Testes Of Fish Research Articles

Effects of decabromodiphenyl ether (BDE‐209) on mRNA transcription of thyroid hormone pathway and spermatogenesis associated genes in Chinese rare minnow (Gobiocypris rarus)

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants, which are ubiquitous environmental contaminant found in both abiotic and biotic environmental samples. Deca-BDE (BDE-209) is the principal component, which is currently used worldwide. In this study, the effect of BDE-209 on the mRNA levels of thyroid hormone (TH) related genes and spermatogenesis associated genes were determined from larvae and adult rare minnow (Gobiocypris rarus) exposed to concentrations 0.01, 0.1, 1, and 10 μg/L for 21 days. The results showed that the type II deiodinase (dio2) and sodium iodide symporter (nis) mRNA levels were significantly up-regulated in the larvae at 10 μg/L treatment. In adult, histopathological observations showed that liver of female fish were degenerated at 10 μg/L treatment, and inhibition of spermatogenesis were observed in testis of male fish. In addition, the thyroid hormone receptor α (trα), dio2, and nis mRNA levels in the liver of male and female fish were significantly up-regulated, whereas dio2 and nis mRNA levels were significantly down-regulated in the brain. These results indicate that exposure to BDE-209 could result in tissue-specific alternations of TH-related genes expression in adults. Moreover, the mRNA levels of the testis-specific apoptosis genes, the spermatogenesis-associated 4 (spata4) and spermatogenesis-associated 17 (spata17), were down-regulated at 10 μg/L treatment in testis of male fish. Our results suggest that BDE-209 may pose threat to normal thyroid and reproductive function in fish.

Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization

Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.

Selective uptake and biological consequences of environmentally relevant antidepressant pharmaceutical exposures on male fathead minnows

Antidepressant pharmaceuticals have been reported in wastewater effluent at the nanogram to low microgram-per-liter range, and include bupropion (BUP), fluoxetine (FLX), sertraline (SER), and venlafaxine (VEN). To assess the effects of antidepressants on reproductive anatomy, physiology, and behavior, adult male fathead minnows ( Pimephales promelas) were exposed for 21 days either to a single concentration of the antidepressants FLX, SER, VEN, or BUP, or to an antidepressant mixture. The data demonstrated that exposure to VEN (305 ng/L and 1104 ng/L) and SER (5.2 ng/L) resulted in mortality. Anatomical alterations were noted within the testes of fish exposed to SER and FLX, both modulators of the neurotransmitter serotonin. Additionally, FLX at 28 ng/L induced vitellogenin in male fish—a common endpoint for estrogenic endocrine disruption. Significant alterations in male secondary sex characteristics were noted with single exposures. Effects of single compound exposures neither carried over, nor became additive in the antidepressant mixtures, and reproductive behavior was not affected. Analysis of brain tissues from the exposed fish suggested increased uptake of FLX, SER and BUP and minimal uptake of VEN when compared to exposure water concentrations. Furthermore, the only metabolite detected consistently in the brain tissues was norfluoxetine. Similar trends of uptake by brain tissue were observed when fish were exposed to antidepressant mixtures. The present study demonstrates that anatomy and physiology, but not reproductive behavior, can be disrupted by exposure to environmental concentrations of some antidepressants. The observation that antidepressant uptake into fish tissues is selective may have consequences on assessing the mode-of-action and effects of these compounds in future studies.

Teleost maturation-inducing hormone, 17,20β-dihydroxypregn-4-en-3-one, peaks after spawning in Tinca tinca

During an eight month study of the reproductive cycle in two age groups, and in both sexes, of tench ( Tinca tinca L.), it was found that plasma concentrations of the presumptive ‘maturation inducing hormone (MIH)’ 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) did not reach a peak during the spawning season, but as much as two months after spawning had ceased. The cessation of the spawning season was confirmed by histological examination of the gonads and by measurement of 11-ketotestosterone and 17β-estradiol in the plasma of males and females, respectively. Measurements were also made of the ‘alternative MIH’ 17,20β,21-trihydroxypregn-4-en-3-one in the older fish. However, this steroid did not show the same pattern as 17,20β-P. An assessment was made of the prevalence of primary spermatocytes in the testes of post-spawned fish – to test an alternative hypothesis that 17,20β-P might be involved in the stimulation of meiosis. However, there was no evidence for any increase in testis differentiation post-spawning. In fact the testes became increasingly undifferentiated as the autumn progressed. The role, if any, of this ‘unseasonal’ peak of 17,20β-P production remains to be determined.

