The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

The effect of gonadal hormones on the gene expression of brain-pituitary in protandrous black porgy, Acanthopagrus schlegelii.

In black porgy (Acanthopagrus schlegelii), the brain-pituitary-testis (Gnrh-Gths-Dmrt1) axis plays a vital role in male fate determination and maintenance, and then inhibiting female development in further (puberty). However, the feedback of gonadal hormones on regulating brain signaling remains unclear. In this study, we conducted short-term sex steroid treatment and surgery of gonadectomy to evaluate the feedback regulation between the gonads and the brain. The qPCR results show that male phase had the highest gths transcripts; treatment with estradiol-17ß (E2) or 17α-methyltestosterone (MT) resulted in the increased pituitary lhb transcripts. After surgery, apart from gnrh1, there is no difference in brain signaling genes between gonadectomy and sham fish. In the diencephalon/mesencephalon transcriptome, de novo assembly generated 283,528 unigenes; however, only 443 (0.16%) genes showed differentially expressed between sham and gonadectomy fish. In the present study, we found that exogenous sex steroids affect the gths transcription; this feedback control is related to the gonadal stage. Furthermore, gonadectomy may not affect gene expression of brain signaling (Gnrh-Gths axis). Our results support the communication between ovotestis and brain signaling (Gnrh-Gths-testicular Dmrt1) for the male fate.

Cellular mechanisms underlying extraordinary sulfide tolerance in a crustacean holobiont from hydrothermal vents.

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulfide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulfide-rich vent systems. Despite the toxicity of hydrogen sulfide, X. testudinatus occupies an ecological niche in a sulfide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulfide detoxification. Here sulfide is oxidized to thiosulfate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulfate and hypotaurine is mediated by the sodium-independent sulfate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulfide-oxidizing endo-symbiotic bacteria, creating an effective sulfide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet.

Effects of Osmotic Stress on the mRNA Expression of prl, prlr, gr, gh, and ghr in the Pituitary and Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii.

In euryhaline teleost black porgy, Acanthopagrus schlegelii, the glucocorticoid receptor (gr), growth hormone receptor (ghr), prolactin (prl)-receptor (prlr), and sodium-potassium ATPase alpha subunit (α-nka) play essential physiological roles in the osmoregulatory organs, including the gill, kidney, and intestine, during osmotic stress. The present study aimed to investigate the impact of pituitary hormones and hormone receptors in the osmoregulatory organs during the transfer from freshwater (FW) to 4 ppt and seawater (SW) and vice versa in black porgy. Quantitative real-time PCR (Q-PCR) was carried out to analyze the transcript levels during salinity and osmoregulatory stress. Increased salinity resulted in decreased transcripts of prl in the pituitary, α-nka and prlr in the gill, and α-nka and prlr in the kidney. Increased salinity caused the increased transcripts of gr in the gill and α-nka in the intestine. Decreased salinity resulted in increased pituitary prl, and increases in α-nka and prlr in the gill, and α-nka, prlr, and ghr in the kidney. Taken together, the present results highlight the involvement of prl, prlr, gh, and ghr in the osmoregulation and osmotic stress in the osmoregulatory organs (gill, intestine, and kidney). Pituitary prl, and gill and intestine prlr are consistently downregulated during the increased salinity stress and vice versa. It is suggested that prl plays a more significant role in osmoregulation than gh in the euryhaline black porgy. Furthermore, the present results highlighted that the gill gr transcript's role was solely to balance the homeostasis in the black porgy during salinity stress.

Localization of the Neuropeptide Arginine Vasotocin and Its Receptor in the Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii: Gills, Kidneys, and Intestines.

