Upregulation of coagulation factor V by glucocorticoid in the preovulatory follicles of zebrafish.

Increased cortisol levels in the preovulatory follicular fluid suggests a role of glucocorticoid in human ovulation. However, the mechanisms through which cortisol regulates the ovulatory process remain poorly understood. In this study, we examined the upregulation of f5 mRNA by glucocorticoid and its receptor (Gr) in the preovulatory follicles of zebrafish. Our findings demonstrate a significant increase in 11ß-hydroxysteroid dehydrogenase type 2 (hsd11b2), a cortisol response gene, in preovulatory follicles. Additionally, hydrocortisone exerts a dose- and time-dependent upregulation of f5 mRNA in these follicles. Importantly, this stimulatory effect is Gr-dependent, as it was completely abolished in gr-/- mutants. Furthermore, site-directed mutagenesis identified a glucocorticoid response element (GRE) in the promoter of zebrafish f5. Interestingly, successive incubation of hydrocortisone and the native ovulation-inducing steroid, progestin (17α,20ß-dihydroxy-4-pregnen-3-one, DHP), further enhanced f5 expression in preovulatory follicles. Overall, our results indicate that the dramatic increase of f5 expression in preovulatory follicles is partially attributable to the regulation of glucocorticoid and Gr.

Structural and ultrastructural aspects of the skin of large yellow croaker Larimichthys crocea.

The skin color of the large yellow croaker (Larimichthys crocea) is a crucial indicator to determine its economic value. However, the location of pigment cells in the skin structure is uncertain. To determine the pigment cell type in the skin, the vertical order and ultrastructure of pigment cells were examined using light microscopy and transmission electron microscopy. Both dorsal and ventral skins comprise the epidermis, dermis, and hypodermis. Xanthophores, melanophores, and iridophores were observed in the dermis of the dorsal skin, whereas the latter two were in the dermis of the ventral skin. Interestingly, the size of xanthophores in the dorsal skin was significantly smaller than that of xanthophores in the ventral skin; however, the density of dorsal xanthophores was significantly higher than that of ventral xanthophores. The type L-iridophores with large crystalline structures were observed in the uppermost area of the upper pigment layer, which contributed to the strikingly metallic luster shown by the ventral skin. The melanophores were exclusively found in the dorsal skin, offering the purpose of camouflage. Taken together, our results indicated that the pigment cells display different arrangement patterns between dorsal and ventral skin, and the golden color in the ventral skin results from the coexistence of light-reflecting iridophores and light-absorbing xanthophores.

Neurotransmitter norepinephrine regulates chromatosomes aggregation and the formation of blotches in coral trout Plectropomus leopardus.

Color changes and pattern formations can represent strategies of the utmost importance for the survival of individuals or of species. Previous studies have associated capture with the formation of blotches (areas with light color) of coral trout, but the regulatory mechanisms link the two are lacking. Here, we report that capture induced blotches formation within 4-5 seconds. The blotches disappeared after anesthesia dispersed the pigment cells and reappeared after electrical stimulation. Subsequently, combining immunofluorescence, transmission electron microscopy and chemical sympathectomy, we found blotches formation results from activation of catecholaminergic neurons below the pigment layer. Finally, the in vitro incubation and intraperitoneal injection of norepinephrine (NE) induced aggregation of chromatosomes and lightening of body color, respectively, suggesting that NE, a neurotransmitter released by catecholaminergic nerves, mediates blotches formation. Our results demonstrate that acute stress response-induced neuronal activity can drive rapid changes in body color, which enriches our knowledge of physiological adaptations in coral reef fish.

A progestin regulates the prostaglandin pathway in the neuroendocrine system in female mudskipper Boleophthalmus pectinirostris.

