Sex reversal of pejerrey (Odontesthes bonariensis), a species with temperature-dependent sex determination, in a seminatural environment.

This study examined the changes in sex ratios and sex reversal rates in pejerrey Odontesthes bonariensis that occur with the progression of the spawning season in a seminatural setting. Four groups of hatchery-produced pejerrey larvae were stocked in floating cages in La Salada de Monasterio lake (Pampas region), a natural habitat of this species, and reared from hatching beyond gonadal sex determination with minimum human interference. Cage 1 was stocked at the beginning of the spring spawning season and the other cages were stocked with monthly delays until cage 4 in early summer. The genotypic (amhy+, XY/YY; amhy-, XX) and phenotypic (testis, male; ovary, female) sex ratios and proportions of genotype/phenotype mismatched individuals were estimated and their relation to water temperature and daylength during the experiment was analysed by generalized linear modelling. Water temperature varied between 11 and 30.5°C, and daylength duration between 11 h 22 min and 14 h 35 min. Sex genotyping revealed nearly balanced sex ratios of XY/YY (46%-49.1%) and XX (50.9%-54%) fish in cages 2-4 whereas the genotypic sex ratio in cage 1 was clearly biased towards XY/YY fish (60.6%). Phenotypic males ranged from 42% to 54.4% in cages 1-3. Cage 4, in turn, had significantly more phenotypic males (66%). The percentage of XX males (phenotypic male/genotypic female) was 23.1% in cage 1, decreased to a minimum of 5.4% in cage 2 and gradually increased in cages 3 and 4 to a maximum of 40.7% in the latter. The percentages of XY/YY females (phenotypic female/genotypic male) were highest in cage 1 (30%) and decreased progressively in the other cages to a significantly lower value (4.3%) in cage 4. These results generally support the findings of laboratory studies on the effect of temperature on the sex determination of this species and also provide novel evidence of a XX genotype-specific masculinizing effect of short daylength.

Effects of Anthropic Pollutants Identified in Pampas Lakes on the Development and Reproduction of Pejerrey Fish Odontesthes bonariensis.

Anthropic activities can seriously affect the health of the organisms inhabiting them, and the observation of any alteration in the reproduction of fish could be associated with the presence of endocrine disruptors. In this manuscript we have collected information on the adverse effects of pollutants (heavy metals, environmental steroids, and agrochemicals), present in Chascomús lake, Argentina, either at environmentally relevant and pharmacological concentrations on reproduction, embryonic development, and larval survival of pejerrey fish Odontesthes bonariensis. During development, it has been reported that 17ß-estradiol (E2) feminized and reduced larval survival, while 17α-ethinyl-estradiol (EE2) not only feminized but also affected both embryo and larval survival. In adult male fish, treatments with EE2 and E2 + EE2 were able to increase mRNA abundance of gnrh3 and cyp19a1b and decreased those of gonadotropin receptors (fshr and lhcgr). Heavy metals such as cadmium, chromium, and copper negatively affected sperm quality, diminishing the motility. Also, a decrease in the percentage of hatching rate and larval survival was also observed with the same metals, highlighting zinc as the most detrimental metal. Furthermore, all these metals altered the expression of hypothalamic and pituitary genes related to reproduction in male pejerrey (gnrh1,2,3; cyp19a1b; fshb; lhb; fshr and, lhcgr). Moreover, in all cases pyknotic cells, corresponding to the degeneration of the germ cells, were observed in the testes of exposed fish. For agrochemicals, exposure of male pejerrey to environmental concentrations of glyphosate did not cause alterations on the endocrine reproductive axis. However, male pejerrey with gonadal abnormalities such as the presence of intersex (testis-ova) gonads were found in other Pampa´s lakes with high concentrations of atrazine and glyphosate associated with soybean and corn crops near their coasts. These types of studies demonstrate that pejerrey, an endemic species with economic importance inhabiting the Pampas shallow lakes, can be used as a sentinel species. It should be noted that increased pollution of aquatic ecosystems and the effects on the reproduction of organisms can lead to a decline in fish populations worldwide. Which, added to overfishing and other external factors such as global warming, could cause an eventual extinction of an emblematic species.

Gonadal development in pejerrey (Odontesthes bonariensis) during spawning season in relation with sex steroids and temperature variation in Gómez lake (Pampas region, Argentina).