The neurohypophysial hormone arginine vasotocin (avt) and its receptor (avtr) regulates ions in the osmoregulatory organs of euryhaline black porgy (Acanthopagrus schlegelii). The localization of avt and avtr transcripts in the osmoregulatory organs has yet to be demonstrated. Thus, in the present study, we performed an in situ hybridization analysis to determine the localization of avt and avtr in the gills, kidneys, and intestines of the black porgy. The avt and avtr transcripts were identified in the filament and lamellae region of the gills in the black porgy. However, the basal membrane of the filament contained more avt and avtr transcripts. Fluorescence double tagging analysis revealed that avt and avtr mRNAs were partially co-localized with α-Nka-ir cells in the gill filament. The proximal tubules, distal tubules, and collecting duct of the kidney all had positive hybridization signals for the avt and avtr transcripts. Unlike the α-Nka immunoreactive cells, the avt and avtr transcripts were found on the basolateral surface of the distal convoluted tubule and in the entire cells of the proximal convoluted tubules of the black porgy kidney. In the intestine, the avt and avtr transcripts were found in the basolateral membrane of the enterocytes. Collectively, this study provides a summary of evidence suggesting that the neuropeptides avt and avtr with α-Nka-ir cells may have functions in the gills, kidneys, and intestines via ionocytes.

Expression and Transcript Localization of star, sf-1, and dax-1 in the Early Brain of the Orange-Spotted Grouper Epinephelus coioides.

We investigated the developmental expression and localization of sf-1 and dax-1 transcripts in the brain of the juvenile orange-spotted grouper in response to steroidogenic enzyme gene at various developmental ages in relation to gonadal sex differentiation. The sf-1 transcripts were significantly higher from 110-dah (day after hatching) and gradually increased up to 150-dah. The dax-1 mRNA, on the other hand, showed a decreased expression during this period, in contrast to sf-1 expression. At the same time, the early brain had increased levels of steroidogenic gene (star). sf-1 and star hybridization signals were found to be increased in the ventromedial hypothalamus at 110-dah; however, dax-1 mRNA signals decreased in the early brain toward 150-dah. Furthermore, the exogenous estradiol upregulated star and sf-1 transcripts in the early brain of the grouper. These findings suggest that sf-1 and dax-1 may have an antagonistic expression pattern in the early brain during gonadal sex differentiation. Increased expression of steroidogenic gene together with sf-1 during gonadal differentiation strongly suggests that sf-1 may play an important role in the juvenile grouper brain steroidogenesis and brain development.

Potential role of DNA methylation of cyp19a1a promoter during sex change in protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen has a pivotal role in early female differentiation and further ovarian development. Aromatase (Cyp19a) is responsible for the conversion of androgens to estrogens in vertebrates. In teleosts, cyp19a1a and it paralog cyp19a1b are mainly expressed in the ovary and hypothalamus, respectively. Decreased plasma estrogen levels and lower cyp19a1a expression are associated with the initiation of female-to-male sex change in protogynous grouper. However, an 17α-methyltestosterone (MT)-induced the sex change from a female to a precocious male is a transient phase, and a reversible sex change (induced male-to-female) occurs after chemical withdrawal. Thus, we used this characteristic to study the epigenetic modification of cyp19a1a promoter in orange-spotted grouper. CpG-rich region with a CpG island is located on the putative regulatory region of distal cyp19a1a promoter. Our results showed that cyp19a1a promoter exhibited tissue-specific methylation status. Low methylation levels of distal cyp19a1a promoter and hypomethylated (0-40%) clones of cyp19a1a promoter region were widely observed in the ovary but not shown in testis and other tissues. In femaleness, higher numbers of hypomethylated clones of cyp19a1a promoter region were observed in the vitellogenic oocyte stage compared to the primary oocyte stage. Furthermore, decreased numbers of hypomethylated clones of cyp19a1a promoter region were associated with the maleness during the female-to-male sex change. DNA methylation inhibitor (5-aza-2'-deoxycytidine) delayed the spermatogenesis process (according to germ cell stage and numbers: by decrease of sperm and increase of spermatocytes) but did not influence the reversed sex change in MT-induced bi-directional sex change. These results suggest that epigenetic modification of cyp19a1a promoter may play an important role during the sex change in orange-spotted grouper.

