389embryogenesis. In stage II, germ plasm bearing specific mRNAs is moving towards
the vegetal pole. Some RNAs like cyclinB1, pou-2, and notch1a are localized to the
animal pole cortex until embryogenesis. They represent the earliest molecular
markers for the animal pole (Howley and Ho 2000 ). At stage III, the oocytes increase
in size due to vitellogenesis (yolk uptake), while the GV remains in the center of the
oocyte. The animal–vegetal asymmetries become more visible with the formation
of the micropylar cell within the follicular layer at the animal pole. The mRNAs
localized at the animal and vegetal pole are distributed underneath the oocyte
plasma membrane and will stay at the cortex until embryogenesis. At stage IV,
oocytes undergo maturation, including GV migration to the future animal pole, sub-
sequent germinal vesicle breakdown (GVBD) and the completion of first meiosis.
After maturation, mature ovulated oocytes in second meiotic metaphase, are trans-
ported through the oviduct, and are now capable of being fertilized (the mature
oocyte is now called egg). The micropylar forms the micropyle, an actin canal,
permitting sperm passage through the chorion (Howley and Ho 2000 ).
Cytoplasm rearrangements: After fertilization, cytoplasm segregates from the
yolk and starts to migrate towards the animal pole to form the blastodisc. Profound
reorganization occurs during this time. Germ plasm mRNAs are redistributed and
positioned at the animal pole, driven by the rearrangement of the cytoskeleton, par-
ticularly through the action of the microtubules and microfilaments (f-actin func-
tion) (Fig. 8.1). Some RNAs, like vasa, dead-end1, and nanos1, become enriched at
the base of the blastodisc. Other components of the germ plasm, including dazl and
bruno-like mRNAs, are delayed at the vegetal cortex, and are not observed at the
blastodisc until almost one hour after fertilization, concomitantly when the first
cleavage furrow starts. These observations suggest an alternative route or mecha-
nism for RNA transport (Hashimoto et al. 2004 , 2006 ). Differential regulation of
dazl and bruno-like mRNAs is likely through binding elements present in their 3′
untranslated regions (3′ UTR). Binding elements present in the 3′ UTR of germ
plasm RNAs play a key role in localization and posttranscriptional regulation dur-
ing oogenesis and embryogenesis (Kosaka et al. 2007 ). Aggregation within the ani-
mal region and translocation from the vegetal cortex lead to the recruitment of
RNAs at the furrow during germ plasm aggregation (Theusch et al. 2006 ). Notably,
germ plasm co-segregation in the furrow presents a nonoverlapping pattern. The
selective localization defines a compartmentalization within the germ plasm, with
dissimilar RNA composition at the furrow. Where a medial domain is enriched in
nos1, vasa, and dead-end (dnd), an intermediate domain that contains both, early
mRNA and dazl, and a distal domain enriched in dazl RNA. As development pro-
ceeds, germ plasm mRNAs are segregated asymmetrically during cell divisions
until the activation of the zygotic genome at gastrula stage (Figs. 8.1 and 8.2).
Germ plasm formation: The Balbiani body (Bb) is a transient and complex assem-
blage of mitochondria, endoplasmic reticulum, Golgi, proteins, and RNA that forms
next to the nucleus. The initial components that will form the Bb originate from the
nucleus and can be detected ultrastructurally as a complex assortment of mRNA and
proteins called nuage (Figs. 8.1 and 8.5a, b). In zebrafish and frogs, the Bb acts as a
vehicle to transport the germ plasm components to the vegetal pole of the oocyte.
8 Mechanisms of Vertebrate Germ Cell Determination