Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A.

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Suppression of adult cytogenesis in the rat brain leads to sex-differentiated disruption of the HPA axis activity.

OBJECTIVES: The action of stress hormones, mainly glucocorticoids, starts and coordinates the systemic response to stressful events. The HPA axis activity is predicated on information processing and modulation by upstream centres, such as the hippocampus where adult-born neurons (hABN) have been reported to be an important component in the processing and integration of new information. Still, it remains unclear whether and how hABN regulates HPA axis activity and CORT production, particularly when considering sex differences. MATERIALS AND METHODS: Using both sexes of a transgenic rat model of cytogenesis ablation (GFAP-Tk rat model), we examined the endocrinological and behavioural effects of disrupting the generation of new astrocytes and neurons within the hippocampal dentate gyrus (DG). RESULTS: Our results show that GFAP-Tk male rats present a heightened acute stress response. In contrast, GFAP-Tk female rats have increased corticosterone secretion at nadir, a heightened, yet delayed, response to an acute stress stimulus, accompanied by neuronal hypertrophy in the basal lateral amygdala and increased expression of the glucocorticoid receptors in the ventral DG. CONCLUSIONS: Our results reveal that hABN regulation of the HPA axis response is sex-differentiated.

Effects of Diethylstilbestrol on Zebrafish Gonad Development and Endocrine Disruption Mechanism.

Environmental estrogen is a substance that functions as an endocrine hormone in organisms and can cause endocrine system disruption. A typical environmental estrogen, diethylstilbestrol (DES), can affect normal sexual function and organism development. However, even though the effects of different exposure stages of DES on the endocrine system and gonadal development of zebrafish juveniles are unknown, sex determination is strongly influenced by endocrine-disrupting chemicals (EDCs). From 10-90 days post fertilization (dpf), juvenile zebrafish were exposed to DES (100 and 1000 ng/L) in three different stages (initial development stage (IDS), 10-25 dpf; gonadal differentiation stage (GDS), 25-45 dpf and gonadal maturity stage (GMS), 45-60 dpf). Compared with that of IDS and GMS, the growth indicators (body length, body weight, and others) decreased significantly at GDS, and the proportion of zebrafish females exposed to 100 ng/L DES was significantly higher (by 59.65%) than that of the control; in addition, the zebrafish were biased towards female differentiation. The GDS is a critical period for sex differentiation. Our results show that exposure to environmental estrogen during the critical gonadal differentiation period not only affects the development of zebrafish, but also affects the population development.

Global expression response of genes in sex-undifferentiated Nile tilapia gonads after exposure to trace letrozole.

The aromatase inhibitor letrozole can be found in rivers, effluents, and even drinking water. Studies have demonstrated that letrozole affects various metabolic pathways and may cause reproductive toxicity, especially in fish exposed during development. However, studies on the effect of a low concentration of letrozole at the whole-gonad transcriptomic level in the early stage of fish sexual development have not been investigated. The aim of our study was to explore the potential effects of a low concentration of letrozole on the gonad transcriptome of Nile tilapia at an early stage of sexual development. In this study, 9 dpf (days postfertilization) Nile tilapia were exposed to trace letrozole for 12 days. Letrozole exposure from 9 dpf to 21 dpf persistently altered phenotypic sex development and induced the male-biased sex ratio. The transcriptome results showed that 1173 differentially expressed genes (DEGs) were present in the female control vs 1.5 µg/L letrozole-treated female comparison group and that 1576 DEGs were present in the 1.5 µg/L letrozole-treated female vs male control comparison group. Differentially expressed gene enrichment analysis revealed several crucial pathways, including the drug metabolism-cytochrome P450 pathway, the ErbB-PI3K/Akt/mTOR pathway, and the calcium signalling pathway. Further analysis of these identified DEGs indicated that some key genes correlated with metabolism and epigenetic regulation were significantly affected by letrozole, such as UDP-glucuronosyltransferase (Ugt), glutathione S-transferase omega-1 (Gsto1), lysine-specific demethylase 6bb (Kdm6bb, original name is Kdm6a), jumonji and AT-rich interaction domain containing 2 (Jarid2b, original name is Jarid2), growth arrest and DNA damage inducible gamma (Gadd45g), and chromobox protein 7 (Cbx7). The qRT-PCR validation results for twelve DEGs showed that the Pearson's correlation of the log10fold change values between the qPCR and RNA-Seq results was 0.90, indicating the accuracy and reliability of the RNA-Seq results. Our study is the first to report the effect of letrozole on the transcriptome of gonads from fish during early-stage sexual development. These findings will be useful for understanding the toxic effects and molecular mechanisms of letrozole exposure at the early stage of gonad development on the sexual development of aquatic organisms.