Involvement of RFamide neuropeptides in polyp contraction of the adult scleractinian corals Euphyllia ancora and Stylophora pistillata.

The distribution and functions of neurons in scleractinian corals remain largely unknown. This study focused on the Arg-Phe amide family of neuropeptides (RFamides), which have been shown to be involved in a variety of biological processes in animals, and performed molecular identification and characterization in the adult scleractinian coral Euphyllia ancora. The deduced amino acid sequence of the identified RFamide preprohormone was predicted to contain 20 potential neuropeptides, including 1 Pro-Gly-Arg-Phe (PGRF) amide and 15 Gln-Gly-Arg-Phe (QGRF) amide peptides. Tissue distribution analysis showed that the level of transcripts in the tentacles was significantly higher than that in other polyp tissues. Immunohistochemical analysis with the FMRFamide antibody showed that RFamide neurons were mainly distributed in the epidermis of the tentacles and mouth with pharynx. Treatment of E. ancora polyps with synthetic QGRFamide peptides induced polyp contraction. The induction of polyp contraction by QGRFamide peptide treatment was also observed in another scleractinian coral, Stylophora pistillata. These results strongly suggested that RFamides play a role in the regulation of polyp contraction in adult scleractinians.

De novo transcriptome assembly from the gonads of a scleractinian coral, Euphyllia ancora: molecular mechanisms underlying scleractinian gametogenesis.

BACKGROUND: Sexual reproduction of scleractinians has captured the attention of researchers and the general public for decades. Although extensive ecological data has been acquired, underlying molecular and cellular mechanisms remain largely unknown. In this study, to better understand mechanisms underlying gametogenesis, we isolated ovaries and testes at different developmental phases from a gonochoric coral, Euphyllia ancora, and adopted a transcriptomic approach to reveal sex- and phase-specific gene expression profiles. In particular, we explored genes associated with oocyte development and maturation, spermiogenesis, sperm motility / capacitation, and fertilization. RESULTS: 1.6 billion raw reads were obtained from 24 gonadal samples. De novo assembly of trimmed reads, and elimination of contigs derived from symbiotic dinoflagellates (Symbiodiniaceae) and other organisms yielded a reference E. ancora gonadal transcriptome of 35,802 contigs. Analysis of 4 developmental phases identified 2023 genes that were differentially expressed during oogenesis and 678 during spermatogenesis. In premature/mature ovaries, 631 genes were specifically upregulated, with 538 in mature testes. Upregulated genes included those involved in gametogenesis, gamete maturation, sperm motility / capacitation, and fertilization in other metazoans, including humans. Meanwhile, a large number of genes without homology to sequences in the SWISS-PROT database were also observed among upregulated genes in premature / mature ovaries and mature testes. CONCLUSIONS: Our findings show that scleractinian gametogenesis shares many molecular characteristics with that of other metazoans, but it also possesses unique characteristics developed during cnidarian and/or scleractinian evolution. To the best of our knowledge, this study is the first to create a gonadal transcriptome assembly from any scleractinian. This study and associated datasets provide a foundation for future studies regarding gametogenesis and differences between male and female colonies from molecular and cellular perspectives. Furthermore, our transcriptome assembly will be a useful reference for future development of sex-specific and/or stage-specific germ cell markers that can be used in coral aquaculture and ecological studies.

Apoptosis in gonadal somatic cells of scleractinian corals: implications of structural adjustments for gamete production and release.

