SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus).

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24-48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Long-day stimulation increases thyroid-stimulating hormone expression and affects gonadal development in chub mackerel.

For seasonal breeders, photoperiodic changes are important signals that mark the start of the breeding season. Thyroid-stimulating hormone (TSH) is a glycoprotein hormone that not only promotes the secretion of thyroid hormone but also plays a key role in regulating seasonal reproduction in birds and mammals. However, whether TSH activation has been implicated as a seasonal indicator in fish breeding has not been fully investigated. In this study, we isolated tshb as a starting point to elucidate the effect of photoperiodic changes on the activation of the reproductive axis of chub mackerel. The isolated tshb was classified as tshba, which is widely conserved in vertebrates. The quantitative PCR results showed that tshb was strongly expressed in the pituitary. When female and male chub mackerel with immature gonads were reared for six weeks under different photoperiodic conditions, the gonads developed substantially in the long-day (LD) reared fish compared to those in the short-day reared fish. Real-time PCR results showed that the expression level of tshb in the pituitary gland was significantly elevated in the LD group. Although there was no difference in the gonadotropin-releasing hormone 1 gene expression level in the preoptic area of the brain, follicle-stimulating hormone and luteinizing hormone gene expression levels in the pituitary were also significantly elevated in the LD group. In conclusion, TSH is a potential mediator of seasonal information in the reproductive endocrine axis and may induce gonadal development during the breeding season of chub mackerel.

Development of sandwich enzyme-linked immunosorbent assays for chub mackerel Scomber japonicus gonadotropins and regulation of their secretion in female reproduction.

The pituitary gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play critical roles in regulating gonadal development and sexual maturation in vertebrates. We developed non-competitive enzyme-linked immunosorbent assays (ELISAs) to measure Fsh and Lh in chub mackerel Scomber japonicus, which is a commercially important scombrid species. Mouse monoclonal antibodies specific for Fsh and Lh, and a rabbit polyclonal antibody against both Gths were produced by immunization with hormones purified from chub mackerel pituitaries. These monoclonal and polyclonal antibodies were used as capture and detection antibodies in the developed sandwich ELISAs. The ELISAs were reproducible, sensitive, and specific for chub mackerel Fsh and Lh. Parallelism between the standard curve and serial dilutions of chub mackerel serum and pituitary extract was observed for both Fsh and Lh ELISAs. Comparison between vitellogenic and immature females revealed that Fsh is secreted during vitellogenesis and Lh is barely released during immaturity. After gonadotropin-releasing hormone analog (GnRHa) injection, vitellogenic females showed increases in serum Lh, whereas serum levels of Fsh did not vary. Moreover, the serum steroid profiles revealed that estradiol-17ß was continuously produced after GnRHa treatment, whereas 17,20ß-dihydroxy-4-pregnen-3-one secretion was transiently induced. These results indicate that, in vitellogenic females, GnRHa stimulates the release of Lh, but not Fsh, which results in acceleration of vitellogenesis and induction of oocyte maturation via steroid production.

Sex Lethal Gene Manipulates Gonadal Development of Medaka, Oryzias latipes, through Estrogenic Interventions.

Germ cells are pivotal for gonadal sexuality maintenance and reproduction. Sex lethal (sxl), the somatic sex determining gene of Drosophila, is the known regulator and initiator of germ cell femininity in invertebrates. However, the role of the Sxl homologue has rarely been investigated in vertebrates. So, we used medaka to clarify the role of sxl in vertebrate gonadogenesis and sexuality and identified two Sxl homologues, i.e., Sxl1a and Sxl1b. We found that sxl1a specifically expresses in the primordial germ cells (PGC), ovary, (early gonia and oocytes), while sxl1b distributions are ubiquitous. An mRNA overexpression of sxl1a accelerated germ cell numbers in 10 DAH XY fish, and sxl1a knockdown (KD), on the other hand, induced PGC mis-migration, aberrant PGC structuring and ultimately caused significant germ cell reduction in XX fish. Using an in vitro promoter analysis and in vivo steroid treatment, we found a strong link between sxl1a and estrogenic germ cell-population maintenance. Further, using sxl1a-KD and erß2-knockout fish, we determined that sxl1 acts through erß2 and controls PGC sexuality. Cumulatively, our study highlights the novel role of sxl1a in germ cell maintenance and sexual identity assignment and thus might become a steppingstone to understanding the commonalities of animal sexual development.

