imply that fish need to achieve an optimal threshold of hormone production, particularly from the kisspeptin/Gnrh/Gth systems, in order to initiate and conclude puberty. However, a long-term restricted feeding regime during the second year of life did not prevent the onset of puberty, thus suggesting that the fish are able to maintain the reproductive function, even at the expense of other functions. Finally, the study of daily hormonal rhythms under different photoperiod regimes revealed the equivalence between their core values and those of seasonal rhythms, in such a way that the daily rhythms could be considered as the functional units of the seasonal rhythms.

Gonadotropins in European sea bass: Endocrine roles and biotechnological applications.

Follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of the gonadal function in vertebrates. They act through specific receptors located in certain cell types found in the gonads. In fish, the differential roles of these hormones are being progressively elucidated due to the development of suitable tools for their study. In European sea bass (Dicentrarchus labrax), isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance to conduct expression studies. Later, to overcome the limitation of using native hormones, recombinant dimeric gonadotropins, which show different functional characteristics depending on the cell system and DNA construct, were generated. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This approach has allowed the development of detection methods for native gonadotropins, with European sea bass being one of the few species where both gonadotropins can be detected in their native form. By administering recombinant gonadotropins to gonad tissues in vitro, we were able to study their effects on steroidogenesis and intracellular pathways. Their administration in vivo has also been tested for use in basic studies and as a biotechnological approach for hormone therapy and assisted reproduction strategies. In addition to the production of recombinant hormones, gene-based therapies using somatic gene transfer have been offered as an alternative. This approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the genes injected were functional and have allowed studies on the action of gonadotropins in spermatogenesis.

Melatonin-induced changes in kiss/gnrh gene expression patterns in the brain of male sea bass during spermatogenesis.

Evidence exists that melatonin may drive the seasonal changes in kisspeptin-expressing cells and GnRH/gonadotropin secretion in mammals, thus modulating their reproductive activity. This study established the influence of long-term melatonin administration (as an implant) on growth performance and reproduction of adult male sea bass. Melatonin reduced the fish weight and condition factor, thus affecting the performance of fish. Melatonin also affected gonadogenesis, as shown by a decrease in the gonadosomatic index after 150 days of treatment and the lower percentage of running males during the spermatogenesis and full spermiation stages of this species. Exogenous melatonin also resulted in lower plasma androgen levels during the reproductive period, and showed a significant decrease in serum Lh and Fsh concentration after 30 and 60 days of treatment, respectively. Thus, melatonin elicited seasonal changes in key reproductive hormones that affected testicular maturity. The hypothalamic expression of kiss1 was significantly higher in melatonin-treated fish than in controls after 30 days of treatment, while a significant increase in kiss2 expression was detected on day 90 of treatment. By contrast, melatonin showed a significant decrease in kisspeptin expression in the dorsal brain on day 150 of treatment and also affected the expression of gnrh-1 and gnrh-3 and gnrhr-II-1a and 2b and the fshß gene in the pituitary. These results suggest that in this species, melatonin evokes changes in the mRNA levels of kisspeptin and gnrh system genes that appear to mirror disturbances in spermatogenesis.

Administration of follicle-stimulating hormone in vivo triggers testicular recrudescence of juvenile European sea bass (Dicentrarchus labrax).

Follicle-stimulating hormone (Fsh) is thought to act early in the process of spermatogenesis; however, its action in fish has not yet been clearly established. In the present work, we analyzed the effects of recombinant Fsh in sea bass (Dicentrarchus labrax) spermatogenesis according to two different approaches: direct injection of recombinant single-chain Fsh hormone (scFSH) and injection of scFSH coding sequence. Both approaches were efficient in increasing plasma Fsh at 7 and 15 days, respectively, after injection. The Fsh increment caused a significant increase in plasma 11-ketotestosterone levels and induced dramatic changes at the testicular level. Fsh-treated groups showed an increase in germ cell proliferation at Day 7, and cysts of spermatocytes and spermatids were observed at the end of the experiment. After treatment with Fsh, a suppression in amh transcripts and an increase of lhr transcripts were detected at Day 7 and Day 15, respectively, and an increment in fshr expression became evident at Day 23. These results show that Fsh initiates germ cell proliferation, triggering spermatogenesis in sea bass via androgen production and regulation of spermatogenesis-related genes.

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids.

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17ß-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function.

Characterization of sea bass FSHß 5' flanking region: transcriptional control by 17ß-estradiol.

