Mother-to-embryo vitellogenin transport in a viviparous teleost Xenotoca eiseni Atsuo Iidaa,b,1, Hiroyuki N. Araib, Yumiko Someyac, Mayu Inokuchic, Takeshi A. Onumad, Hayato Yokoie, Tohru Suzukie, Eiichi Hondoa, and Kaori Sanof aDepartment of Animal Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan; bDepartment of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Shogo-in Kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan; cDepartment of Life Sciences, Toyo University, Itakura, Gunma 374-0113, Japan; dDepartment of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan; eLaboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8577, Japan; and fDepartment of Chemistry, Faculty of Science, Josai University, Sakado, Saitama 350-0295, Japan Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved September 19, 2019 (received for review July 30, 2019) Vitellogenin (Vtg), a yolk nutrient protein that is synthesized in the in nutrient incorporation during gestation. That consists of ab- livers of female animals, and subsequently carried into the ovary, sorptive epithelium, blood vessels, and mesenchymal connective contributes to vitellogenesis in oviparous animals. Thus, Vtg levels tissues (12, 13). Previous studies hypothesized that the ovarian are elevated during oogenesis. In contrast, Vtg proteins have been fluid components are absorbed into the epithelial cells and then genetically lost in viviparous mammals, thus the yolk protein is not carried into the capillary (12, 14). In some goodeid species, the involved in their oogenesis and embryonic development. In this ovarian fluids include multiple protein components in a similar study, we identified Vtg protein in the livers of females during the pattern to that in the blood serum (15, 16). In Xenotoca eiseni,the gestation of the viviparous teleost, Xenotoca eiseni. Although vi- viviparous goodeid species used in this study, the intraovarian tellogenesis is arrested during gestation, biochemical assays embryo develops for approximately 5 wk before birth. The em- revealed that Vtg protein was present in ovarian tissues and lu- bryo’s growth depends on matrotrophic nutrition absorbed from men fluid. The Vtg protein was also detected in the trophotaeniae the trophotaeniae for most of its intraovarian development (17– of the intraovarian embryo. Immunoelectron microscopy revealed 19). The trophotaeniae are regressed by apoptosis during the PHYSIOLOGY that Vtg protein is absorbed into intracellular vesicles in the epi- latest stage of gestation (20); they are no longer required in the thelial cells of the trophotaeniae. Furthermore, extraneous Vtg postnatal growth phase when oral food intake occurs. The com- protein injected into the abdominal cavity of a pregnant female ponents of the maternal nutrients absorbed from the tropho- was subsequently detected in the trophotaeniae of the intraovarian taeniae have not been identified but are hypothesized to be embryo. Our data suggest that the yolk protein is one of the matro- secreted blood serum proteins. trophic factors supplied from the mother to the intraovarian embryo Vitellogenin (Vtg) is a glycolipophosphoprotein typically X. eiseni during gestation in . present in females, but also in minor amounts in males, that is conserved in nearly all oviparous species including fish, am- Goodeidae | reproduction | viviparity | trophotaeniae phibians, reptiles, birds, monotremes, and most invertebrates (21–24). In these vertebrates, vtg genes are typically expressed iviparity is a form of reproductive system in which fertiliza- and synthesized in female liver, then the protein product is Vtion and embryonic development progress inside the moth- transported to the ovary via the bloodstream. In the ovary, Vtg er’s body prior to the birth, as opposed to oviparity where such protein is cleaved into subdomains and absorbed into the egg developmental events are completed outside of the parent. In some viviparous vertebrates, the embryo receives nutrients from Significance the mother in addition to those in the yolk sac. The nutrient supply and embryonic growth in the mother’s body are important Viviparity is a type of reproductive system in which the embryo factors influencing survival in the habitat environment after the utilizes a maternal nutrient supply until birth. Viviparous species birth (1). In eutherian mammals, maternal nutrients including occur in many taxa including bony fish. The mother fish raises blood plasma pass into the fetus via a placenta and umbilical her offspring in the ovarian lumen or oviduct during gestation. cords (2). Viviparous species also occur in