Metabolomic and transcriptomic profiles after immune stimulation in the zebrafish testes.

Fish farms are prone to disease outbreaks and stress due to high-density rearing conditions in tanks and sea cages, adversely affecting growth, reproduction, and metabolism. To understand the molecular mechanisms affected in the gonads of breeder fish after an immune challenge, we investigated the metabolome and the transcriptome profiles in zebrafish testes after inducing an immune response. After 48 h of the immune challenge, ultra-high-performance liquid chromatography (LC-MS) and transcriptomic analysis by RNA-seq (Illumina) resulted in 20 different released metabolites and 80 differentially expressed genes. Among these, glutamine and succinic acid were the most abundant metabolites released and 27,5% of the genes belong to either the immune or reproduction systems. Pathway analysis based on metabolomic and transcriptomic crosstalk identified cad and iars genes that act simultaneously with succinate metabolite. This study deciphers interactions between reproduction and immune systems and provides a basis to improve protocols in generating more resistant broodstock.

Cylindrospermopsin impairs zebrafish (Danio rerio) embryo development.

Cyanotoxins are among common contaminants that can impair human, animal, and environmental health. Cylindrospermopsin (CYN) is an abundant form of cyanotoxins elevated following algal bloom in the water worldwide. Previous studies have described CYN effects on several organs in mammals. However, little is known about its toxicity mechanisms in other vertebrates. This study aims to characterize the developmental effects of CYN using zebrafish larvae as an aquatic model organism. A wide range of CYN concentrations (0-2000 µg/L) was tested using a morphometric approach for survival, hatching, various growth and developmental abnormalities. We also investigated the expression of genes related to oxidative stress, osmoregulation, and thyroid function. Exposure to CYN resulted in decreased growth, increased developmental abnormalities such as pericardial and yolk sac edema as well as swim bladder absence. In addition, CYN increased tr1a, and decreased dio1 and dio3 transcript levels which are involved in thyroid-mediated function. It also increased transcript levels related to oxidative stress, including hsp70, ahr1a, cyp1a, gpx and cat. Lastly, CYN exposure increased aqp3a and decreased dab2, which are involved in osmoregulation with a threshold of 10 µg/L. The present study demonstrates multiple effects of exposure to environmentally relevant CYN concentrations in zebrafish embryos.

Image-guided percutaneous cutting needle biopsy in difficult locations.

During the last 30 years, the techniques of percutaneous biopsy have evolved dramatically and its indications have expanded accordingly. Additionally, recent treatments in oncology have made it necessary to use larger cutting needles in biopsies for tissue analysis. These developments force radiologists to perform biopsies in areas they are not very familiar with. However, lesions in such organs and regions are traditionally considered to be dangerous because of the risk of bleeding and trauma to the critical structures along the needle tract. As a result, radiologists are generally reluctant to perform image-guided biopsies in such locations particularly using cutting needles. We present the images of nine cases from our archive, in whom we performed image-guided percutaneous cutting needle biopsy (PCNB) for lesions located in larynx, tongue, intestines, stomach, deep pelvis, uterus, pancreas, penis and spleen. For each specific location, we also mention alternative biopsy methods, the indications of PCNB, its potential risks and special techniques to avoid complications.

Quantitative assessment of temporomandibular disc and masseter muscle with shear wave elastography.