Gómez lake (34 ° 39 'S 61 ° 01' W) is a typical shallow lake of Pampas region placed in the upper area of the Salado river. The most abundant fish species in this lake is the pejerrey (Odontesthes bonariensis) valued due to the quality of its flesh and its attractiveness as a game fish. The aim of this study was to describe for the first time in this pejerrey wild population the gonadal stages during three consecutive spawning seasons (August to December) in relation with sexual steroids and temperature in this lake. In general, pejerrey gonadal development, the gonadosomatic index and the plasma levels of estradiol and testosterone varied in relation to air temperature. During the sampling period, pejerrey females started to ovulate in early August (winter), with a peak in October and ending in December with some of them with atretic oocytes. For males, it was possible to find spermiating animals during the whole spawning season and some arrested animals only in December. Our results confirm the relationship with pejerrey maturation and temperature and can be useful for decision making in the management of this natural resource.

Effects on Ultrastructure, Composition and Specific Surface Area of the Gills of Odontesthes bonariensis Under Subchronic Glyphosate-Based Herbicide Exposure.

Gills represent one of the major sites of gas exchange of fish, consequently they are in continuous close contact with the aquatic environment and its pollutants. In the present study the effects on gills of pejerrey fish, Odontesthes bonariensis, under glyphosate-based herbicide subchronic exposure were analyzed. Adult animals were exposed to sublethal concentrations of a glyphosate-based commercial formulation (1 and 10 PMG mg L-1, PMG: glyphosate active ingredient) for 15 days, while control group was maintained in rearing water. Ultrastructural changes in gills were observed by scanning electron microscopy (SEM). The composition of the surface epithelium and specific surface area were determined by energy dispersive spectroscopy (EDS) and N2 (g) adsorption-desorption isotherms, respectively. The herbicide exposure induced severe alterations in gill ultrastructure, as shown in the SEM micrographs. Accordingly, an increase in surface area of the gills of exposed animals was determined. These results support that gills parameters of freshwater fish are sensitive morphological biomarkers for glyphosate exposure.

Effects of metals on sperm quality, fertilization and hatching rates, and embryo and larval survival of pejerrey fish (Odontesthes bonariensis).

Some species of fish have been used as bioindicators of aquatic environmental pollution all over the world. Pejerrey (Odontesthes bonariensis) was selected for the current study due to its sensitivity to pollutants and because is one of the emblematic fish species that inhabits shallow lakes of the Pampa region (Argentina). Recently, in Chascomús lake were recorded concentrations of Cd, Cr, Cu and Zn with values above the Argentine National Guidelines for the Protection of the Aquatic life. Regarding this, the aim of the present study was to investigate the effects of environmental concentrations of these metals on the sperm quality, fertilization and hatching rates, and embryo and larval survival of pejerrey. Also, the same endpoints were analyzed with concentrations ten times higher to simulate a polluted worst-case scenario. The results showed that the presence of some metals in aquatic environments reduced pejerrey sperm motility (in ~50%) and velocity (in ~30%). These results were obtained using a computer assisted sperm analyzer enforcing the application of this analysis as a tool or bioindicator of aquatic pollution. In addition, fertilization rate was diminished (in ~40%) for all treatments. Besides, the hatching rate, and embryo and larval survival were drastically affected being zero for the highest metal concentrations assessed. All together these results, showed that even lower metal concentrations can negatively affect different reproductive parameters of one of the most emblematic fish species of the Argentinean water bodies.

Effects of heavy metals identified in Chascomús shallow lake on the endocrine-reproductive axis of pejerrey fish (Odontesthes bonariensis).

