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-β (FSH-β, LH-β), 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-β in both sexes, LH-β 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.     

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Warm water induces apoptosis, gonadal degeneration, and germ cell loss in subadult pejerrey Odontesthes bonariensis (Pisces, Atheriniformes)

Abstract Recent studies have shown that exposure to warm water can trigger gonadal degeneration and germ cell loss in fish of both sexes, but the mechanism behind this pathology is still not understood. This study was designed to characterize this process histologically and determine whether apoptosis plays any role during high temperature-induced gonadal cell degeneration in subadult pejerrey (Odontesthes bonariensis). For this purpose, fish were reared continuously at constant temperatures of 24 degrees C (control) and 29 degrees C (prolonged heat stress) or exposed for 36 h to 31 degrees C and then returned to 24 degrees C (short heat stress). Gonads were sampled at various times (hours, days, weeks) after the start of the experiment and were analyzed by light microscopy and stereometry for histological integrity/degeneration and germ cell counts, as well as by acridine orange fluorescence microscopy, TUNEL, and caspase activity assay for histochemical and biochemical signs of apoptosis. The results clearly implicate apoptosis in heat-induced somatic and germ cell degeneration in pejerrey and revealed that the dynamics and severity of this process were proportional to the magnitude of the thermal stress. Even a 36-h exposure to 31 degrees C induced significant increases in caspase-3 activity and number of apoptotic cells in both sexes, but males were shown to be more sensitive to heat stress than females.     

Thermal threshold and histological process of heat-induced sterility in adult pejerrey (Odontesthes bonariensis): a comparative analysis of laboratory and wild specimens

Elevated water temperature has been found to cause gonadal degeneration in fish, including the partial or complete loss of germinal elements, and might impair fertility and reproductive performance. Germ cell-deficient and even completely sterile pejerrey Odontesthes bonariensis have been found in two lagoons in Argentina, and exposure to warm water is one of the possible causes of these abnormalities. This experiment was conducted (a) to compare the histological characteristics of the abnormal gonads from wild pejerrey with those of animals exposed to heat in the laboratory and (b) to examine whether short-term pulses of heat similar to diurnal temperature variations in natural environments during summer can trigger gonadal cell degeneration in adult pejerrey. Wild fish with gonadal abnormalities were obtained from the San Miguel del Monte and Lacombe Lagoons (Buenos Aires Province, Argentina). Laboratory specimens were obtained by exposure of adult pejerrey to five thermal regimes (constant 24 degrees and 29 degrees C and 12-h cycles of 27 degrees -31 degrees, 28 degrees -30 degrees, and 28 degrees -31 degrees C) for up to 16 wk. Germ cell-deficient specimens for histological comparison with wild animals were also obtained by exposing larvae and juveniles for 8-12 wk to 29 degrees C and rearing until they became adults. The histological characteristics of the abnormal gonads of wild pejerrey closely resembled those of fish partially or completely sterilized by high water temperature in the laboratory. The results indicate that fluctuating (high) thermal regimes triggered germ cell disappearance in a manner comparable to a constant temperature of 29 degrees C. These results support the notion that high temperature during unusually warm summers might trigger germ cell degeneration and could be the cause of the observed gonadal abnormalities in wild pejerrey.     

Sexually dimorphic expression of pituitary glycoprotein hormones in a sex-changing fish (Pseudolabrus sieboldi)

It is widely accepted that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in socially controlled sex-changing fish. However, the specific secretion profiles of pituitary gonadotropins (GtHs) in this type of fish are not known. To address this fundamental question, we demonstrated that the diurnal secretion patterns of GtHs differ distinctly between males and females in a socially controlled sex-changing fish. We analyzed the pituitary mRNA levels of glycoprotein hormone subunits (i.e., the common alpha-subunit and specific beta-subunits follicle-stimulating hormone beta, luteinizing hormone beta, and thyroid-stimulating hormone beta) in the wrasse Pseudolabrus sieboldi, which is a model fish that exhibits accurate diurnal rhythms of gametogenesis in both males and females. Northern blots clearly showed that each subunit gene exhibits a diurnal rhythm of expression in the pituitary and that the expression patterns differ distinctly between the sexes. Our results suggest that oogenesis and spermatogenesis in this hermaphroditic fish are regulated differentially through the distinct secretion patterns of pituitary glycoprotein hormones. This study also provides direct evidence of the sexual plasticity of pituitary GtH secretion in a socially controlled sex-changing fish.     