Sex hormones regulate the reproductive cycle through brain-pituitary axis, but the molecular mechanism is still enigmatic. In the reproductive season, the mudskipper Boleophthalmus pectinirostris possesses a semilunar periodicity spawning rhythm, which coincides with the semilunar periodicity variations in 17α-hydroxyprogesterone, the precursor of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), a sexual progestin in teleosts. In the present study, we investigated in vitro the brain transcriptional differences between DHP-treated tissues and control groups using RNA-seq. Differential expression analysis revealed that 2700 genes significantly differentially expressed, including 1532 up-regulated and 1168 down-regulated genes. The majority of prostaglandin pathway-related genes were dramatically up-regulated, especially the prostaglandin receptor 6 (ptger6). Tissue distribution analysis revealed that ptger6 gene was ubiquitously expressed. In situ hybridization results showed that ptger6, nuclear progestin receptor (pgr), and DHP-induced c-fos mRNA were co-expressed in the ventral telencephalic area, the ventral nucleus of ventral telencephalic area, the anterior part of parvocellular preoptic nucleus, the magnocellular part of magnocellular preoptic nucleus, the ventral zone of periventricular hypothalamus, the anterior tubercular nucleus, the periventricular nucleus of posterior tuberculum, and the torus longitudinalis. DHP significantly enhanced promoter activities of ptger6 via Pgr. Together, this study suggested that DHP regulates the prostaglandin pathway in the neuroendocrine system of teleost fish.

Cytoarchitecture of Mudskipper (Boleophthalmus pectinirostris) Brain.

Mudskippers are intertidal burrowing fish with unique living habits. So far, studies on the cytoarchitecture of the brain in fish with such behaviors remain limited. Therefore, documenting the neuroanatomy of this animal is of interest because of its unique characteristics. In this study, we examined the cytoarchitecture of mudskipper (Boleophthalmus pectinirostris) brain and investigated whether it has any peculiarities in its brain structures. In general, the basic composition, morphology, and organization of mudskipper brain do not vary markedly from those of other teleosts. The main differences appear in the telencephalon and diencephalon. In addition to Nissl staining, immunostainings for catecholaminergic and cholinergic systems were performed to help identify certain nuclei. The results showed that the number of subdivisions of the central division of pallium, lateral division of pallium, and medial division of pallium were different with other teleost species. In addition, some diencephalic nuclei, including the nucleus subglomerulosus, lateral thalamic nucleus, and intermediate superficial pretectal nucleus, were absent, which suggests the corresponding functions, such as visual or gustatory function, are less developed or specialized in B. pectinirostris. These results will provide a fundamental neuroanatomical basis for future studies on neuroendocrine regulation of behavior in intertidal burrowing fish.

Ultrastructure and regulation of color change in blue spots of leopard coral trout Plectropomus leopardus.

The leopard coral trout generally exhibited numerous round, minute blue spots covering its head (about the size of nostril) and body (except ventral side). This is a characteristic that distinguishes them from similar species. Recently, however, we found the leopard coral trout with black spots. Here, the distribution and ultrastructure of chromatophores in the blue and black spots were investigated with light and transmission electron microscopies. The results showed that in the blue spots, two types of chromatophores are present in the dermis, with the light-reflecting iridophores located in the upper layer and the aggregated light-absorbing melanophores in the lower layer. Black spots have a similar chromatophore composition, except that the melanosomes within the melanophores disperse their dendritic processes to encircle the iridophores. Interestingly, after the treatment of forskolin, a potent adenylate cyclase activator, the blue spots on the body surface turned black. On the other hand, using the skin preparations in vitro, the electrical stimulation and norepinephrine treatment returned the spots to blue color again, indicating the sympathetic nerves were involved in regulating the coloration of blue spots. Taken together, our results revealed that the blue spots of the leopard coral trout can change color to black and vice versa, resulting from the differences in the distribution of melanosomes, which enriches our understanding of the body color and color changes of fishes.

Nuclear Progestin Receptor-mediated Linkage of Blood Coagulation and Ovulation.

Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, ~3145-fold) and tissue factor (f3a, ~120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wild-type preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting-triggered ovulation in vertebrates.

Robust host source tracking building on the divergent and non-stochastic assembly of gut microbiomes in wild and farmed large yellow croaker.