Apoptosis is an evolutionarily conserved process of programmed cell death. Here, we show structural changes in the gonads caused by apoptosis during gametogenesis in the scleractinian coral, Euphyllia ancora. Anatomical and histological analyses revealed that from the non-spawning to the spawning season, testes and ovaries increased in size due to active proliferation, differentiation and development of germ cells. Additionally, the thickness and cell density of the gonadal somatic layer decreased significantly as the spawning season approached. Further analyses demonstrated that the changes in the gonadal somatic layer were caused by apoptosis in a subpopulation of gonadal somatic cells. The occurrence of apoptosis in the gonadal somatic layer was also confirmed in other scleractinian corals. Our findings suggest that decreases in thickness and cell density of the gonadal somatic layer are structural adjustments facilitating oocyte and spermary (male germ cell cluster) enlargement and subsequent gamete release from the gonads. In animal reproduction, apoptosis in germ cells is an important process that controls the number and quality of gametes. However, apoptosis in gonadal somatic cells has rarely been reported among metazoans. Thus, our data provide evidence for a unique use of apoptosis in animal reproduction.

The plasticity of gonad development of sexual reproduction in a scleractinian coral, Porites lichen.

Metazoans have evolved a complexity of sexual system and gonad development, however, sexual reproduction of scleractinian corals is not well understood. This study aimed to address the sexual system and gametogenesis in Porites lichen, a common species in the Indo-West Pacific. This study represents the first description of sexual system, which were determined by histological analysis of the samples collected in northern Taiwan. In addition, female and hermaphroditic colonies were separately cultured in aquarium to further monitor the release of eggs/larvae and thereby confirm the breeding system. The results demonstrate that P. lichen is a polygamodioecious brooder and displays seasonal gametogenesis and embryogenesis that ends in late summer. In hermaphroditic colonies, male polyps are predominant and hermaphroditic polyps make up a very small percent (1%-19.3%). In addition, two new gametogenic features were observed from the histological analysis: 1) oocytes developed within the spermaries in hermaphroditic polyps during the early stage of gametogenesis and 2) melanin granular cells were clustered in spermaries in both male and hermaphroditic colonies. This study demonstrated the plasticity of gametogenesis and melanin related cells appeared in corals, which provides an important information to explore hormones and molecular mechanism involving in gonadal arrangement and production of melanin for further studies.

Elevated estradiol-17ß levels inhibit final oocyte maturation via G protein-coupled estrogen receptor (Gper) in yellowfin porgy, Acanthopagrus latus.

Yellowfin porgy a protandrous teleost, exhibits asynchronous oocyte development and multiple spawning. Seasonal profiles of plasma estradiol-17ß (E2) levels showed a peak in three-year-old females during the spawning season, when batches of fully-grown oocytes undergo final oocyte maturation (FOM). Because E2 has been shown to inhibit FOM via the G protein-coupled estrogen receptor (Gper) in several teleost species, we investigated the role of this "paradoxical" increase in E2 during FOM in yellowfin porgy. In vivo treatment with a GnRH-agonist stimulated germinal vesicle breakdown (GVBD) and increased E2 plasma levels, and ovarian cyp19a1a transcripts, confirming the increase in E2 production at the time of FOM. Ovarian transcripts of gper peaked at the time of FOM, indicating an increase in ovarian responsiveness to Gper-mediated E2 effects. In vitro, E2 and the Gper agonist, G-1, inhibited the stimulatory effect of maturation-inducing steroids (MIS) on GVBD, while an aromatase inhibitor enhanced the MIS effect, in agreement with a physiological inhibitory role of E2 on FOM via Gper. Immunohistological studies showed that the Gper protein was specifically located on the oocyte plasma membrane. Ovarian membranes displayed high-affinity and limited-capacity specific [3H]-E2 receptor binding which was displaced by G-1, characteristic of Gper. Expression of gper increased at the time of FOM in mid-vitellogenic oocytes, but not in larger oocytes undergoing GVBD. These results suggest increases in both E2 production and E2 responsiveness via Gper upregulation in mid-vitellogenic oocytes, may maintain meiotic arrest in this oocyte stage class during the period when full-grown oocytes are undergoing FOM. This study indicates a critical involvement of E2 in the control of asynchronous oocyte maturation and the multiple spawning pattern in Sparidae.

Basal teleosts provide new insights into the evolutionary history of teleost-duplicated aromatase.

