Cellular defense mechanisms against benzo[a]pyrene in testicular Leydig cells: implications of p53, aryl-hydrocarbon receptor, and cytochrome P450 1A1 status.

Leydig cells of the mammalian testis produce testosterone and support spermatogenesis, and thereby their role in male function is fundamental. Although benzo[a]pyrene (B[a]P) has been known to exhibit carcinogenic, apoptogenic, and endocrine-disrupting activities, its potential signaling system in Leydig cells remains to be discovered. In the present study, using the TM3 Leydig cell line and primary Leydig cells, we showed that Leydig cells do not die by exposure to B[a]P and found that an increased level of X chromosome-linked inhibitor of apoptosis protein may be associated with the antiapoptotic process. The Leydig cells were shown to express p53, but its translational level was extremely low. Although a high level of p53 protein was not necessary for apoptosis induced by B[a]P-7,8-diol-9,10-epoxide (a final B[a]P metabolite) in Leydig cells, the apoptosis of primary Leydig cells appears to be p53 independent. This indicates the lack of p53 function in primary Leydig cells. Furthermore, Leydig cells were found to retain insignificant levels of endogenous aryl-hydrocarbon receptor and AhR nuclear transporter proteins in nature. Exposure to B[a]P did not result in a significant increase in aryl-hydrocarbon receptor proteins that are required for CYP1A1 transcription. CYP1A1 expression was present in Leydig cells but at levels insufficient to exhibit its activity. Finally, we have demonstrated that overexpression of CYP1A1 in Leydig cells sensitizes the cells to exhibit its activity in the presence of B[a]P and, thus, induction of apoptosis. Together, these results indicate that the deficiency of CYP1A1 activity might be a decisive condition rendering Leydig cells secure from exogenous polycyclic aromatic hydrocarbons such as B[a]P.

Doxazosin-induced clusterin expression and apoptosis in prostate cancer cells.

The purpose of this study was to correlate temporal expression of clusterin and apoptosis in androgen-independent human prostate cancer cells (PC-3) treated with 25 microM doxazosin. DNA fragmentation, reverse transcriptase polymerase chain reaction, and terminal transferase-mediated biotinylated 16-desoxy-uridene triphosphate nick-end labeling (TUNEL) assays were used to assess degree of apoptosis and temporal and spatial expression of clusterin mRNA and protein. DNA fragmentation was significant at 48 hours. Clusterin mRNA expression was 3-fold higher than control at 9 hours and was maintained over 48 hours. The TUNEL assay showed increasing percentage of apoptotic cells and presence of clusterin after doxazosin treatment. During doxazosin-induced apoptosis in PC3 cells, clusterin appeared to initially accumulate in the cytoplasm and protect against apoptosis; later, after its transport to the nucleus, clusterin was no longer able to suppress apoptosis.

Nonylphenol and octylphenol-induced apoptosis in human embryonic stem cells is related to Fas-Fas ligand pathway.

Human embryonic stem (hES) cells have been proposed as a source of various cell types for cell replacement therapy. Besides their potential in therapeutic uses, ES cells also have other potential applications, such as in drug discovery and in vitro screening assays of various toxicants. Nonylphenol (NP) and octylphenol (OP) are common environmental contaminants, known to disrupt the reproductive and endocrine system. However, little is known about their toxicological effects on early embryonic development in humans. In this study, we used undifferentiated hES cells and the neural progenitor cells derived from them to investigate the potential toxicity of NP and OP. Our results show that the cytotoxic effects of NP and OP involve DNA fragmentation, the major characteristic of apoptosis. The NP- and OP-induced apoptosis was concomitant with the increased activity of Caspase-8 and -3. Moreover, both Fas and Fas ligand (FasL) protein expressions were markedly increased in the NP- or OP-exposed hES cells. These results suggest that NP and OP are able to trigger apoptosis in hES cells via a pathway dependent on caspase activation and Fas-FasL interaction. In particular, hES cell-derived neural progenitor cells had a higher sensitivity to the toxicants than undifferentiated hES cells, thereby suggesting that the toxic stress response may differ depending on the developmental stage. These findings offer new perspectives for understanding the fundamental mechanisms in chemical-induced apoptosis in hES cells.

Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle.

Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.

Cloning of Ki-ras and Ha-ras cDNAs from the hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and its expression after exposure to 4-nonylphenol.