Vitellogenin Induction and Histo-metabolic Changes Following Exposure of Cyprinus carpio to Methyl Paraben

Methyl paraben (MP), which is used as a preservative in pharmaceutical and cosmetic (shampoo) products, foods and beverages, enters into the aquatic environment and can pose a potential fish health hazard. In this experiment, effects of MP were evaluated in adult male common carp (Cyprinus carpio) by exposing them to fractions (1/143 rd to 1/29th) of the LC50 dose with every change of water for 28 days. Vitellogenin induction, metabolic enzymes, somatic indices and bioaccumulation were studied at weekly intervals. The 96 th h LC50 of MP in fingerlings was 120 mg/L. Compared to the control, except for increases (p<0.01) in alkaline phosphatase (EC 3.1.3.1), alanine aminotransferase (EC 2.6.1.2) and liver size, there were decreases (p<0.01) in activity of acid phosphatase (EC 3.1.3.2), aspartate aminotransferase (EC 2.6.1.1), and testiculosomatic index following exposure to any dose of MP. Vitellogenin induction was significantly higher (p<0.01) in exposed than unexposed (control) fish. The bioaccumulation of MP in testis, liver, brain, gills and muscle tissues of fish increased significantly (p<0.01) with increase of dose from 0.84 ppm to 1.68 ppm. Dose and duration of exposure (p<0.01) indicated that an exposure period of 1 to 2 weeks was sufficient to cause changes in the quantifiable parameters studied. Fish exposed to 4.2 ppm MP became lethargic after the 26 th d. Histologically, degeneration, vacuolization and focal

The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish ( Danio rerio) eleuthero-embryos and adult males

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 μg/L. In eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ß3, cyp19b cyp19a, hhex and pax8 were induced at 3000 μg/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.

Prevention of cytogenetic, histochemical and biochemical alterations in Oreochromis niloticus by dietary supplement of sorbent materials

The current study was conducted to evaluate the ability of Egyptian bentonite (EB) and montmorillonite (EM) for the prevention of genotoxicity, histochemical and biochemical changes induced by aflatoxin B 1 (AFB 1) using the micronucleus (MN) assay, chromosomal aberrations and DNA fragmentation analysis in Tilapia fish. Six groups of fish were treated for 3 weeks and included the control group, AFB 1-treated group and the groups treated with EB or EM alone or in combination with AFB 1. At the end of experiment period, blood samples were collected for MN, testosterone and biochemical assays. Chromosomal aberrations were determined in kidney tissues, DNA fragmentation test was determined in liver and testis, whereas histochemical study was carried out on liver, testis and gills. The results indicated that a significant decrease in total protein, albumin, globulin, testosterone and DNA content in liver, gills and testis accompanied with a significant increase in number of micronucleated erythrocytes (MnRBCs), total chromosomal aberrations in kidney and DNA fragmentation in testis and liver of fish received AFB 1 alone. Fish treated with EB or EM alone were comparable to the control regarding the biochemical parameters except testosterone in EB-treated group which was significantly decreased. Both clays did not induce any significant differences in number of MnRBCs, chromosomal aberrations in the kidney, DNA fragmentation in testis, but not in liver of EB-treated group. The combined treatment with AFB 1 and EB or EM succeeded to improve all the tested parameters towards the control values although it did not normalize them. Moreover, the improvement was pronounced in the group received EM plus AFB 1. It could be concluded that EB and EM have the ability to tightly bind AFB 1 in the gastrointestinal tract of fish resulting in decreasing its bioavailability. Moreover, the two tested clays were safe and can be used as potential aflatoxin binders in animal feed.

