Thermal and endocrine regulation of gonadal apoptosis during sex differentiation in pejerrey Odontesthes bonariensis.

The fate of the differentiating gonads in pejerrey Odontesthes bonariensis is determined by the environmental water temperature experienced by the larvae during the critical period of sex determination. We previously reported a link between apoptosis, temperature and sex differentiation in this species. To clarify this link, we subjected larvae to thermal and endocrine treatments between hatching and the onset of histological sex differentiation of the gonads and assessed the patterns of gonadal development and apoptosis by light microscopic histology and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, respectively. Apoptotic labeling was widespread among somatic cells of the anterior region of the right gonads of fish reared at the male-producing temperature (MPT) and part of the fish at the mixed sex-producing temperature prior to sex differentiation. In contrast, TUNEL-positive cells were rarely observed in gonads at the female-producing temperature (FPT). Administration of exogenous estrogen completely prevented MPT-induced masculinization, induced feminization and reduced gonadal apoptosis, whereas an aromatase inhibitor (fadrozole) induced TUNEL signals in the gonads of FPT-reared larvae. These results provide strong evidence that apoptosis in somatic cells in the right lobe of the gonads might play a key role in testicular differentiation in pejerrey and that estrogens are involved in the regulation of this process.

Electroporation enhances permeation of cryoprotectant (dimethyl sulfoxide) into Japanese whiting (Sillago japonica) embryos.

Survival after cryopreservation has never been achieved with fish embryos, presumably because of insufficient cryoprotectant permeation before cooling. The objective was to determine the relative efficiency of electroporation for incorporation of cryoprotectant into Japanese whiting embryos and survival of electroporated embryos in DMSO after freeze-thawing. Embryos (somites stage) subjected to electroporation at 100, 200, or 300 V in artificial sea water had similar hatching rates (94%-96%) as untreated control embryos (0 V; 97%) and those treated with voltages between 400 and 900 had survival rates of 88% to 0%. Embryos (somites stage) electroporated at 300 V in 10%, 20%, or 30% DMSO/artificial sea water solutions had hatching rates of 94%, 88%, and 85%, respectively, and DMSO contents of 10, 30, and 78 mM. Embryos treated with higher voltages had higher DMSO uptake (up to 84 mM), but reduced survival (62%-6%). Pre-exposure of embryos to 10% DMSO for 20 minutes before electroporation improved DMSO uptake (116 mM). Embryos treated with DMSO and electroporated under the best conditions determined in this study did not resume development after attempted vitrification. We concluded that electroporation enhanced DMSO uptake by fish embryos, but concentrations obtained with this procedure alone were apparently insufficient to prevent internal ice formation during cooling and thawing.

Analysis of sexually dimorphic expression of genes at early gonadogenesis of pejerrey Odontesthes bonariensis using a heterologous microarray.

The process of morphological development of a differentiated gonad from an undifferentiated primordium is a very important step of gonadogenesis. Studies on sexually dimorphic gene expression are important to increase our understanding of this process and to investigate how environmental factors such as temperature can regulate gonadal development. The aim of this study was to identify putative genes involved in sex differentiation in pejerrey (Odontesthes bonariensis) reared at male- and female-producing temperatures (MPT and FPT, respectively) using a microarray heterologous from the medaka (Oryzias latipes), a closely phylogenetic species. Genes related to numerous processes presented higher expression at MPT, including those involved in muscular contraction, metabolic pathways, developmental processes, and reproduction. Genes induced by FPT were classified under the gene ontology terms of response to stimulus, transport and proteolysis. From genes selected for validation, at MPT ndrg3 expression was observed in the somatic cells, whereas pen-2 was detected in germ cells in the caudal portion of the gonads, where no apoptotic signals were observed. Finally, hsp90 was highly expressed in somatic cells of the gonads at the FPT. The results suggest that the interplay of pro-apoptotic and anti-apoptotic genes is important during the masculinization process and for the prevention of sterility following exposure to warm temperatures.

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos.

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl(2) and CaCl(2)). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl(2) and 0.125 M CaCl(2) compared to sea water. A combination of treatments with trehalose and MgCl(2) was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl(2)/CaCl(2) (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.

