Host and geography impact virus diversity in New Zealand's longfin and shortfin eels.

The fishing and aquaculture industry is vital for global food security, yet viral diseases can result in mass fish die-off events. Determining the viromes of traditionally understudied species, such as fish, enhances our understanding of the global virosphere and the factors that influence virome composition and disease emergence. Very little is known about the viruses present in New Zealand's native fish species, including the shortfin eel (Anguilla australis) and the longfin eel (Anguilla dieffenbachii), both of which are fished culturally by Maori (the indigenous population of New Zealand) and commercially. Through a total RNA metatranscriptomic analysis of longfin and shortfin eels across three different geographic locations in the South Island of New Zealand, we aimed to determine whether viruses had jumped between the two eel species and whether eel virome composition was impacted by life stage, species, and geographic location. We identified nine viral species spanning eight different families, thereby enhancing our understanding of eel virus diversity in New Zealand and the host range of these viral families. Viruses of the family Flaviviridae (genus Hepacivirus) were widespread and found in both longfin and shortfin eels, indicative of cross-species transmission or virus-host co-divergence. Notably, both host specificity and geographic location appeared to influence eel virome composition, highlighting the complex interaction between viruses, hosts, and their ecosystems. This study broadens our understanding of viromes in aquatic hosts and highlights the importance of gaining baseline knowledge of fish viral abundance and diversity, particularly in aquatic species that are facing population declines.

Low dose of propyl-pyrazole-triol, an agonist of estrogen receptor alpha, administration stimulates the Coolidge effect in fadrozole-treated male rats.

Estrogen receptor (ER) α is involved in male sexual function. Here, we aim to investigate how ERα activation influences sexual satiety and the Coolidge effect (i.e., when a rat, that has reached sexual satiety, experiences an increased arousal after exposure to a novel sexual partner) in estrogen-deprived male rats. Male rats (8 per group) were treated daily for 29 days with either saline (Control group) or fadrozole dissolved in saline (1 mg/kg/day) 1 h before mating. On Days 13 and 29, rats treated with fadrozole received either no additional treatment (fadrozole group) or a single injection of propyl-pyrazole-triol (ERα-agonist group, dissolved in sesame oil, 1 mg/kg). Rats mated until reaching sexual satiety on Days 13 and 29. In these sessions, the Control group displayed higher frequency of intromission and ejaculation than the other groups. The ERα-agonist group mounted more frequently but reached sexual satiety sooner than the Control group. On Day 29, when exposed to a new sexual partner, the fadrozole-treated rats were less likely to display intromission than the other groups, or ejaculation than the Control group, or mounting than the ERα-agonist group. The Control group showed more ejaculatory behavior and shorter ejaculation latency than the other groups. Body weights, testosterone levels, estradiol levels, and ERα-immunoreactive cell counts in brain regions for sexual behavior were comparable between groups after 29 days of treatments. Our data suggest that estrogen helps regulate sexual satiety and the Coolidge effect in male rats.

Lipoprotein receptors in ovary of eel, Anguilla australis: molecular characterisation of putative vitellogenin receptors.

Lipoprotein receptors, including low-density lipoprotein receptor (LDLr) relatives (Lrs) and LDLr-related proteins (Lrps), belong to the LDLr supergene family and participate in diverse physiological functions. In this study, novel sequences of lr and lrp genes expressed in the ovary of the short-finned eel, Anguilla australis, during early gonadal development are presented. The genes encoding the LDLr-like, Lrp1-like, Lrp1b-like, Lrp3, Lrp4-like, Lrp5-like, Lrp6, Lrp10, Lrp11, Lrp12-like, and Lr11-like proteins were found and identified by sequence and structure analysis, in addition to phylogenetic analysis. Genes encoding proteins previously implicated in follicle development and vitellogenin (Vtg) uptake in oviparous vertebrates were also identified, i.e. lr8 (including lr8 + and lr8- variants) and lrp13; their identification was reinforced by conserved synteny with orthologues in other teleost fish. Compared to other lr/lrp genes, the genes encoding Lr8 + , Lr8-, and Lrp13 were highly expressed in ovary during early development, decreasing as oocyte development advanced when induced by hypophysation. Furthermore, lr8 + , lr8-, and lrp13 were dominantly expressed in the ovary when compared with 17 other tissues. Finally, this study successfully detected the expression of both lr8 variants, which showed different expression patterns to those reported in other oviparous vertebrates and provided the first characterisation of Lrp13 in Anguilla sp. We propose that lr8 + , lr8-, and lrp13 encode putative Vtg receptors in anguillid eels.