reptiles, amphibians, Embryos of the viviparous bony fish Xenotoca eiseni (family and fish (3–5). The embryos of some amphibians and sharks eat Goodeidae) utilize nutrients secreted into the ovarian lumen. each other and develop within the mother’s body (6, 7). A vi- However, the source of the maternal nutrients and their mother- viparous reptile possesses a placenta-like structure similar to to-embryo transport have not been experimentally demon- those found in eutherians (8). Some viviparous chondrichthyans strated. In this study, we focus on the yolk nutrient protein raise their offspring in the uterus filled with a nutrient-rich liquid vitellogenin (Vtg) as a matrotrophic factor. Our results are of during gestation (9, 10). In viviparous teleosts, the mother fish fundamental importance to the investigation of viviparous sys- maintains her embryo in the ovarian lumen or oviduct (5). Di- tems in teleosts and in other vertebrates and invertebrates. verse mechanisms of embryo growth among viviparous verte- brates have been described. However, the nature of the maternal Author contributions: A.I. and K.S. designed research; A.I., H.N.A., Y.S., M.I., T.A.O., and nutrients in viviparous teleosts has not been identified. E.H. performed research; H.Y., T.S., and K.S. contributed new reagents/analytic tools; A.I. The teleost family Goodeidae is currently known to include 42 analyzed data; and A.I. wrote the paper. viviparous species distributed in the lakes and rivers of México The authors declare no competing interest. (11). Their embryo increases in dry mass in the mother’s body This article is a PNAS Direct Submission. during the gestation, suggesting that maternal nutrients are This open access article is distributed under Creative Commons Attribution-NonCommercial- supplied in addition to those in the yolk sac (5). In the mid to late NoDerivatives License 4.0 (CC BY-NC-ND). gestation stages, the goodeid embryo possesses a pseudoplacenta, 1To whom correspondence may be addressed. Email: [email protected]. called the trophotaeniae. The trophotaeniae form through hy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pertrophic development of elongated processes that extend from 1073/pnas.1913012116/-/DCSupplemental. the hindgut out through the cloaca and are thought to be involved First published October 8, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1913012116 PNAS | October 29, 2019 | vol. 116 | no. 44 | 22359–22365 Downloaded by guest on September 27, 2021 yolk (25, 26). In eutherian mammals, vtg genes have been ge- arrested at previtellogenesis stages, thus there are no mature eggs netically lost through coevolution with casein genes, thus few in the ovarian lumen (Fig. 1B) and the ovary is dedicated to raising yolk nutrients are included in their eggs (22, 27). In contrast, the embryo via nutrient supply. To investigate expression of some viviparous fish species have maintained vtg genes, and a vitellogenin genes (vtgA, vtgB,andvtgC)inX. eiseni, RT-PCR large amount of yolk nutrient is contained in their eggs (28, 29). analysis was performed using cDNA purified from the liver and In these fish, Vtg protein is a potential candidate for one of the gonad. In vitellogenic females, but not in male fish, vtgA and B maternal nutrients supplied into the intraovarian embryo. were strongly expressed in the liver. In contrast, there were no However, there is no definitive evidence that maternal Vtg detectable signals in testis and ovary. Expression of vtgC was protein is transported into the intraovarian embryo. In this study, minimal in all tissues examined in this study (SI Appendix, Fig. we investigated Vtg transport from mother to embryo in the S1A). Expression of the vtg genes in the liver was not decreased in goodeid species, X. eiseni, and our results suggest that the yolk pregnant females, despite the cessation of vitellogenesis (Fig. 1C). protein lost in eutherian mammals is one of the matrotrophic In the ovary, vtg genes were not expressed during gestation, as for factors supplied to the intraovarian embryo during gestation in the vitellogenesis stage (Fig. 1C and SI Appendix, Fig. S1A). VtgA the viviparous teleost. and B proteins were detectable by immunohistochemistry in the nonpregnant female liver (SI Appendix,Fig.S1B). The strong Results signals for Vtg proteins in female liver were replicated using an- Production and Distribution of Vitellogenin Proteins during Gestation. other antibody, anti-Vtg#1 (SI Appendix,Fig.S2A and Table S1). At the onset of gestation in goodeid species including X. eiseni In contrast, specific signals for Vtg protein in male liver were