Comparative biological effects and potency of 17α- and 17β-estradiol in fathead minnows

17β-Estradiol is the most potent natural estrogen commonly found in anthropogenically altered environments and has been the focus of many toxicological laboratory studies. However, fewer aquatic toxicological data on the effects of 17α-estradiol, a diastereoisomer of 17β-estradiol, exists in the literature even though it has been found in the aquatic environment, sometimes at higher concentrations than 17β-estradiol. The central objective of this study was to determine how the anatomical, physiological, and behavioral effects of exposure to 17α-estradiol compare to the well-documented effects of 17β-estradiol exposures in aquatic vertebrates. A 21-day flow-through exposure of mature male and female fathead minnows to three concentrations each of 17α- and 17β-estradiol (averaged measured concentrations 27, 72, and 150 ng/L for 17α-estradiol, and 9, 20, and 44 ng/L for β-estradiol, respectively) yielded significant, concentration-dependent differences in plasma vitellogenin concentrations among estradiol-exposed males when compared to fish from an ethanol carrier control. Interstitial cell prominence in the testis of fish was elevated in all estradiol treatments. Aggressiveness of male fish to defend nest sites appeared depressed in many of the higher concentration estradiol treatments (albeit not significantly). No clear effects were observed in female fish. Based on plasma vitellogenin data, it appears that 17β-estradiol is 8–9 times more potent than 17α-estradiol and that the lowest observable effect concentration (LOEC) for 17α-estradiol in fathead minnows is greater than 25 ng/L and may be less than 75 ng/L.

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

BackgroundGerm cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan.Methodology/Principal FindingsUsing two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods.ConclusionTherefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species.

Bcl-2 and Caspase 3 mRNA levels in the testes of gudgeon, Gobio gobio, exposed to ethinylestradiol (EE2)

Apoptosis inhibition has been reported in the male reproductive tract of teleost fish exposed to 17β-estrogen or estrogen-like compounds. In order to understand the molecular mechanisms of cell death inhibition, this study examined 2 genes involved in the apoptotic pathway, Bcl-2 and Caspase 3, an anti-apoptotic and a pro-apoptotic genes, respectively. Partial cDNA sequences of Bcl-2 and Caspase 3 were cloned from gudgeon (Gobio gobio), a common European cyprinid fish. To follow mRNA levels of Bcl-2 and Caspase 3 under xenoestrogen exposure, we first performed an in vitro experiment on fish testis exposed to the most potent xenoestrogen found in the environment, ethinylestradiol (EE2). We further studied mRNA expression of both genes in the testis of fish exposed to xenoestrogens in situ. In the in vitro experiment, fragments of gudgeon testis were exposed for 21 days to 10−3, 10−2, 10−1, 1 and 10μg/L of EE2, as well as to positive (10−1μg/L of E2) and ethanol control medium. Results showed a significant induction of Bcl-2 mRNA at 10−1μg/L (p<0.05). Surprisingly, Caspase 3, a cell death effector, displayed the same profile as observed for the anti-apoptotic gene Bcl-2. In the experiment on wild gudgeon exposed from birth to an estrogenic sewage treatment plant effluent, the mRNA expression of Bcl-2 and Caspase 3 in feminized fish (ovotestis) was not significantly different due to high variability of expression between individuals. At the current state of knowledge on spermatogenesis disruption in teleost fish, in vitro studies seem better adapted than in situ investigations to enlighten the molecular pathway of apoptosis inhibition in testis exposed to xenoestrogens.

Divergent expression of 11β-hydroxysteroid dehydrogenase and 11β-hydroxylase genes between male morphs in the central nervous system, sonic muscle and testis of a vocal fish

The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1 h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman’s two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11β-hydroxylase (11βH) and 11β-hydroxysteroid dehydrogenase (11βHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11βH and 11βHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11βHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology.