Gene Expression of Takifugu rubripes Gonads During AI- or MT-induced Masculinization and E2-induced Feminization.

Elucidating the global molecular changes that occur during aromatase inhibitor (AI)- or 17α-methyltestosterone (MT)-induced masculinization and estradiol-17ß (E2)-induced feminization is critical to understanding the roles that endocrine and genetic factors play in regulating the process of sex differentiation in fish. Here, fugu larvae were treated with AI (letrozole), MT, or E2 from 25 to 80 days after hatching (dah), and gonadal transcriptomic analysis at 80 dah was performed. The expression of dmrt1, gsdf, foxl2, and other key genes (star, hsd3b1, cyp11c1, cyp19a1a, etc.) involved in the steroid hormone biosynthesis pathway were found be altered. The expression of dmrt1, gsdf, cyp19a1a, and foxl2 was further verified by quantitative polymerase chain reaction. In the control group, the expression of dmrt1 and gsdf was significantly higher in XY larvae than in XX larvae, while the expression of foxl2 and cyp19a1a was significantly higher in XX larvae than in XY larvae (P < .05). AI treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 and gsdf in XX larvae. MT treatment suppressed the expression of foxl2, cyp19a1a, dmrt1, and gsdf in XX larvae. E2 treatment suppressed the expression of dmrt1 and gsdf, but did not restore the expression of foxl2 and cyp19a1a in XY larvae. The shared response following AI, MT, and E2 treatment suggested that these genes are essential for sex differentiation. This finding offers some insight into AI or MT-induced masculinization, and E2-induced femininization in fugu.

Methyl farnesoate regulatory mechanisms underlying photoperiod-dependent sex determination in the freshwater crustacean Daphnia magna.

Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

Exposure to 4-nonylphenol induces a shift in the gene expression of gsdf and testis-ova formation and sex reversal in Japanese medaka (Oryzias latipes).

The branched isomer mixture 4-nonylphenol (4-NP) has been used worldwide as a surfactant, and can have endocrine-disrupting effects on aquatic organisms. For instance, 4-NP induces the formation of testis-ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine-disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4-NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 µg/L 4-NP, testis-ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4-NP exposure at 1-100 µg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 µg/L-exposed group was significantly lower than that in the controls. The 4-NP concentration at which gsdf expression was suppressed was equal to that at which testis-ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.

Histological and transcriptional effects of androstenedione in adult zebrafish (Danio rerio).

As a natural androgen, androstenedione (AED) may pose potential risks to aquatic organisms due to its ubiquitousness in aquatic environments. Here we assessed the adverse effects of AED on histology of gonads, as well as mRNA expression levels of 34 genes concerned with hypothalamic-pituitary-gonadal (HPG) axis, germ-cell differentiation and sex differentiation in zebrafish (Danio rerio). Adult zebrafish were exposed to solvent control and three measured concentrations of 0.2, 2.3 and 23.7 µg/L AED for 60 days. The results showed that AED did not induce any obvious histological effects in the ovaries and testes. Of the investigated genes, transcriptional expression levels of amh and cyp11c1 genes in the ovaries of females were significantly increased by AED at 2.3 or 23.7 µg/L. However, different exposure concentrations of AED significantly inhibited mRNA expression of gnrh3, atf4b1 and cyp19a1b in the brain of males. In the testes of males, AED at 2.3 µg/L led to a significant induction of sox9b gene, but it at 23.7 µg/L down-regulated nr5a1b gene. These observed transcriptional changes indicated that AED could pose potential androgenic effects in zebrafish.

