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.

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.

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 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.

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.

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.

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.

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.

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.

The effects of migratory stage and 11-ketotestosterone on the expression of rod opsin genes in the shortfinned eel (Anguilla australis).

The androgen 11-ketotestosterone (11KT) can induce many of the changes associated with silvering, i.e., the transformation of a non-migrating 'yellow' eel into a migrating 'silver' eel. We posited that plasticity in spectral sensitivity of the eye, accompanied by expression of different opsins in the retina during silvering, is controlled by 11KT. To test this hypothesis, mRNA levels of freshwater (fwo) and seawater (swo) opsins and of the two androgen receptors (ara and arb) in retinas of wild-caught female shortfinned eels, Anguilla australis were compared. Swo expression was much higher (3-4 orders of magnitude) and fwo expression substantially lower in silver than in yellow eels, whereas mRNA levels of both ars did not differ between stages. Yellow eel retinas exposed to 11KT in vitro exhibited a robust dose-dependent increase in swo, but weak decreasing effects on fwo transcript abundance were inconsistent. Similarly, increased retinal swo expression was seen after in vivo treatment of yellow eels with 11KT implants, whereas expression of fwo remained unaffected. Lastly, co-treatment with 11KT and the androgen receptor blocker flutamide was undertaken to determine whether 11KT exerts its effects through nuclear androgen receptors. Flutamide did not block 11KT-affected expression of any target gene, neither in vivo nor in vitro. We conclude that 11KT greatly increases the abundance of swo, identifying the androgen as an important regulator of the opsin switch during silvering in freshwater eels.