Effects of cortisol on female-to-male sex change in a wrasse.

Sex change occurs as a usual part of the life cycle for many teleost fish and the modifications involved (behavioural, gonadal, morphological) are well studied. However, the mechanism that transduces environmental cues into the molecular cascade that underlies this transformation remains unknown. Cortisol, the main stress hormone in fish, is hypothesised to be a key factor linking environmental stimuli with sex change by initiating gene expression changes that shift steroidogenesis from oestrogens to androgens but this notion remains to be rigorously tested. Therefore, this study aimed to experimentally test the role of cortisol as an initiator of sex change in a protogynous (female-to-male) hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). We also sought to identify potential key regulatory factors within the head kidney that may contribute to the initiation and progression of gonadal sex change. Cortisol pellets were implanted into female spotty wrasses under inhibitory conditions (presence of a male), and outside of the optimal season for natural sex change. Histological analysis of the gonads and sex hormone analyses found no evidence of sex change after 71 days of cortisol treatment. However, expression analyses of sex and stress-associated genes in gonad and head kidney suggested that cortisol administration did have a physiological effect. In the gonad, this included upregulation of amh, a potent masculinising factor, and nr3c1, a glucocorticoid receptor. In the head kidney, hsd11b2, which converts cortisol to inactive cortisone to maintain cortisol balance, was upregulated. Overall, our results suggest cortisol administration outside of the optimal sex change window is unable to initiate gonadal restructuring. However, our expression data imply key sex and stress genes are sensitive to cortisol. This includes genes expressed in both gonad and head kidney that have been previously implicated in early sex change in several sex-changing species.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

Sperm quality of artificially matured shortfinned eel is not affected by human chorionic gonadotropin dose and route of administration.

Background: Acquisition of high quality sperm is key to the artificial propagation of eels in captivity, but fertility drugs are expensive and repeated handling is stressful to the fish. An interrupted treatment regime (an initial hormone injection to stimulate spermatogenesis, followed several weeks later by weekly booster injections to induce sperm maturation) for acquisition of sperm in captive male eels has promise for high sperm quality on the one hand, and animal welfare benefits on the other. To further develop this approach for shortfinned eel, Anguilla australis, we evaluated the efficacy of (i) different initial doses of human chorionic gonadotropin (hCG) and (ii) route of administration. Methods: Male eels were artificially induced to mature with a single injection of 0, 250, 500 or 1,000 IU/fish of hCG, administered either intramuscularly (IM) or intraperitoneally (IP). Sperm maturation was induced with 150 IU hCG/fish from week 5 onwards and sperm collected for evaluation of quality by computer-assisted sperm analysis. Results: Control males did not mature and hence, sperm could not be retrieved and analysed, but all other treatments were effective in inducing testicular maturation. Milt volume tended to be higher for fish injected IM compared to those injected IP, whereas hCG dose had no effect. Conversely, the concentration of spermatozoa tended to be higher for several sperm collection time points in IP-injected than in IM-injected fish. Sperm quality, represented by percent motility, percent progressive motility and curvilinear velocity, was equal in fish given an initial dose of 250 IU hCG to those given higher initial doses of hCG. Conclusions: We recommend that an initial dose of 250 IU hCG/fish be administered to induce spermatogenesis in male A. australis, and, after a period of 4-5 weeks, weekly booster injections of ∼150 IU hCG/fish be administered in the day prior to sperm collection; both routes of administration (IM or IP) are equally effective. We contend that an interrupted treatment regime has notable benefits for induced maturation in male anguillids, as it reduces fish handling and manipulation and reduces the resources required to produce high quality sperm.

Reproductive development of female wallago catfish (Wallago attu) in captivity.