17α-ethinylestradiol induces an imbalance between apoptosis and cell proliferation to sex steroid disruption in a testis culture of gudgeon,Gobio gobio

The aim of this study was to investigate the effect of the most potent xenoestrogen currently found in the environment, ethinylestradiol (EE2), on some physiological events occurring during early spermatogenesis of gudgeon (Gobio gobio), a common European fish species. Physiological pathways studies were apoptosis, cell proliferation, and steroidogenesis on sex steroids (testosterone [T], 11-ketotestosterone [11-KT], and 17beta-estradiol [E2]). Testis pieces were cultured in vitro during 21 d at 10(-4), 10(-3), 10(-2), 10(-1), 1 and 10 microg/L of EE2 as well as in positive (10(-1) microg/L of E2) and ethanol control medium. Apoptosis and cell proliferation displayed opposite responses related to the EE2 concentration. When apoptosis inhibition was observed, cell proliferation was induced at 10(-2) and 10(-1) microg/L of EE2 as well as in the positive control. In contrast, a massive cell death was detected for high EE2 concentrations (1 and 10 microg/L). Steroidogenesis was also disrupted in a dose-related manner. 11-Ketotestosterone was depressed at 10(-2) and 10(-1) microg/L of EE2 whereas E2 was detectable in the medium only at 10(-3), 10(-2), and 10(-1) microg/L of EE2. High concentrations of T were detected in the medium at 10(-3), 10(-2), and 10(-1) microg/L of EE2 but depressed at 1 and 10 microg/L of EE2. In conclusion, intermediate EE2 concentrations (10(-2) and 10(-1) microg/L) used in this experimental design have obviously disrupted early spermatogenesis, leading to an imbalance between cell death and cell proliferation in a sex steroid environment toward E2. The results of the present study could be the basis conditions for oocyte development within the testis of a common teleost fish under xenoestrogen exposure.

Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation

In non-mammalian vertebrates, estrogens are key players in ovarian differentiation, but the mechanisms by which they act remain poorly understood. The present study on rainbow trout was designed to investigate whether estrogens trigger the female pathway by activating a group of early female genes (i.e. cyp19a1, foxl2a, foxl2b, fst, bmp4, and fshb) and by repressing early testicular markers (i.e. dmrt1, nr0b1, sox9a1 and sox9a2). Feminization was induced in genetically all-male populations using 17alpha-ethynylestradiol (EE2, 20 mg/kg of food during 2 months). The expression profiles of 100 candidate genes were obtained by real-time RT-PCR and 45 expression profiles displayed a significant differential expression between control populations (males and females) and EE2-treated populations. These expression profiles were grouped in five temporally correlated expression clusters. The estrogen treatment induced most of the early ovarian differentiation genes (foxl2a, foxl2b, fst, bmp4, and fshb) and in particular foxl2a, which was strongly and quickly up-regulated. Simultaneously, Leydig cell genes, involved in androgen synthesis, as well as some Sertoli cell markers (amh, sox9a2) were strongly repressed. However, in contrast to our initial hypothesis, some genes considered as essential for mammalian and fish testis differentiation were not suppressed during the early process of estrogen-induced feminization (dmrt1, nr0b1, sox9a1 and pax2a) and some were even strongly up-regulated (nr0b1, sox9a1and pax2a). In conclusion, estrogens trigger male-to-female transdifferentiation by up-regulating most ovarian specific genes and this up-regulation appears to be crucial for an effective feminization, but estrogens do not concomitantly down-regulate all the testicular differentiation markers.