Exogenous Oestrogen Impacts Cell Fate Decision in the Developing Gonads: A Potential Cause of Declining Human Reproductive Health.

The increasing incidence of testicular dysgenesis syndrome-related conditions and overall decline in human fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact testis and ovary function and development. Oestrogen is the critical driver of ovarian differentiation in non-mammalian vertebrates, and in its absence a testis will form. In contrast, oestrogen is not required for mammalian ovarian differentiation, but it is essential for its maintenance, illustrating it is necessary for reinforcing ovarian fate. Interestingly, exposure of the bi-potential gonad to exogenous oestrogen can cause XY sex reversal in marsupials and this is mediated by the cytoplasmic retention of the testis-determining factor SOX9 (sex-determining region Y box transcription factor 9). Oestrogen can similarly suppress SOX9 and activate ovarian genes in both humans and mice, demonstrating it plays an essential role in all mammals in mediating gonad somatic cell fate. Here, we review the molecular control of gonad differentiation and explore the mechanisms through which exogenous oestrogen can influence somatic cell fate to disrupt gonad development and function. Understanding these mechanisms is essential for defining the effects of oestrogenic EDCs on the developing gonads and ultimately their impacts on human reproductive health.

Tissues injury and pathological changes in Hyla intermedia juveniles after chronic larval exposure to tebuconazole.

Tebuconazole (TBZ), an azole pesticide, is one of the most frequently detected fungicides in surface water. Despite its harmful effects, mainly related to endocrine disturbance, the consequences of TBZ exposure in amphibians remain poorly understood. Here, we investigated the adverse and delayed effects of TBZ chronic exposure on a native anuran species, often inhabiting cultivated areas, the Italian tree frog (Hyla intermedia). To disclose the multiple mechanisms of action through which TBZ exerts its toxicity we exposed tadpoles over the whole larval period to two sublethal TBZ concentrations (5 and 50 µg/L), and we evaluated histological alterations in three target organs highly susceptible to xenobiotics: liver, kidney, and gonads. We also assessed morphometric and gravimetric parameters: snout-vent length (SVL), body mass (BM), liver somatic index (LSI), and gonad-mesonephros complex index (GMCI) and determined sex ratio, gonadal development, and differentiation. Our results show that TBZ induces irreversible effects on multiple target organs in H. intermedia, exerting its harmful effects through several pathological pathways, including a massive inflammatory response. Moreover, TBZ markedly affects sexual differentiation also by inducing the appearance of sexually undetermined individuals and a general delay of germ cell maturation. Given the paucity of data on the effects of TBZ in amphibians, our results will contribute to a better understanding of the environmental risk posed by this fungicide to the most endangered group of vertebrates.

High temperature stress response is not sexually dimorphic at the whole-body level and is dependent on androgens to induce sex reversal.

The understanding of the molecular and endocrine mechanisms behind environmentally-induced sex reversal in fish is of great importance in the context of predicting the potential effects of climate change, especially increasing temperature. Here, we demonstrate the global effects of high temperature on genome-wide transcription in medaka (Oryzias latipes) during early development. Interestingly, data analysis did not show sexual dimorphic changes, demonstrating that thermal stress is not dependent on genotypic sex. Additionally, our results revealed significant changes in several pathways under high temperature, such as stress response from brain, steroid biosynthesis, epigenetic mechanisms, and thyroid hormone biosynthesis, among others. These microarray data raised the question of what the exact molecular and hormonal mechanisms of action are for female-to-male sex reversal under high temperatures in fish. Complementary gene expression analysis revealed that androgen-related genes increase in females (XX) experiencing high water temperature. To test the involvement of androgens in thermal-induced sex reversal, an androgen antagonist was used to treat XX medaka under a high-temperature setup. Data clearly demonstrated failure of female-to-male sex reversal when androgen action is inhibited, corroborating the importance of androgens in environmentally-induced sex reversal.