The wallago catfish (Wallago attu) is a new potential fish for aquaculture in Vietnam. Data related to the reproductive cycle of W. attu in captivity are, however, not available. To provide reliable indicators for oocyte maturation (OM) and the spawning season of the captive W. attu, this study investigated the temporal variation in hepatosomatic and gonadosomatic indices, oocyte diameter and color (greenish vs yellowish), germinal vesicle migration, and plasma concentrations of estradiol-17ß (E2) and vitellogenin (Vtg) in female broodstock in association with changes in light density, temperature and amount of rainfall during the reproductive cycle. The results of this study displayed a clear seasonality in all the investigated parameters. The highest concentration of E2 (2.6 ± 3.5 ng/mL) was found in April, followed by a peak of Vtg (543 ± 43 ng/mL) in June. Meanwhile, the largest mean oocyte diameter (1.70 ± 0.02 mm) was observed in June. The shortest distance between the germinal vesicle and the edge of the oocyte (0.20 ± 0.01 mm) was recorded in July. Correspondingly, the amount of rainfall increased remarkably in July from 43.9 mm to over 200 mm in August. Taken together, we conclude that OM and the onset of the spawning season of captive W. attu occur in July and August, respectively. The percentage of greenish oocytes increased significantly over sampling time points. The changes in the color of oocytes combined with oocyte diameter could, therefore, be considered as promising indicators to predict the OM and spawning season of captive W. attu.

Effects of gonadotropins, 11-ketotestosterone, and insulin-like growth factor-1 on target gene expression and growth of previtellogenic oocytes from shortfinned eels, Anguilla australis, in vitro.

Pituitary gonadotropins, metabolic hormones, and sex steroids are known factors affecting the advanced stages of ovarian development in teleost fish. However, the effects of these hormones and of the interactions between them on the growth of previtellogenic ovarian follicles are not known. In order to address this void in understanding, previtellogenic ovarian fragments from eel, Anguilla australis, were incubated in vitro with recombinant Japanese eel follicle-stimulating hormone (rec-Fsh), human chorionic gonadotropin (hCG), or 11-ketotestosterone (11-KT) in the presence or absence of recombinant human insulin-like growth factor-1 (IGF1). The results of long-term in vitro culture (21 days) demonstrated that rec-Fsh and 11-KT, rather than hCG, caused significant increases in the diameter of previtellogenic oocytes. Meanwhile, only 11-KT induced a significant increase in lipid accumulation. Moreover, a greater effect on oocyte growth was observed when IGF1 supplementation was combined with 11-KT rather than with rec-Fsh or hCG. For short-term culture (24 h), treatment with 11-KT in the presence or absence of IGF1 had no significant effects on mRNA levels of target genes (lhr, cyp19, cyp11b, lpl, and ldr) except for upregulation of fshr. There were no significant effects of rec-Fsh on expression of any target gene, whereas hCG downregulated the expression of these genes. There was no evidence for any interaction between the gonadotropins and IGF1 that resulted in growth of previtellogenic oocytes. Taken together, these results suggest that hormones from both the reproductive and the metabolic axes regulate the growth of previtellogenic oocytes in Anguilla australis.

Are Cell Junctions Implicated in the Regulation of Vitellogenin Uptake? Insights from an RNAseq-Based Study in Eel, Anguilla australis.

At the onset of puberty, ovarian follicles become competent to incorporate large amounts of vitellogenin (Vtg). Using an RNAseq-based approach, transcriptomes from pre-vitellogenic (PV) and early vitellogenic (EV) ovaries from wild-caught eel, Anguilla australis, were compared to investigate the expression of specific genes encoding cell junction proteins that could be involved in regulating Vtg uptake. Partial support was found for the mechanical barrier hypothesis proposing that the access of Vtg to the oolemma is restricted by a tight junction (TJ) network within the granulosa cell layer, which changes between the PV and EV stage. Among 25 genes encoding TJ-constituting proteins, five were down-regulated and two were up-regulated. A chemical barrier hypothesis stating that gap junctions (GJs) are involved in modulating Vtg uptake was not supported, as only five GJs were found to be expressed in the ovary with no significant changes in expression between stages. Furthermore, the endocytic pathway was found to be up-regulated during the PV-EV transition. Finally, the study showed that gene expression patterns may help identify suitable candidates involved in the regulation of Vtg uptake, and provided novel sequence data for A. australis, including putative Vtg receptors corresponding to Lr8 and Lrp13 members of the low-density lipoprotein receptor family.