Temperature affects male and female potential reproductive rates differently in the sex-role reversed pipefish, Syngnathus typhle

The differences in potential reproductive rate between the sexescan be used to predict the operational sex ratio and the patternsand intensity of mating competition and hence sexual selectionin a population. This article describes how one environmentalcomponent, temperature, affects potential reproductive ratesof the two sexes in the paternally brooding, sex-role reversedpipefish (Syngnathus typhle). Males brooded embryos much longer(on average 58 days) in cold water (about 10°C) than inwarmer water (35 days at about 15°C). As a consequence,the potential reproductive rate (number of eggs brooded perday) of males was significantly higher in warm water. In females,however, potential reproductive rate, i.e., number of eggs producedper day given an unlimited access to mates, was not significantlydifferent between temperatures. In both sexes, potential reproductiverate was positively related to body size. At both temperatures,females had the potential to reproduce faster than males. Asa result, the operational sex ratio will become female biasedand sex-roles reversed, as is the case in this species. Sincetemperature differently influenced the potential reproductiverates of males and females, with the sexual difference largerat lower temperatures, more intense female-female competitionis predicted at low temperatures.     

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°C, FPT), both sexes (25°C, MixPT), and only males (29°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. Mol. Reprod. Dev. 77: 521–532, 2010. © 2010 Wiley‐Liss, Inc.     

Influence of testosterone and/or luteinizing hormone releasing hormone analogue on precocious sexual development in the juvenile rainbow trout

The influence of testosterone, luteinizing hormone releasing hormone (LHRH) agonist and combinations of these hormones on gonadotropic hormone (GtH) levels in the sexually immature trout was investigated. Both the steroid and releasing hormone preparations, testosterone in Silastic capsules and cholesterol-pelleted LHRH-A, were formulated for sustained release and long-term biological action following a single hormone implantation. Marked increases in pituitary GtH followed testosterone and/or testosterone and LHRH analogue treatment combined, but the low pituitary GtH level in controls remained unchanged after LHRH analogue administration alone. Plasma GtH titers increased with time after testosterone treatment, indicating a positive steroid feedback effect by androgen on GtH in the juvenile rainbow trout. When combined with testosterone treatment, LHRH analogue augmented plasma GtH levels compared to fish receiving testosterone treatment alone. In males the elevated plasma GtH levels were associated with testes stimulation and onset of spermatogenesis; in females, however, no significant stimulation of the ovaries was observed. It can be concluded from these studies that the testosterone stimulus is sufficient to induce onset of sexual development in immature males but not females. Whereas LHRH analogue releases GtH from the testosterone-primed trout pituitary, LHRH treatment alone under these conditions fails to stimulate the juvenile trout reproductive system.     

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.     

Embryonic temperature affects metabolic compensation and thyroid hormones in hatchling snapping turtles.

Temperature acclimation of adult vertebrates typically induces changes in metabolic physiology. During early development, such metabolic compensation might have profound consequences, yet acclimation of metabolism is little studied in early life stages. We measured the effect of egg incubation temperature on resting metabolic rate (RMR) and blood thyroid hormone levels of hatchling snapping turtles (Chelydra serpentina). Like many reptiles, snapping turtles have temperature-dependent sex determination (TSD), in which embryonic temperature determines sex. Therefore, we designed the experiments to separately measure effects of temperature and of sex on the response variables. We incubated eggs in the laboratory at 21. 5 degrees, 24.5 degrees, 27.5 degrees, and 30.5 degrees C, producing both sexes, all males, both sexes, and all females, respectively. Hatchling RMR, when measured at a common temperature (either 25 degrees or 31 degrees C), was negatively correlated with egg temperature in both males and females, such that RMR of turtles from 21.5 degrees C-incubated eggs averaged 160% that of turtles from 30.5 degrees C-incubated eggs. These results indicate that egg temperatures induced positive metabolic compensation in both sexes. Thyroid hormone levels of hatchlings showed similar correlations with egg temperature; thyroxine level of turtles from 21.5 degrees C-incubated eggs averaged 220% that of turtles from 30.5 degrees C-incubated eggs. To examine the possibility that thyroid hormones contribute to positive metabolic compensation, we added triiodothyronine to eggs during mid-incubation. RMR of hatchlings from these treated eggs averaged 131% that of controls, consistent with the previous possibility. Moreover, the effects of embryonic temperature on metabolic physiology, in combination with effects on sex, can result in differences in RMR and thyroid hormone levels between male and female hatchling turtles. Such differences may be important to the ecology and evolution of TSD.     

Dependence of kinetic constants of intact human lymphocyte beta2-adrenoreceptors on the nature of the radioligands used

An analysis of human peripheral blood intact mononuclear lymphocyte beta 2-adrenoreceptors showed that the hydrophilic radioligand 3H-CGP-12177 binds at various temperatures only to surface receptors. The density of receptors as determined by the binding of the lipophilic antagonist 125I-CYP at 37 degrees C is 2 times as high as that at 4 degrees C. The affinity of lymphocyte beta 2-adrenoceptors for 1-isoproterenol measured by the labeled ligand displacement at low (4 degrees C) temperatures is by two or three orders of magnitude higher than that at 37 degrees C. The thiol-alkylating agent, N-ethylmaleimide (NEM) causes oppositely directed changes in the density of beta 2-adrenoceptors on intact lymphocytes depending on their original density. NEM increases the affinity of beta 2-adrenoreceptors for the hormone (1.5-12-fold). The state and regulation of human peripheral blood lymphocyte beta 2-adrenoreceptors depend on the hormonal status of patients at the moment of blood sample collection.     