Effects of LPXRFamide peptides on chub mackerel gonadotropin secretion†.

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide, suppresses gonadotropin (GTH) secretion in birds and mammals. In fish, the GnIH homolog LPXRFamide (LPXRFa) produces mature peptides with species-dependent effects on sexual reproduction. Here, we investigated the effects of LPXRFa on GTH secretion in the chub mackerel (cm; Scomber japonicus). We cloned cmlpxrfa (603 bp) and cmlpxrfa-r (1416 bp). Additionally, we isolated lpxrfa from the bluefin tuna (Thunnus orientalis) to confirm the conservation of the LPXRFa mature sequence. Phylogenetic analysis showed that the LPXRFa precursor protein produces three mature peptides, LPXRFa-1, -2, and -3, in both species. Reverse transcription-quantitative polymerase chain reaction revealed that cmlpxrfa is expressed in the hypothalamus and thalamus and midbrain (T.MB), and sexual differences were observed. Receptor expression was observed in the preoptic area, hypothalamus, T.MB, and pituitary. Female hypothalamic lpxrfa expression did not change during puberty. Reporter gene assay showed that LPXRFa induced receptor activation via the CRE and SRE signaling pathways. However, in the presence of forskolin, an intracellular cyclic AMP enhancer, none of the LPXRFa could suppress receptor activity. The in vitro bioassay results showed that gonadotropin-releasing hormone-1 (GnRH1) had no effect on follicle-stimulating hormone (FSH) secretion, whereas the three LPXRFa significantly increased FSH secretion in pituitary cells from male chub mackerel. Contrarily, GnRH1 and three LPXRFa significantly increased luteinizing hormone secretion. The in vivo administration of LPXRFa had no effect on fshb and lhb expression in prepubertal and mature male chub mackerel. Overall, cmLPXRFa lacks the ability to suppress GTH secretion but can promote GTH secretion.

In vitro action of leptin on gonadotropin secretion in pre-pubertal male chub mackerel.

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln1 and Asp2 did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe8 can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cmKissR1, bftKissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.

Leptin stimulates gonadotropin release and ovarian development in marine teleost chub mackerel.

Leptin transmits information about energy stored in the periphery to the reproductive axis and is an essential signal for puberty initiation in mammals; however, to date, few studies have focused on the direct effects of leptin stimulation on reproductive factors in fish. This study demonstrated the effect of leptin stimulation on important reproductive factors and ovarian development in the marine teleost chub mackerel (Scomber japonicus). We prepared recombinant leptin and conducted functional analyses through in vitro bioassays using primary pituitary cells, long-term leptin treatment administered to pre-pubertal females, and intracerebroventricular (ICV) administration. The results showed that leptin stimulation strongly induced gonadotropin (follicle-stimulating hormone: FSH and luteinizing hormone: LH) secretion from pituitary cells collected from pre-pubertal females, and that long-term leptin treatment significantly promoted ovarian development and triggered pubertal onset. Furthermore, ICV administration of leptin did not affect kisspeptin gene expression but significantly upregulated gonadotropin-releasing hormone 1 (gnrh1), fshb and lhb gene expression in sexually immature females. These results strongly suggest leptin as an important signal for reproductive-axis activation in chub mackerel.

Kiss1 hexadecapeptide directly regulates gonadotropin-releasing hormone 1 in the scombroid fish, chub mackerel.

Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1- and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH2) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.

Two vitellogenins in the loliginid squid Uroteuthis edulis: Identification and specific expression in ovarian follicles.

Vitellogenenesis is a physiological process common in oviparous animals. The molecular profile, modifications, and utilization of vitellogenin (VTG), a precursor of yolk protein, have been characterized in various taxa to understand oogenesis within different modes of reproduction. Hormonal regulation of VTGs has been investigated in invertebrates, such as insects and crustaceans; conversely, little is known for cephalopods. In this study, we isolated two VTG genes (ue-VTG1 and ue-VTG2) from the loliginid swordtip squid, Uroteuthis edulis, via a comprehensive survey of a transcriptome database and subsequent cDNA cloning. Structural analysis of the two ue-VTGs revealed their unique features, namely the absence of two domains usually found in VTGs from other organisms: the von Willebrand factor D domain (vWD) and the domain of unknown function 1943 (DUF1943). Levels of ue-VTG1 and ue-VTG2 transcripts in the ovary, specifically in follicular cells, increased during the late-vitellogenic phase, suggesting that yolk accumulation progresses via paracrine interactions involving follicular cells and oocytes. N-terminal amino acid sequencing of biochemically purified yolk protein revealed its origins from these two VTGs, indicating that both are functional precursors of yolk protein. These results provide information that is essential to understanding the physiological pathway of yolk synthesis, accumulation, and storage in loliginid squids.