Spatiotemporal expression of activin receptor-like kinase-5 and bone morphogenetic protein receptor type II in the ovary of shortfinned eel, Anguilla australis.

In the eel ovary, the expression of growth differentiation factor-9 (Gdf9) appears to be largely confined to the germ cell in early stages of oogenesis. However, both the target tissue and the function of Gdf9 in fish remain unknown. This study aimed to describe the abundance and localization of activin receptor-like kinase-5 (Alk5) and bone morphogenetic protein receptor type II (Bmpr2), which together mediate the Gdf9 signal, in the ovary of a basal teleost, the shortfinned eel, Anguilla australis, during early folliculogenesis. The cDNA encoding eel alk5 and bmpr2 genes were cloned, characterized and the transcript abundances of these receptors quantified by quantitative real-time PCR. Ovarian transcript abundance for both receptors, along with that of gdf9 and of its paralogue bmp15, increased from the previtellogenic to early vitellogenic stage. Localization of receptor mRNAs by in situ hybridization revealed that these receptors are located in the somatic cells surrounding the oocyte. Furthermore, tissue distribution analysis showed that the expression of alk5 and bmpr2 were highest in ovary and thyroid, respectively. Unexpectedly, however, bmpr2 mRNA levels were lower in the ovary than in any of the other 17 tissues examined, and indeed, lower than ovarian gdf9 transcript abundance. These findings, together with the ovarian expression pattern of Gdf9, suggest that Gdf9, and conceivably, Bmp15, from the oocyte can signal through receptors that are located on the somatic cells surrounding the oocyte; this, in turn, facilitates elucidation of the function of these growth factors during oogenesis in teleost fish.

An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achieved in vivo through social manipulation, inhibition of aromatase activity, or steroid treatment. However, the induction of sex change in vitro has been largely unexplored. In this study, we established an in vitro culture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17ß-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e., sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex change in vitro. The in vitro gonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

Evaluating the impact of hydropower on downstream migrating anguillid eels: Catchment-wide and fine-scale approaches to identify cost-effective solutions.

Hydropower is an increasingly popular source of renewable and 'green' (in terms of emissions) energy, but reduced longitudinal connectivity and diverting flow through turbines can have negative impacts on catadromous anguillid eel species that have declined globally. There is an urgent need for environmental managers to perform remediation actions, such as protecting flows for migratory fish and providing passage solutions at infrastructure, under increasing legislative pressure. To deliver this, a more comprehensive understanding of eel migration in catchments with hydropower is required. Here, we illustrate the importance of catchment-wide and fine-scale acoustic telemetry, coupled with the influence of eel maturation (i.e. sex steroid levels), to determine the impact of Wairua run-of-river Power Station (WPS) on downstream migrating shortfin eels (Anguilla australis; n = 25) in Wairua River, New Zealand. Migration speed through the unregulated reach upstream of WPS was positively correlated with flow, but not eel length or sex steroids. Three eels passed a diversion weir (DW) to follow the natural watercourse and eight entered the WPS canal. Eels predominantly entered (95.2%) and were last detected (85.7%) in WPS forebay during hours of darkness. Eleven (52%) of the 21 eels that entered WPS forebay were impinged or entrained, all when three or four turbines were in operation (power generation >3.04 MW). Ten (48%) passed WPS spillway during significantly higher spill than impinged or entrained eels, with four passing during no turbine operation, after experiencing high flows near the intake (multiple receivers in WPS forebay used to quantify fine-scale behaviour). On average, eels were impinged or entrained at WPS significantly quicker (6.40 ± 11.13 days) than eels that entered the spillway (25.17 ± 15.12 days), but eel length and sex steroids did not significantly influence fate. Of the eels that migrated through the entire 55 km study reach, passage time at DW and WPS equated to 0.01-0.02% and 47.62-92.17% of their migration, respectively. Mitigation for WPS (and similar power schemes) should focus on operational or physical changes at DW to minimise eels entering power station forebay(s). Turbine shutdowns, ensuring WPS spillway is available and the provision of a bypass channel in WPS forebay are also discussed as ways to conserve the species with the potential to save costs for water resource managers.

