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

Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the 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 trohotaenia 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.

New Insights on Folliculogenesis and Follicular Placentation in Marine Viviparous Fish Black Rockfish (Sebastes Schlegelii)

Background: In viviparous fish, a considerable degree of variation in placental structures have been described. However, no distinct structures are reported in Scorpaenidae.Results: In this study, we demonstrate a new type of folliculogenesis and follicular placentation in Sebastes schlegelii. Before copulation, the germinal epithelium gradually surrounds the oocytes and develops to individually follicles with a stalk-like structure hanging on the ovigerous lamella, which ensures each follicle have access to spermatozoa after copulation. From stage V to early gestation, the cyp17-I highly expressed accompanied by cyp19a1a signals disappearance, and 11-ketotestosterone level keeps rising and peaks at blastula stage, while 17β-estradiol declines to the bottom. Meanwhile, the theca cells rapidly proliferate and invade outwards forming a highly hypertrophied and folded microvillous placenta. This unbalance of hormone might be an important factor driving the theca cells proliferation and invasion. Additionally, some conserved genes related to mammalian placentation are significantly high expression in follicular placenta suggesting the high convergence in vertebrate placenta evolution.Conclusions: This finding provided a new type of placentation pattern for viviparous teleost between the intrafollicular gestation and intraluminal gestation.

Embryonic Onset of Sexually Dimorphic Heart Rates in the Viviparous Fish, Gambusia holbrooki

In fish, little is known about sex-specific differences in physiology and performance of the heart and whether these differences manifest during development. Here for the first time, the sex-specific heart rates during embryogenesis of Gambusia holbrooki, from the onset of the heart rates (HRs) to just prior to parturition, was investigated using light cardiogram. The genetic sex of the embryos was post-verified using a sex-specific genetic marker. Results reveal that heart rates and resting time significantly increase (p < 0.05) with progressive embryonic development. Furthermore, both ventricular and atrial frequencies of female embryos were significantly higher (p < 0.05) than those of their male sibs at the corresponding developmental stages and remained so at all later developmental stages (p < 0.05). In concurrence, the heart rate and ventricular size of the adult females were also significantly (p < 0.05) higher and larger respectively than those of males. Collectively, the results suggest that the cardiac sex-dimorphism manifests as early as late-organogenesis and persists through adulthood in this species. These findings suggest that the cardiac measurements can be employed to non-invasively sex the developing embryos, well in advance of when their phenotypic sex is discernible. In addition, G. holbrooki could serve as a better model to study comparative vertebrate cardiovascular development as well as to investigate anthropogenic and climatic impacts on heart physiology of this species, that may be sex influenced.

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

Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the 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 trohotaenia 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.

New insights on folliculogenesis and follicular placentation in marine viviparous fish black rockfish (Sebastes schlegelii)

In viviparous fish, a considerable degree of variation in placental structures have been described. However, no distinct structures are reported in Scorpaenidae. In this study, we demonstrate a new type of folliculogenesis and follicular placentation in Sebastes schlegelii. Before copulation, the germinal epithelium gradually surrounds the oocytes and develops to individually follicles with a stalk-like structure hanging on the ovigerous lamella, which ensures each follicle have access to spermatozoa after copulation. From stage V to early gestation, the cyp17-I highly expressed accompanied by cyp19a1a signals disappearance, and 11-ketotestosterone level keeps rising and peaks at blastula stage, while 17β-estradiol declines to the bottom. Meanwhile, the theca cells rapidly proliferate and invade outwards forming a highly hypertrophied and folded microvillous placenta. This unbalance of hormone might be an important factor driving the theca cells proliferation and invasion. Additionally, some conserved genes related to mammalian placentation are significantly high expression in follicular placenta suggesting the high convergence in vertebrate placenta evolution.

Multiple Fetal Nutritional Patterns Before Parturition in Viviparous Fish Sebastes schlegelii (Hilgendorf, 1880)

Sebastes schlegelii is a commercially important fish with a special viviparous reproductive system that is cultured in near-shore seawater net cages in East Asia. In the gonadal development of the species, the gonad of males mature before those of females, which mature after mating. Mating in male/female fishes occurs in October of each year. Then, females undergoing oocyte maturation complete fertilization using stored sperm in March of the following year. The pregnancy is completed when larvae are produced in the ovary. It has been reported that embryonic nutrient supply originates entirely from the female viviparous reproductive systems. However, until now, the nutritional patterns and the processes of nutrient provision in S. schlegelii before parturition have not been clear. The goal of this research was to study the embryos, larvae and juveniles of S. schlegelii during pregnancy. Anatomical observations, light microscopy and scanning electron microscopy were used to study the developmental characteristics of early embryos and larvae and the connecting structures between the mother and the fetus. The results showed the following: (1) Placental-like structures were found during the process of embryonic development in S. schlegelii, and these placental-like structures proliferated after fertilization. (2) The embryos of S. schlegelii were encased by a saclike structure composed of blood vessels, connective tissue, and surface epithelial cells. The vessels near the embryo existed in the thecal layer. Vascularized proliferation was detected following embryonic development. (3) Starting in the gastrula stage, connections between the embryo and surrounding cells loosened, and ovarian fluid became abundant. In addition, a large number of small holes and cristae were observed on the surface of the embryo. We speculate that embryos may be able to absorb nutrients from the ovarian fluid. (4) Yolk was present throughout embryo development. (5) Two types of nutritional modes were observed, lecithotrophic and matrotrophic during embryonic development. Three forms of placental analogs may exist in S. schlegelii: (1) external epithelial absorptive surfaces; (2) trophonemata, with modifications of the ovarian epithelia for absorbing the histotroph; and (3) a follicular pseudoplacenta, with close apposition between follicle cells and embryonic absorptive epithelia.

Reproductive cycle of native viviparous fish species (Actinopterygii: Cyprinodontiformes: Goodeidae) in a subtropical Mexican lake

Reproductive tactics and strategies contribute to the persistence and maintenance of long-term populations in fish species. Members of the subfamily Goodeinae are a group of small-bodied freshwater fish with specialized reproduction (viviparity-matrotrophy). They are found in the highlands of central Mexico, most of them endemic. The aim of this study was to conduct a comprehensive investigation to evaluate the annual reproductive cycle of seven species of goodeines (splitfins). We carried out our study in the subtropical Lake Zacapu, Mexico, with bi-monthly sampling from May 2019 to March 2020. We obtain the fertility, size at first maturity (L50), sex ratio, and gonadosomatic index. Our result shows that populations of goodeines have high fertility compared to other populations of the same species in other aquatic systems and also to other species of goodein. We found that males mature at smaller sizes than females, the observed proportion of females was greater than males in all the goodeines. Lake Zacapu goodeines have two reproductive peaks, one in spring (April to June) and another in fall (September to November). These tactics (fertility rates, sex ratio, reproductive period) and strategies (viviparity-matrotrophy) favor reproductive success in this environmentally stable subtropical lake in the highlands of Mexico.