Neural and Hormonal Control of Sexual Behavior.

Gonadal hormones contribute to the sexual differentiation of brain and behavior throughout the lifespan, from initial neural patterning to "activation" of adult circuits. Sexual behavior is an ideal system in which to investigate the mechanisms underlying hormonal activation of neural circuits. Sexual behavior is a hormonally regulated, innate social behavior found across species. Although both sexes seek out and engage in sexual behavior, the specific actions involved in mating are sexually dimorphic. Thus, the neural circuits mediating sexual motivation and behavior in males and females are overlapping yet distinct. Furthermore, sexual behavior is strongly dependent on circulating gonadal hormones in both sexes. There has been significant recent progress on elucidating how gonadal hormones modulate physiological properties within sexual behavior circuits with consequences for behavior. Therefore, in this mini-review we review the neural circuits of male and female sexual motivation and behavior, from initial sensory detection of pheromones to the extended amygdala and on to medial hypothalamic nuclei and reward systems. We also discuss how gonadal hormones impact the physiology and functioning of each node within these circuits. By better understanding the myriad of ways in which gonadal hormones impact sexual behavior circuits, we can gain a richer and more complete appreciation for the neural substrates of complex behavior.

Key role of estrogen receptor ß in the organization of brain and behavior of the Japanese quail.

Estrogens play a key role in the sexual differentiation of the brain and behavior. While early estrogen actions exert masculinizing effects on the brain of male rodents, a diametrically opposite effect is observed in birds where estrogens demasculinize the brain of females. Yet, the two vertebrate classes express similar sex differences in the brain and behavior. Although ERα is thought to play a major role in these processes in rodents, the role of ERß is still controversial. In birds, the identity of the estrogen receptor(s) underlying the demasculinization of the female brain remains unclear. The aim of the present study was thus to determine in Japanese quail the effects of specific agonists of ERα (propylpyrazole triol, PPT) and ERß (diarylpropionitrile, DPN) administered at the beginning of the sensitive period (embryonic day 7, E7) on the sexual differentiation of male sexual behavior and on the density of vasotocin-immunoreactive (VT-ir) fibers, a known marker of the organizational action of estrogens on the quail brain. We demonstrate that estradiol benzoate and the ERß agonist (DPN) demasculinize male sexual behavior and decrease the density of VT-ir fibers in the medial preoptic nucleus and the bed nucleus of the stria terminalis, while PPT has no effect on these measures. These results clearly indicate that ERß, but not ERα, is involved in the estrogen-induced sexual differentiation of brain and sexual behavior in quail.

Peroxisome proliferator-activated receptor alpha is involved in the temperature-induced sex differentiation of a vertebrate.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Rapid Induction of Female-to-Male Sex Change in Adult Zebrafish by Injection of an Aromatase Inhibitor.

Previously, we examined whether aromatase inhibitor (AI) treatment induces a sex change in adult female zebrafish. A 5-month AI treatment regime resulted in the retraction of the ovaries and testis formation. Eight weeks after changing the diet to AI-free food, a large number of normal sperm were obtained. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of zebrafish. However, >7 months of treatment was necessary; thus, pairing was unsuccessful. In this study, we tried to induce sex change through the injection of an AI to shorten the time course of sex change. When the AI solution was directly injected into the abdomen of zebrafish, retraction of the ovary was induced within 2 months. The natural mating of sex-changed females with normal females was successful at 3 months. Although the fertilization rate was low, juveniles resulting from these matings developed normally. We succeeded in establishing a method for inducing sex changes in adult zebrafish within 3 months. The procedure will support the study of how sexual plasticity persists in adult zebrafish following sex differentiation and the identification of undifferentiated stem cells.

Expression profile and estrogenic regulation of Amh during gonadal sex differentiation in northern snakehead (Channa argus).