Changes in lipid biology during ovarian development in farmed beluga sturgeon, Huso huso L.

The present study was conducted to understand key biochemical, physiological, and molecular changes associated with ovarian growth and with lipid transfer and/or accumulation into the ovary during oogenesis in captive beluga sturgeon. Plasma levels of triacylglycerides, cholesterol, phospholipid, and sex steroid hormones were determined and all were found to increase notably throughout development from the perinucleolar to the tertiary yolk stage. Using fast protein liquid chromatography, we recognized three major lipoprotein peaks in chromatograms from all samples. These peaks were characterized as containing very low-density lipoprotein (Vldl), low-density lipoprotein/high-density lipoprotein (Ldl/Hdl), and plasma proteins. While Ldl/Hdl represented the most abundant lipoprotein fraction, the relative abundance of different lipoprotein classes did not change with the stage of oogenesis. Eluted lipoproteins were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and sequenced. The peptide sequence spectra for 66-kDa, 205-kDa, 29-kDa, and 70-kDa bands matched with albumin, vitellogenin (Vtg) AB2b, immunoglobulin light-chain precursor, and immunoglobulin heavy-chain, respectively. The large amount of albumin in the plasma protein peak and the confined presence of Vtg AB2b to within Ldl/Hdl reinforce the lipoprotein classification. Lastly, transcript levels of genes encoding ovarian lipoprotein lipase (lpl), apolipoprotein E (apoe), very low-density lipoprotein receptors (vldlr), and low-density lipoprotein receptor-related protein 8-like (lrp8) were estimated using quantitative RT-PCR. The high mRNA levels of lpl, apoe, and lipoprotein receptors vldlr and lrp8 in previtellogenic females suggest that sturgeon oocytes need to be prepared to accept and traffic Vtg and lipids internally, before the start of vitellogenesis.

Effects of estradiol and 11-ketotestosterone pre-treatment on artificial induction of maturation in silver female shortfinned eels (Anguilla australis).

Our previous work documented significant advancements in steroid-induced progression of oogenesis, demonstrating that co-treatment of female eels with 11-ketotestosterone (11KT) and estradiol-17ß (E2) successfully induced uptake of vitellogenin by oocytes. Here we evaluate the effects of this steroid co-treatment on subsequent time to ovulation and egg quality in shortfinned eels artificially matured by hypophysation. Co-treatment with 11KT (1 mg) and E2 (0.2 or 2 mg) significantly reduced time to ovulation and therefore, the amount of pituitary homogenate required, without any detrimental effects on gonadosomatic index, oocyte diameter or the total weight of stripped eggs. E2 treatment resulted in promising increases in fertilization rates. These indicators suggest that co-treatment with 11KT and E2 holds promise for future artificial maturation practices in terms of minimising fish handling and stress, and of reducing the need for expensive pituitary preparations.

Maternal transcripts in good and poor quality eggs from Japanese eel, Anguilla japonica-their identification by large-scale quantitative analysis.

Our understanding of maternal control of development in vertebrates remains incomplete. In this study, we investigated levels of maternal transcripts in good and poor quality eggs from artificially matured Japanese eel, using RNA-Seq and quantitative polymerase chain reaction (qPCR), to identify candidate maternal transcripts related to development. De novo assembly or mapping of reads to the eel draft genome yielded 619,029 contigs and 85,906 transcripts, respectively; normalized read counts to these assemblies were calculated using reads (RPKM) or fragments (FPKM) per kilobase of transcript per million mapped reads. In silico screening identified 1,594 contigs and 150 transcripts with lower RPKM or FPKM in poor than in good quality eggs, 245 contigs, and 85 transcripts of which could be annotated by BLASTx, respectively. From selected contigs or transcripts, six genes (dnajb4, gnpat, card14, pdp1, fcgbp, ttn) had significantly lower messenger RNA levels in poor than in good quality eggs by qPCR. Multiple regression analysis showed that five genes (gnpat, b4galnt1, acsl6, rtkn, trim24) significantly correlated with hatchability. Taken together, 10 genes were identified as candidate maternal transcripts, regulating development in Japanese eel. Our results contribute to understanding the molecular basis for maternal control of development in vertebrates.

Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses.

Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae)-a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, gonadal sex change was preceded by downregulation of cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and accompanied by upregulation of amh (encoding anti-müllerian hormone that primarily regulates male germ cell development), and these genes may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.

Expressional regulation of gonadotropin receptor genes and androgen receptor genes in the eel testis.

Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11ß-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro, and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish.

11-Ketotestosterone induces oocyte growth, but does not affect oocyte cytology in pre-vitellogenic captive beluga, Huso huso L.

An effect of 11-ketotestosterone (11-KT) on growth of previtellogenic (PV) ovaries of eel, salmon and Atlantic cod has been demonstrated. The purpose of this study was to investigate the effects of 11-KT treatment (in vivo) on ovarian growth, on hormonal and biochemical changes in blood, and on ovarian mRNA levels of lipidation-related genes in captive beluga with PV oocytes. In addition, the potential involvement of lipoprotein lipase (Lpl), an important enzyme for extracellular hydrolysis of lipoprotein-associated lipids, was evaluated. Twelve beluga (4-year olds) were treated with an intraperitoneal slow-release implant of either 11-KT (2.5 mg) or a compressed matrix (control). Ovarian biopsy was done to obtain pre- (day 0: T0) and post-treatment (day 21: T21) data on histology and target gene expression. Three weeks of exposure resulted in an increase in serum 11-KT levels from 2.2 ng/mL to 83 ng/mL but did not yield significant changes in serum levels of triacylglycerides and cholesterol. Furthermore, 11-KT implantation increased oocyte diameters from 259 µm (T0) to 309 µm by T21. Regardless of the increase in oocyte size, ovaries remained in the PV stage, mostly as late perinucleolar oocytes. Meanwhile, at the molecular level, the expression of lipidation-related transcripts [lpl, apolipoprotein E (apoe), very low density lipoprotein receptors (vldlr), low-density lipoprotein receptor-related protein 8-like (lrp8)] was significantly up-regulated after three weeks. Immunostaining for Lpl by Western blotting indicated three immunoreactive bands (70, 58 and 37 kDa) in ovarian homogenates from beluga, but signal intensity was not affected by treatment. Altogether, the administration of 11-KT increased 11-KT serum levels, oocyte size, and the expression of genes associated with lipid uptake. However, this treatment did not advance ovarian development beyond the PV stage.

Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

Estradiol-17ß (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

Expression of gonadotropin subunit and gonadotropin receptor genes in wild female New Zealand shortfinned eel (Anguilla australis) during yellow and silver stages.

Despite tremendous importance of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) as primary controllers of reproductive development, information on the expression profiles of the genes encoding gonadotropin subunits and gonadotropin receptors (Fshr and Lhr) in wild eels are essentially non-existent. This study investigated pituitary fshb and lhb mRNA levels and ovarian fshr and lhr mRNA levels of wild shortfinned eels, Anguilla australis at different stages of oogenesis. Protein expression of Fsh in the pituitary was also quantified and visualized using slot blot and immunohistochemistry. Pituitary fshb and lhb mRNA levels showed a differential expression pattern, fshb mRNA levels increasing significantly from the perinucleolus (PN) to the oil droplet stage (OD) before slightly decreasing (not significantly) in the early vitellogenic stage (EV). A similar trend was observed in relative Fsh protein levels analyzed by slot blot and immunohistochemistry, but this trend was not reflected in the plasma levels of sex steroids. In contrast, pituitary lhb mRNA levels increased significantly from the PN to EV stage. A higher expression of Fsh at both mRNA and protein levels in the pituitary of eels at the OD stage compared to other investigated stages suggests that synthesis of Fsh production in the pituitary may reach a peak at the OD stage. In the ovary, transcript abundances of fshr and lhr gradually increased during previtellogenic follicle growth, but markedly and significantly increased thereafter. Taken together, our data suggest i) that Fsh release may be very limited, or absent, prior to onset of puberty in shortfinned eels and ii) that Lh is not functionally important in this fish during the EV stage.