BACKGROUND: Given the lack of genetic background, the source tracking unknown individuals of fish species with both farmed and wild populations often cannot be robustly achieved. The gut microbiome, which is shaped by both deterministic and stochastic processes, can serve as a molecular marker of fish host source tracking, particularly as an alternative to the yet-to-be-established host genetic marker. A candidate for testing the feasibility is the large yellow croaker, Larimichthys crocea, which is carnivorous and ranks the top mariculture fish in China. Wild resource of this fish was depleted decades ago and might have potential problematic estimation because of escaping of farmed individuals. RESULTS: The rectums of wild (n = 212) and farmed (n = 79) croakers from multiple batches were collected for the profiling of their gut bacterial communities. The farmed individuals had a higher alpha diversity and lower bacterial load than the wild individuals. The gut microbiota of the two sources exhibited divergence and high inter-batch variation, as featured by the dominance of Psychrobacter spp. in the wild group. Predicted functional capacity of the gut microbiome and representative isolates showed differences in terms of host source. This difference can be linked to the potential diet divergence between farmed and wild fishes. The non-stochastic distribution pattern of the core gut microbiota of the wild and farmed individuals supports the feasibility of microbiota-based host source tracking via the machine learning algorithm. A random forest classifier based on the divergence and non-stochastic assembly of the gut microbiome was robust in terms of host source tracking the individuals from all batches of croaker, including a newly introduced batch. CONCLUSIONS: Our study revealed the divergence of gut microbiota and related functional profiles between wild and farmed croakers. For the first time, with representative datasets and non-stochastic patterns, we have verified that gut microbiota can be robustly applied to the tracking of host source even in carnivorous fish. Video abstract.

Expressions of melanopsins in telencephalon imply their function in synchronizing semilunar spawning rhythm in the mudskipper Boleophthalmus pectinirostris.

The mudskipper Boleophthalmus pectinirostris inhabits intertidal mudflats, exhibiting semilunar reproductive rhythms. To investigate whether melanopsin is possibly involved in the synchronization of the semilunar spawning rhythm in the female mudskipper, we first cloned all four melanopsin subtypes (opn4m1, opn4m3, opn4x1, opn4x2) in B. pectinirostris. Results from RTq-PCR showed that significantly higher transcription levels of all four melanopsin subtypes were observed in the eyes rather than other tissues. In brain, all four melanopsin subtypes were also detectable in different regions, including the telencephalon, in which the expression of melanopsin has not been reported in other teleosts. The transcription levels of opn4m3 and opn4x1 in the telencephalon exhibited a daily fluctuation pattern. When females entered the spawning season, opn4m1 and opn4x1 transcript levels increased significantly in the telencephalon. During the spawning season, the transcript levels of opn4m3 and opn4x1 in the telencephalon appeared to have a cyclic pattern associated with semilunar periodicity, exhibiting two cycles with a peak around the first or the last lunar quarters. Results from ISH showed that, opn4x1 mRNA was localized in the medial of dorsal telencephalic area, dorsal nucleus of ventral telencephalic area (Vd), ventral nucleus of ventral telencephalic area (Vv), anterior part of parvocellular preoptic nucleus, magnocellular part of the magnocellular preoptic nucleus (PMmc), habenular and ventral zone of hypothalamus. Intriguingly, gnrh3 mRNA was also located in Vd, Vv and PMmc. Taken together, our results suggested that melanopsins, e.g. opn4x1, expressed in the telencephalon might mediate semilunar spawning activity in the female mudskipper.

GFP expression pattern in pituitary and gonads under the control of nuclear progesterone receptor promoter in transgenic zebrafish.

BACKGROUND: The nuclear progesterone receptor (Pgr) is a ligand-dependent transcription factor primarily responsible for mediating progesterone actions relevant for reproduction across vertebrates. Information on the cellular localization of Pgr expression in the reproductive system is required for developing a comprehensive approach to elucidate the role of Pgr in reproduction. RESULTS: We generated transgenic zebrafish Tg(pgr:eGFP) that express enhanced green fluorescent protein (eGFP) driven by promoter sequence of pgr gene. The tissue distribution pattern of egfp mRNA is consistent with the pgr mRNA expression in Tg(pgr:eGFP). In the pituitary, GFP signals are found in the proximal pars distalis. In order to better discern the cellular localization of GFP signals in gonads, Tg(pgr:eGFP) line was crossed with Tg(gsdf:nfsB-mCherry) line, specifically expressing nitroreductase-mCherry fusion protein in granulosa and Sertoli cells in ovary and testis, respectively. Imaging of testis tissue showed that GFP expression was confined to Leydig cells. In the ovary, GFP expression colocalized with the mCherry signal in granulosa cells. Intriguingly, we also identified some non-granulosa cells close to where blood vessels branched, expressing stronger GFP signals than granulosa cells. CONCLUSIONS: Analyzing Tg(pgr:eGFP) expression in zebrafish provided leads toward new routes to study the role of Pgr in reproduction.