BACKGROUND: Anti-Müllerian hormone (Amh) plays a critical role in both early sex determination and later gonad development in vertebrate species. However, it remains unknown in northern snakehead (Channa argus), which is economically important freshwater fish with sexual dimorphism. OBJECTIVE: This study aimed to identify the expression profiles and estrogenic regulation of CaAmh during gonadal sex differentiation in C. argus. METHODS: The cDNA and genomic DNA sequences of CaAmh were identified by PCR and RACE techniques. The expression patterns of CaAmh were detected by qRT-PCR during the gonadal sex differentiation and after 17α-ethinyloestradiol (EE2) treatments. RESULTS: CaAmh is composed of seven exons and six introns, and its full-length cDNA is 2413 bp in length, with 1635 bp open reading frame (ORF) that encodes a 544 amino acid protein. Tissues expression patterns revealed that CaAmh display the highest expression in testis of XY males (40.36 folds, p < 0.01). The spatio-temporal expression patterns during gonadal sex differentiation indicated that CaAmh expression differed between XX females and XY males at 30 day after hatching (dah), and reached to the peak (36.03 folds, p < 0.01) at 90 dah in XY gonads. However, CaAmh expression in XX gonads remained low throughout the sampling period. Furthermore, CaAmh expression in the gonads (ovaries) of the sex-reversed XY fish (XY-F) by the administration of estrogen EE2 was downregulated to low level, similar to that in ovaries of normal XX females (XX-F). CONCLUSIONS: These results show that Amh plays a critical role in testicular differentiation of C. argus and it is apparently modulated by estrogens in this species.

Agrochemicals disrupt multiple endocrine axes in amphibians.

Concern over global amphibian declines and possible links to agrochemical use has led to research on the endocrine disrupting actions of agrochemicals, such as fertilizers, fungicides, insecticides, acaricides, herbicides, metals, and mixtures. Amphibians, like other species, have to partition resources for body maintenance, growth, and reproduction. Recent studies suggest that metabolic impairments induced by endocrine disrupting chemicals, and more particularly agrichemicals, may disrupt physiological constraints associated with these limited resources and could cause deleterious effects on growth and reproduction. Metabolic disruption has hardly been considered for amphibian species following agrichemical exposure. As for metamorphosis, the key thyroid hormone-dependent developmental phase for amphibians, it can either be advanced or delayed by agrichemicals with consequences for juvenile and adult health and survival. While numerous agrichemicals affect anuran sexual development, including sex reversal and intersex in several species, little is known about the mechanisms involved in dysregulation of the sex differentiation processes. Adult anurans display stereotypical male mating calls and female phonotaxis responses leading to successful amplexus and spawning. These are hormone-dependent behaviours at the foundation of reproductive success. Therefore, male vocalizations are highly ecologically-relevant and may be a non-invasive low-cost method for the assessment of endocrine disruption at the population level. While it is clear that agrochemicals disrupt multiple endocrine systems in frogs, very little has been uncovered regarding the molecular and cellular mechanisms at the basis of these actions. This is surprising, given the importance of the frog models to our deep understanding of developmental biology and thyroid hormone action to understand human health. Several agrochemicals were found to have multiple endocrine effects at once (e.g., targeting both the thyroid and gonadal axes); therefore, the assessment of agrochemicals that alter cross-talk between hormonal systems must be further addressed. Given the diversity of life-history traits in Anura, Caudata, and the Gymnophiona, it is essential that studies on endocrine disruption expand to include the lesser known taxa. Research under ecologically-relevant conditions will also be paramount. Closer collaboration between molecular and cellular endocrinologists and ecotoxicologists and ecologists is thus recommended.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause male-biased sex differentiation in zebrafish.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used pharmaceuticals to treat pain, fever and inflammation. NSAIDs are also known to have many side effects including adverse effects on reproduction in both humans and animals. As NSAIDs usage is not regulated they are frequently detected at high concentrations in the environment. In order to understand the effect of NSAIDs on zebrafish sex differentiation, we used seven different NSAIDs which were either Cox-1 selective, Cox-1 biased, non-selective or COX-2 selective. We show that at higher concentration, NSAIDs are toxic to zebrafish embryo as they lead to mortality and hatching delay. Gene expression analysis following short term exposure of NSAIDs led to downregulation of female specific genes including zp2, vtg2 foxl2 and wnt4. Long term exposure of larvae to environmentally relevant concentrations of Cox-2 selective and non-selective NSAIDs resulted in male-biased sex ratio which confirmed the qRT-PCR analysis. However, the Cox-1 selective acetylsalicylic acid and the Cox-1 biased ketoprofen did not alter sex ratio. The observed male-biased sex ratio could also be due to induction of apoptosis process as the genes including p21 and casp8 were significantly upregulated following exposure to the Cox-2 selective and the non-selective NSAIDs. The present study indicates that NSAIDs alter sex differentiation in zebrafish, primarily through inhibition of Cox-2. This study clearly demonstrates that the use of NSAIDs and their release into the aquatic environment should be carefully monitored to avoid adverse effects to the aquatic organisms.