Implications of climate change for the reproductive capacity and survival of New World silversides (family Atherinopsidae).

The New World silversides (family Atherinopsidae) are found in marine, estuarine and inland waters of North, Central and South America, where they are ecologically important as forage fishes and sometimes economically important for commercial and recreational fisheries. This report reviews the knowledge of the reproductive attributes of temperate and subtropical atherinopsids in relation to temperature and discusses the potential effects of climate change on their reproduction and adaptive responses. Their reproductive cycles are primarily entrained by photoperiod with high temperature acting as a limiting factor. They are generally multiple spawners which release successive batches of eggs in spring, but some species can spawn also in autumn and even summer when temperatures do not increase excessively. The decoupling of temperature patterns and photoperiod with further global warming and associated asymmetric thermal fluctuations could lead to spawning at times or temperatures that are unsuitable for larval development and growth. Many members of this family show temperature-dependent sex determination (TSD), where the phenotypic sex of an individual is determined partly or wholly by the temperature experienced during gonadal sex differentiation, and high-temperature induced germ cell degeneration and decreased fertility. The predicted short-term reproductive responses of atherinopsids to climate change therefore include acceleration, shortening or overall disruption of spawning activity, and also more subtle, but nonetheless equally population-threatening, dysfunctions such as highly skewed sex ratios and partial or total loss of fertility. In the case of species with TSD, asymmetric thermal fluctuations could also cause larvae to encounter temperatures lower than normal during early development and be feminized. Such dysfunctions have been documented already in natural populations but are confined so far to landlocked, inland water habitats, perhaps because they impose more severe thermal fluctuations and limitations to migration and dispersal. The severity and recurrence of these dysfunctions with further climate change will depend both on the magnitude, speed and pattern of change and on how much (or how fast) physiological and behavioural traits can evolve to match the new conditions imposed by the climate, which is largely unknown. In this regard, compelling evidence is shown that numerous traits, including the sex determination system, are capable of rapid evolution and could mitigate the negative effects of temperature increases on population viability in atherinopsids.

Vulnerability of pejerrey Odontesthes bonariensis populations to climate change in pampean lakes of Argentina.

The vulnerability of the pejerrey Odontesthes bonariensis population in Lake Chasicó was assessed under different climate change conditions. During the sampling period, the water temperature was adequate for fish reproduction and to sustain an adequate sex ratio. Climate-driven higher temperatures, however, may severely distort population structure and cause drastic reduction or local extinction of stocks. Lake Chasicó can be classified as eutrophic with clear waters and cyanobacteria that regularly cause fish mortality were identified as Nodularia spumigena and Oscillatoria sp. Global warming may strengthen the effects of eutrophication (e.g. toxic blooms or anoxia). Since many Cyanophyta species tolerate higher temperatures better than other algae, toxic blooms could increase. Furthermore, cyanobacteria have low nutritional value and could decouple the low-diversity food web. Lake Chasicó has currently the salinity optimum (c. 20) for the development of the early life-history stages of O. bonariensis. Climate change, however, is likely to amplify the intensity of droughts or inundations. Floods can endanger O. bonariensis development due to its sub-optimal growth at low salinity and droughts could increase lake salinity and also temperature and nutrient concentration. In order to reduce some of the effects of climate change on the O. bonariensis population in Lake Chasicó, integrated basin management based on an eco-hydrological approach is proposed.

Establishment of a strain inheriting a sex-linked SNP marker in Patagonian pejerrey (Odontesthes hatcheri), a species with both genotypic and temperature-dependent sex determination.