Upregulation of adamts9 by gonadotropin in preovulatory follicles of zebrafish.

Previously we had identified adamts9 as a downstream target of Pgr, which is essential for ovulation in zebrafish. The primary goal of this study is to determine whether human chorionic gonadotropin (hCG, LH analog) also regulate adamts9 expression prior to ovulation. The expression of adamts9 was induced by hCG in a dose and time dependent manner in zebrafish preovulatory follicles in vitro. Interestingly, the stimulatory effect of hCG on adamts9 expression was not blocked in pgr-/- follicles but blocked in lhcgr-/-. This effect of hCG was via Lhcgr and its associated cAMP and PKC signaling pathways. Reduced fecundity and reduced expression of adamts9 were also found in lhcgr-/- females in vivo. Therefore, we have provided the first evidence of gonadotropin (hCG) regulated adamts9 in zebrafish, which could be important for ovulation.

Paralogues From the Expanded Tlr11 Gene Family in Mudskipper (Boleophthalmus pectinirostris) Are Under Positive Selection and Respond Differently to LPS/Poly(I:C) Challenge.

Toll-like receptors (TLRs) are major molecular pattern recognition receptors, which are essential for triggering a series of innate immune responses against invading pathogens by recognizing their evolutionary conserved molecular patterns. The mudskipper, Boleophthalmus pectinirostris is exceptional among fishes due to its amphibious lifestyle and adaptation to living on mudflats. The whole-genome sequencing of B. pectinirostris has revealed that this species possesses an expansion of Tlr11 family [12 Tlr11 family genes (one tlr21, 4 tlr22, and 7 tlr23)] that we focused on in the present study. The full-length cDNA sequences of the 12 tlrs in B. pectinirostris were cloned and their deduced amino acid sequences possessed a typical TLR domain arrangement. Likelihood tests of selection revealed that these 12 Tlr11 family genes are under diversifying selection. A total of 13 sites were found to be positively selected by more than one evolution model, of which 11 were located in the ligand-binding ectodomain. The observed non-synonymous substitutions may have functional implications in antigen and pathogen recognition specificity. These 12 tlrs were highly expressed in immune-related tissues, i.e. spleen and kidney. Tlr21 and tlr22b transcripts were significantly up-regulated by LPS, whereas tlr22a, tlr22d, tlr23b, tlr23e, tlr23g were significantly up-regulated by poly(I:C) in the spleen or/and kidney, which implies that the expanded Tlr11 family genes may play roles in protecting the fish from the invasion of gram-negative bacteria and double-stranded RNA viruses. The results from the present study suggested that the expansion of Tlr11 family genes in B. pectinirostris may recognize ligands from various pathogens found in the intertidal zone.

Androgen induces olfactory expression of prostaglandin E2 receptor Ep1 in the burrow-living fish Bostrychus sinensis.