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHß 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHß gene. Analysis of the ~3.5 kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5 kb of the sbFSHß 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LßT2) cell lines. The sbFSHß 5' FR was efficiently expressed under basal conditions in LßT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHß transcriptional activity, in vitro treatments with 17ß-estradiol were carried out on primary cultures of pituitary cells and LßT2 cells transiently expressing luciferase under the control of sbFSHß 5' FR. Overall, these results demonstrate that 17ß-estradiol inhibits sbFSHß gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHß 5'FR (~3.5 kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article.

Luteinizing hormone plasmid therapy results in long-lasting high circulating Lh and increased sperm production in European sea bass (Dicentrarchus labrax).

The present work aimed at evaluating the potential of intramuscular injection of a hormone-coding gene as an approach for gene therapy in fish. A plasmid containing luteinizing hormone (Lh) in a single-chain (sc) form, pCMV-scLh, was chosen as the coding gene, and sea bass was chosen as the target species. In vivo injection of pCMV-scLh in muscle of juvenile sea bass rendered plasma Lh levels higher than 50 ng/ml in 40% of the injected fish, while these Lh levels were only detected in 4% of controls. Injections performed on spermiating broodstock demonstrated that this strategy produced an active Lh able to increase sperm production without affecting its quality, in terms of density. Compared with the injection of a recombinant single-chain Lh, plasmid injection provoked longer-lasting and higher plasma Lh levels. These results show that sea bass skeletal muscle is able to uptake plasmid DNA and to secrete the encoded protein to the bloodstream. Therefore, we propose somatic gene transfer as a realistic approach for hormone therapy of dysfunctions due to low hormone levels in fish or just to synchronize spawning.

Longitudinal HIV-1 gp120-C2V3C3 phylogenetic surveillance and tropism evolution in patients under HAART.

This 8-year longitudinal study was aimed to analyze the HIV-1 gp120-C2V3C3 sequence dynamics, their phylogenetic relationships and tropism evolution in patients under HAART. Such viral analysis comprised two compartments: plasma and PBMC. Fifty gp120-C2V3C3 genomic sequences were characterized from 16 patients: 41 from plasma when viremia was measurable and 9 from PBMCs if plasma viral load was undetectable. The vast majority of HIV isolates (43 out of 50) were ascribed to BF subtype, irrespective of the compartment (plasma or mononuclear-cells) analyzed. A statistically well-supported clustering phenomenon was observed for each patient sampling data. Each cluster comprised viral sequences from both compartments analyzed. In the vast majority of cases, the viral sequences obtained along active production periods were intermingled with those identified from proviral sequences. A substantial stability of co-receptor tropism for the HIV BF subtype was observed over an 8-year followup.

Isolation and characterization of Ff1 and Gsdf family genes in European sea bass and identification of early gonadal markers of precocious puberty in males.

Puberty represents the transition from an immature to a mature reproductive stage. The mechanisms underlying the onset of normal or precocious puberty have not yet been elucidated. With the goal of gaining an understanding of early events that occur in the testes of precocious animals during this process, a hemigonadectomy was performed on male juvenile sea bass and expression levels of candidate mRNAs were determined through quantitative real-time RT-PCR. For this purpose, the gonadal soma-derived factors gsdf1 and gsdf2, the nuclear receptor 5 subfamily members nr5a1a (ff1b), nr5a1b (ff1d), nr5a2 (ff1a) and nr5a5 (ff1c) and the proliferating cell nuclear antigen or pcna, genes with a putative role in the beginning of spermatogenesis, were isolated and cloned. Hemigonadectomy proved to be a suitable strategy for the study of gonadal stages prior to the appearance of histological differences between precocious and non-precocious fish, as it allowed the subsequent classification of these gonads. The upregulation of the gene encoding the steroidogenic acute regulatory protein (Star) in precocious testes indicates that sex steroids could play a role in the onset of spermatogenesis in sea bass. In contrast, the downregulation observed in ff1b expression indicates that this initial surge in star expression is not the result of Ff1b transactivation, suggesting an alternative pathway for this transcriptional activation. Finally, a decrease in gsdf1 expression in precocious animals suggests that this gene may play a role in the onset of puberty, while its correlation with ff1b expression points to gsdf1 as a putative target for Ff1b-mediated transactivation.

Development of a homologous enzyme-linked immunosorbent assay for European sea bass FSH. Reproductive cycle plasma levels in both sexes and in yearling precocious and non-precocious males.