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17°C and 23°C. However, sex ratios become female-biased at <15°C and male-biased at 25°C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.     

Seasonal changes in daily torpor patterns of free-ranging female and male Daubenton’s bats (Myotis daubentonii)

Daily torpor can provide significant energy and water savings in bats during cold ambient temperatures and food scarcity. However, it may reduce rates of foetal and juvenile development. Therefore, reproductive females should optimize development by minimizing times in torpor. To test this hypothesis, the use of torpor by female and male free-ranging Daubenton’s bats (Myotis daubentonii) during reproduction (gestation, lactation, and post-lactation period) was investigated in 1998 and 1999. Temperature-sensitive radio transmitters were attached to the bats to measure skin temperature. Simultaneously, ambient temperature was recorded. While both sexes became torpid during daytime, male bats used daily torpor (>6°C below individual active temperature) significantly more often during reproductive period (mean: 78.4 % of day time in May and 43 % in June) than females. Female bats went into daily torpor, particularly in late summer when juveniles were weaned (mean: 66.6 % of daytime). Lowest skin temperatures occurred in a female bat with 21.0°C during post-lactation. Skin temperatures of male bats fluctuated from 16.8°C in torpor to 37.2°C during times of activity. There was a significant effect of reproductive period on skin temperature in females whereas mean ambient temperature had no significant effect. However, mean ambient temperature affected mean skin temperatures in males. Our findings indicate that female Daubenton’s bats adopt their thermoregulatory behaviour in particular to optimize the juvenile development.     

Influence of temperature on developmental rate,wing length,and larval head capsule size of pestiferous midge Chironomus crassicaudatus (Diptera: Chironomidae)

Larvae of Chironomus crassicaudatus Malloch were reared individually at nine constant temperatures from 12.5 to 32.5 degrees C (2.5 degrees C increments) for 120 d. Duration of immature stages (egg, four instars, and pupa), head capsule width of fourth instars, and wing length were recorded. Some adults emerged at all temperatures, except at 12.5 degrees C where individuals developed to fourth instars during the experiment. Sharpe and DeMichele's four-parameter model with high-temperature inhibition described the temperature-dependent developmental rates. The slowest development was observed at 15 degrees C, with developmental rate peaking between 25 and 27.5 degrees C. Developmental rate increased rapidly with increasing temperature up to 20 degrees C, slowed between 20 and 27.5 degrees C, and decreased at temperatures >27.5 degrees C. No developmental inhibition at high temperatures was observed in eggs. The most apparent high-temperature inhibition of development was recorded in fourth instars, which comprised the largest proportion of developmental time. Males developed faster than females, but females had wider larval head capsules and longer wings than males. Adult size was negatively related with temperature in both sexes, but this relationship was steeper in males than in females. Larval size peaked at 20 degrees C, whereas the head capsule width was reduced at temperatures higher and lower than 20 degrees C.     

Histological process and dynamics of germ cell degeneration in pejerrey Odontesthes bonariensis larvae and juveniles during exposure to warm water

Elevated temperature causes degeneration and disappearance of the germ cells in the males of scrotal mammals. It was recently shown that heat-induced germ cell degeneration occurs also in fish but, unlike in mammals, it occurs not only in males but also in females. The purpose of this study was to clarify the histological process and dynamics of heat-induced germ cell disappearance in pejerrey Odontesthes bonariensis larvae and juveniles. Monosex and mixed-sex fish produced by thermal manipulation of sex (temperature-dependent sex determination) were subjected to 29 degrees C for periods between 1 and 12 weeks, and used to analyze, by histological methods, the changes in gonadal size and the number of normal and degenerating germ cells. Groups exposed to 29 degrees C for 8-12 weeks were subsequently transferred to 24 degrees C to verify if any gonadal damage would be permanent. Germ cell degeneration, histologically characterized by nuclear pyknosis or eosinophilia and cytoplasmic eosinophilia, was observed with increasing frequency at higher temperatures (29>24> 17 degrees C) and more in males than in females. Clear degenerative changes in the germinal epithelium usually began within one week of exposure to 29 degrees C and appeared clearer in females than in males. Complete loss of germ cells was observed only in individuals exposed for periods of 8-12 weeks to 29 degrees C but no treatment produced 100% sterile fish. Germ cells that remained in the gonads after exposure to 29 degrees C retained the capacity to rapidly recolonize germ cell-depleted areas, suggesting that the associated somatic cells in the gonads are little or not affected by this temperature.