Blastocyst development after fertilization with in vitro spermatids derived from nonhuman primate embryonic stem cells

Sujittra Khampang, In Ki Cho, Kanchana Punyawai, Brittany Gill, Jacqueline N. Langmo, Shivangi Nath, Katherine W. Greeson, Krista M. Symosko, Kristen L. Fowler, Siran Tian, John P. Statz, Alyse N. Steves, Rangsun Parnpai, Michael A. White, Jon D. Hennebold, Kyle E. Orwig, Calvin R. Simerly, Gerald Schatten, Charles A. Easley

    Research output: Contribution to journalArticlepeer-review

    1 Scopus citations

    Abstract

    Objective: To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. Design: Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. Setting: Multiple academic laboratory settings. Patient(s): Not applicable. Intervention(s): Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measure(s): Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. Result(s): Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. Conclusion(s): This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.

    Original languageEnglish (US)
    Pages (from-to)365-375
    Number of pages11
    JournalF and S Science
    Volume2
    Issue number4
    DOIs
    StatePublished - Nov 2021

    Keywords

    • In vitro spermatogenesis
    • TET3
    • blastocysts
    • round spermatids

    ASJC Scopus subject areas

    • Reproductive Medicine
    • Embryology

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