Poushali Das, Int PBS, Assignment

Oogenesis In Zebrafish
Oogenesis is a dynamic process where the oocyte undergoes different stages of development.
Depending on the pattern of development of the oocyte, ovary and the oogenesis itself can be
classified into three different types :

synchronic oogenesis : here all the oocytes develop at the same time, ovulation also being
simultaneous.
synchronous oogenesis : it consists of at least two populations of the oocytes at different
developmental stages.
asynchronic oogenesis : different development stages of the oocyte maturation and
ovulation in groups may be found within the ovaries.

The oogenesis in Zebrafish is found to be asynchronic.

Stages Of Oogenesis : The structure of the ovary of zebrafish when investigated by using the
histological, histomorphological, histochemical and transmission electron microscopy methods after
the application of different fixatives and stains at both light and electron microcopy level, show four
morphological stages of oocyte development.

1) Primary growth stage : In the first growth phase, multiple nucleoli are observed in the nucleus of
the oocyte. The oocyte diameters vary between 0.08- 0.16 mm during the first growth phase of the
oogenesis. There is a direct relationship between the oocyte growth phase in the oogenesis and the
volume of the ovary or oocytes. The layers (zona radiata) around the follicle are neither thick nor
completely present in the growth phase.

2)Cortical alvelolus stage : here, the granular structures in the ooplasm increase in number.As the
oocytes grow, the cortical alveoli proliferate and follicle increases in the size.The oocyte becomes
opaque in the area that surrounded the nucleus. This stage has oocytes with diameters of 0.16- 0.28
mm. During this stage the nucleus enlarges, and the nucleoli are pushed from the nuclear envelope.
In this phase the vitelline envelope (zona radiata) begin to form and the follicle epithelium becomes
thicker. Invaginations of the nucleus membrane are infrequently accompanied to the irregular
structure of the nucleus.

3)Vitellogenic stage (Vitellogenesis) : In this stage the vitelline membrane begin to develop. The
oocyte size increases. Vitellogenesis or the development of the vitelline envelope occurs in follicles
with diameters ranging between 0.28-0.74 mm. There is the appearance of round , cortical vesicles
in the periphery of the cytoplasm. The number and size of the yolk vesicle increase. Lipids and
protein granules accumulate. The granular structures that appeared in the cortical alveolar phase
are now larger and the nucleus was irregular in the shape.

4)Mature oocytes : At this stage ,the nucleus can no longer be observed as the granular structures
fill up the entire cytoplasm. The oocyte diameter reaches 0.74-0.76 mm, which is the maximum size
of the oocytes during the oogenesis. The membrane of the nucleus dissolves. The lipid and protein
particles fuse and attain a homogeneous appearance. The vesicles gradually join and become larger.
The vitelline envelope which appears in the cortical alveolar phase and continued its development is
clearly evident in this stage. Outside of the envelope, the follicle epithelium cells are present with
their uniformly arranged nuclei. The structure of the vitelline envelope can be monitored clearly by
using the optical and electron microscope.

During the oogenesis process, two inclusion formations could be considered as significant- lipid and
protein formation in the vitellogenesis phase. As oogenesis progresses, the amounts and sizes of the
protein granules and the lipid droplets increase and these inclusion particles eventually fuse. There
may be a relationship between the amount of vitellogenesis and oocyte size. The vitellogenic oocyte
proceed through final maturation coalescence of the yolk globules and oil droplets.

Genetic Control : The process of oogenesis maybe controlled at different stages by a large number
of internal and external factors. The first of these controls is possibly the control at genetic level. The
nme family of genes have been known to be associated with oocyte development. Analysis of the
expression of several nme genes in the zebrafish gonads, with special attention for the nme
transcripts that are maternally inherited, indicates that these might participate in the determination
of oocyte developmental competence.

Reported Genes : nme2b1, nme3, nme4, and nme6 are highly expressed in the ovary and present in
the zebrafish oocyte throughout oogenesis. While the four transcripts are maternally inherited,
nme3 and nme6 display a typical maternal profile and are detected in the zebrafish early embryo. In
contrast to nme3, nme6 abundance exhibits a sharp decrease during early embryogenesis .During
early development, nme2b1, nme2b2, nme3 and nme6 are found to be significantly expressed in the
zebrafish embryo. In contrast, nme2a, nme4, nme5, and nme7 are either not detected or expressed
at very low levels. Significant levels of nme3 and nme6 transcripts are detected in fertilized zebrafish
eggs, thus demonstrating their maternal origin. In contrast, nme2b1 and nme2b2 are not
significantly detected in fertilized eggs. All ovarian nme genes display a decreasing expression profile
during oogenesis, the decrease in oocyte expression levels being more progressive for nme6 and
nme3 in comparison to nme2b1 and nme4. This suggests that nme2b1 and nme4 are translated
during oogenesis. It is thus possible that the corresponding proteins are present in the mature
oocyte and are subsequently maternally-inherited. nme3 displays a typical maternal RNA profile as
it is expressed in the oocytes during oogenesis and present in the embryo during early development.
During the first hours of development, abundance levels are stable. nme6 also displays a typical
maternal mRNA profile during zebrafish early development. However, in contrast to nme3 that is
stable during the first 6 hour post-fertilization, nme6 abundance displays a rapid decrease during
the first cell cycles. This decrease of nme6 transcript abundance suggests an important translational
activity and an important role in the very first steps of development. nme6 is actively transcribed
and its expression is mainly localized in the brain, the eyes and in the somites.

