Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey,a Teleost Fish with Temperature-Dependent Sex Determination?

Background

Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals “sense” environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation.

Methodology/Principal Findings

Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29°C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17°C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a “sexually neutral” temperature (24°C) caused significant increases in the proportion of males.

Conclusions/Significance

Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination.     

Where does gonadal sex differentiation begin? Gradient of histological sex differentiation in the gonads of pejerrey,Odontesthes bonariensis (Pisces,Atherinidae)

This study investigated the possibility that the histological process of gonadal sex differentiation in pejerrey (Odontesthes bonariensis), a fish with marked temperature-dependent sex determination (TSD), occurs through a predictable gradient of differentiation as opposed to simultaneous or random differentiation throughout the gonad. For this purpose, fish reared at 17 degrees, 24 degrees, and 29 degrees C from hatching were sampled weekly for 11 weeks, fixed, and prepared for histological observation of serial cross-sections of the gonads. The thermal manipulation and sampling procedure ensured the availability of males and females at various degrees of gonadal sex differentiation. The location of the differentiated area(s) was estimated in the right and left gonads of 17 females and 14 males selected among the available specimens so as to represent increasing degrees of differentiation. The analysis revealed that sex differentiation followed a gradient from the anterior to posterior areas of the gonads regardless of sex. Furthermore, plotting of the degree of sex differentiation in the right gonad as a function of the degree of differentiation of the left gonad clearly showed that sex differentiation only begins in the right gonad when 10-30% of the length of the left gonad has already differentiated. The mean rostral edge of the differentiated areas in females was 9% and 10.8% for the left and right gonads, respectively, while for males these values were 7.3% and 7.0%, respectively. Thus, it was established that ovarian and testicular differentiation in pejerrey follow both a cephalocaudal and a left-to-right gradient. Possible explanations for this gradient and its relevance for TSD in pejerrey, that is, as a mechanism to prevent discrepant differentiation of male and female features within the same gonad, are discussed.     

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.     

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.     

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.     

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.     

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.     

Temperature sex-reversal in amphibians and reptiles

The sexual differentiation of gonads has been shown to be temperature-sensitive in many species of amphibians and reptiles. In two close species of salamanders, Pleurodeles poireti and P. waltl, both displaying a ZZ/ZW mechanism of genotypic sex determination (GSD), the rearing of larvae at high temperatures (30 degrees-32 degrees C) produces opposite effects: ZZ genotypic males of Pleurodeles poireti become phenotypic females whereas ZW genotypic females of P. waltl become phenotypic males. Sex-reversal of these individuals has been irrefutably demonstrated through genetic, cytogenetic, enzymatic and immunological studies. In many turtles, both sexes differentiate only within a critical range of temperature: above this range, all the individuals become phenotypic females, whereas below it, 100% become phenotypic males. The inverse occurs in some crocodiles and lizards. In many species of these three orders of reptiles, females are obtained at low and high temperatures, and males at intermediate ones. Preliminary studies in turtles (Emys orbicularis) indicate that within the critical range of temperature, sexual phenotype conforms with GSD, but that above and below this range, GSD is overriden. Temperature shifts during larval development in salamanders and during embryonic development in reptiles allowed the determination of thermosensitive stages for gonadal differentiation. Estrogens synthesized in the gonads at these stages appear to be involved in their sexual differentiation, higher levels being produced at feminizing temperatures than at masculinizing ones. The phenomenon of temperature sensitivity of gonadal differentiation occurs in species showing a very early stage in the evolution of sex chromosomes. Its adaptive value, chiefly in reptiles, remains an open question.     

Role of peanut-lectin-affinity molecules (PLAM) on primordial germ cells in the initial determination of sex in anura

Peanut lectin (PNA) or N-acetylgalactosamine (galNA, a part of the disaccharide unit which is recognized by PNA) was injected into the coelomic cavity of anuran larvae at the developmental stages during which the genital ridges were growing, and the effect of these compounds on the initial determination of gonadal sex was examined. The treatment with PNA tended to inhibit (or perturb) the expression of feminizing gene(s) in Rana japonica, and of both feminizing and masculinizing genes in R. nigromaculata. In contrast, treatment with galNA suppressed the expression of masculinizing gene(s) considerably. In terms of the initial determination of gonadal sex during normal development, these results suggest that the PNA-affinity molecule (PLAM) of primordial germ cells acts as a trigger for the expression of genes that control sexual differentiation of somatic cells. Furthermore, the somatic cells (perhaps mesenchymal and/or epithelial cells), which respond to the stimulus via the PLAM of primordial germ cells, may differ in terms of the threshold for such a response between genetic males and females. This result suggests the mesenchymal and/or epithelial cells are not sexually predetermined, but rather that sexual determination follows the response to some signal(s) mediated by the PLAM.     

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.     

