Owing to the colocalization between mitochondria and the

Owing to the colocalization between mitochondria and the germ plasm of some animals, models whereby the Balbiani body selects the healthiest mitochondria for the next generation have been proposed (Cox and Spradling, 2003; Pepling and Spradling, 1998). Early as well as more recent studies thus examined the distribution of mitochondria in oocytes using reporters of mitochondrial respiration to identify more active mitochondria (Wilding et al., 2001; Tworzydlo et al., 2016; Zhang et al., 2008). Consistent with a role in selecting the “most fit” mitochondria, these studies revealed an enrichment of the most active mitochondria within the Balbiani body (Wilding et al., 2001; Tworzydlo et al., 2016; Zhang et al., 2008), but how individual mitochondria are selected remains an open question. It is possible that sequestration of mitochondria within the Balbiani could lead to differential marking of the mitochondria. For example, this could occur by promoting posttranslational modification of a mitochondrial protein, facilitating novel associations with proteins that are spatially restricted to the Balbiani body, or by preventing the sequestered mitochondria from receiving a destruction mark. The question of whether the mitochondria are presorted and then selected based on their activity, or only become activated once localized within the Balbiani body remains to be determined. Moreover, whether the proteins of mitochondria within the Balbiani body are otherwise differentially marked from those outside the Balbiani body has not been demonstrated. Single cell sequencing approaches allow the variation in individual oocytes to be determined and thus provide a picture of population level selection. To capture selection at the level of the organelle in oocytes one would need to pinpoint precisely when the bottleneck occurs and analyze mitochondrial content at stages just before and after the bottleneck. A recent study by Otten and colleagues measured mitochondrial copy number to identify the timing of mitochondrial DNA protease inhibitors in zebrafish (Otten et al., 2016). This work revealed distinct bottleneck events in germline and nongermline cells, and provided evidence that in the female germline a bottleneck occurs in stage I oocytes (Otten et al., 2016). In zebrafish, stage I encompasses oocytes that overlap with the period prior to and when the Balbiani body is present (Fig. 2). In the Otten study, these early stage oocytes were not subdivided according to meiotic stage and oogonia were mixed with meiotic stages from zygotene to diplotene; therefore, it was not possible to pinpoint the precise stage when the bottleneck occurs. If the bottleneck mechanism includes selection of mitochondria by sequestration or exclusion from the Balbiani-body localized, then future analysis and comparison of mitochondrial DNA copy number and variation in oocytes sorted based on meiotic stage and phase of Balbiani body development (e.g. prior to formation, after formation, during translocation and after reaching the cortex) may prove to be informative and should provide sufficient resolution to pinpoint the timing of the oocyte bottleneck. In addition, isolation and proteomic analysis of mitochondrial proteins of the Balbiani body associated mitochondria and cytoplasmic mitochondria should provide insight into whether distinct posttranslational modifications are associated with these mitochondrial populations. Zebrafish mutants and transgenic oocytes that are mutant for or overexpress Bucky ball protein, a vertebrate specific protein with uncharacterized protein domains, disrupt Balbiani body formation and fail to localize mitochondria at diplotene stage (Marlow and Mullins, 2008; Bontems et al., 2009; Heim et al., 2014). We have previously demonstrated that Bucky ball protein is asymmetrically localized at zygotene stage before the Balbiani body forms (Heim et al., 2014), raising the possibility that oocyte polarization was initiated earlier than had been previously thought. We and others recently provided evidence that this indeed appears to be the case. These studies used electron microscopy to examine and quantify mitochondria distribution in the female germline during this period, and found that mitochondria are asymmetrically distributed and proximal to ring canals in stages prior to Balbiani body formation (Marlow and Mullins, 2008; Elkouby et al., 2016). This is one of the stages represented within the early ooctye bottleneck identified by Otten and colleagues, raising the possibility that a mechanism to enrich mitochondria, and thus a potential purifying selection could occur prior to or at zygotene stage, when asymmetric Bucky ball protein is first detected (Heim et al., 2014) (Fig. 2). Alternatively, it is possible that an earlier mitochondrial bottleneck could function in a Bucky ball and Balbiani body independent manner because mitochondria are initially asymmetrically distributed at earlier stages in bucky ball mutants although mitochondria are not sequestered in bucky ball mutants at later stages (Marlow and Mullins, 2008; Bontems et al., 2009; Heim et al., 2014; Elkouby et al., 2016). If selection occurs prior to or independent of Balbiani body formation then the mitochondria composition of bucky ball mutants would be expected to resemble the variation in wild-type reported by Otten and colleagues. In contrast, if the Balbiani body is required for purifying selection then mitochondria of bucky ball mutants might show more variation compared to wild-type.