Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Vitellogenin uptake activity in the intestinal ducts of intraovarian embryos in a viviparous teleost Xenotoca eiseni.

In the viviparous teleost species belonging to the family Goodeidae, intraovarian embryos absorb maternal supplements while they grow during the gestation period. They take up the components via trophotaeniae, a hindgut-derived placental structure. Our previous study using a goodeid species Xenotoca eiseni revealed that intraovarian embryos absorb the yolk protein vitellogenin (Vtg) via the trophotaenia. However, another group indicated yolk components accumulate in the intestinal lumen of X. eiseni embryos. Here, we investigated whether the intestinal duct is capable of protein uptake, as is the trophotaenia. Immunohistochemical studies indicated that endogenous vitellogenin is detected in the intestinal epithelial cells of the intraovarian embryo. Tracer analysis using FITC-Vtg also indicated that intestinal tissues can take up protein. The endocytosis-related genes expressed in trophotaenia were also detected in the intestinal tissues of the embryo. Lipid transporter genes which are not expressed in the trophotaenia were detected in the embryonic intestine. This evidence suggests that the intraovarian embryo of X. eiseni possesses two distinct sites for uptake of the maternal proteins. However, the presumed functions of the embryonic intestine and trophotaenia might be not identical. The study provides a new perspective on how mother-to-embryo matrotrophic interactions have changed in the evolution of viviparous teleosts.

Mother-to-embryo vitellogenin transport in a viviparous teleost Xenotoca eiseni.

Vitellogenin (Vtg), a yolk nutrient protein that is synthesized in the livers of female animals, and subsequently carried into the ovary, contributes to vitellogenesis in oviparous animals. Thus, Vtg levels are elevated during oogenesis. In contrast, Vtg proteins have been genetically lost in viviparous mammals, thus the yolk protein is not involved in their oogenesis and embryonic development. In this study, we identified Vtg protein in the livers of females during the gestation of the viviparous teleost, Xenotoca eiseni Although vitellogenesis is arrested during gestation, biochemical assays revealed that Vtg protein was present in ovarian tissues and lumen fluid. The Vtg protein was also detected in the trophotaeniae of the intraovarian embryo. Immunoelectron microscopy revealed that Vtg protein is absorbed into intracellular vesicles in the epithelial cells of the trophotaeniae. Furthermore, extraneous Vtg protein injected into the abdominal cavity of a pregnant female was subsequently detected in the trophotaeniae of the intraovarian embryo. Our data suggest that the yolk protein is one of the matrotrophic factors supplied from the mother to the intraovarian embryo during gestation in X. eiseni.

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost (Xenotoca eiseni).

Nutrient transfer from mother to embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni (family Goodeidae), the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trophotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Expression and antimicrobial activity of liver-expressed antimicrobial peptides in the ovaries of the viviparous teleost Xenotoca eiseni.

The mechanism via which the mothers of viviparous animals regulate the internal environment of pregnancy-associated organs for maintaining offspring growth is poorly understood. Environmental niches in organs contain fluid components for supporting embryonic growth; however, they may serve as nutrients for microbes. Therefore, microbial control is essential in viviparous animals to reduce the risk of infection in the ovarian lumen. Its importance may be higher than that in the case of oviparous animals. In this study, we investigated the antimicrobial factors in a viviparous teleost, Xenotoca eiseni. Four transcripts of the liver-expressed antimicrobial peptide (LEAP) were identified via RNA-Seq analysis. Some of the genes were expressed in the ovaries or intraovarian embryos of the fish. In particular, high expression of leap1a was detected in the ovaries of both pregnant and non-pregnant fish. Moreover, the ovary extracts from X. eiseni and transformed leap genes exhibited antimicrobial activity against Escherichia coli. Our results suggest that viviparous teleosts utilize antimicrobial peptides to reduce the risk of infection in the ovarian lumen.