Since the late 1980s, gonadotropins have been isolated and characterized in several fish species, but specific immunoassays for the follicle-stimulating hormone (FSH) have only been developed for a few. The present study reports the development and use of a specific and homologous competitive ELISA for measuring FSH in European sea bass (Dicentrarchus labrax) using a recombinant FSH and its specific antiserum. Recombinant European sea bass FSHß and FSH heterodimer were produced in the methylotrophic yeast Pichia pastoris and a baculovirus expression system, respectively. Specific polyclonal antibodies, generated by rabbit immunization against recombinant FSHß, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166 µg/ml). The sensitivity of the assay was 0.5 ng/ml [B(0)-2SD], and the intra- and inter-assay coefficients of variation were 2.12% (n=10) and 5.44% (n=16) (B(i)/B(0) ∼45%), respectively. A high degree of parallelism was observed between the standard curve and serially diluted plasma and pituitary samples of European sea bass. The ELISA developed was used to study the plasma FSH profiles of mature males and females during the reproductive cycle, and those of immature juvenile males under different light regimes. The analysis showed that FSH increased significantly during the intermediate stages of spermatogenesis and during vitellogenesis. Analyses in immature juvenile males showed that the continuous light photoperiod significantly reduced plasma FSH levels, and consequently, testicular growth and precocious puberty. In conclusion, the immunoassay developed has proven to be sensitive, specific and accurate for measuring European sea bass FSH, and it represents a valuable tool for future studies on the reproductive endocrinology of this species.

Comparative insights of the kisspeptin/kisspeptin receptor system: lessons from non-mammalian vertebrates.

Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species.

Crossbow injuries: A case of suicide.

We are reporting the case of a 65 year-old man who suffered a severe depression and committed suicide using a crossbow. The death happened at his home. The suicide victim was on his knees, with of a crossbow leveled at his thorax and located facing him on the sofa. He shot it by pulling the trigger with the bended end of a ramrod. The arrow entered between the third and the fourth rib on the left side, with a downwards and slightly outwards direction. It went through the lung and the tip of the arrow came out the back. The arrow was removed when the body was lifted, and the wounds had a three-pointed star shape which corresponded to the head of the arrow crossbow. Police enquiry and forensic investigation confirmed a suicidal manner of death. This paper presents different issues of the case, such as the attestation of tears in clothing, the morphology of the wounds or the arrow track. The case is compared with other cases in the medical forensic literature involving the use of crossbows. Finally, it is highlighted how easy it is to purchase these weapons despite their obvious power and accuracy, factors that seriously recommend legislative regulation to be increased and its use more restricted.

Determination of Fsh quantity and bioactivity during sex differentiation and oogenesis in European sea bass.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone that plays a key role in the regulation of gonadal functions in vertebrates. The present study reports the monitoring of pituitary and plasma Fsh levels during sex differentiation and oogenesis in European sea bass (Dicentrarchus labrax) using a homologous immunoassay and an in vitro bioassay. Both assays were used complementarily for the first time in a fish species. High levels of Fsh bioactivity in plasma were found during the initial phases of sexual differentiation. Plasma and pituitary Fsh (quantity and bioactivity) levels and biological to immunological (B:I) ratios were higher in females than in males, suggesting sexual dimorphism in the synthesis and potency of Fsh. In females, the B:I ratios in adult were lower than during sex differentiation indicating that Fsh would be less biopotent in the adult stage. Plasma Fsh bioactivity levels increased during vitellogenesis, suggesting that Fsh would be involved in the regulation of the midphases of oogenesis, whereas luteinizing hormone would be responsible for the final events.

Metal content in stranded pelagic vs deep-diving cetaceans in the Canary Islands.

The Canary Islands are home to many cetacean species, many of which are resident species. The present work aims to analyze, for the first time to the best of the authors' knowledge, the macronutrients, micronutrients and trace elements and toxic heavy metals in muscle and liver tissue of six species of stranded cetaceans in the Canary Islands. The study species were: Tursiops truncatus, Stenella frontalis, Delphinus delphis, Grampus griseus, Globicephala macrorynchus and Physeter macrocephalus. Statistical analysis studied the significant differences between the concentrations in muscle and liver tissues, with the differences in element content depending on the type of diving and length of the species. The results indicate that there are differences between muscle and liver for Ca, Cd, Co, Cu, K, Mg, Mn, Mo, Ni, Pb, Sr, V and Zn. Deep-diving animals differ in their concentrations of Cr, Cu, Mg, Mn, Mo, and Zn with respect to shallow-diving animals in muscle and in liver in Al, B, Cr, K, Mn and Mo. As for the differences between sex, the males present differences in their concentrations of B, Cd, K and Mg in muscle tissue with respect to the females, while differences in the liver were only detected in the Fe content. The study of the correlations shows that as the size of the animal increases, the concentration of Cd increases while the concentrations of Al, Cu and Zn decrease. The specimens foraging in shallower waters had the highest concentration of the macronutrient.