It is well documented that androgens modify olfactory processing in vertebrates. In fish, several lines of evidence indicate that androgens increase olfactory sensitivity to prostaglandin pheromone, but the molecular mechanism is still unclear. Our previous studies showed that prostaglandin E2 (PGE2) is a sex pheromone in the burrowing-living fish Chinese black sleeper (Bostrychus sinensis) and that the PGE2 receptor 1 (Ep1) in the olfactory rosette is a candidate receptor for sensing sex pheromone PGE2. In the present study, we found that testosterone (T) and 11-ketotestosterone (11-KT) exhibited stimulatory effects on the expression of ep1 in the olfactory rosette in vivo and ex vivo. Moreover, the androgen receptor (Ar) agonist R1881 had similar effects to 11-KT on the expression of ep1 ex vivo, suggesting the up-regulatory effect is mediated by Ar. The amount of arα transcripts (˜1500 copies/100 ng total RNA) was greater than that of arß (˜300 copies/100 ng total RNA) in the olfactory rosette, and the expression levels of arα increased with spermatogenesis and peaked at late meiosis stage. Moreover, activated Arα but not Arß transactivated a 2k bp ep1 promoter in HEK293T cell, and some OSNs exhibited co-localization of arα mRNA and Ep1 protein signals. Taken together, our results suggest that Arα, but not Arß, plays a crucial role in mediating the androgen-induced up-regulation of ep1 expression in B. sinensis. The present study is the first to shed light on the molecular mechanisms whereby androgens enhance responsiveness to prostaglandin sex pheromones in teleosts.

Progestin and Nuclear Progestin Receptor Are Essential for Upregulation of Metalloproteinase in Zebrafish Preovulatory Follicles.

Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr -/-) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and adamts1 in preovulatory follicles at different times within the spawning cycle in pgr -/- and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr -/-, whereas expression of adamts1 was not affected in pgr -/- zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr -/-. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20ß-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.

Androstenedione and 17α-methyltestosterone induce early ovary development of Anguilla japonica.

Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d-1 body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 µg L-1 AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhß increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhß expression.

Involvement of Membrane Progestin Receptor Beta (mPRß/Paqr8) in Sex Pheromone Progestin-Induced Expression of Luteinizing Hormone in the Pituitary of Male Chinese Black Sleeper (Bostrychus Sinensis).

Our previous studies showed that 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) acted as a sex pheromone to induce reproductive success in Chinese black sleeper (Bostrychus sinensis), but its functional mechanism remains unclear. In the present study, we cloned the cDNAs of the gonadotropin subunits (cgα, fshß, and lhß), and found that, in exposure to 5 nM DHP, transcript levels of lhß significantly increased in the pituitary at 6 h post exposure; plasma 11-KT levels increased at 24 h post exposure in mature male fish. In contrast, DHP exposure failed to increase the transcript levels of lhß in the pituitary of immature male fish, suggesting that the responsiveness to DHP depends on reproductive status. Interestingly, expression of progestin and adipoQ receptor 8 (paqr8, also known as mPRß) and progesterone receptor membrane component 2 significantly increased in the olfactory rosette of male fish at late meiosis stage following a co-injection of human chorionic gonadotropin (HCG) and luteinizing hormone releasing hormone-A3 (LHRH-A3), while no increases of other progestin receptors were observed. Moreover, Paqr8 protein was localized in the dendritic knobs of the olfactory sensory neurons, which were activated following the in vivo exposure to DHP. The DHP-induced expression of lhß in pituitary was not inhibited by RU486, an antagonist of nuclear progesterone receptor. Taken together, our results suggested that sex pheromone DHP increased the expression of lhß transcript in the pituitary and plasma 11-KT levels of mature male, important for reproduction; and Paqr8 might be involved in responding to sex pheromone DHP in the olfactory rosette of male B. sinensis.

The complete mitochondrial genome of the palette surgeonfish, Paracanthurus hepatus (Perciformes: Acanthuridae).

In this study, the complete mitogenome sequence of the palette surgeonfish, Paracanthurus hepatus (Perciformes: Acanthuridae), has been sequenced by next-generation sequencing method. The assembled mitogenome was 16 498 bp in length, consisted of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes. The overall base composition of palette surgeonfish was 28.6% for A, 28.6% for C, 16.3% for G, 26.4% for T and showed 87% identities to somber surgeonfish Zebrasoma flavescens. The complete mitogenome of the palette surgeonfish provides essential and important DNA molecular data for further phylogeography and evolutionary analysis for surgeonfish's phylogeny.

The complete mitochondrial genome of the Royal dottyback Pictichromis paccagnellae (Perciformes: Pseudochromidae).