The Patagonian pejerrey Odontesthes hatcheri is an atherinopsid species presenting genotypic sex determination (GSD) at intermediate temperatures and temperature-dependent sex determination at the low and high ranges of thermal tolerance. A recent study revealed the presence of a sex-linked SNP marker in some males of this species, but a strain which inherits the marker faithfully has not been established. This research was conducted to develop such a strain, for use as a tool to study the molecular mechanisms of gonadal sex differentiation and sexual dimorphism, and to obtain basic information on the GSD mode in this species. For these purposes, we performed backcrosses and full-sibling crosses using males and females whose presumptive genotypic sex was inferred from the presence of the sex-linked SNP marker. Four backcrosses between SNP(-) daughters and their SNP(+) father generated balanced sex ratios with the phenotypic sex matching the genotypic sex in most cases (98.21%) at an intermediate, sexually neutral temperature (21 degrees C). Full-sibling crosses between these four SNP(-) females and their SNP(+) brothers produced three progenies with balanced sex ratios and one with 94.4% males. The results of this study confirm that a strain inheriting the sex-linked SNP marker was successfully developed. Moreover, the inheritance pattern of the marker and the sex ratios of the progenies provide strong evidence that the GSD mode in O. hatcheri is the XX-XY system.

Effects of light and temperature conditions on the expression of GnRH and GtH genes and levels of plasma steroids in Odontesthes bonariensis females.

In this study we examined the endocrine mediation between environmental factors (temperature and photoperiod) and the brain-pituitary-gonadal axis in females of pejerrey Odontesthes bonariensis. Changes in the expression of brain gonadotropin-releasing hormones (GnRHs) and gonadotropin (GtH) subunit [follicle stimulating-beta (FSH-beta), luteinizing hormone-beta (LH-beta), glycoprotein hormone-alpha (GPH-alpha)] genes, plasma gonadal steroids [estradiol (E(2)) and testosterone (T)], gonadal histology, and gonadosomatic index (GSI) in adult females exposed to combinations of short-day (8 h) or long-day (16 h) photoperiods and low (12 degrees C) or high (20 degrees C) temperatures after winter conditions (8 h light, 12 degrees C) were analyzed. Pejerrey females kept under the short photoperiod had low GSIs, and their ovaries contained only previtellogenic oocytes regardless of the experimental temperature. In contrast, females exposed to the long photoperiod had high GSIs and ovaries with vitellogenic oocytes at both temperatures. These fish also showed a significantly higher expression of sGnRH, pjGnRH, cGnRH-II (the three different GnRH variants found to date in the pejerrey brain), FSH-beta, LH-beta and GPH-alpha genes and plasma E(2 )levels than those at the shorter photoperiod. No significant changes were observed in plasma T levels. Based on these results, we concluded that the increase in day length but not that of temperature triggers the maturation of pejerrey females after the winter period of gonadal rest and that this occurs by an integrated stimulation of the various components of the brain-pituitary-gonad axis.

mRNA expression of GnRH variants and receptors in the brain, pituitary and ovaries of pejerrey (Odontesthes bonariensis) in relation to the reproductive status.

The present study examined the differential mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition, suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.

Experimentally induced depletion of germ cells in sub-adult Patagonian pejerrey (Odontesthes hatcheri).

Germ cell (GC) transplantation (GCT) is a novel reproductive technology with application in seed production and conservation of endangered species. This study examined the suitability of treatment with Busulfan, a cytotoxic agent, and warm water, known to cause GC degeneration, for depletion of endogenous GCs in sub-adult Patagonia pejerrey Odontesthes hatcheri intended as hosts in GCT. In two experiments, fish were treated with six combinations of temperature (intermediate and high, 20 and 25 degrees C, respectively) and Busulfan (0, 20, and 40 mg/kg body weight), given intraperitoneally (ip) as a single (0 week) or repeated (0 and 4 week) dose. The effectiveness of the treatments was assessed by gonado-somatic index, histology, and (germ cell-specific) vasa gene expression after 8 weeks. Fish were allowed to recover at 17 degrees C for 4-8 weeks after the treatments to ascertain the permanency of the effects. The high temperature (25 degrees C) alone induced only incipient gonadal degeneration and germ cell loss, but was highly effective in combination with double administration of 40 mg/kg Busulfan. Males tolerated Busulfan better and were more easily depleted of germ cells than females. Animals treated for 8 weeks were severely devoid of germ cells, but were still capable of gametogenesis. Thus, the combination of Busulfan and high water temperature appeared to be efficient for depletion of GCs in adult fish; and the treated gonads retained the ability to support GC proliferation and differentiation. Furthermore, quantitative analysis of vasa transcript levels was found to be an useful to monitor the degree of gonad sterility during treatment.

