Mitochondrial matters: Mitochondrial bottlenecks, self-assembling structures, and entrapment in the female germline

Abstract

Mitochondrial replacement therapy, a procedure to generate embryos with the nuclear genome of a donor mother and the healthy mitochondria of a recipient egg, has recently emerged as a promising strategy to prevent transmission of devastating mitochondrial DNA diseases and infertility. The procedure may produce an embryo that is free of diseased mitochondria. A recent study addresses important fundamental questions about the mechanisms underlying maternal inheritance and translational questions regarding the transgenerational effectiveness of this promising therapeutic strategy. This review considers recent advances in our understanding of maternal inheritance of mitochondria, implications for fertility and mitochondrial disease, and potential roles for the Balbiani body, an ancient oocyte structure, in mitochondrial selection in oocytes, with emphasis on therapies to remedy mitochondrial disorders.

Figure. 1. MitoDNA transfer procedure/timing relative to oocyte bottlenecks

A) Schematic depicting the stages of oogenesis and the timing of mitochondrial bottlenecks and amplification in oocytes and early embryos. B) Schematics depict ideal mitochondrial replacement procedure to generate a healthy embryo free of diseased mitochondria, and the observed mitochondrial contamination during the transfer, which can lead to heteroplasmy and cell type specific disease or reversion to homoplasmy.

Figure. 2. Germline development and asymmetries in early oogenesis and potential mitochondrial bottleneck in zebrafish

A) Cartoons depicting the stages of germ cell development beginning with the primordial germ cells (PGCs) in larvae. Cellular asymetries have been observed in mitotic oogonia of female zebrafish. Asymmetric Bucky ball protein (light blue) is detected at zygotene stage, and is required for Balbiani body formation during prophase I (this period corresponds to stage I oocytes in zebrafish). The Balbiani body translocated to the prospective vegetal cortex and then disassembles in stage II ooctyes. B) Schematic graph depicting potential mitochondrial bottleneck and purifying selection stages in zebrafish. Grey dashed lines indicate stages/periods of oogenesis analyzed by Boke and colleagues. In their study, they observed a dramatic increase in mitochondrial DNA copy number between PGC stages and stage I oocytes, a smaller increase between stage I and stage II oocytes and mature oocytes, and a decline in embryos (black lines). Because stage I oocytes are diverse in terms of cytological stage of prophase I and phase of Balbiani body development the precise stage when the bottleneck and purifying selection, and how it relates to polarity in mitochondrial distribution in oogonia (red line), and asymmetric enrichment of Bucky ball protein (purple line) and Balbiani body formation and enrichment of mitochondria there is not known.

Figure. 3. Comparison of Bucky ball/XVelo/Oskar functional domains

A) Schematic depicting the protein domains of Xenopus Velo and zebrafish Bucky ball. The amino terminus harbors a prion-like domain (PLD), also known as the BUVE (Bucky ball Velo domain) that is essential for assembly of an amyloid-like matrix. The Carboxy terminus is K/R rich and has properties suggestive of RNA binding function. The C-terminus is required for Balbiani body assembly in zebrafish, including recruiting RNAs and mitochondria, and for entrapment of mitochndria and RNAs in XVelo assembled amyloid-like matrix in Xenopus oocytes and reconstitution assays. The PLD and KR rich region are separated by an intrinsically disorded region. B) Schematic depicting long and short forms of Drosophila Oskar proteins. The Amino termimal extension (NTE) of the long isoform is essential for nucleating actin filaments and anchoring of germ plasm components to the posterior cortex in oocytes. The LOTUS domain mediates Oskar dimerization and interactions with protein binding partners, like Vasa. The actin binding protein Lasp binds to the disordered region. The OSK domain is composed of basic and hydrophilic amino acids and is thought to mediate interactions with the RNA effectors of Oskar. Short Oskar activity and stabiity are regulated by posttranslational modifications, including phosphorylation and ubiquitination (not shown). Although long Oskar has the LOTUS and OSK domains it does not bind to Vasa nor does it bind to RNAs. The NTE is thought to inhibit these activities.

Figure. 4. Mitochondria asymmetry, Balbiani body formation, and germ plasm assembly as potential oocyte bottlenecks

A) Schematic depicts an early zebrafish oocyte prior to Balbiani body formation. Buc protein is asymmetric next to the nucleus, and mitochondria are asymmetrically distributed in oocytes in a Buc-independent manner at this stage. Buc is required for Balbiani body assembly and recruitment of the most active mitochondria (green) there. RNAs and proteins, including RNA binding proteins (like Dazl and Rbpms2) are also recruited to the Balbiani body at this time. Although the mechanism of recruitment is not understood, a mechanism whereby Buc forms a self-assembling network that can recruit germ plasm (GP) components directly, and through conserved interactions with RNA binding proteins has been proposed for Balbiani body assembly in zebrafish and Xenopus oocytes. Analysis of zebrafish maternal-effect mutants and overexpression assays indicates that Buc/XVelo coordinate recruitment, possibly by entrapment, of mitochondria and RNAs to the nonmembrane bound Balbiani body. B) In Drosophila, one of 16 cyst cells is specified as the oocyte. Mitochondria are transferred to the oocyte in a Kinesin and Milton dependent manner to form the Balbiani body. At this stage, oskar RNA is present, but is translationally repressed; therefore, if recruitment of mitochondria at this stage requires oskar this would be an RNA function rather than a coding function of oskar. In mid-oogenesis, oskar RNA is anchored at the posterior pole and is translated there. Once translated, short Oskar mediates germ plasm assembly components, including mitochondria via self-assembly and interactions with its RNA and protein effectors, including RNA binding proteins that lead to entrapment of components within the germ plasm. Tudor (Tud) binds to mitochondria and RNA, but is not required to localize mitochondria to the posterior pole. Therefore, in Drosophila recruitment of mitochondria and germ plasm RNAs to nonmembrane bound compartments can be uncoupled by Oskar downstream effectors. Balbiani body formation is a conserved feature of oocyte development and analysis of mitochondria indicates bottlenecks within these stages; however, it remains to be determined if selection at either of these stages influences mitochondrial content of oocytes or the embryonic germline.