PURPOSE: To assess the normal stiffness values of temporomandibular joint disc (TMJ) and masseter muscle by shear wave elastography (SWE) in a healthy adult population. This may be helpful in diagnosing and understanding the mechanism of temporomandibular disorders (TMD). METHODS: This prospective study evaluated a total of 160 healthy adult volunteers (77 males, 83 females). The stiffness values of anterior, intermediate and posterior parts of the TMJ disc and masseter muscle were measured. Study cohort was classified into four groups based on age. Additionally, we investigated the changes in mean shear wave elasticity (kPa) and shear wave velocity(m/s) values of disc parts and masseter muscle in closed and open mouth positions, and the correlation of these SWE values with age and gender. RESULTS: Mean stiffness values of the disc were 37.02 ± 23.75 kPa and 3.28 ± 1.09 m/s in the anterior part, 30.47 ± 18.89 kPa and 2.97 ± 1.04 m/s in the intermediate part, 22.61 ± 13.97 kPa and 2.55 ± 0.88 m/s in the posterior part. Stiffness values showed significant decrease in the posterior part compared to the rest of the disc both in males and females. No significant differences in mean stiffness values of masseter muscle related to mouth position, age or gender. CONCLUSION: This study provides the normative quantitative elasticity values of disc and masseter muscle which could be a reference point for upcoming studies. Disc elasticity values are higher in women than men. Maybe this is one of the reason why TMD is more common in women. TMJ disc stiffness was significantly lower in the posterior part. SWE is a useful imaging method that can be used with routine ultrasonography in evaluation of the TMJ disc and masticatory muscles .

Diagnosis of COVID-19 in symptomatic patients: An updated review.

A group of pneumonia patients was detected in Hubei Province, in China in December 2019. The etiology of the disease was unknown. Later, the researchers diagnosed the novel Coronavirus as the causal agent of this respiratory disease. On February 12th 2020, the World Health Organization (WHO) officially named this disease Coronavirus disease 2019 (COVID-19). Consequently, the disease spread globally and became a pandemic. As there is no specific treatment for the symptomatic patients and several vaccines are approved by WHO, the efficacy and effectiveness of these vaccines are not fully understood yet and the availability of these vaccines are very limited. In addition, new variants and mutants of SARS-CoV-2 are thought to be able to evade the immune system of the host. So, diagnosis and isolation of infected individuals is advised. Currently, real-time reverse transcription-polymerase chain reaction (RT-PCR) is considered the gold standard method to detect novel Coronavirus, however, there are few limitations associated with RT-PCR such as false-negative results. This demanded another diagnostic tool to detect and isolate COVID-19 early and accurately. Chest computed tomography (CT) became another option to diagnose COVID-19 patients accurately (about 98% sensitivity). However, it did not apply to the asymptomatic carriers and sometimes the results were misinterpreted as from other groups of Coronavirus infection. The combination of RT-PCR and chest CT might be the best option in detecting novel Coronavirus infection early and accurately thereby allowing adaptation of measures for the prevention and control of the COVID-19.

En diciembre de 2019 se detectó un grupo de pacientes con neumonía en la provincia de Hubei, China, desconociéndose la etiología de la enfermedad. Posteriormente, los investigadores señalaron al nuevo coronavirus como agente causal de esta enfermedad respiratoria. El 12 de febrero de 2020, la Organización Mundial de la Salud (OMS) la designó oficialmente como enfermedad por coronavirus de 2019 (COVID-19). A continuación, dicha enfermedad se propagó a nivel global, y se convirtió en una pandemia. No existe tratamiento específico para los pacientes sintomáticos, y la OMS ha aprobado diversas vacunas. Sin embargo, la eficacia y la efectividad de las mismas no se comprende plenamente aún, siendo muy limitada su disponibilidad. Además, se piensa que las diferentes variantes y mutaciones del SARS-CoV-2 son capaces de evadir el sistema inmune del huésped. Por tanto, se recomienda el diagnóstico y aislamiento de las personas infectadas. Actualmente se considera la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) a tiempo real el método de referencia para detectar el nuevo coronavirus. Sin embargo, existen algunas limitaciones asociadas a RT-PCR tales como los resultados falso-negativos. En consecuencia, ello ha demandado otra herramienta diagnóstica para detectar y aislar la COVID-19 de manera temprana y precisa. La tomografía computarizada (TC) de tórax se ha convertido en otra opción para diagnosticar de manera precisa a los pacientes con COVID-19 (cerca del 98% de sensibilidad). Sin embargo no se aplica a los portadores asintomáticos, y a veces se han malinterpretado los resultados como en el caso de otros grupos de infección por coronavirus. La combinación de RT-PCR y TC de tórax podría ser la mejor opción para detectar la nueva infección por coronavirus de manera temprana y precisa, permitiendo, por tanto, la adaptación de las medidas para la prevención y el control de la COVID-19.