Dimorphic expression of dmrt1 and cyp19a1 (ovarian aromatase) during early gonadal development in pejerrey, Odontesthes bonariensis.

The pejerrey (Odontesthes bonariensis) is a teleost fish with strong temperature-dependent sex determination (TSD). Several studies have shown that dmrt1 and gonadal aromatase (cyp19a1) are implicated in the sex differentiation process in teleosts but little is known on the expression balance and endocrine regulation of these two genes during TSD. This study was designed to clarify the expression patterns of both genes during gonadal sex differentiation of pejerrey reared at female-, male- and mixed-sex-producing temperatures (FPT, MPT, and MixPT, respectively). The expression of dmrt1 was found to be significantly higher during gonadal sex differentiation at MPT compared to FPT. Conversely, cyp19a1 expression clearly increased during differentiation at FPT but not at MPT. The expression of both genes at MixPT showed a dimorphic profile with individual values resembling either those at the MPT or FPT. Administration of exogenous 17beta-estradiol down- and up-regulated the expression of dmrt1 and cyp19a1, respectively, regardless of temperature, and rescued the female phenotype at the MPT. However, treatment with the aromatase inhibitor Fadrozole caused masculinization without changing the pattern of gene expression. These results are strong evidence of the involvement of both genes in the gonadal differentiation process of pejerrey. The involvement of estradiol is discussed.

The significance of cortisol on acclimation to salinity in pejerrey Odontesthes bonariensis

The role of cortisol on the osmoregulation of pejerrey Odontesthes bonariensis at different salinities was investigated in adult fish injected with 0.7mg hydrocortisone per 100g body weight of fish, and transferred to 0, 5 and 20ppt of NaCl. Blood cortisol was 566ng/ml at the beginning of the experiment (0h) but surged to 1250ng/ml within 3h in cortisol-injected fish. Cortisol levels were influenced not only by treatment but also by time, being higher at 3h compared to 24h. Salinity level, time of exposure and their interaction, but not cortisol treatment, significantly affected plasma osmolality and the concentration of ions Cl- and Na+. This study showed that exogenous cortisol does not seem to play a significant role on the regulation of plasma osmolality and concentration of individual ions in pejerrey

Investigou-se a participação do cortisol na osmoregulação de peixe-rei Odontesthes bonariensis, em diferentes salinidades, em peixes adultos injetados com 0,7mg hidrocortisona por 100g de peso corporal, e transferidos para 0, 5 e 20ppt de NaCl. No inicio do experimento (0h), o cortisol encontrado no plasma foi de 566ng/ml, aumentando para 1250ng/ml em 3h em peixes injetados com cortisol. A concentração de cortisol foi influenciada não somente pelo tratamento, mas também pelo tempo, sendo maior 3h após a inoculação, comparada à 24h. A salinidade, o tempo de exposição e a interação desses dois fatores, mas não o tratamento com o cortisol, afetaram significativamente a osmolaridade e a concentração dos íons Cl- e Na+ do plasma. Este estudo mostrou que, o cortisol exógeno não influi significativamente na regulação da osmolaridade e da concentração de íons Na+ e Cl- no plasma em peixe-rei

Temperature-dependent sex determination in Hd-rR medaka Oryzias latipes: gender sensitivity, thermal threshold, critical period, and DMRT1 expression profile.