Duplicated cyp19a1 genes (cyp19a1a encoding aromatase a and cyp19a1b encoding aromatase b) have been identified in an increasing number of teleost species. Cyp19a1a is mainly expressed in the gonads, while cyp19a1b is mainly expressed in the brain, specifically in radial glial cells, as largely investigated by Kah and collaborators. The third round of whole-genome duplication that specifically occurred in the teleost lineage (TWGD or 3R) is likely at the origin of the duplicated cyp19a1 paralogs. In contrast to the situation in other teleosts, our previous studies identified a single cyp19a1 in eels (Anguilla), which are representative species of a basal group of teleosts, Elopomorpha. In the present study, using genome data mining and phylogenetic and synteny analyses, we confirmed that the whole aromatase genomic region was duplicated in eels, with most aromatase-neighboring genes being conserved in duplicate in eels, as in other teleosts. These findings suggest that specific gene loss of one of the 3R-duplicated cyp19a1 paralogs occurred in Elopomorpha after TWGD. Similarly, a single cyp19a1 gene was found in the arowana, which is a representative species of another basal group of teleosts, Osteoglossomorpha. In eels, the single cyp19a1 is expressed in both the brain and the gonads, as observed for the single CYP19A1 gene present in other vertebrates. The results of phylogenetic, synteny, closest neighboring gene, and promoter structure analyses showed that the single cyp19a1 of the basal teleosts shared conserved properties with both teleost cyp19a1a and cyp19a1b paralogs, which did not allow us to conclude which of the 3R-duplicated paralogs (cyp19a1a or cyp19a1b) was lost in Elopomorpha. Elopomorpha and Osteoglossomorpha cyp19a1 genes exhibited preserved ancestral functions, including expression in both the gonad and brain. We propose that the subfunctionalization of the 3R-duplicated cyp19a1 paralogs expressed specifically in the gonad or brain occurred in Clupeocephala, after the split of Clupeocephala from Elopomorpha and Osteoglossomorpha, which represented a driving force for the conservation of both 3R-duplicated paralogs in all extant Clupeocephala. In contrast, the functional redundancy of the undifferentiated 3R-duplicated cyp19a1 paralogs in elopomorphs and osteoglossomorphs would have favored the loss of one 3R paralog in basal teleosts.

Resveratrol Induces Expression of Metabolic and Antioxidant Machinery and Protects Tilapia under Cold Stress.

Exposures to low ambient temperature require ectothermic fish to not only adjust their metabolic machinery but also to mount protective responses against oxidative stress. In this study, we tested whether diets supplemented with resveratrol (RSV), a naturally occurring polyphenol known to stimulate metabolic and protective responses in various animals, would be beneficial to tilapia (Oreochromis mossambicus) under hypothermic challenge. Feeding tilapia with RSV-supplemented diet promoted liver expression of sirtuins and their known targets, including metabolic/antioxidative enzymes. After exposure to 15 °C cold conditions for three days, the oxygen-nitrogen (O:N) ratio was decreased in the control-diet-fed tilapia but not in their RSV-fed counterparts. Moreover, at 27 °C, RSV-fed tilapia showed significantly higher prolonged swim speed compared with controls. RSV feeding produced no significant effect on upper and bottom layer preference between the control- and RSV-treated tilapia at either 27 °C or 15 °C. Together, these findings suggest that RSV stimulates beneficial metabolic/antioxidative adjustments in teleosts and may serve as a valuable feed supplement for tropical fish exposed to cold stress during winter.

Testicular somatic cells in the stony coral Euphyllia ancora express an endogenous green fluorescent protein.