Maternal mRNAs : Large numbers of maternal RNAs are deposited in oocytes and are reserved for
later development. Translational regulation of these maternal RNAs in early oogenesis is extremely
important. Zar1 is a maternal RNA binding protein. Translational repression of ZP proteins by Zar1 is
crucial for early development of oocyte. Zebrafish Zar1 mutants are found to undergo oocyte
apoptosis. Loss of Zar1 causes marked upregulation of zona pellucida (ZP) family proteins, while
overexpression of ZP proteins in oocytes causes upregulation of stress related activating
transcription factor 3 (atf3) which hinders oogenesis. Thus, tightly controlled translation of ZP
proteins by Zar1 and other factors, is essential for ER homeostasis during early oogenesis.

Zar1 maybe one of many proteins involved in the repression and de-repression based translational
control system of the developing oocyte. These regulations are still very poorly understood. The
critical stages of life before protein synthesis can begin from the zygotic nucleus,are sustained by the
maternally inherited RNAs. Their regulation must be extremely specialised since there is a limited
supply, and hence are completely distinct from the translational regulation in the adult.

Mitochondrial Arrangement : Mitochondria are vital cell organelles and they perform many
important functions. During oogenesis they contribute to redox and Ca2+ homeostasis, provide
intermediary metabolites and most importantly, organize and transport the germ plasm
.Mitochondria in zebrafish oocytes actively participate in germ plasm formation and translocation
and are highly active.The close relationship of mitochondria with germ plasm during early oogenesis
may be related to preservation of mitochondrial genome integrity.In zebrafish ,from the late
oogonia stage to early stage I oocytes, the germ plasm, including the germ line determining RNAs,
such as Xlsirts, Xwnt-11, and Xcat2, move out of the nucleus and become intimately associated with
mitochondria. Together they form the mitochondrial cloud (MC) (Kloc et al. 1993; Kloc & Etkin 1994,
1995, 1998; Forristall et al. 1995; Biliñski et al. 2004; Kosaka et al. 2007). The MC, along with the
germ plasm gradually moves from the perinuclear area to the vegetal cortex during early oogenesis.
This process is microtubule- and microfilament-independent (Kloc & Etkin 1995; Kloc et al. 1996),
which is different from other cellular systems in which mitochondria translocation typically depends
on microtubules.In the early stage I oocytes (diameter about 10–40 µm) most mitochondria
aggregated into cluster masses of different sizes and remained dispersed into the entire ooplasm
with higher density near the nucleus . It gradually translocated from the perinuclear area to the
vegetative cortex and then dispersed within the vegetative cortex as the oocyte developed further
into the mid-stage II. As the oocyte grew bigger (> 40 µm in diameter) more mitochondria typically
aggregated around the nucleus, forming a ‘necklace-like’ circle around the nucleus.

This mitochondrial arrangement in the oocyte is extremely interesting and perhaps could be used to
explain differentiation and morphogenesis in later stages. As all those are organised processes, they
should require energy. If the localisation of the mitochondria can be linked with the energy profiles
of separate so-called routes of morphogenesis in the future embryo, maybe a clearer idea of
development will be obtained.

Adhesion : In zebrafish, homologues of αE-catenin and plakoglobin have been reported, these
proteins, together with their complex partners in adherens junctions—i.e., β-catenin and an E-
cadherin equivalent—are abundantly synthesized and stored during oogenesis, either in complexed
forms with cadherins or as free cytoplasmic pools.These proteins can be localized to specific
junctional structures which connect the oocyte with the follicle epithelial cells. The maintenance of
these proteins in the unfertilized egg suggests an additional function as a maternal pool
contributing, among other things, to junction formation during early embryogenesis.

Exploring the transcriptional and translational regulation of these adhesion proteins, along with
their function in the ‘oocyte-follicle’ junction may help better understand the preparatory stages
prior to fertilization.