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

Abstract

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.

Keywords: Goodeidae; reproduction; trophotaeniae; viviparity.

Fig. 1.

Vitellogenin synthesis and supply to ovary during gestation. (A) Whole body photograph of adult X. eiseni. The adult male fish exhibits sex-specific characteristics, such as a high body arch and red color in the tail. (Scale bar, 10 mm.) (B) HE-stained transverse sections of vitellogenesis (Upper) and gestation (Lower) stages of an X. eiseni female ovary. In the nonpregnant ovary, mature eggs including yolk were observed in the ovarian lumen, and some postvitellogenic oocytes invaginate to the stroma in the ovarian luminal epithelium. In the pregnant ovary, developing embryos filled the ovarian lumen, and there were no mature eggs with yolk. Some immature oocytes can still be observed in the ovarian luminal epithelium. Arrowheads indicate immature oocytes. (Scale bar, 1 mm [whole ovary and embryo] and 200 µm [immature egg].) (C) RT-PCR for vtg genes in female X. eiseni. All vtg genes were detected in the liver during vitellogenesis. The hepatic expression was also observed in the third week of gestation. No vtg expression was observed in any ovary samples. Nicotinamide adenine dinucleotide (nadh) was used as an internal control. M, size marker. Vit., vitellogenesis. Ges., gestation. (D) Fluorescence immunohistochemistry for VtgA or B in lobe edges of female liver in the third week of gestation. (Scale bar, 20 µm.) (E) Fluorescence immunohistochemistry for VtgA or B in the ovarian septum in the third week of gestation. (Scale bar, 20 µm.) See also SI Appendix, Figs. S1 and S2.

Fig. 2.

Vitellogenin distribution in the trophotaeniae of the intraovarian embryo. (A) RT-PCR for vtg genes in X. eiseni intraovarian embryo in the third week postfertilization. There was no vtg expression in whole embryo and trophotaeniae. Female liver was used as a positive control for the reaction. Nicotinamide adenine dinucleotide (nadh) was used as an internal control. (B) Fluorescence immunohistochemistry for VtgA or B in the trophotaeniae of the intraovarian embryo in the third week postfertilization. E, epithelial layer. C, capillary. M, mesenchyme. (Scale bar, 20 µm.) (C) Immunoelectron microscopy using anti-Vtg#1 for the trophotaeniae of the intraovarian embryo in the third week postfertilization. Enlarged image for epithelium shows specific signals against Vtg in vesicles (arrowheads). Dark spots in the cytoplasm marked with asterisks indicate mitochondria. Enlarged image for mesenchyme shows specific signals on the mesenchymal cell (arrowheads). (Scale bar, 1 µm.) (D) Dual-fluorescence immunohistochemistry for Vtg and fibronectin (a marker for extracellular matrix). (Scale bar, 20 µm.) See also SI Appendix, Fig. S2.

Fig. 3.

Detection of vitellogenin fragments in ovarian fluid and trophotaeniae. (A) Electrophoresis and CBB staining of ovarian fluid and intraovarian embryo lysate extracted from pregnant female fish in the third week after mating. The ovarian fluid showed 2 major protein bands of 240- and 75-kDa (arrow and arrowhead). The 75-kDa band was also observed in the lysate from whole embryo and trophotaeniae. The 240-kDa protein was not detected in the embryo samples. Ova., ovarian. Vit., vitellogenesis. Ges., gestation. (B) Western blotting (using anti-Vtg#1 antibody) of ovarian fluid and intraovarian embryo lysate extracted from pregnant female fish in the third week after mating. The ovarian fluid indicated 2 major signals against Vtg at 240- and 75-kDa (arrow and arrowhead). In the gestation sample, 6 specific signals (240-, 100-, 75-, 55-, 50-, and 45-kDa) were observed as distinct bands. Magenta boxes indicate the signals against Vtg detected in the both ovarian fluid and trophotaeniae. Blue boxes indicate the signals detected only in ovarian fluid. GAPDH was used to check for contamination of cellular components into the ovarian fluid and nonspecific degradation of extracted proteins during the sample collection. See also SI Appendix, Fig. S3.

Fig. 4.

Tracing for mother-to-embryo vitellogenin transfer in live fish. (A) A scheme for the tracing analysis. Mating: To prepare a pregnant female, a pair of mature X. eiseni were mated, and then the male fish was removed after mating. i.p. injection: At 3 wk after mating, fluorescent Vtg solution was injected into the abdominal cavity of the pregnant female. Extraction & observation: At 18 h after injection, the embryo was extracted from the female fish and observed under a fluorescent microscope. (B) Fluorescent microscopy for the injected fluorescent solution in the female fish. The solutions were injected into the abdominal cavity from the gravid spot of pregnant female fish (arrowhead). The blood vessels in the caudal fin were visualized by a green fluorescence for the FITC-Vtg. (Scale bar, 1 mm.) (C) Fluorescent microscopy of the intraovarian embryo. Green fluorescence was detected in the epithelial cell layer of trophotaeniae in the extracted embryo (arrowheads). The green fluorescence was merged to signals for anti-FITC (arrow). E, epithelial layer. M, mesenchyme. (Scale bar, 10 µm.) See also SI Appendix, Figs. S4 and S5.

Fig. 5.

A model for mother-to-embryo vitellogenin transfer during gestation. In the pregnant female, vitellogenin genes are strongly expressed in the liver, and then the proteins are carried to the ovary via the bloodstream. In the ovarian lumen, full-length and fragmented vitellogenin proteins are secreted into the ovarian fluid, and then absorbed into the embryo via the epithelium of the trophotaeniae. In the epithelium of the trophotaeniae, the fragmented vitellogenin proteins are imported into the epithelial cells and carried as macromolecules via vesicle trafficking.