GABAergic input through GABAB receptors is necessary during a perinatal window to shape gene expression of factors critical to reproduction such as Kiss1.

Lack of GABAB receptors in GABAB1 knockout mice decreases neonatal ARC kisspeptin 1 (Kiss1) expression in the arcuate nucleus of the hypothalamus (ARC) in females, which show impaired reproduction as adults. Our aim was to selectively impair GABAB signaling during a short postnatal period to evaluate its impact on the reproductive system. Neonatal male and female mice were injected with the GABAB antagonist CGP 55845 (CGP, 1 mg/kg body wt sc) or saline from postnatal day 2 (PND2) to PND6, three times per day (8 AM, 1 PM, and 6 PM). One group was killed on PND6 for collection of blood samples (hormones by radioimmunoassay), brains for gene expression in the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), and ARC micropunches [quantitative PCR (qPCR)] and gonads for qPCR, hormone contents, and histology. A second group of mice was injected with CGP (1 mg/kg body wt sc) or saline from PND2 to PND6, three times per day (8 AM, 1 PM, and 6 PM), and left to grow to adulthood. We measured body weight during development and parameters of sexual differentiation, puberty onset, and estrous cycles. Adult mice were killed, and trunk blood (hormones), brains for qPCR, and gonads for qPCR and hormone contents were obtained. Our most important findings on PND6 include the CGP-induced decrease in ARC Kiss1 and increase in neurokinin B (Tac2) in both sexes; the decrease in AVPV/PeN tyrosine hydroxylase (Th) only in females; the increase in gonad estradiol content in both sexes; and the increase in primordial follicles and decrease in primary and secondary follicles. Neonatally CGP-treated adults showed decreased ARC Kiss1 and ARC gonadotropin-releasing hormone (Gnrh1) and increased ARC glutamic acid decarboxylase 67 (Gad1) only in males; increased ARC GABAB receptor subunit 1 (Gabbr1) in both sexes; and decreased AVPV/PeN Th only in females. We demonstrate that ARC Kiss1 expression is chronically downregulated in males and that the normal sex difference in AVPV/PeN Th expression is abolished. In conclusion, neonatal GABAergic input through GABAB receptors shapes gene expression of factors critical to reproduction.

The Administration of Cortisol Induces Female-to-Male Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides.

In this study, we injected cortisol into the protogynous orange-spotted grouper (Epinephelus coioides) to investigate the role of this hormone in sex change. Following injection, we evaluated gonadal changes, serum levels of steroid hormones, and sex-related gene expression during the processes of cortisol-induced sex change and cortisol withdrawal in the orange-spotted grouper. Cortisol treatment caused the degeneration of oocytes and induced sex change in a dose-dependent manner. Over the long-term, we observed a significant increase in serum 11-ketotestosterone (11-KT) levels in all cortisol-treated groups, although levels of 17ß-estradiol did not change significantly. Consistent with the elevation of serum 11-KT levels, the expression of genes related to testicular development was also significantly up-regulated in the cortisol-treated groups. Based on our results, we propose that cortisol may trigger masculinization by inducing the synthesis of 11-KT and by directly activating the expression of sex-related genes. Furthermore, we found that cortisol-induced sex change was not permanent and could be reversed after the withdrawal of cortisol treatment.