Previous studies on ras proto-oncogene genes in fish have been focused on chemical-associated carcinogenesis, and the expression of fish ras genes was not well-characterized. We investigated Ki- and Ha-ras genes from the hermaphroditic fish Rivulus marmoratus to understand better their expression patterns in specific tissues, as well as their responses to endocrine-disrupting chemicals such as 4-nonylphenol (4-NP). By investigating expression patterns, we found that the R. marmoratus Ki-ras (Rm Ki-ras) gene showed an alternative splicing event between exons 4A and 4B according to tissue types, which is different from the expression pattern of mammalian Ki-ras genes. In the Rm Ki-ras gene, there were two different expressed types, with exons 1-2-3-4A-4B (long form) and with exons 1-2-3-4B (short form). In the Rm Ki-ras gene, the long form was expressed strongly in the gonad and intestine, and the short form was expressed ubiquitously, except for a low level of expression in the liver. Following 4-NP exposure (300 microg/L), the Rm Ki-ras long form in the liver was significantly expressed, while it was expressed moderately in the ovaries. However, the Rm Ha-ras gene was significantly over-expressed in the brain, while its expression in the gonad was down-regulated. In relation to these modulations after 4-NP exposure, we searched the Rm Ha- and Ki-ras promoter regions and found several ERE-half sites, that may be involved in the modulation of ras gene expression following 4-NP exposure. These genes could be applicable as new biomarker genes for assessing exposure to endocrine-disrupting chemicals (EDCs). Further, this implies the disturbance of ras-dependent signal transduction following EDC exposure.

Cloning and characterization of glutathione S-transferase gene in the intertidal copepod Tigriopus japonicus and its expression after exposure to endocrine-disrupting chemicals.

To investigate the impacts of marine pollution on aquatic organisms, we tested the intertidal copepod Tigriopus japonicus as a model species. To analyze the copepods' responses to endocrine-disrupting chemicals (EDCs), we exposed them to two different chemicals: 4,4'-octylphenol (4,4'-OP, 12.5-100 microg/L for 2 h) and polychlorinated biphenyl (PCB, 6.25-25 microg/L for two days). 4,4'-OP was toxic, although exposure time was limited to 2h. After extracting total RNA from the exposed T. japonicus, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) to determine gene expression patterns following chemical exposure. To analyze the gene expression of T. japonicus, we used glutathione S-transferase with GAPDH as an internal control. Of the genes tested using EDC-exposed samples, 4,4'-OP induced upregulation of the glutathione S-transferase (GST) gene, while PCB caused downregulation of the GST gene. These results suggest that the two EDCs act in different manners in T. japonicus.

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary.

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary.

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.

Amifostine has an inhibitory effect on the radiation-induced p53-branched cascade in the immature mouse ovary.

The organic thiophosphate, amifostine, is a promising pharmacological compound showing selective protection in many tissues against the toxic side-effects of radiation and cytotoxic drugs. The aim of the present study was to assess the radioprotective effects of amifostine on ovarian follicles. Three-week-old female mice, with or without pretreatment with amifostine, were irradiated with 6.42 Gy of gamma-ray. Reduced proliferation of granulosa cells was verified with BrdU staining and the incidences of follicular degeneration increased in ovarian follicles in the gamma-ray-irradiated mice compared to that of the control or amifostine-treated group. Biochemical changes caused by gamma-irradiation provoked a rise of p53 and Bax protein and a decline of the inactive form in caspase-3 and PARP protein. Caspase-3 and PARP cleaved into active peptides during apoptosis. This process was confirmed by the result of this study, which was that the amount of the stable form decreased immediately after irradiation. In the amifostine treatment group before irradiation, the increased rate of p53 and Bax was suppressed, particularly in the LDs-treated group. The relationship between PARP and caspase-3 levels showed the effect of amifostine exposure before irradiation. In conclusion, amifostine had an inhibitory effect on ovarian programmed cell death induced by gamma-ray, affecting the expression of apoptotic signaling molecules and the level of proliferation of the granulosa cells.

cDNA cloning and expression of ferritin heavy chain 1, ferritin heavy chain 2 and hemoglobin genes from the fire-bellied frog Bombina orientalis.

We cloned two Bombina orientalis ferritin heavy chains (ferritin heavy chains 1 and 2) and one hemoglobin beta-chain gene from a B. orientalis oviduct cDNA library, and the length of transcripts was 882, 858 and 611 bp encoding 177, 177 and 148 aa, respectively. B. orientalis ferritin heavy chain genes showed high similarity to those of amphibia (88-93%), mammals (70-71%), and fishes (70-72%), and the hemoglobin beta-chain gene showed moderate similarity to amphibian (65-68%) and mammalian (54-57%) hemoglobin beta-chain genes, respectively. Based on phylogenetic analysis, the genes were clustered to the same clade in amphibia. The two B. orientalis ferritin heavy chain genes showed different tissue-specific gene expression patterns. Thus, ferritin heavy chain 1 gene was highly expressed in intestine and oviduct but ferritin heavy chain 2 gene was ubiquitously expressed in most of the examined tissues. The hemoglobin beta-chain gene was more highly expressed in liver than in oviduct. These findings indicate that the genes may play different roles in different tissues. In this paper, we discuss the basic characteristics of B. orientalis ferritin heavy chain genes and hemoglobin beta-chain gene.