Effects of steroid sex hormones on chick embryo gonads in organ culture, with special reference to hormonal control of gonadal sex differentiation

At the initial stages of sex differentiation (7.5 and 8.5 days of incubation), chick embryo gonads were treated directly with testosterone or estradiol-17 beta in organ cultures. Chemically-defined media containing cholesterol as a steroid precursor were used. The differentiation of gonads in the 10 to 12-day controls, cultured in media containing no hormones, was close to that of gonads of equivalent age in ovo. Testosterone added to the medium exerted an inhibitory effect on the cortex of the female gonad and a masculinizing one on its medulla. The results of estradiol treatment confirmed the known feminizing effect of that hormone on the male gonad, the meiotic prophase in the genetically male germ cells being initiated in the induced cortex. These data may be interpreted in favour of a bihormonal theory of gonadal sex differentiation in birds, where the predominantly-synthesized male or female hormone in the gonad determines the male or female pattern of development of the corresponding gonad.     

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.     

Temperature effects on sex differentiation of two South American atherinids, Odontesthes argentinensis and Patagonina hatcheri

Synopsis The present study investigated the effects of water temperature (18, 21, and 25 °C) on the histological process of gonadal sex differentiation of two commercially important atherinid fishes from South America, Odontesthes argentinensis (sea pejerrey) and Patagonina hatcheri (Patagonian freshwater pejerrey). In both species, female gonadal sex differentiation began with the formation of lateral stromal cell outgrowths and the appearance of meiotic oocytes. The male gonads remained quiescent for about twice as long as the female gonads, with differentiation becoming evident by the formation of the main sperm duct and of cysts of germ cells at the periphery of the gonads. Meiosis in males occurred relatively long after somatic differentiation of the testis. The ovaries of O. argentinensis differentiated at 28 days (20.3 mm) at 25 °C, 42 days (24.0 mm) at 21 °C, and 56 days (23.8 mm) at 18 °C. In the males, differentiation was observed at 98 days at 25 and 21 °C (39.4 mm and 40.4 mm, respectively), but at 112 days under 18 °C (40.7 mm). In P. hatcheri, differentiation of females occurred at 21 days (17.8 mm) at 25 °C, 28 days (20.8 mm) at 21 °C, and 35 days (23.2 mm) at 18 °C. Male differentiation became evident at 56 days under 25 and 21 °C (30.8 and 32.7 mm, respectively), and at 70 days (37.7 mm) at 18 °C. The sex-ratios of O. argentinensis reared at 18 or 21 °C were female-biased whereas those at 25 °C were not; groups reared at 18 °C had significantly more females than groups from the same progeny reared at 25 °C. In contrast, the sex-ratios in all groups of P. hatcheri did not differ significantly from 1:1 and no significant differences were found between groups of the same progeny reared at different temperatures. These results suggest the occurrence of thermolabile sex determination (TSD) in O. argentinensis whereas in P. hatcheri gonadal sex appears to be strongly genetically determined.     

Environmental sex determination in a reptile varies seasonally and with yolk hormones

Most hypotheses that have been put forward in order to explain the persistence of environmental sex determination (ESD) in reptiles assume a relatively fixed association of sex with temperature-induced phenotype and no maternal influence on offspring sex. Here we demonstrate the association of maternally derived yolk hormone levels with the offspring sex ratio and describe two new aspects of temperature-dependent sex determination (TSD), i.e. seasonal variation in both thermal response and yolk steroid levels. Eggs from painted turtles (Chrysemys picta) were incubated at 28 degrees C. The hatchling sex ratio at 28 degrees C (i.e. the phenotypic reaction norm for sex at 28 degrees C) shifted seasonally from ca. 72% male to ca. 76% female. Yolk oestradiol (E2) increased seasonally while testosterone (T) decreased. The proportion of males in a clutch decreased as E2 levels increased and the E2:T ratio increased. These new findings are discussed in relation to heritability and adaptive explanations for the persistence of ESD in reptiles. Maternally derived yolk hormones may provide a mechanism for the seasonal shift in the sex ratio which in turn may help explain the persistence of ESD in reptiles. They may also explain those clutches of other reptiles with TSD that fail to yield only males at maximally masculinizing conditions.     

Evidence of thermolabile sex determination in pejerrey*

Temperature regimes of 17 ± 1°C and 21 ±1°C early in development of pejerrey Odontesthes bonariensis produced nearly all females, whereas at 25 ± 1°C variable, sometimes male-biased sex-ratios were obtained. The critical period of thermolabile sex determination seemed to occur between 25 and 50 days post-hatch (about 11 and 21 mm s.i.) at low temperatures (17–20°C) and between 0 and 25 days (about 7 and 15 mm) at high temperatures (22–25°C). The likelihood of expression of temperature-dependent sex determination in natural populations and the possible adaptive significance of environmental sex determination in pejerrey are discussed.