Effects of temperature on the reproductive physiology of female elasmobranchs: The case of the narrownose smooth-hound shark (Mustelus schmitti).

The knowledge of how temperature influences elasmobranchs reproductive physiology allows a better understanding of their reproductive patterns. This study describes the relationship between temperature fluctuations and the plasmatic changes of the sex steroids related to reproduction: testosterone (T), estradiol (E2) and progesterone (P4), throughout the female reproductive cycle of the shark Mustelus schmitti. A total of 123 adult females were bi-monthly sampled in Buenos Aires, Argentina, coastal waters. Bottom temperatures were recorded at each sampling point and blood samples were taken from each female for plasma sex steroids measurement. Sex steroid plasma levels were analyzed in relation with maximum follicular diameter (MFD), uterosomatic index (USI, as indicator of pregnancy) and temperature using Generalized Additive Models. Plasmatic E2 and T increased during follicular growth until MFD reached 1.34 and 1.46 cm, respectively. Peak of T occurred at the follicular stage associated with parturition (MFD, 1.4-1.6 cm), just prior to final maturation and ovulation (MFD, 1.6-2.0 cm). Progesterone significantly increased at this last ovarian phase, while T and E2 decreased. The increase of USI with pregnancy was associated to a decrease in T and mainly E2 levels, while P4 remained unaffected. Prior to ovulation, T plasma levels decreased with temperature below to 13 °C and then increased progressively with a pronounced elevation above 17 °C, while E2 presented an opposite pattern. Progesterone plasma levels changed with temperature showing a similar pattern to that observed for T. Using M. schmitti shark as model species, this study shows a clear picture of how seawater temperature variations can affect the reproductive physiology in elasmobranch females. A hypothetical mechanism (based on T elevation driven by temperature increase and its connection by feedback with a P4 rise and parturition/ovulation induction) is proposed as evidence to support that the increase in temperature can trigger reproductive events in elasmobranchs. In addition to its ecological scope, this work contributes to reinforce the relatively scarce general knowledge of elasmobranchs reproductive physiology.

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.

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.

Effects of global warming on fish reproductive endocrine axis, with special emphasis in pejerrey Odontesthes bonariensis.

The ongoing of global warming trend has led to an increase in temperature of several water bodies. Reproduction in fish, compared with other physiological processes, only occurs in a bounded temperature range; therefore, small changes in water temperature could significantly affect this process. This review provides evidence that fish reproduction may be directly affected by further global warming and that abnormal high water temperature impairs the expression of important genes throughout the brain-pituitary-gonad axis. In all fishes studied, gonads seem to be the organ more readily damaged by heat treatments through the inhibition of the gene expression and subsequent synthesis of different gonadal steroidogenic enzymes. In view of the feedback role of sex steroids upon the synthesis and release of GnRH and GtHs in fish, it is possible that the inhibition observed at brain and pituitary levels in treated fish is consequence of the sharp decrease in plasma steroids levels. Results of in vitro studies on the inhibition of pejerrey gonad aromatase expression by high temperature corroborate that ovary functions are directly disrupted by high temperature independently of the brain-pituitary axis. For the reproductive responses obtained in laboratory fish studies, it is plausible to predict changes in the timing and magnitude of reproductive activity or even the total failure of spawning season may occur in warm years, reducing annual reproductive output and affecting future populations.

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.

Extrahypophyseal expression of gonadotropin subunits in pejerrey Odontesthes bonariensis and effects of high water temperatures on their expression.

It has been traditionally accepted that the gonadotropins (GtHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), are synthesized and secreted only by the pituitary. However, the presence of theses hormones in extrapituitary tissues has been demonstrated in mammals, and more recently also in fish. In this study, we cloned the cDNAs and characterized the expression of FSH-ß, LH-ß, and glycoprotein hormone α (GPH-α) subunits from brain and gonads of male and female pejerrey Odontesthes bonariensis at different stages of gonadal maturation. In situ hybridization revealed that, in addition to their classical location in pituitary cells, the three GtH transcripts were also located in the gonads. FSH-ß and GPH-α subunits were found in the cytoplasm of oogonia, previtellogenic and vitellogenic oocytes in ovaries. LH-ß expression was detected in previtellogenic and vitellogenic oocytes but not in oogonia. In males, the three subunits were expressed in spermatogonia and to a lesser extent in spermatocytes. Exposure of fish to high water temperatures that impair pejerrey reproduction also induced a decrease of extrahypophyseal expression of GtH subunits.