Cylindrospermopsin directly disrupts spermatogenesis in isolated male zebrafish testis.

Cylindrospermopsin (CYN) is a cytotoxin, and its documented effects in mammals include damage to several organs. CYN also has hormone-disrupting properties, including estrogenic activity, progesterone production inhibition, and apoptosis induction. While CYN has been reported to exert reproductive toxicity in mice, little is known about its effect on fish reproductive function. Using ex vivo organ culture, we investigated the direct action of CYN on the male reproductive system. Isolated zebrafish testis was exposed to 250, 500, and 1000 µg/L CYN for 24 h and 7 d, followed by histo-morphological analysis. The results demonstrate that exposure to CYN led to a decrease in cell types from all three phases of spermatogenesis in zebrafish testis. There were also significant changes in fshr, lhr, and igf3 transcript levels, as well as testosterone secretion following exposure to CYN. In summary, this study provides novel information on the adverse effects of CYN on testicular spermatogenesis and male reproduction in zebrafish. These results provide a framework for a better understanding of CYN toxicity and the mechanism underlying the adverse action of CYN on male reproduction in fish.

Cylindrospermopsin impairs tubular transport function in kidney cells LLC-PK1.

Cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. Studies have demonstrated that the kidney is the most affected organ. CYN exposure leads to low-molecular-weight proteinuria and increased excretions of the tubular enzymes in mice, suggesting the damage caused by CYN is mainly tubular. However, the mechanism involved in CYN nephrotoxicity remains unknown. Thus, in order to evaluate the effects of CYN exposure (0.1, 0.5 and 1.0 µg/mL) on tubular renal cells LLC-PK1 distinct mechanisms were analyzed by assessing cell death using flow cytometry, albumin uptake by fluorescence analysis, Na+/K+-ATPase activity by a colorimetric method, RT-qPCR of genes related to tubular transport and function as well as internalization of CYN by ELISA. In this study, CYN was found to induce necrosis in all concentrations. CYN also decreased albumin uptake as well as downregulated megalin and dab2 expression, both proteins involved in albumin endocytosis process. Moreover, CYN appears to be internalized by renal tubular cells through a receptor-mediated endocytosis. Finally, the present study demonstrates that CYN is responsible for disrupting tubular cell transport and function in LLC-PK1 cells.

Efficacy of Thermostable Newcastle Disease Virus Strain I-2 in Broiler Chickens Challenged with Highly Virulent Newcastle Virus.

Newcastle disease (ND) is a major threat to poultry industry production throughout developing countries. The Newcastle disease viruses (NDVs) infecting industrialized and indigenous poultry in Iran are velogenic strains and responsible for the frequent outbreaks of ND in poultry farms even in vaccinated flocks causing serious economic losses in the commercial and indigenous poultry. However, vaccination is the only way to protect against endemic ND, and the conventional vaccines are not heat stable and consequently require complex cold-chains to be transferred to users leading to not much resistance. The present study aimed to evaluate the efficacy of thermostable NDV strain I-2 in broiler chickens vaccinated via drinking water and coated on oiled wheat grain. The horizontal transmission of I-2 strain and transmission of disease from vaccinated to unvaccinated chickens were also evaluated in this study. The obtained results showed that both routes of administration, following primary and/or secondary dose, provoked the production of necessary antibody titer and adequate protective immunity in broiler chickens. Moreover, the horizontal transmission of I-2 strain from vaccinated to unvaccinated chickens housed together induced an antibody response and protected unvaccinated chickens against a local field isolate of a virulent strain of NDV (The intravenous pathogenicity index 2.46, mean death time 59 h). Nevertheless, all unvaccinated and Newcastle challenged broilers chickens against the NDV died in this study. It is noteworthy that the transmission of the virus from challenged broiler chickens was very low to induce clinical signs in susceptible chickens. The obtained results of this study revealed the efficacy of NDV strain I-2 coated on the oiled wheat and via drinking water as it protects broiler chickens from highly virulent NDV.