Some heavy metals related to human activities were measured in the water of Chascomús lake. The maximum concentrations were: 0.23 µg/L for Cd, 4.28 µg/L for Cr, 22.09 µg/L for Cu, 2.49 µg/L for Ni, 3.24 µg/L for Pb and 210.76 µg/L for Zn. The values of Cd, Cr, Cr, Pb and Zn were above the Argentine National Guidelines for the Protection of the Aquatic life. The analysis of gonadal condition of pejerrey fish (Odontesthes bonariensis) from this lake did not revealed any reproductive damages. However, exposures with environmental concentrations of Cd, Cr, Cu and Zn under laboratory conditions of pejerrey males (14 days), caused a significant increase of the expression of the three variants of gnrh in the brain (within Cd exposure) and a decrease in cyp19a1b mRNA (within Cu exposure). Furthermore, at pituitary level, a decrease in fshb transcript levels was observed in the fish exposed to Cd and Cr and a decrease in the expression of both gonadotropin receptors at gonadal level in Zn exposure. Moreover, the gonads of the fish exposed to all the tested metals suffered structural damages showing shortness of the spermatic lobules, fibrosis, testis ova and the presence of piknotic cells. All these findings alert that heavy metals pollution affects the expression of key reproductive genes and gonadal structure of fish species that represent the predominant group of organisms and are considered sentinel species in the aquatic ecosystems.

Sex hormone binding globulin during an annual reproductive cycle in the hepatopancreas and ovary of pejerrey (Odontesthes bonariensis).

In the present study, we determined the hepatopancratic shbg transcript abundance and ovarian immunoreactive Shbg (ir-Shbg) localization in pejerrey females at different gonadal stages during an annual ovarian cycle. In the hepatopancreas, shbg expression remains was constant in pre-vitellogenic stages, decreased at final vitellogenesis to increase again in final maturation and atretic stages to previous levels at post-vitellogenic stages. Related to this, also we found a negative significant relation between sex steroid and shbg expression. On the other hand, in the ovary we found ir-Shbg inside of cortical alveoli, from previtellogenic stages to final maturation. This localization of Shbg in a teleost fish ovary suggests a new role for Shbg in oocytes, that may also extend the oocyte fertilization/development process.

Kisspeptins and their receptors in the brain-pituitary-gonadal axis of Odonthestes bonariensis: Their relationship with gametogenesis along the reproductive cycle.

In vertebrates, the reproduction is controlled by the brain-pituitary-gonadal (BPG) axis and kisspeptin has emerged as a key player of this axis. In this study, we analyzed changes in the expression levels of kiss1, kiss2, and their receptors, kissr2 and kissr3 during gametogenesis in the BPG axis of feral Odontesthes bonariensis. In females, levels of brain kiss1 showed an increase at final maturation (Fm), while kiss2 levels were shown to be high at primary growth (Pg) stage, with no differences in the expression of their receptors. In the pituitary, kiss1 and kiss2 peaked at the cortical alveoli (Ca) stage, and kissr3 at initial vitellogenesis. In parallel, there was an increase of kiss1, kissr2 and kissr3 in the ovary during the Ca stage and both receptors again at Fm stage. In males, the four genes were highly expressed in the brain at the arrested (A) stage. In the pituitary, kiss2 peaked at spermatogonial (SG) and spermatocytary (SC) stages; while kissr3 reached a peak at the spermiogenic stage (SP). In testes, kiss1 and kiss2 significantly increased during the SG and SC stages; meanwhile, kissr2 increased at SG and SC, whereas kissr3 levels were significantly high at SC and SP stages. Taken together these results showed that the kisspeptin system in pejerrey is expressed in the three levels of the BPG axis with different expression profiles during the gonadal cycle. These findings pointed that kisspeptins have different roles in gametogenesis in this species.

Exposure to E2 and EE2 environmental concentrations affect different components of the Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes bonariensis).

The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.

Effects of estradiol and ethinylestradiol on sperm quality, fertilization, and embryo-larval survival of pejerrey fish (Odontesthes bonariensis).

17ß-Estradiol (E2) and synthetic 17α-Ethinylestradiol (EE2) are estrogenic compounds present in surface waters as a consequence of municipal sewage discharges. The aim of this study was to evaluate the effects of E2, EE2 and its mixtures on different reproductive parameters and embryo-larval survival in pejerrey fish (Odontesthes bonariensis). In order to analyze the effect of these compounds on sperm quality, fertilization%, embryo-larval survival (%), and the point of no return (PNR), different assays were performed using concentrations 175, 350, 700 and 1400 ng/L of E2; 22.5, 45, 90 and 180 ng/L of EE2 and mixtures M1 (175 E2+22.5 EE2, ng/L), M2 (350 E2+45 EE2, ng/L), M3 (700 E2+90 EE2, ng/L) and M4 (1400 E2+180 EE2 ng/L). No significant differences in motility parameters were observed between E2 and EE2 treatments and the control group. However, a significant decrease in motility% was recorded for all mixtures tested compared with the control samples. For fertilization%, only sperm activated with M4 showed a significant decrease compared with the control group. In the case of embryo survival, there was only a significant decrease in the highest concentration of EE2 compared with the control group. For the mixtures, M3 is the one that had the most adverse effect on embryo survival. In larval survival, there was a significant decrease in concentration 175 and 700 ng/L of E2 compared with the control group. In EE2 treatments, the ones with a significant reduction in larval survival were concentration 45 and 90 ng/L. And for the mixture treatments, M1, M3 and M4 had a significantly lower larval survival than the control group. In comparison to other treatments, M1 demonstrated a significant difference in PNR when compared with the control group. The results obtained demonstrated that the exposure to mixtures of E2 and EE2 affected fish sperm motility, fertilization% and, embryo and larval survival even at relevant environmental concentrations highlighting the necessity of considering the effects of pollutants mixtures in ecotoxicological studies.