In a variety of organisms, adult gonads contain several specialized somatic cells that regulate and support the development of germline cells. In stony corals, the characteristics and functions of gonadal somatic cells remain largely unknown. No molecular markers are currently available that allow for the identification and enrichment of gonadal somatic cells in corals. Here, we showed that the testicular somatic cells of a stony coral, Euphyllia ancora, express an endogenous green fluorescent protein (GFP). Fluorescence microscopy showed that, in contrast to the endogenous expression of the red fluorescent protein of E. ancora ovaries that we have previously reported, the testes displayed a distinct green fluorescence. Molecular identification and spectrum characterization demonstrated that E. ancora testes expressed a GFP (named EaGFP) that is a homolog of the GFP from the jellyfish Aequorea victoria and that possesses an excitation maximum of 506 nm and an emission maximum of 514 nm. Immunohistochemical analyses revealed that the testicular somatic cells, but not the germ cells, expressed EaGFP. EaGFP was enclosed within one or a few granules in the cytoplasm of testicular somatic cells, and the granule number decreased as spermatogenesis proceeded. We also showed that testicular somatic cells could be enriched by using endogenous GFP as an indicator. The present study not only revealed one of the unique cellular characteristics of coral testicular cells but also established a technical basis for more in-depth investigations of the function of testicular somatic cells in spermatogenesis in future studies.

Activation of the brain-pituitary-gonadotropic axis in the black porgy Acanthopagrus schlegelii during gonadal differentiation and testis development and effect of estradiol treatment.

Previous studies revealed an estradiol (E2)-dependent peak in brain activity, including neurosteroidogenesis and neurogenesis in the black porgy during the gonadal differentiation period. The brain-pituitary-gonadotropic axis is a key regulator of reproduction and may also be involved in gonadal differentiation, but its activity and potential role in black porgy during the gonadal differentiation period is still unknown. The present study analyzed the expression of regulatory factors involved in the gonadotropic axis at the time of gonadal differentiation (90, 120, 150 days after hatching [dah]) and subsequent testicular development (180, 210, 300 dah). In agreement with previous studies, expression of brain aromatase cyp19a1b peaked at 120 dah, and this was followed by a gradual increase during testicular development. The expression of gonadotropin subunits increased slightly but not significantly during gonadal differentiation and then increased significantly at 300 dah. In contrast, the expression of brain gnrh1 and pituitary gnrh receptor 1 (gnrhr1) exhibited a pattern with two peaks, the first at 120 dah, during the period of gonadal differentiation, and the second peak during testicular development. Gonad fshr and lhcgr increased during gonadal differentiation period with highest transcript level in prespawning season during testicular development. This suggests that the early activation of brain gnrh1, pituitary gnrhr1 and gths, and gonad gthrs might be involved in the control of gonadal differentiation. E2 treatment increased brain cyp19a1b expression at each sampling time, in agreement with previous studies in black porgy and other teleosts. E2 also significantly stimulated the expression of pituitary gonadotropin subunits at all sampling times, indicating potential E2-mediated steroid feedback. In contrast, no significant effect of E2 was observed on gnrh1. Moreover, treatment of AI or E2 had no statistically significant effect on brain gnrh1 transcription levels during gonadal differentiation. This indicated that the early peak of gnrh1 expression during the gonadal differentiation period is E2-independent and therefore not directly related to the E2-dependent peak in brain neurosteroidogenesis and neurogenesis also occurring during this period in black porgy. Both E2-independent and E2-dependent mechanisms are thus involved in the peak expression of various genes in the brain of black porgy at the time of gonadal differentiation.

Dmrt1 (doublesex and mab-3-related transcription factor 1) expression during gonadal development and spermatogenesis in the Japanese eel.

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.

The germline-specific expression of Foxl3a and its paralogous Foxl3b are associated with male gonadal differentiation in the Japanese eel, Anguilla japonica.