Multifactorial control of reproductive and growth axis in male goldfish: Influences of GnRH, GnIH and thyroid hormone.

Reproduction and growth are under multifactorial control of neurohormones and peripheral hormones. This study investigated seasonally related effects of GnIH, GnRH, and T3 on the reproductive and growth axis in male goldfish at three stages of gonadal recrudescence. The effects of injection treatments with GnRH, GnIH and/or T3 were examined by measuring serum LH and GH levels, as well as peripheral transcript levels, using a factorial design. As expected, GnRH elevated serum LH and GH levels in a seasonally dependant manner, with maximal elevations of LH in late stages of gonadal recrudescence (Spring) and maximal increases in GH in the regressed gonadal stage (Summer). GnIH injection increased serum LH and GH levels only in fish at the regressed stage but exerted both stimulatory and inhibitory effects on GnRH-induced LH responses depending on season. T3 treatment mainly had stimulatory effects on circulating LH levels and inhibitory effects on serum GH concentrations. In the liver and testes, we observed seasonal differences in thyroid receptors, estrogen receptors, vitellogenin, follicle-stimulating hormone receptor, aromatase and IGF-I transcript levels that were tissue- and sex-specific. Generally, there were no clear correlation between circulating LH and GH levels and peripheral transcript levels, presumably due to time-related response and possible direct interaction of GnRH and GnIH at the level of liver and testis. The results support the hypothesis that GnRH and GnIH are important components of multifactorial mechanisms that work in concert with T3 to regulate reciprocal control of reproduction and growth in goldfish.

Apparent diffusion coefficient measurement of ovarian stroma: A potential tool for the diagnosis of polycystic ovary syndrome.

PURPOSE: The purpose of this study was to assess the diagnostic value of measuring the apparent diffusion coefficient (ADC) of ovarian stroma in polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: This study was approved by the institutional review board and informed consent was obtained from each women. A total of 17 women (mean age, 22.2 years±5.1 [SD]; range, 16-33 years) with a diagnosis of PCOS according to the Androgen Excess and PCOS Society criteria and 18 healthy women (mean age, 22.8 years±5.2 [SD]; range, 16-31 years) who served as a control group were included in this prospective study. ADC values of the ovarian stroma during the early follicular phase were calculated by two observers in the two groups. Comparisons were performed using the Student t-test. RESULTS: The mean ADC value in woman with PCOS (1.29±0.27×10-3 mm2/s; range: 0.59×10-3 - 1.88×10-3 mm2/s) was significantly lower than that in the control group (1.48±0.17×10-3 mm2/s; range: 1.12×10-3 - 1.86×10-3 mm2/s) (P<0.001). The ADC cutoff value for the determination of PCOS with maximum accuracy was 1.38×10-3 mm2/s (AUC: 0.720; 95% CI: 0.597, 0.843), yielding 78% sensitivity and 62.9% specificity. CONCLUSION: ADC values of ovarian stroma are lower in patients with PCOS than in control subjects. ADC measurement of ovarian stroma in women with PCOS might help improve the diagnosis of PCOS.

Seasonal effects of GnIH on basal and GnRH-induced goldfish somatotrope functions.