Subcellular energy balance of Odontesthes bonariensis exposed to a glyphosate-based herbicide.

Water pollution by agrochemicals is currently one of the most critical problems for the conservation of aquatic ecosystems. Glyphosate [N-(phosphonomethyl) glycine); PMG] is the main broad-spectrum post emergence herbicide used for the control of a wide range of pests in soybean crops. Adenylate energy charge (AEC) reflects the energy balance of the cells, a measure of the energy available from the adenylate pool: adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Background adenylates, phosphagens and the AEC index of two year old Odontesthes bonariensis were determined in some tissues by HPLC, and the impact on subcellular energy balance of sublethal glyphosate-based herbicide exposure was analyzed. The doses used were 0 (control tank), 1 or 10mg PMGL(-1), trials were carried out during 15 days. AEC values in brain, liver and muscle from control fish were 0.37 ± 0.02, 0.49 ± 0.05 and 0.56 ± 0.03, respectively (means ± SEM). While brain ATP concentrations were undetectable (hence low values of AEC), the muscle tissue showed the highest concentrations of the more energetic molecules: 0.18 µmole ATP g(-1) and 8 µmole phosphocreatine g(-1) (PCrg(-1)). In the brain, no significant changes were detected in exposed fish compared to controls. Instead, in both the liver and muscle of animals exposed to the highest concentration of the herbicide, significant changes in the AEC (reduction of 26% and 15%, p<0.05) with respect to the control group were determined. Chronic exposure (15 days) of Odontesthes bonariensis to 1 and 10mgL(-1) of formulated glyphosate did not affect brain AEC. However, the highest concentration of the herbicide produced a significant decrease in liver and muscle AEC manifesting adverse sublethal effects on the energy metabolism. These results suggest the usefulness of AEC as a biomarker of fish glyphosate exposure.

Seasonal changes and endocrine regulation of pejerrey (Odontesthes bonariensis) spermatogenesis in the wild.

The endocrine mechanisms that regulate spermatogenesis and their interaction with environmental cues have been poorly studied compared with oogenesis in fish. The aim of this work was to study the spermatogenesis in pejerrey under the influence of photoperiod and water temperature fluctuation in the wild, evaluating the transcript levels of brain Gnrh variants and cyp19a1b, pituitary Gth subunits, gonadal Gth receptors, 11ß-hsd, and 11-KT plasma levels. Males at spermiogenic stage were observed during spring and autumn, under a photoperiod above 11h of light and a water temperature below 23 °C. Most arrested males were observed in summer when water temperatures increased above 23 °C. Males at spermatogonial stage were mainly observed in autumn, while most males at spermatocytary stage were caught in winter. An increase of gnrh-I, cyp19a1b, fshb, gpha and 11ß-hsd transcripts and 11-KT plasma levels was observed during spermatogonial and/or spermatocytary stage (early spermatogenesis). The spermiogenic stage was associated to the maximum gnrh-I gene expression level and a significant increase of Gth receptors transcripts, being this fact more evident for lhcgr. During this last gonadal stage, cyp19a1b transcript level remained high, while fshb mRNA and 11-KT plasma levels showed a significant decreased compared to that occurred at the spermatocytary stage. Also, gphα and 11ß-hsd gene expression levels fell during spermiation up to similar values to those observed in arrested males. A significant correlation between 11-KT and gnrh-I, cyp19a1b, gphα, fshb, 11ß-hsd transcripts, and the number of spermatocytes was observed during spermatogenesis. All these findings suggested that in pejerrey, the spermatocyte proliferation occurs mainly during winter under the stimulation of 11-KT induced by FSH through the stimulation of specific enzymes, including the 11ß-hsd while spermiation occurs after photoperiod increase and with temperatures of the water below 23 °C, through the stimulation of gnrh-I, cyp19a1b and lhcgr.