Distinct interferon response in bat and other mammalian cell lines infected with Pteropine orthoreovirus.

Bats serve as natural hosts of Pteropine orthoreovirus (PRV), an emerging group of bat-borne, zoonotic viruses. Bats appear to possess unique innate immune system responses that can inhibit viral replication, thus reducing clinical symptoms. We examined the innate immune response against PRV and assessed viral replication in cell lines derived from four bat species (Miniopterus fuliginosus, Pteropus dasymallus, Rhinolophus ferrumequinum, and Rousettus leschenaultii), one rodent (Mesocricetous auratus), and human (Homo sapiens). The expression levels of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferons (IFNB1 and IFNL1) were higher and PRV replication was lower in cell lines derived from M. fuliginosus, R. ferrumequinum, and R. leschenaultii. Reduction of IFNB1 expression by the knockdown of PRRs in the cell line derived from R. ferrumequinum was associated with increased PRV replication. The knockdown of RIG-I led to the most significant reduction in viral replication for all cell lines. These results suggest that RIG-I production is important for antiviral response against PRV in R. ferrumequinum.

Sharply Angled Lateral Curvature Modification in Anal Fin of a Viviparous Fish, Xenotoca variata (Cyprinodontiformes: Goodeidae).

Almost all viviparous species possess male external genitalia; for example, the mammalian penis is an intromittent organ. Some live-bearing bony fish use their anal fins to assist in mating and internal fertilization. We previously reported a male-specific asymmetric curvature at the posterior end of the anal fin in Xenotoca eiseni, a viviparous fish of the family Goodeidae. However, three other goodeid species, Xenotoca melanosoma, Zoogoneticus quitzeoensis, and Chapalichthys pardalis, examined in that study possessed lesser anal fin curvature modifications as compared to those in the anal fin of X. eiseni. Here, we report the second case of acute-angled curvature modification of the male anal fin in the family Goodeidae. We obtained a dead specimen of the goodeid species Xenotoca variata from a city zoo in Japan, and the morphological and histological analyses indicated an acute-angled asymmetric curvature of the posterior end of the anal fin in X. variata, similar to that observed in X. eiseni in the previous study. However, in our previous report, obtuse-angled modification was only observed in one other Xenotoca species, X. melanosoma, and two species belonging to other genera, Z. quitzeoensis and C. pardalis. Therefore, our findings suggest that the acute-angled curvature in the male anal fin has been developed in the genus Xenotoca.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Generation and evaluation of a transgenic zebrafish for tissue-specific expression of a dominant-negative Rho-associated protein kinase-2.

The Ras homologous (Rho) proteins are a family of small GTPases, which regulate the cytoskeleton and are related to stress fibers and focal adhesion. The Rho-associated protein kinases (ROCK) constitute part of the Rho effectors that regulate cell shape and movement via phosphorylation of the myosin light chain and actin depolymerizing factor/cofilin. ROCK members are widely expressed and play roles in various cell types during vertebrate development and morphogenesis; therefore, ROCK-knockout animals exhibit multiple defects mostly initiated at the embryonic stage. Analyzing the distinct roles of ROCK in cell shape and movement during the embryonic stages using live mammalian models is difficult. Here, we inhibited the Rho/ROCK pathway in zebrafish, which is a small fish that can be conveniently used as a developmental animal model in place of mammals. To inhibit the Rho/ROCK pathway, we designed a dominant-negative ROCK-2 (dnROCK-2) that lacked the kinase domain and was under the control of an upstream activation sequence (UAS). To evaluate the effects of expression of dnROCK-2, transgenic zebrafish lines were generated by mating strains expressing the construct with counterpart strains expressing the Gal4 activator in target tissues. In this study, we crossed the dnROCK-2-expressing line with two such Gal4-expressing lines; (1) SAGFF(LF)73A for expression in the whole body, and (2) Tg(fli1a: Gal4FF)ubs4 for endothelial cell-specific expression. The phenotypes of the fish obtained were observed by fluorescent stereomicroscopy or confocal microscopy. Overexpression of dnROCK-2 in the whole body resulted in an inhibition of development, notably in cephalic formation, at 1-day post-fertilization (dpf). Confocal microscopy revealed that Hensen's zone became unclear in the trunk muscle fibers expressing dnROCK-2. Endothelial cell-specific expression of dnROCK-2 caused abnormalities in cardiovascular formation at 2-dpf. These results suggest that dnROCK-2 can act as a dominant negative construct of the Rho/ROCK pathway to affect regulation of the cytoskeleton. This construct could be a convenient tool to investigate the function of ROCK members in other vertebrate cell types.