Molecular characterization and functional analysis of pituitary GnRH receptor in a commercial scombroid fish, chub mackerel (Scomber japonicus).

The gonadotropin-releasing hormone (GnRH) is essential during pubertal onset, for its regulation of the synthesis and release of pituitary gonadotropins. Its action is mediated by GnRH receptors (GnRHRs) in the pituitary gonadotrophs. Our previous study demonstrated that the chub mackerel brain expresses three GnRH forms (gnrh1, gnrh2, and gnrh3), and that only GnRH1 neurons innervate anterior pituitary regions. Furthermore, chub mackerel gnrh1 mRNA exhibited a significant increase at pubertal onset. The present study aimed to isolate the functional GnRHR form involved in chub mackerel puberty. The open reading frame of our cloned receptor encodes 428 amino acids and contains seven transmembrane domains. Phylogenetic analysis also indicated clustering with other teleost-type IIB GnRHRs, mainly those involved in reproduction. Reporter gene assay results showed that all four synthetic peptides (GnRH1, GnRH2, GnRH3, and GnRH analogue) bind to the cloned receptor. Three deduced GnRH ligands stimulated luteinizing hormone (LH) release from cultured pituitary cells in vitro. Receptor gene expression was mainly detected in the pituitary and showed an increasing trend in the developing gonadal stages of both sexes during the pubertal process; this process was synchronous with previous studies of follicle-stimulating hormone beta (fshß) and lhß gene expression in chub mackerel. These results suggest that the cloned receptor is likely involved in the regulation of pubertal onset in this species. Therefore, we have designated the receptor cmGnRHR1.

Possible role of the leptin system in controlling puberty in the male chub mackerel, Scomber japonicus.

Leptin directly regulates kisspeptin neurons in the hypothalamus and gonadotropin secretion from the pituitary, making it a central player in the onset of mammalian puberty. Recently, we identified two leptin genes (lepa and lepb) and a single leptin receptor (lepr) in the marine perciform fish chub mackerel; however, the expression of these genes did not correlate with the expression of important reproductive genes or ovarian stage during female puberty. Here, we expand upon these initial observations by evaluating the expression of lepa, lepb, and lepr during pubertal transition and under differential feeding conditions in the male chub mackerel. Reverse transcription-polymerase chain reaction (RT-PCR) showed that lepa was primarily expressed in the liver of pubertal and gonadal recrudescence adults, as well as in the brain of adult fish; lepb was primarily expressed in the brain of all fish tested; and lepr was widely expressed in a variety of tissues. qRT-PCR analyses revealed significant increases in the hepatic expression of lepa in accordance with testicular stage, whereas pituitary follicle-stimulating hormone (fshß) expression increased in unison with hepatic lepa. In contrast, expression of both brain lepa and lepb dramatically decreased during pubertal transition, with brain kisspeptin 1 (kiss1) expression strongly correlating with leptin expression patterns. In pre-pubertal males, lepa, lepb, and lper gene expression in the brain, pituitary gland, and liver decreased in fish given a high feed diet, relative to the controlled feeding group. Taken together, these results indicate high sexual specificity of leptin expression, suggesting a possible role for leptin signaling in endocrine and neuroendocrine functions during spermatogenesis in the pubertal male chub mackerel.

Expression changes of mRNAs encoding kisspeptins and their receptors and gonadotropin-releasing hormones during early development and gonadal sex differentiation periods in the brain of chub mackerel (Scomber japonicus).

