Structural and functional organization of germ plasm condensates.

Reproductive success of metazoans relies on germ cells. These cells develop early during embryogenesis, divide and undergo meiosis in the adult to make sperm and oocytes. Unlike somatic cells, germ cells are immortal and transfer their genetic material to new generations. They are also totipotent, as they differentiate into different somatic cell types. The maintenance of immortality and totipotency of germ cells depends on extensive post-transcriptional and post-translational regulation coupled with epigenetic remodeling, processes that begin with the onset of embryogenesis [1, 2]. At the heart of this regulation lie germ granules, membraneless ribonucleoprotein condensates that are specific to the germline cytoplasm called the germ plasm. They are a hallmark of all germ cells and contain several proteins and RNAs that are conserved across species. Interestingly, germ granules are often structured and tend to change through development. In this review, we describe how the structure of germ granules becomes established and discuss possible functional outcomes these structures have during development.

RNA Granules: A View from the RNA Perspective.

RNA granules are ubiquitous. Composed of RNA-binding proteins and RNAs, they provide functional compartmentalization within cells. They are inextricably linked with RNA biology and as such are often referred to as the hubs for post-transcriptional regulation. Much of the attention has been given to the proteins that form these condensates and thus many fundamental questions about the biology of RNA granules remain poorly understood: How and which RNAs enrich in RNA granules, how are transcripts regulated in them, and how do granule-enriched mRNAs shape the biology of a cell? In this review, we discuss the imaging, genetic, and biochemical data, which have revealed that some aspects of the RNA biology within granules are carried out by the RNA itself rather than the granule proteins. Interestingly, the RNA structure has emerged as an important feature in the post-transcriptional control of granule transcripts. This review is part of the Special Issue in the Frontiers in RNA structure in the journal Molecules.

The Folding of Germ Granule mRNAs Controls Intermolecular Base Pairing in Germ Granules and Maintains Normal Fly Development.

Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration, creating an elevated potential for intermolecular base pairing. However, the type and abundance of intermolecular base pairing in mRNA clusters is poorly characterized. Using single-molecule super-resolution microscopy, chemical probing for base accessibility, phase separation assays, and simulations, we demonstrated that mRNAs remain well-folded upon localization to germ granules. While most base pairing is intramolecular, mRNAs still display the ability for intermolecular base pairing, facilitating clustering without high sequence complementarity or significant melting of secondary structure. This base pairing among mRNAs is driven by scattered and discontinuous stretches of bases appearing on the surface of folded RNAs, providing multivalency to clustering but exhibits low probability for sustained interactions. Notably, engineered germ granule mRNAs with exposed GC-rich complementary sequences (CSs) presented within stable stem loops induce sustained base pairing in vitro and enhanced intermolecular interactions in vivo. However, the presence of these stem loops alone disrupts fly development, and the addition of GC-rich CSs exacerbates this phenotype. Although germ granule mRNAs contain numerous GC-rich CSs capable of stable intermolecular base pairing, they are primarily embedded by RNA folding. This study emphasizes the role of RNA folding in controlling the type and abundance of intermolecular base pairing, thereby preserving the functional integrity of mRNAs within the germ granules.

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

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. PATIENTS: Not applicable. INTERVENTIONS: Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. MAIN OUTCOME MEASURES: 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. RESULTS: 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. CONCLUSIONS: 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.