To understand how gonadotropin-inhibitory hormone (GnIH) regulates goldfish GH cell functions, we monitored GH release and expression during early, mid-, and/or late gonadal recrudescence. In vivo and in vitro responses to goldfish (g) GnIH were different, indicating direct action at the level of pituitary, as well as interactions with other neuroendocrine factors involved in GH regulation. Injection of gGnIH consistently reduced basal serum GH levels but elevated pituitary gh mRNA levels, indicating potential dissociation of GH release and synthesis. Goldfish GnRH (sGnRH and cGnRHII) injection differentially stimulated serum GH and pituitary gh mRNA levels with some seasonal differences; these responses were reduced by gGnIH. In contrast, in vitro application of gGnIH during 24-h static incubation of goldfish pituitary cells generally elevated basal GH release and attenuated sGnRH-induced changes in gh mRNA, while suppressing basal gh mRNA levels at mid- and late recrudescence but elevating them at early recrudescence. gGnIH attenuated the GH release responses to sGnRH during static incubation at early, but not at mid- and late recrudescence. In cell column perifusion experiments examining short-term GH release, gGnIH reduced the cGnRHII- and sGnRH-stimulated secretion at late recrudescence but inhibited tha action of cGnRHII only during mid-recrudescence. Interestingly, a reduction of basal GH release upon perifusion with gGnIH during late recrudescence was followed by a rebound increase in GH release upon gGnIH removal. These results indicate that gGnIH exerts complex effects on basal and GnRH-stimulated goldfish GH cell functions and can differentially affect GH release and mRNA expression in a seasonal reproductive manner.

Pathogenesis of H9N2 virus in Chukar partridges.

Low-pathogenic avian influenza virus (H9N2) is circulating in the poultry industry of many countries in the Middle East and Asia, causing serious economic damage. In this study the clinical signs, antibody response, viral shedding and efficacy of oil emulsion vaccines in Chukar partridges were investigated until 9 days post inoculation (d.p.i.). Seventy-five Chukar partridges (Alectoris chukar) were divided randomly in three groups of challenged (Group C), vaccinated and challenged (Group VC) and control (non-vaccinated and non-challenged [Group NC]), 25 birds/group. Groups C and VC were inoculated with 0.4 ml allantoic fluid containing 10(7) median embryo infective dose/bird of A/Chicken/Iran/772/1998(H9N2) avian influenza virus. Clinical signs, antibody response, viral shedding and vaccine efficacy were evaluated and compared among these groups over 9 days. Clinical signs such as coughing and sneezing with depression and decreased feed and water consumption were observed in Group C. In Group VC only a slight decrease in food and water consumption was observed. Both Groups C and VC showed maximum antibody titre at 9 d.p.i. At 1 d.p.i. the virus was detected from all tissues in challenged group, but the virus was not detected from the spleen and caecal tonsil of Group VC. Group C showed the longest period of viral shedding in the trachea and kidney.

Seasonal effect of gonadotrophin inhibitory hormone on gonadotrophin-releasing hormone-induced gonadotroph functions in the goldfish pituitary.

We have shown that native goldfish gonadotrophin inhibitory hormone (gGnIH) differentially regulates luteinsing hormone (LH)-ß and follicle-stimulating hormone (FSH)-ß expression. To further understand the functions of gGnIH, we examined its interactions with two native goldfish gonadotrophin-releasing hormones, salmon gonadotrophin-releasing hormone (sGnRH) and chicken (c)GnRH-II in vivo and in vitro. Intraperitoneal injections of gGnIH alone reduced serum LH levels in fish in early and mid gonadal recrudescence; this inhibition was also seen in fish co-injected with either sGnRH or cGnRH-II during early recrudescence. Injection of gGnIH alone elevated pituitary LH-ß and FSH-ß mRNA levels at early and mid recrudescence, and FSH-ß mRNA at late recrudescence. Co-injection of gGnIH attenuated the stimulatory influences of sGnRH on LH-ß in early recrudescence, and LH-ß and FSH-ß mRNA levels in mid and late recrudescence, as well as the cGnRH-II-elicited increase in LH-ß, but not FSH-ß, mRNA expression at mid and late recrudescence. sGnRH and cGnRH-II injection increased pituitary gGnIH-R mRNA expression in mid and late recrudescence but gGnIH reduced gGnIH-R mRNA levels in late recrudescence. gGnIH did not affect basal LH release from perifused pituitary cells and continual exposure to gGnIH did not alter the LH responses to acute applications of GnRH. However, a short 5-min GnIH treatment in the middle of a 60-min GnRH perifusion selectively reduced the cGnRH-II-induced release of LH. These novel results indicate that, in goldfish, gGnIH and GnRH modulate pituitary GnIH-R expression and gGnIH differentially affects sGnRH and cGnRH-II regulation of LH secretion and gonadotrophin subunit mRNA levels. Furthermore, these actions are manifested in a reproductive stage-dependent manner.