Expression pattern of prmt5 in adult fish and embryos of medaka, Oryzias latipes

DNA methylation is an important biochemical epigenetic determinant of gene expression in cells and therefore actively involved in gene regulation, chromosomal conformation, and protein activity. Protein arginine methyltransferases (PRMTs) play a major role in the methylation of proteins that have an arginine residue, catalyzing both the asymmetric dimethylation of arginine (aDMA) and symmetric dimethylation of arginine (sDMA). PRMT5, a type II PRMT which catalyzes sDMA, has been shown to have a pivotal role in pole plasm assembly and germ cell development in Drosophila and also to be an associate factor of Blimp1 for germ cell development in mouse. Here, we report a homolog of prmt5 identified in medaka, Oryzias latipes, which was detected in the brain, gill, muscle, heart, liver, spleen, intestine, testis and ovary of adult fish by reverse transcriptase-PCR. The expression of prmt5 in the gonads is restricted to oocytes of the ovary, spermatogonia, and spermatocytes of testis. The prmt5 transcripts were detected as early as the one-cell stage and in all the tissues of embryos during embryogenesis. In summary, prmt5 is a maternal determinant factor of embryogenesis of medaka, possibly playing an important role in oogenesis and spermatogenesis in adult medaka.

A role for matrix metalloproteinases in granulocyte infiltration and testicular remodelation in a seasonal breeding teleost

The testicular cystic structure and the abrupt morphological changes that the fish testis undergoes during the reproductive cycle (RC) make it an interesting model for studying the regulation of spermatogenesis, in particular the role of matrix metalloproteinases (Mmps). The gilthead seabream is a seasonal breeding teleost whose testis undergoes drastic remodeling events, especially during the post-spawning stage when a massive infiltration of a immune cell type, the acidophilic granulocytes, occurred. Bearing this in mind, we studied the gilthead seabream testis gelatinolytic activities involved in migration and tissue remodeling and its regulation by endocrine, immune and tissue stimuli. Thus, we demonstrated that the germinal epithelium of the testis showed gelatinolytic activity during spermatogenesis and post-spawning but not during resting, when only scarce interstitial cells were stained. Moreover, the precursor and mature forms of two gelatinases, Mmp2- and Mmp9-like, were active in the gonad, whose activities were up-regulated by 17β-estradiol (E 2) but not by lipopolysaccharide (LPS)/bacterial DNA (VaDNA) in testicular cell suspensions. E 2 and LPS/VaDNA also up-regulated a variety of cytokines and chemokines. We also cloned mmp9, mmp13, tissue inhibitors of Mmps ( timp)- 2a and timp2b genes and found that all of them were expressed in the gonad in a RC stage-dependent manner. Interestingly, mmps and timps were highly expressed by the testicular acidophilic granulocytes. Moreover, in these cells, the gelatinolytic activity seemed to correspond to the precursor and mature forms of putative Mmp2 and Mmp9 gelatinases, while the main gelatinolytic activity seemed to correspond to the mature form of Mmp2 in head–kidney acidophilic granulocytes. Finally, although none of the stimuli used were able to induce the gelatinolytic activity of Mmp9-like in head–kidney acidophilic granulocytes, the expression of mmp9, timp2a and timp2b were all up-regulated by LPS/VaDNA in these cells, while only mmp9 and timp2a expression increased upon stimulation with gelatin.

The Kisspeptin/Gonadotropin-Releasing Hormone Pathway and Molecular Signaling of Puberty in Fish1