The developmental time and thermal threshold for temperature-dependent sex determination (TSD), gender differences in temperature sensitivity, the fertility of thermally sex reversed fish, and the effect of temperature on the expression of two major sex determination/differentiation genes (DMY/DMRT1bY and DMRT1) were examined in the Hd-rR strain of medaka, Oryzias latipes. Fertilized eggs were exposed from either shortly after fertilization (8-16 cells; embryonic stages 5-6) or from middle embryogenesis (heart development stage; stage 36) until hatching to temperatures ranging from 17 degrees C to 34 degrees C. Secondary sexual characteristics, gonadal histology, progeny testing, sex-linked body coloration and gene expression were used to determine phenotypic and genotypic sex. Sex determination was unaffected by low or high temperatures in genotypic (XY) males. In contrast, genotypic (XX) females treated from stages 5-6 showed increasing rates of sex reversal into phenotypic males at temperatures above 27 degrees C up to 100% at 34 degrees C. Thermal manipulation of sex was ineffective after stage 36, indicating that gonadal fate in medaka is determined considerably earlier than histological differentiation (stage 39). High temperature induced DMRT1 expression in genotypic females, which was observed already from stage 36. Sex-reversed males had histologically normal testes, were capable of sexual courtship and, with the exception of fish from 34 degrees C, sired viable progeny when mating with fertile females. These results clarify the pattern of TSD in medaka and provide important clues to understand the mechanism of sex determination in this species. They also suggest that a brief exposure to high temperature early in life could impair the fertility of medaka as adults.

Immunocytochemical identification of GtH1 and GtH2 cells during the temperature-sensitive period for sex determination in pejerrey, Odontesthes bonariensis.

The ontogeny of gonadotropin 1 (GtH1) and 2 (GtH2) cells and its possible link to gonadal sex differentiation were studied in pejerrey, Odontesthes bonariensis, by immunocytochemistry using anti-chum salmon beta-GtH1 and beta-GtH2 antisera. In adults, GtH1 cells were found in the proximalis pars distalls (PPD) close to the neurohypophysis, whereas GtH2 cells were identified surrounding GtH1 cells, at the external layer of the PPD and in the pars intermedia. Essentially the same distribution was observed in larvae. In pejerrey, the phenotypic sex is governed by the temperature during the critical period of sex determination (temperature-dependent sex determination, TSD). Female proportions vary gradually from 100% at 15-19 degrees to 0% at 29 degrees, and the critical time of TSD has been estimated to be 3-5, 2-4, and 1-4 weeks after hatching at 17, 19, and 27 degrees, respectively. Thus, the expression of both GtHs was examined weekly in larvae reared from hatching to week 11, at 17, 24, and 29 degrees. The proportion of females at 17, 24, and 29 degrees was, 100%, 78%, and 0%, respectively. Histological ovarian differentiation was noticed at 7 and 4 weeks at 17 and 24 degrees, whereas testicular differentiation occurred at 7 and 4 weeks at 24 and 29 degrees. GtH1 cells were first observed at week 3 at 29 degrees and at week 4 at 17 and 24 degrees. These cells increased in number until week 4 and then decreased, disappearing after week 6 at all temperature regimes. GtH2 cells appeared at week 2 at 24 and 29 degrees and at week 3 at 17 degrees. GtH2 cell number increased until week 3 at 29 degrees and until week 4 at 17 and 24 degrees and then temporarily decreased, thereafter increasing again. These results strongly suggest that GtH1 and GtH2 are expressed by different cells. The fact that GtH1 and GtH2 cells appear just before histological gonadal differentiation at all temperatures, together with the peak of GtH1 and GtH2 cell number during the temperature-sensitive period, suggests that GtHs are related to sex differentiation or TSD in O. bonariensis.

Heat-induced germ cell deficiency in the teleosts Odontesthes bonariensis and Patagonina hatcheri.

Pathological or experimental elevation of testicular temperature is known to trigger degeneration and disappearance of germ cells in scrotal mammals. In contrast, there are no reports of heat-induced germ cell deficiency in males of non-scrotal mammals and other vertebrate taxa, nor in females of any species. This study describes the induction of germ cell deficiency up to complete sterility in the teleosts Patagonina hatcheri and Odontesthes bonariensis by rearing larvae and juveniles for prolonged periods at incipient lethal, high temperatures (27-28.5 and 29 degrees C, respectively). It was shown that female germ cells are also heat-sensitive and disappear under high temperatures. The potential implications of these findings for physiological, ecological, and environmental studies and the usefulness of heat-induced germ cell-deficient fish in research and animal production are discussed.