Quality of pejerrey (Odontesthes bonariensis) eggs and larvae in captivity throughout spawning season

The aim of this work was to assess the quality of pejerrey eggs and larvae throughout its spawning season. Fertilized eggs were taken on September, October, November, and December from a captive broodstock. The egg diameter, yolk diameter, and oil droplets area decreased along the spawning season, with higher values in September. Fertilization and hatching rates decreased throughout this period, with highest values in September (88.0%; 55.2%) and the lowest values on December (43.0%; 25.2%). The larvae hatched from eggs obtained on October were the heaviest and longest (1.57 mg; 8.24 mm). The survival rate at 30 days post hatching (dph) was similar in larvae from September and October eggs (66.1%; 62.9%) with a sharp decrease in larvae from November and December eggs (22.4%; 23.3%). Furthermore, the highest body weight (15.1 mg) and total length (15.25 mm) at 30 dph were obtained in larvae from October eggs. The results obtained showed that overall eggs quality was better at the beginning of the spawning period, influencing the larvae performance...

O objetivo deste trabalho foi avaliar a qualidade de ovos de peixe-rei ao longo do período reprodutivo. Ovos fertilizados foram amostrados em setembro, outubro, novembro e dezembro de reprodutores em cativeiro. O diâmetro do ovo e do vitelo, como também o tamanho das gotículas de óleo diminuíram ao longo do período reprodutivo, com valores maiores em setembro. Taxas de fertilização e eclosão diminuíram ao longo deste período, apresentando valores maiores em setembro (88,0%, 55,2%) e menores em dezembro (43,0%, 25,2%). As larvas eclodidas dos ovos, obtidas em outubro, eram maiores e apresentavam maior peso (8,24 mm; 1,57 mg). A taxa de sobrevivência aos 30 dias pós-eclosão foi maior nos meses de setembro e outubro, apresentando valores similares (66,1%, 62,9%), no entanto, houve uma diminuição acentuada na sobrevivência das larvas de ovos em novembro e dezembro (22,4%, 23,3%). Além disso, o maior peso corporal (15,1 mg) e o comprimento total (15,25 mm) aos 30 dias pós-eclosão foram obtidos a partir de larvas de ovos durante o mês de outubro. Os resultados obtidos mostraram que a qualidade dos ovos em geral foi melhor no início do período reprodutivo, influenciando o desempenho das larvas...

Seasonal changes and endocrine regulation of pejerrey (Odontesthes bonariensis) oogenesis in the wild.

The goal of this study was to evaluate the essential components controlling the brain-pituitary-gonad axis during pejerrey (Odontesthes bonariensis) oogenesis in the wild. Ovarian developmental stages from vitellogenesis up to ovulation were associated with increasing day length and water temperatures below 21°C (winter and beginning of spring). Gonadal regression was observed when water temperature exceeded this value or when photoperiod decreased. Most females were arrested at primary growth stage during summer (high temperature) or at cortical alveoli stage between autumn and beginning of winter (short photoperiod). Plasma E2 and transcript levels of fshr, cyp19a1b and cyp19a1a increased during vitellogenesis, while fshb remained high at all vitellogenic stages. A significant correlation between plasma sex steroids (T and E2) and cyp19a1b as well as lhcgr transcript levels was observed during vitellogenesis, suggesting a steroid positive feedback. Gnrh-I, Gth subunits and lhcgr transcript levels increased significantly during late vitellogenesis and final maturation. Present results suggest that pejerrey vitellogenesis is controlled by Fsh/Fshr, stimulating gonadal aromatase and estradiol synthesis. Moreover, the increase of testosterone and estradiol during final vitellogenesis could induce coordinately the functioning of the Gnrh/Lh system (perhaps through brain P450 aromatase stimulation and brain estradiol increase) and the gonadal Lhcgr synthesis to promote the final maturation of oocytes. All these stimulation mechanisms of gonadal development would be possible only under permissive environmental conditions.