In recent years, brain kisspeptin system has been shown to be involved in diverse reproductive function, including sexual differentiation in vertebrates. Our previous reports demonstrated that the chub mackerel (Scomber japonicus) brain expresses two kisspeptin (kiss1, kiss2), two kisspeptin receptor (kissr1, kissr2) and three gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) genes. In the present study, using quantitative real-time PCR (qRT-PCR) assays, we analysed expression changes of these genes during early development (0-30dphs) and gonadal sex differentiation periods (37-60dphs). Absolute expression level of kiss-kissr-gnrh in the whole head was higher between 0 and 15dphs, in comparison to later developmental periods. Histological analyses revealed presence of sexually differentiated males and females with testicular and ovarian features at 37, 45, and 60dphs. In both males and females, kiss2, kissr1, and kissr2 levels were higher at 37dph, in comparison to 45 and 60dphs, with kiss1 showing no significant differences. Levels of all three gnrh mRNAs were higher at 45dph, in comparison to 60dph. Changes in the expression level of kiss-kissr-gnrh mRNAs in different brain regions of sexually differentiated males and females indicated differences in their regional distribution. These results suggest possible involvement of Kiss-KissR-GnRH systems during early development and gonadal sex differentiation in the chub mackerel.

Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): Molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages.

Leptin is a hormone produced by fat cells that regulates the amount of fat stored in the body and conveys nutritional status to the reproductive axis in mammals. In the present study we identified two subtypes of leptin genes (lepa and lepb) and a leptin receptor gene (lepr) from chub mackerel (Scomber japonicus) and there gene expression under different feeding conditions (control and high-feed) and pubertal development stages was analyzed using quantitative real-time PCR. The protein lengths of LepA, LepB and LepR were 161 amino acids (aa), 163 aa and 1149 aa, respectively and both leptin subtypes shared only 15% similarity in aa sequences. In pubertal females, lepa was expressed in the brain, pituitary gland, liver, adipose tissue and ovary; however, in adult (gonadal maturation after the second in the life) females, lepa was expressed only in the liver. lepb was expressed primarily in the brain of all fish tested and was expressed strongly in the adipose tissue of adults. lepr was characterized by expression in the pituitary. The high-feed group showed a high conditioning factor level; unexpectedly, hepatic lepa and brain lepr were significantly more weakly expressed compared with the control-feed group. Furthermore, the expression levels of lepa, lepb and lepr genes showed no significant differences between pre-pubertal and post-pubertal fish. On the other hand, pituitary fshß and lhß showed no significant differences between different feeding groups of pre-pubertal fish. In contrast, fshß and lhß expressed abundantly in the post-pubertal fish of control feed group. Based on these results, whether leptin plays an important role in the nutritional status and pubertal onset of chub mackerel remains unknown.

mRNA levels of kisspeptins, kisspeptin receptors, and GnRH1 in the brain of chub mackerel during puberty.

Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared chub mackerel.

Gonadal development and gonadotropin gene expression during puberty in cultured chub mackerel (Scomber japonicus).

Understanding puberty is important for establishing aquaculture in fish. In this study, we analyzed the timing and completion of pubertal development along with changes in pituitary gonadotropin genes (fshb and lhb) in cultured chub mackerel (Scomber japonicus). At 45 days post-hatching (dph), gonadal sex differentiation was observed. The onset of puberty occurred at 192 dph in females with the start of vitellogenesis, whereas it occurred at 164 dph in males, with the beginning of spermatogenesis (proliferation and differentiation of germ cells). The completion of puberty was at 326 dph in females when vitellogenesis completed, and it was at 338 dph in males during spermiation. All fish sampled during the spawning season completed pubertal development. In the pituitary of female fish, fshb expression was activated during early secondary growth and was maintained high throughout vitellogenesis, whereas lhb expression was highest at the completion of vitellogenesis. In male fish, fshb and lhb expression were activated from the onset of spermatogenesis and further activated during late pubertal development; fshb remained high between late spermatogenesis and spermiation, whereas lhb was highest during spermiation.

Exposure effects of benzalkonium chloride (BAC) on gonadal physiology and fertility suppression in medaka (Oryzias latipes).