Unlike its paralog Foxl2, which is well known for its role in ovarian development in vertebrates, the function of Foxl3 is still unclear. Foxl3 is an ancient duplicated copy of Foxl2. It is present as a single copy in ray-finned fish. But, due to repeated losses, it is absent in most tetrapods. Our transcriptomic data, however, show that two Foxl3s (Foxl3a and its paralog Foxl3b) are present in Japanese eel. Foxl3a is predominantly expressed in the pituitary, and Foxl3b is predominantly expressed in the gills. Both Foxl3s show a sex-dimorphic expression, being higher expression in testes than in ovaries. Moreover, Foxl3a and Foxl3b were exclusively expressed during gonadal differentiation in control eels (100% male). Conversely, Foxl3a and Foxl3b significantly decreased after gonadal differentiation in E2-treated eels (100% female). Furthermore, in accordance the difference in adhesive ability between somatic cells and germline cells in testes, Foxl3s showed a high expression in suspension cells (putative germline cells) and low expression in adhesive cells (putative somatic cells). In situ hybridization further showed that Foxl3a and Foxl3b were expressed in the testicular germline cells. In addition, Foxl3s expression was not changed by sex steroids in in vitro testes culture. Taken together, our results suggest that the teleost-specific Foxl3 paralog was repeatedly lost in most fish after the third round of whole genome duplication. The two germline-expressed Foxl3s had higher expression levels in males than in females during gonadal differentiation in Japanese eel. These results demonstrated that Foxl3s might play an important role in germline sexual fate determination from ancient fish to modern fish.

Two distinct vitellogenin genes are similar in function and expression in the bigfin reef squid Sepioteuthis lessoniana.

Unlike vitellogenin, which is the sole major precursor of yolk protein in all oviparous vertebrates, a variety of major precursor of yolk proteins are found among oviparous invertebrates. Sea urchins have a transferrin-like yolk protein, while all other major precursors of yolk proteins in oviparous invertebrates belong to the superfamily of large lipid transfer proteins (LLTPs). However, a comprehensive understanding of vitellogenesis is absent in cephalopods. To understand control of vitellogenesis by the LLTPs gene, two vitellogenins (VTG1 and VTG2), two apolipophorins (APOLP2A and APOLP2B), and a cytosolic large subunit of microsomal triglyceride transfer protein (MTTP) found in the bigfin reef squid. Only the two VTGs showed high levels of expression in mature females compared to males. We further analyzed the expression profile and localization of both VTGs/VTGs during ovarian development. Our data showed that VTGs/VTGs expressions were correlated to the female reproductive cycle. Ovarian VTG1 and VTG2 were localized in the follicle cells but not in oocytes. In addition, VTG1 and VTG2 were represented in follicle cells and oocytes. Thus, our results showed that both VTGs were synthesized by follicle cells and are then delivered to oocytes. In addition, we demonstrated that VTGs were the major precursor of yolk protein in bigfin reef squid. We also found differential proteolytic cleavage processes of VTG1 and VTG2 during VTGs accumulation in oocytes. Therefore, our data shed light on the molecular mechanism of the yolk accumulation pathway in cephalopods.

Primary males guide the femaleness through the regulation of testicular Dmrt1 and ovarian Cyp19a1a in protandrous black porgy.

Controlling the development of the sexes is critically important for the broodstock management in aquaculture. Sex steroids are widely used for sex control of fish. However, hermaphroditic fish have a plastic sex, and a stable sex is difficult to maintain with sex steroids. We used the black porgy (Acanthopagrus schlegelii) as a model to understand the possible mechanism of sexual fate decision. Low exogenous estradiol (E2) induced male development. In contrast, high exogenous E2 induced the regression of the testis and the development of the ovary and resulted in an unstable expression of femaleness (passive femaleness, with ovaries containing only the primary oocytes). The removal of testicular tissue by surgery resulted in the early development of vitellogenic oocytes and active femaleness. Our data also demonstrated that the male-to-female sex change is blocked by the maintenance of male function with gonadotropin-induced dmrt1 expression in the testis. Furthermore, our data also indicated that ovarian cyp19a1a expression is regulated by the testis through epigenetic modifications. Therefore, the primary male guides the femaleness in the protandrous black porgy and the transition of sexual fate from male to female is determined by the status of the testicular tissue.