In this study, the complete mitogenome sequence of the Royal dottyback, Pictichromis paccagnellae (Perciformes: Pseudochromidae), has been sequenced by the next-generation sequencing method. The assembled mitogenome was of 16 976 bp in length, consisting of 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs genes. The overall base composition was 27.4% for A, 28.1% for C, 17.3% for G, and 27.2% for T and showed 76% identities to Fire-tail devil Labracinus cyclophthalmus in the same family. The complete mitogenome of P. paccagnellae provides essential and important DNA molecular data for further phylogeography and evolutionary analysis for Pseudochromidae.

Silver nanoparticles induce oocyte maturation in zebrafish (Danio rerio).

Public concern regarding silver nanoparticles (AgNPs) in the environment has been increasing since they can cause adverse effects in some aquatic species. However, few data are actually available on the effects of AgNPs on the germ cells. In the present study, we used the zebrafish ovarian follicle as a model to assess the potentially adverse effects of AgNPs on oocyte maturation (germinal vesicle breakdown, GVBD) in vitro. Similar to the maturation inducing hormone (17α, 20ß-dihydroxy-4-pregnen-3-one), AgNPs induced GVBD, and reduced the total cyclic adenosine monophosphate (cAMP) concentration in zebrafish ovarian follicles. The results from transmission electron microscope observation and Hoechst 33342 staining clearly indicated that AgNPs induced apoptosis in ovarian follicle cells surrounding the oocyte. Similar to AgNPs, AgNO3 also induced GVBD, decreased cAMP concentration and induced apoptosis of ovarian follicle cells. However, the results from gene expression analysis showed that transcript levels of oxidative stress related genes were more sensitive to AgNPs than AgNO3. Further more, H2O2 has an ability to induce zebrafish oocytes maturation by induction of apoptosis in ovarian follicle cells. Taken together, the results from our study indicated that oxidative stress appeared to be one of important mechanisms in AgNP induced apoptosis in ovarian follicle cells, which further triggered the GVBD.

Cloning and expression of two hepcidin genes in the mudskipper (Boleophthalmus pectinirostris) provides insights into their roles in male reproductive immunity.

The mudskipper Boleophthalmus pectinirostris is a burrow-dwelling fish inhabiting intertidal mudflats. During the spawning season, in a spawning chamber located at the center of their burrow, a pair of male and female fish mate and fertilized eggs adheres onto the inner walls and ceiling with filamentous attachments. During 5 days of incubation, the fertilized eggs are kept clean and hatch with a very high hatching rate under the natural conditions filled with microorganisms. This suggests that the male and/or female reproductive tract may synthesize antimicrobial substances to offer protection against microorganisms that may be deleterious to fertility. To study the antimicrobial strategy of this fish in the spawning season, we first cloned the two hepcidin isoforms from B. pectinirostris, and designated them as Hepcidin-1 and Hepcidin-2 based on phylogenetic analyses. Both of these hepcidin isoforms were highly expressed in the liver, but only Hepcidin-1 showed significant change in response to iron overload. Interestingly, these two hepcidin isoforms were expressed in male reproductive tracts, i.e. the testes and seminal vesicles. The monthly expression pattern indicated that Hepcidin-1 transcript levels showed a peak point only in March (before spawning) in the seminal vesicle, while Hepcidin-2 transcript levels were correlated with male reproductive status and reached their highest level in May (the peak spawning period). Under experimental conditions, the expression of these two hepcidin isoforms showed no response to iron overload in the male gonad. However, after lipopolysaccharide injection, the Hepcidin-1 transcript level was significantly up-regulated in the testes and seminal vesicle 6 h post injection, while Hepcidin-2 transcript levels exhibited a clear time-course dependent upregulation pattern and reached the highest levels 24 h post injection. More interestingly, after injection with LHRH-A3, the expression of Hepcidin-2 was significantly up-regulated in both testes and seminal vesicle. Results from in situ hybridization showed that Hepcidin-2 was expressed in the Leydig cells of the testes and in the epithelium of the seminal vesicle. Taken together, the results from our study indicated that these two hepcidin isoforms in the mudskipper may have different functions: Hepcidin-1 may play a dual role in both iron metabolism regulation in the liver and a short antimicrobial response in male reproductive tracts, while Hepcidin-2 is more specialized in reproductive immunity in male reproductive tracts.