Benzalkonium chloride (BAC), a commonly used quaternary ammonium compound in various products like antiseptics, cosmetics, and disinfectants, has raised concerns due to its potential to contaminate aquatic environments and subsequently affect the reproductive performance of the organisms within those ecosystems. The article underscores a critical concern regarding the impact of BAC on aquatic ecosystems, particularly its effect on fish reproductive quality, using medaka (Oryzias latipes) as a model organism. Firstly, while measuring lethal dose of BAC in adult medaka, we observed a dose dependent mortality in BAC treated fish (100 and 200 ppm: 100%; 60 ppm: 51.7%; 30 ppm or less: no mortality at 24 h post treatment (hpt)) and calculated the LD50 at 96 hpt as 39.291 ppm (95% confidence interval: 28.817-53.570 ppm). Further, we assessed the molecular, cellular and histological changes through long-term exposure. Enlarged sperm pockets and reduced spermatocyte were seen in BAC exposed testis while no significant structural changes were observed in the ovaries. Following BAC exposure, drastic alterations in the gene expression and cellular localization related to sex, estrogen signaling, and autophagy were also noted from gonads and liver. Subsequently, using a short-term exposure analysis, we confirmed the sex and time responsive transcriptional kinetics and found that BAC sequentially affected the gonadal somatic cells followed by germ cell differentiation. Finally, using reproductively competent male and female medaka, we conducted progeny production and performance analysis and depicted a drastic reduction in fecundity, and fertilization and hatching rate, indicating adverse effects of BAC on reproductive success. Cumulatively, these findings emphasize the consequences of widespread use of BAC on reproductive security of aquatic animals and highlights the need for further research to comprehend the potential harm posed by such compounds to aquatic animal health and ecosystem integrity.

Modeling the SDF-1/CXCR4 protein using advanced artificial intelligence and antagonist screening for Japanese anchovy.

SDF-1/CXCR4 chemokine signaling are indispensable for cell migration, especially the Primordial Germ Cell (PGC) migration towards the gonadal ridge during early development. We earlier found that this signaling is largely conserved in the Japanese anchovy (Engraulis japonicus, EJ), and a mere treatment of CXCR4 antagonist, AMD3100, leads to germ cell depletion and thereafter gonad sterilization. However, the effect of AMD3100 was limited. So, in this research, we scouted for CXCR4 antagonist with higher potency by employing advanced artificial intelligence deep learning-based computer simulations. Three potential candidates, AMD3465, WZ811, and LY2510924, were selected and in vivo validation was conducted using Japanese anchovy embryos. We found that seven transmembrane motif of EJ CXCR4a and EJ CXCR4b were extremely similar with human homolog while the CXCR4 chemokine receptor N terminal (PF12109, essential for SDF-1 binding) was missing in EJ CXCR4b. 3D protein analysis and cavity search predicted the cavity in EJ CXCR4a to be five times larger (6,307 Å³) than that in EJ CXCR4b (1,241 Å³). Docking analysis demonstrated lower binding energy of AMD3100 and AMD3465 to EJ CXCR4a (Vina score -9.6) and EJ CXCR4b (Vina score -8.8), respectively. Furthermore, we observed significant PGC mismigration in microinjected AMD3465 treated groups at 10, 100 and 1 × 105 nM concentration in 48 h post fertilized embryos. The other three antagonists showed various degrees of PGC dispersion, but no significant effect compared to their solvent control at tested concentrations was observed. Cumulatively, our results suggests that AMD3645 might be a better candidate for abnormal PGC migration in Japanese anchovy and warrants further investigation.

Characterization, localization, and stage-dependent gene expression of gonadotropin receptors in chub mackerel (Scomber japonicus) ovarian follicles.

The pituitary gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of gametogenesis in teleosts. However, little is known about the physiological mechanisms by which GtHs regulate asynchronous oocyte development in multiple-spawning marine fishes. We cloned cDNAs encoding GtH receptors (FSHR and LHR) from chub mackerel (Scomber japonicus). FSH and LH were purified by anion-exchange chromatography, gel filtration, and concanavalinA-agarose. When expressed in mammalian cells, FSHR and LHR responded strongly to their own ligands. By separating LH into two subunits by the use of reverse-phase chromatography, we found that the beta-subunit is responsible for signal transduction and the alpha-subunit may be important for holding hormone-receptor complex. In situ hybridization showed that only fshr was expressed in prefollicle and granulosa cells in oocytes at the perinucleolus and cortical alveolus stages, suggesting that FSH is involved in the primary and early secondary growth of oocytes. In ovarian follicles during vitellogenesis, both fshr and lhr were expressed in granulosa and thecal cells, and lhr was strongly expressed during germinal vesicle migration (GVM). Real-time PCR analysis of stage-dependent fshr and lhr expression showed that fshr expression was high in ovarian follicles throughout vitellogenesis and decreased during GVM, whereas lhr expression was low in early vitellogenesis, but increased markedly in the late phase of vitellogenesis, remaining high during GVM. These findings suggest that switching of the expression of FSHR to LHR controls the effects of FSH and/or LH on vitellogenesis and final oocyte maturation via steroid production in granulosa and thecal cells.