Circadian expression of Mel1a and PL-II genes in placenta: effects of melatonin on the PL-II gene expression in the rat placenta.

In the mammal, melatonin regulates the seasonal and/or circadian rhythm of PRL levels. Since several members of the PRL gene family are expressed during late pregnancy, we investigated the relationship between the expression of placental lactogen (PL)-II-one member of the PRL family- and melatonin, as well as the placental expression of one of the receptors for melatonin, melatonin receptor 1a (Mel(1a())). Herein we provide the first demonstration that Mel(1a) is not only expressed in the rat placenta, but that it is spatially and temporally regulated throughout late pregnancy. In situ hybridization and Northern blot analyses show that Mel(1a) mRNA is localized in the rat placenta on gestational day 19, and is mainly restricted to the spongiotrophoblast and trophoblast giant cells. Interestingly, the junctional zone of the placenta at this time showed the strongest gene expression when the tissue was obtained at 16:00 h (daytime) and showed the least expression when it was obtained at 04:00 h (night-time). In contrast, the labyrinth zone showed the strongest expression in tissue obtained at night and showed the least expression in tissue obtained during the day. PL-II gene expression also exhibited a circadian rhythm but the direction of the fluctuation was exactly opposite to that of the Mel(1a) gene, such that at night the junctional zone had the strongest expression, while the labyrinth zone had the weakest. In vitro treatment of placental tissue with an melatonin agonist, chloromelatonin, greatly decreased PL-II mRNA levels. That Mel(1a) plays a regulatory role in the expression of PL-II in the late-pregnancy rat placenta is strongly suggested by the pattern of its own spatial and temporal expression.

Effects of Aroclor 1254 on the expression of rat placental PRL-family genes.

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.

Selective processing of a follicular matrix metalloproteinase-2 isoform by human oviducal fluid.

The present study demonstrates that a unique isoform of matrix metalloproteinase (MMP)-2 present in human follicular fluid (FF) can be processed selectively by human oviducal fluid (OF). A gelatin zymogram of untreated FF showed distinct 88-, 84- and 62-kDa gelatinases. Treatment of FF with EDTA resulted in the appearance of 110-kDa gelatinase (GA110). Most gelatinases, except for the 88- and 84-kDa gelatinases, were abolished by pretreatment with EDTA or phenanthroline, but not by pretreatment with a serine/threonine protease inhibitor. When EDTA-pretreated FF was mixed with OF, the GA110 of the FF was specifically reduced. The reduction in GA110 was dependent upon the amount of OF protein and the incubation period after mixing. Treatment of FF with aminophenylmercuric acetate reduced GA110 activity, but this reduction was accompanied by a concomitant increase of 62-kDa gelatinase activity. Anti-human MMP-2 antibody strongly reacted with both GA110 and 62-kDa gelatinases of FF, but only GA110 immunoreactivity was abolished when FF was mixed with OF. The results suggest that the GA110 of FF is an MMP-2 isoform that can be processed selectively by OF.

Evaluation of caffeine as a radioprotector in whole-body irradiated male mice.

Caffeine is the main psychoactive ingredient of coffee, tea and even colas. This study investigated the radioprotection of caffeine in mice. Mice were irradiated with 6.5 Gy. A caffeinetreated group was administered 80 mg/kg-body weight by an i.p injection at 1 hour before irradiation. At 6 hours after irradiation, the animals were sacrificed for the study. Hormonal analysis was performed with radioimmunoassay on serum. The expression kinetics of the apoptotic genes after irradiation was evaluated with RT-PCR. The weight of body and organ and H and E-stained slides did not show differences between groups. The circulating testosterone significantly decreased in the irradiated group. The expression of Fas antigen, p21, p53, bax and bcl2 related to radiation- induced apoptosis was comparable to that of the caffeine-untreated group. Bax mRNA dramatically increased in the irradiated group, but not in the irradiated caffeine -treated group. The results indicate that caffeine protects an early apoptotic initiation after irradiation.

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes.

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.

Genomic cloning and expression of vitellogenin gene from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae).

We cloned the vitellogenin gene from the self-fertilizing fish Rivulus marmoratus, and sequenced 12,326 bp. The number of exons of R. marmoratus and rainbow trout vitellogenin genes were different, and also the splicing junctions are different throughout most of the exons and introns but the amino acid similarity of R. marmoratus vitellogenin gene to other species was rather high. In promoter region of R. marmoratus vitellogenin gene, there were several E2 binding sites and the estrogen response element (ERE). We discuss here the gene structure and expression of R. marmoratus vitellogenin gene.