Role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and Japanese encephalitis virus infection.

Bats are potential natural hosts of Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV). Bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. Here, kidney epithelial cell lines derived from four bat species (Pteropus dasymallus, Rousettus leschenaultii, Rhinolophus ferrumequinum, and Miniopterus fuliginosus) and two non-bat species (Homo sapiens and Mesocricetus auratus) were infected with EMCV and JEV. The replication of EMCV and JEV was lower in the bat cell lines derived from R. leschenaultii, R. ferrumequinum, and M. fuliginosus with a higher expression level of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferon-beta (IFN-ß) than that in the non-bat cell lines and a bat cell line derived from P. dasymallus. The knockdown of TLR3, RIG-I, and MDA5 in Rhinolophus bat cell line using antisense RNA oligonucleotide led to decrease IFN-ß expression and increased viral replication. These results suggest that TLR3, RIG-I, and MDA5 are important for antiviral response against EMCV and JEV in Rhinolophus bats.

The complete genomic sequence of Rhinolophus gammaherpesvirus 1 isolated from a greater horseshoe bat.

In a comprehensive research project on bat viruses, we successfully isolated a novel herpesvirus from the spleen of a greater horseshoe bat (Rhinolophus ferrumequinum) in Japan using a cell line established from the kidney of the same bat. This herpesvirus was a novel gammaherpesvirus (Rhinolophus gammaherpesvirus 1; RGHV-1), which belonged to the genus Percavirus. The whole RGHV-1 genome (147,790 bp) showed that 12 of the 84 genes predicted to contain open reading frames did not show any homology to those of other herpesviruses.

Isolation of Pteropine orthoreovirus from Pteropus vampyrus in Garut, Indonesia.

Flying foxes belonging to the genus Pteropus are known to be reservoirs of zoonotic viruses. In this study, we describe the isolation of Pteropine orthoreovirus (PRV) from rectal swab samples of Pteropus vampyrus in Indonesia. PRV is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. Rectal swabs (n = 91) were screened by PCR for PRV and 10 (11%) were positive. Phylogenetic analysis based on nucleotide sequences indicated that the S2, S3, S4, M3, L2, and L3 segments of one isolate (Garut-69) were closely related to previously isolated strains in Indonesia. The remaining gene segments showed both similarity and genetic divergence with other PRV strains, suggesting that re-assortment events had occurred. This is the first report of PRV infection to P. vampyrus in West Java, Indonesia.

The LIF-mediated molecular signature regulating murine embryo implantation.

The establishment of a receptive uterus is the prime requirement for embryo implantation. In mice, the E2-induced cytokine leukemia inhibitory factor (LIF) is essential in switching the uterine luminal epithelium (LE) from a nonreceptive to a receptive state. Here we define the LIF-mediated switch using array analysis and informatics to identify LIF-induced changes in gene expression and annotated signaling pathways specific to the LE. We compare gene expression profiles at 0, 1, 3, and 6 h, following LIF treatment. During the first hour, the JAK-STAT signaling pathway is activated and the expression of 54 genes declines, primarily affecting LE cytoskeletal and chromatin organization as well as a transient reduction in the progesterone, TGFbetaR1, and ACVR1 receptors. Simultaneously 256 genes increase expression, of which 42 are transcription factors, including Sox, Kfl, Hes, Hey, and Hox families. Within 3 h, the expression of 3987 genes belonging to more than 25 biological process pathways was altered. We confirmed the mRNA and protein distribution of key genes from 10 pathways, including the Igf-1, Vegf, Toll-like receptors, actin cytoskeleton, ephrin, integrins, TGFbeta, Wnt, and Notch pathways. These data identify novel LIF-activated pathways in the LE and define the molecular basis between the refractory and receptive uterine phases. More broadly, these findings highlight the staggering capacity of a single cytokine to induce a dynamic and complex network of changes in a simple epithelium essential to mammalian reproduction and provide a basis for identifying new routes to regulating female reproduction.