New insights into thyroid hormone function and modulation of reproduction in goldfish.

A number of studies have provided evidence for a link between thyroid hormones and physiological or pathophysiological conditions associated with reproduction. Most of the information available is based on clinical observations in human or research in mammals. There are also a number of studies in non-mammalian species, primarily investigating thyroid and reproductive endocrinology in isolation. The findings demonstrate that hyperthyroidism or hypothyroidism are associated with altered fertility due to changes in the levels and activities of hormones of the brain-pituitary-gonadal axis. There appears to be a consistent pattern based on a number of studies in mammalian and non-mammalian species, linking thyroid with reproduction. Results obtained in goldfish suggest that increased levels of thyroid hormones may reduce overall reproductive function. Since thyroid hormones influence metabolism and are known to stimulate growth in most species, it is likely that increased thyroid hormone levels may divert energy from reproduction and promote somatotropic functions. This is particularly important in oviparous species such as fish since energy investment in females during reproductive season is very significant, and increasing thyroid hormone levels after ovulation may be a contributing factor in promoting growth response. Thyroid hormones will likely work in concert with other hormones to influence reproduction in fish and other vertebrates.

Seasonal effect of GnIH on gonadotrope functions in the pituitary of goldfish.

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release in birds and mammals. To investigate its role in teleosts, we examined the effects of synthetic goldfish (g)GnIH on pituitary LH-ß and FSH-ß subunit, and gGnIH receptor (gGnIH-R) mRNA levels and LH secretion in goldfish. Intraperitoneal injections of gGnIH increased pituitary LH-ß and FSH-ß mRNA levels at early to late gonadal recrudescence, but reduced serum LH and pituitary gGnIH-R mRNA levels, respectively, at early to mid-recrudescence and later stages of recrudescence. Static incubation with gGnIH elevated LH secretion from dispersed pituitary cell cultures from prespawning fish, but not at other recrudescent stages; suppressed LH-ß mRNA levels at early recrudescence and prespawning but elevated LH-ß at mid-recrudescence; and consistently attenuated FSH-ß mRNA in a dose-specific manner. Results indicate that in goldfish, regulation of LH secretion and gonadotropin subunit mRNA levels are dissociated in the presence of gGnIH and dependent on maturational status and administration route.

Administration of heparanase-III improves the survival and angiogenesis of rat skin autografts.

AIM: Heparanase, a glycohydrolase enzyme, cleaves heparan sulfate in the tissue matrix. Heparan sulfate degradation causes the release of angiogenic and growth factors, leading to angiogenesis. The aim of this paper was to evaluate the angiogenic effect of heparinase-III administration on skin autograft healing in rat. METHODS: Four groups of 14 adult male Charles River rats were enrolled. Full thickness skin autografts (15 mm in diameter) were made on the interscapular region of each rat. After 24 hours, 0.1 cc of heparanase, at the three concentrations of 0.1, 0.2, and 0.4 units, were injected intradermally into the grafts of each of the three case groups. The control group received an equal volume of the vehicle (buffered phosphate solution). After 5 days, biopsy specimens from skin grafts of 5 randomly selected rats of each group were submitted for histological studies. RESULTS: The concentration of vessels (with 5-50 mm in diameter) in the grafts of the groups receiving 0.2 and 0.4 units of heparanase-III was significantly more than that of the control group (100.43+/-11.24 and 95.85+/-12.44 vs 71.42+/-5.22 vessels/mm(2), P<0.05). The graft survival time of the group that received 0.2 U heparanase-III was significantly longer than that of the control group (15.43+/-0.72 vs 13.23+/-0.69 days; P<0.05). CONCLUSIONS: Heparanase-III administration improves the healing of rat skin autografts through induction of angiogenesis.