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos.

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.

CDNA cloning and expression of ATP synthase subunit b from the fire-bellied frog Bombina orientalis (Anura: Discoglossidae).

We cloned the Bombina orientalis adenosine triphosphate (ATP) synthase subunit b gene from a B. orientalis oviduct cDNA library. The transcript was 997 bp long and encoded 250 amino acid residues. It showed high similarity to amphibian (84-85%), mammalian (56-62%) and invertebrate (46-50%) sequences. In phylogenetic analyses, it clustered with other amphibian sequences. This gene was highly expressed in brain, intestine and oviduct but not in muscle and liver. In this paper, we report the basic characteristics of B. orientalis ATP synthase subunit b gene.

Bisphenol A exposure during adulthood causes augmentation of follicular atresia and luteal regression by decreasing 17ß-estradiol synthesis via downregulation of aromatase in rat ovary.

BACKGROUND: Bisphenol A (BPA) has been detected in human body fluids, such as serum and ovarian follicular fluids. Several reports indicated that BPA exposure is associated with the occurrence of several female reproductive diseases resulting from the disruption of steroid hormone biosynthesis in the adult ovary. OBJECTIVE: We hypothesized that long-term exposure to low concentrations of BPA disrupts 17ß-estradiol (E2) production in granulosa cells via an alteration of steroidogenic proteins in ovarian cells. METHODS: Adult female rats received BPA for 90 days by daily gavage at doses of 0, 0.001, or 0.1 mg/kg body weight. We determined serum levels of E2, testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). We also analyzed the expressions of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase isomerase (3ß-HSD), and aromatase cytochrome P450 (P450arom) in the ovary. RESULTS: Exposure to BPA significantly decreased E2 serum concentration, which was accompanied by augmented follicular atresia and luteal regression via increase of caspase-3-associated apoptosis in ovarian cells. After BPA exposure, P450arom and StAR protein levels were significantly decreased in granulosa cells and theca-interstitial (T-I) cells, respectively. However, P450scc and 3ß-HSD protein levels remained unchanged. The increase in LH levels appeared to be associated with the decreased synthesis of T in T-I cells after BPA exposure via homeostatic positive feedback regulation. CONCLUSIONS: BPA exposure during adulthood can disturb the maintenance of normal ovarian functions by reducing E2. The steroidogenic proteins StAR and P450arom appear to be targeted by BPA.

Bisphenol A modulates expression of sex differentiation genes in the self-fertilizing fish, Kryptolebias marmoratus.

Endocrine disrupting chemicals (EDCs) have been a major concern in the normal reproduction and development of aquatic organisms. In the teleost, steroid hormones are synthesized via the steroidogenesis pathway, and play a key physiological role in the regulation of gonadal sex differentiation. The protogynous hermaphroditic fish, Kryptolebias marmoratus is the only vertebrate capable of reproducing through internal self-fertilization. To uncover the effect of bisphenol A (BPA) on sex differentiation genes on transcription, we investigated the expression patterns of several sex differentiation-related genes such as dax1, dmrt1, mis, sf1, figlα, StAR and wt1 after BPA exposure with controls (E2 and TMX). In response to 17ß-estradiol (E2) exposure, a testis-specific gene, dmrt1 mRNA was down-regulated in the gonad of the secondary male but the expression of the female-specific gene, dax1 mRNA was significantly elevated in the brain and gonad. A high level of StAR mRNA was detected in the brain and gonad of both hermaphrodite and secondary males, suggesting that the elevated expression of dax1 and StAR genes would be involved in E2 exposure. As expected, upon BPA exposure, the dmrt1 and MIS mRNA level decreased in both hermaphrodite and secondary males, while the female-specific gene, figlα mRNA level increased in the gonad of both genders. BPA showed an opposite mode of action on the expression of dax1 (induction, P>0.05) and sf1 mRNA (inhibition, P>0.05) in the brain and gonad against both genders. The sensitivity of dax1 to BPA on expression was relatively high in the secondary male. The wt1 mRNA was up-regulated in most tissues except in the liver of BPA-exposed secondary males. Regarding the time course study, the figlα mRNA level increased at 6 h after BPA exposure. In addition, BPA elevated the expression of StAR, dax1, and wt1 mRNA but repressed sf1 mRNA. In this paper, we demonstrated that BPA may modulate the expression of sex differentiation and steroidogenesis pathway genes, and this finding would provide a better understanding on the modulation of transcription upon BPA exposure in steroidogenesis and sex differentiation in the hermaphroditic fish, K. marmoratus.