Osmotic induction marking with Alizarin Red S on juveniles of pejerrey, Odontesthes bonariensis (Atherinopsidae)

Juveniles of pejerrey, Odontesthes bonariensis, were exposed to 0.1% Alizarin Red S (ARS) alone or with a previous immersion in 2.2% saline solution (Osmotic Induction, OI) to enhance the ARS marking method. Fish were marked in the field and immediately released in 1 m3 cages in "La Salada de Monasterio" lagoon, Chascomús, Buenos Aires , Argentina. After 73 days, clear marks were observed in the otoliths, caudal fin rays and scales with both treatments, being the intensity of the signal in the scales of OI+ARS treated fish higher. On the other hand, no marks were observed in the control group on the same structures. Approximately one year post-treatment (385 days), only marks in caudal fin rays were found clearly in OI+ARS treated fish. After this period, no significant differences in total length or weight between marked or control fish were observed and the mortality ranged between 30-40 % in all cages. These results provide strong evidence for the potential applicability of this cost-effective marking technique in differentiation of wild and hatchery-produced pejerrey. The success in the caudal fin rays marking is also important because it is easy to do and does not require the sacrifice of fish.

Juvenis de pejerrey, Odontesthes bonariensis, foram expostos a Vermelho de Alizarina S (ARS) 0,1% de duas formas, sozinho ou com uma imersão anterior em 2,2% de solução salina (Indução osmótica, IO) para melhorar o método de marcação ARS. Os procedimentos foram realizados no campo e os peixes foram liberados em gaiolas (tanques-rede) de 1 m3 na lagoa "La Salada de Monasterio", Chascomús, Buenos Aires, Argentina. Após 73 dias, marcas claras foram observadas nos otólitos, raios da nadadeira caudal e escamas em ambos os tratamentos, sendo que a intensidade do sinal nas escalas de IO + ARS de peixe tratado foi superior. Por outro lado, não foram observados marcas no grupo controle sobre as mesmas estruturas. Aproximadamente um ano pós-tratamento (385 dias), apenas marcas nos raios da nadadeira caudal foram encontrados claramente nos peixes tratados com IO+ARS. Entre os peixes observados, após este período, não houve diferenças significativas no comprimento total ou peso entre o grupo controle e marcados, ademais, a mortalidade variou entre 30-40% em todas as gaiolas. Estes resultados fornecem fortes evidências e um grande potencial para aplicação desta técnica rentável de marcação que diferencia o pejerrey selvagem e o produzido em cativeiro. O sucesso na marcação dos raios da nadadeira caudal também é de grande importância, pois sua verificação é fácil e não requer o sacrifício de peixes.

Ultrastructure of fresh and post thawed sperm of pejerrey Odontesthes bonariensis (Atheriniformes)

In the present study it was showed for the first time the ultrastructural morphology of O. bonariensis sperm using electron microscopy techniques. Different kinds of abnormalities were described in fresh and post thawed sperm caused by crogenic protocols. Pejerrey spermatozoon is uniflagellated and is differentiated into three parts: a small roundish head (~1.80µm in length and 1.67µm in width), a midpiece or transitional region (~1.11µm in length and 1.56µm in width), and a long tail or flagellum (~29.08µm). Samples of fresh and post thawed sperm showed evidence of morphological anomalies affecting various intracellular compartments. Spermatozoa with swollen, ruptured, or absent membranes in the head showing excess of cytoplasm, and with alteration of the spatial orientation of the mitochondria were observed. A swollen flagellum was observed containing cytoplasmic vesicles, distributed along the whole length or concentrated in a restricted part of the tail. It was also found a high level of abnormalities (60%) in frozen sperm when compared with normal sperm (18%) reflecting the damage provoked by cryopreservation procedures.