The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration

BackgroundLeukocytes are found within the testis of most, if not all, mammals and are involved in immunological surveillance, physiological regulation and tissue remodelling. The testis of seasonal breeding fish undergoes a regression process. In the present study, the second reproductive cycle (RC) of the protandrous seasonal teleost fish, gilthead seabream, was investigated and the presence of leukocytes analysed. Special attention has been paid to the testicular degenerative process which is particularly active in the last stage of the second RC probably due to the immediacy of the sex change process.MethodsSexually mature specimens (n = 10–18 fish/month) were sampled during the second RC. Some specimens were intraperitoneally injected with bromodeoxyuridin (BrdU) before sampling. Light and electron microscopy was used to determine the different stages of gonadal development and the presence of leukocytes and PCR was used to analyse the gene expression of a testis-differentiating gene and of specific markers for macrophages and B and T lymphocytes. Immunocytochemistry and flow cytometry were performed using a specific antibody against acidophilic granulocytes from the gilthead seabream. Cell proliferation was detected by immunocytochemistry using an anti-BrdU antibody and apoptotic cells by in situ detection of DNA fragmentation.ResultsThe fish in the western Mediterranean area developed as males during the first two RCs. The testis of all the specimens during the second RC underwent a degenerative process, which started at post-spawning and was enhanced during the testicular involution stage, when vitellogenic oocytes appeared in the ovary accompanied by a progressive increase in the ovarian index. However, only 40% of specimens were females in the third RC. Leukocytes (acidophilic granulocytes, macrophages and lymphocytes) were present in the gonad and acidophilic granulocyte infiltration occurred during the last two stages. At the same time DMRT1 gene expression decreased.ConclusionsThe results demonstrate that innate and adaptive immune cells are present in the gonads of gilthead seabream. Moreover, the whole fish population underwent a testicular degenerative process prior to sex change, characterized by high rates of apoptosis and necrosis and accompanied by an infiltration of acidophilic granulocytes and a decrease in DMRT1 levels.

The Effects of Gokshura, Tribulus terrestris on Sex Reversal of Guppy, Poecilia reticulata

This study examined the effects of Tribulus terrestris (TT) on sex reversal in guppy, Poecilia reticulata. The objective of this study was to introduce a new environmentally friendly method for masculinization in P. reticulata. Since male guppy has higher commercial value than female. TT is a natural, non-toxic herb which helps enhance testosterone levels in human and animals. It was prepared in a laboratory in France. Different concentration (0.0, 0.05, 0.1 and 0.15 g L(-1)) of TT was investigated for sex reversal in the Poecilia reticulata. TT extract was administered by immersion of newly born offspring once weekly for two months. Among the dosages used in the present study 0.15 g L(-1) TT was the most effective dosage that ensured maximum male ratio (80%, p < 0.01). Although, sex ratios of 0.05 and 0.1 g L(-1) TT were not significantly different from the expected 1:1 ratio, in these two groups treatment with TT also result in higher number of males (58.25 and 59.77%, respectively), than control (p > 0.05). Total survival rates in all treatments and control were uniformly high ranging from 83 to 87% (p > 0.05). It is concluded that TT has no negative effect on survival rate of P. reticulata. All groups of TT-treated fish exhibited successful growth acceleration comparing to the control group, but only TT treatment at the concentration of 0.15 and 0.1 g L(-1) TT significantly improved growth rate of P. reticulata (p < 0.01). Histological examinations revealed that testes of fish treated with TT-extract contained all stages of spermatogenesis. Sex reversal in P. reticulata demonstrated that TT treated new-born progenies showed successful sex reversal, spermatogenesis and better growth rate than untreated progenies.

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish ( Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.

Feminization of Japanese medaka ( Oryzias latipes) exposed to 17β-estradiol: Formation of testis–ova and sex-transformation during early-ontogeny

Gonad histological changes were examined in Japanese medaka exposed to 17β-estradiol (E2) during early-life stages. Two experiments were conducted at different concentrations of E2 (33.5 and 140.6 ng/L, mean value of measurement) and larvae and juveniles were observed for histological changes in the gonad. Differentiation of ovary and testis in control fish was apparent 12 days post-hatch (dph). At 12 dph, normal testes were observed in male fish that had been exposed to 33.5 ng/L E2, but at 14 and 20 dph, testis–ova was recognized in male fish. Male fish exposed to 140.6 ng/L E2 had testis–ova at 12 dph and gradual transformation to ovary was observed in male fish until 20 dph. In both experiments, the ovarian tissue in testis of male fish exposed to E2 was frequently distributed along the central transverse axis of the gonad, expanding into the transverse axis. The results indicated that 17β-estradiol can induce testis–ova in male medaka during the larval period and sex-transformation is more frequent at higher (140.6 ng/L) than lower concentrations (33.5 ng/L) of estradiol. The results also demonstrated that testis–ova first appear in the central area of the transverse axis of testis.