Kisspeptin neurons mediate reflex ovulation in the musk shrew (Suncus murinus).

The present study investigated whether kisspeptin-G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew (Suncus murinus), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29-induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1-expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1-expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1-positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin-GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats.

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Electric organ discharge from electric eel facilitates DNA transformation into teleost larvae in laboratory conditions.

Background: Electric eels (Electrophorus sp.) are known for their ability to produce electric organ discharge (EOD) reaching voltages of up to 860 V. Given that gene transfer via intense electrical pulses is a well-established technique in genetic engineering, we hypothesized that electric eels could potentially function as a gene transfer mechanism in their aquatic environment. Methods: To investigate this hypothesis, we immersed zebrafish larvae in water containing DNA encoding the green fluorescent protein (GFP) and exposed them to electric eel's EOD. Results and Discussion: Some embryos exhibited a mosaic expression of green fluorescence, in contrast to the control group without electrical stimulation, which showed little distinct fluorescence. This suggests that electric eel EOD has the potential to function as an electroporator for the transfer of DNA into eukaryotic cells. While electric eel EOD is primarily associated with behaviors related to sensing, predation, and defense, it may incidentally serve as a possible mechanism for gene transfer in natural environment. This investigation represents the initial exploration of the uncharted impact of electric eel EOD, but it does not directly establish its significance within the natural environment. Further research is required to understand the ecological implications of this phenomenon.

Glucose absorption activity and gene expression of sugar transporters in the trophotaenia of the viviparous teleost Xenotoca eiseni.

In viviparous reproductive systems, nutrient transfer from mother to embryo plays a critical role in the generation of offspring. Herein, we investigated the mother-to-embryo nutrient transfer machinery in the viviparous teleost Xenotoca eiseni, which belongs to the family Goodeidae. The intraovarian embryo absorbs maternal supplements via the hindgut-derived placental structure termed the trophotaenia. Tracer analysis indicated that the trophotaenia can take up glucose analogs in ex vivo cultured embryos. The candidate genes for absorption, sglt1, glut2, atp1a, and atp1b, were determined from published transcriptomes. These genes were expressed in the trophotaenia of X. eiseni embryos. Fluorescent immunohistochemistry of Na+/K+ ATPase indicated the polarity of epithelial cells in the trophotaenia. The presented evidence suggests that the epithelial cell layer transports monosaccharides from the apical membrane of epithelial cells in a basolateral direction. Taken together, this study provides insight into how maternal fish maintain their offspring during gestation and will aid in the development of strategies to improve offspring generation in these fish.

The complete control of murine pregnancy from embryo implantation to parturition.

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17ß-estradiol (E(2)). Continuous exposure to E(2) was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E(2) and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E(2) after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived placenta of the viviparous teleost Xenotoca eiseni.

Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotaenia, hindgut-derived placental structure, to receive the maternal supplement. However, research on the molecular mechanisms underlying viviparous species is scarce in non-mammalian vertebrates, including teleosts. Thus, we conducted this study to investigate the mechanism for nutrient absorption and degradation in trophotaeniae of X. eiseni. A tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of the trophotaeniae. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in the trophotaeniae. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in the trophotaeniae. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived placenta.