No presente estudo mostrou-se pela primeira vez a morfologia estrutural dos espermatozoides de O. bonariensis utilizando técnicas de microscopia eletrônica. Diferentes tipos de anormalidades foram descritas para sêmen fresco e descongelado. O espermatozoide de Pejerrey é uniflagelado e dividido em três partes: uma cabeça pequena e arredondada (~1.80µm de comprimento e 1.67µm de largura), uma parte intermediária ou região de transição (~1.11µm de comprimento e 1.56µm de largura) e uma cauda longa ou flagelo (~29.08µm). Amostras de sêmen fresco e descongelado mostraram evidências de anormalidades morfológicas afetando vários compartimentos intracelulares. Na cabeça haviam espermatozoides com membranas dilatadas, rompidas ou ausentes, mostrando excesso de citoplasma e alteração na orientação espacial das mitocôndrias. Um flagelo dilatado foi observado contendo vesículas citoplasmáticas, as quais estavam distribuídas ao longo de todo o seu comprimento ou concentradas em uma parte restrita da cauda. Também foi encontrado um alto nível de anormalidades (60%) em sêmen congelado em comparação com o sêmen normal (18%), refletindo os danos provocados pelos procedimentos de criopreservação.

Effects of short periods of warm water fluctuations on reproductive endocrine axis of the pejerrey (Odontesthes bonariensis) spawning.

The aim of this study was to assess fluctuations in daily water temperature in Chascomús Lagoon during one year, and to evaluate whether the highest temperature recorded during pejerrey spawning season can produce an endocrine disruption on brain-pituitary-gonads axis. Fish were subjected to daily temperature fluctuations: 17 °C to 19 °C (reproductive control), 19 °C to 25 °C, and 19 °C to 27 °C. After 8 days, ten fish per treatment were sacrificed and gene expression of gonadotropin-releasing hormone (GnRH-I, GnRH-II, GnRH-III), gonadotropin subunits-ß (FSH-ß, LH-ß), glycoprotein hormone-α (GPH-α), gonadotropin receptors (FSH-R, LH-R), and gonadal aromatase (cyp19a1a) was analyzed. Also, plasma levels of sexual steroids and gonadal reproductive status were studied. Fish exposed to high temperature fluctuations quit spawning, presenting clear signs of gonadal regression. Fish recovered its spawning activity 11 weeks after heat treatment. At endocrine level, GnRH-I and FSH-ß in both sexes, LH-ß and GPH-α in males and FSH-R, LH-R and cyp19a1a in females decreased significantly in treated fish. Also, a strong reduction in plasma sex steroid levels was found for both sexes. This study demonstrated that pulses of warm water in natural environment during pejerrey spawning season can disrupt all levels of the reproductive axis, impairing reproduction.

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature-dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17 degrees C, FPT), both sexes (25 degrees C, MixPT), and only males (29 degrees C, MPT). The pejerrey Fshr cDNA had 3,069 bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936 bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey.

High water temperatures impair the reproductive ability of the pejerrey fish Odontesthes bonariensis: effects on the hypophyseal-gonadal axis.

The aim of this research was to study how high water temperatures impair the reproductive activity of pejerrey Odontesthes bonariensis, an inland-water atherinopsid fish species from the pampasic region of Argentina. Adult pejerrey of both sexes were kept under a control-temperature regime (19 degrees C) and two experimental temperatures (23 degrees and 27 degrees C) for 8 d. The effect of elevated temperature on the pituitary-gonad axis was analyzed in terms of the expression of gonadotropin (GtH) subunits: follicle-stimulating hormone beta, luteinizing hormone beta (LH-beta), glycoprotein hormone alpha, and GtH receptors FSH-R and LH-R by semiquantitative reverse transcriptase polymerase chain reaction, plasma levels of sexual steroids by radioimmunoassay, and reproductive status by gonadal histology. The results of this work clearly indicate that short periods of exposure to high water temperatures disrupt pejerrey reproduction. This effect was observed in spawning activity, at the histological level, and in the reduction of plasma estradiol in females and testosterone in males. The mRNA levels of GtH subunits and GtH receptors generally decreased in proportion to the increase in temperature for both sexes. However, the differences between groups were statistically significant only for LH-beta and for FSH-R expression in pejerrey females. Thus, the gonads of pejerrey appear to be the primary target of high water temperature. Analysis of the air temperature in this region over the past 40 yr indicated an increase of 1.74 degrees C in mean annual temperature. This increase, coupled with the mechanism of high-temperature sensitivity shown in this study, may be one of the reasons for the decline in pejerrey populations observed in this region over recent decades.

Characterization of the cDNAs encoding three GnRH forms in the pejerrey fish Odontesthes bonariensis (Atheriniformes) and the evolution of GnRH precursors.

Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.