Homologous regulation of estrogen receptor subtypes in goldfish (Carassius auratus).

While considerable information is available on the physiological effects of estrogen, much less is known about the regulation of estrogen receptor (ER) subtypes, particularly in non-mammalian vertebrates. Using goldfish as primary experimental model, we investigated sex- and tissue-specific homologous regulation of ER subtypes (ERalpha, ERbetaI, and ERbetaII) by estradiol in vivo, in the liver and gonads. Treatment with estradiol, significantly upregulated transcript levels for all three types of ERs (ERalpha, ERbetaI, and ERbetaII) in the goldfish ovary and testis. In the goldfish liver, treatment with estradiol significantly increased ERalpha, ERbetaI transcript levels without affecting ERbetaII. In all cases increased ER transcript level was correlated with increased ER protein level determined by Western blot analysis, although we are not able to distinguish between ER subtypes. The results provide strong support for the hypothesis that homologous regulation of ERs is tissue- and gender-specific, and may be a mechanism for estrogen-mediated regulation of reproduction in goldfish.

Role of gonadotropin-releasing hormone (GnRH) in the regulation of gonadal differentiation in the gilthead seabream (Sparus aurata).

It has been proposed that gonadotropin-releasing hormone (GnRH) plays an autocrine/paracrine regulatory role in mammalian and fish ovaries. The marine teleost gilthead seabream is an interesting model since, during the life span of the fish, gonadal tissues develop first as testes, which then regress allowing the development of ovarian follicles. Recent studies carried out in ovaries of the gilthead seabream have demonstrated that various GnRH transcripts as well as GnRH splicing variants are expressed. The mRNA level of several GnRH forms in the female and male areas of the switching gonad, and their possible role in this process, were further investigated. The results here reported show that sGnRH, cGnRH-II, and sbGnRH transcripts are locally expressed during gilthead seabream gonadal differentiation; the expression of the three GnRH forms was found to differ among the morphologically defined areas of the switching gonad, as demonstrated by applying reverse transcription-polymerase chain reaction (RT-PCR), together with in situ hybridization, and semiquantitative PCR analyses. Moreover, the hypothesis that GnRH forms may regulate testicular regression via an apoptotic mechanism was investigated by analyzing the different areas of switching gonads for caspase-3 activity as a measure of apoptosis. Our results showed a marked increase of caspase-3 activity in the area corresponding to the regressing testes in which a significant decrease of testosterone production was also found. The present findings demonstrate that the changes in the endogenous GnRH transcripts could be related with the gonadal differentiation in gilthead seabream, and that exogenous GnRH plays a role by stimulating apoptosis in the degenerating testis.

Molecular characterization and sex-related seasonal expression of thyroid receptor subtypes in goldfish.

The thyroid hormones, acting through the nuclear thyroid receptors (TRs), play important roles in the growth and development of vertebrates. The present study investigated the molecular structure and season-related expression of the TR isoforms in the male and female goldfish pituitary, brain, liver, gonads, gut, heart, and muscle. Based on sequence alignment with other species, the results demonstrate the presence of: (1) a TRalpha (TRalpha-1) consisting of 1496 nucleotides encoding a 466 amino acid protein, (2) a novel splice variant of TRalpha (TRalpha-2) containing an out-of-frame deletion of 246 nucleotides in the ligand-binding domain consisting of 1251 nucleotides encoding a 378 amino acid protein, (3) a novel transcript resembling TRalpha, except for non-homology in the hinge region and a premature stop codon prior to the ligand-binding domain (TRalpha-truncated; 1418 nucleotides, 206 amino acid protein), and (4) TRbeta consisting of 1823 nucleotides encoding a 395 amino acid protein. The findings provide the first demonstration of the presence of a truncated TR isoform in non-mammalian vertebrates. In goldfish, the expression patterns for all TRs subtypes were found to be remarkably similar in both male and female, changing significantly before and during reproductive season. The results provide a frame work for better understanding of the functional significance of novel TR forms and TR subtypes in fish and other vertebrates.