Sperm and egg

The sea urchin sperm contains two centrioles. One serves as the basal body of the
flagellum and has a prominent electron-dense cap on the proximal end where it is firmly
anchored in a hof at the base of the sperm nucleus (Fig. 1a, b). In cross-section the basal body has a canonical nine-triplet structure. The
second centriole, located between the mitochondrion and the nucleus (Fig. 1a, b), also has a canonical nine-triplet structure that is embedded in an electron-dense,
ring-shaped matrix (Fig. 1c). This distal centriole in the nomenclature of 4] is short with an aspect ratio reminiscent of a “Life Saver” candy.

Fig. 1. Ultrastructure of sea urchin sperm basal bodies and distal centrioles. a Longitudinal section of a sperm head. At the base of the head one can see the basal
body—flagellar apparatus, the mitochondrion, and the short distal centriole lying
between the mitochondrion and the nucleus. Here, the distal centriole in tangential
section appears as an electron-dense patch to the left of the basal body (arrow). Scale bar 1 ?m. b Longitudinal section of the head of a sperm treated with gluconate-glycine buffer.
The dense cap on the proximal end of the basal body is located where it is mechanically
attached to the hof in the base of the nucleus. The mitochondrion has moved to the
side of the nucleus along with the distal centriole seen in longitudinal section (arrow). Scale bar 1 ?m. c Cross-section of an isolated distal centriole. Scale bar 0.1 ?m

When a female sea urchin is induced to spawn, the eggs are shed in a post-meiotic
state with a haploid interphase nucleus. The unfertilized egg is believed to be devoid
of centrioles as none have been reported from ultrastructural studies. When the cell
cycle is activated in unfertilized eggs, there is no activity that organizes a duplicating
centrosome and no centrioles are assembled 5]. Centrosome inheritance in echinoderms is paternal; both centrioles in the sperm
are contributed to the zygote and after duplication they organize the centrosomes
used in development.

Zygote

Within approximately 15–30 min after fertilization, the two sperm centrioles have
organized a sperm aster and duplicate at this time of DNA synthesis 6]. The female pronucleus moves toward the focus of the sperm aster and fuses with the
male pronucleus to form the zygote nucleus. Later the mother–daughter centriole pairs
organize asters that separate around the zygote nucleus prior to first mitosis. During
the first embryonic mitoses, all centrioles have a canonical nine-triplet structure
(Fig. 2b) and sometimes appear to be slightly separate within a cloud of fibro-granular pericentriolar
material during mitosis (see serial section series in 2], 7]). During the early zygote mitoses, there is no indication that the pericentriolar
material preferentially accumulates around the older or mother centriole, as is the
case for mammalian somatic cells. During mitosis, the mother–daughter centrioles do
not always exhibit a tight orthogonal arrangement which raises questions about the
extent to which they are mechanically and functionally engaged at this point in the
cell cycle. In telophase, the centrosome flattens and DNA synthesis promptly begins—even
before all the karyomeres (individual chromosomes or groups of chromosomes around
which nuclear envelopes have assembled) have fused to form an interphase nucleus 8]. The centrioles duplicate at this time 9].

Fig. 2. a Enucleated sea urchin zygote followed in vivo to characterize centrosome duplication.
Each duplicated centrosome organizes a birefringent aster as visualized with a polarization
microscope. The birefringence and appearance of the asters indicate that this enucleated
zygote was in mitosis when the photograph was taken. The refractile sphere, slightly
out of focus in the center of the cell, is a drop of the mineral oil used to cap the
micropipette employed to enucleate the zygote. Scale divisions are 10 ?m apart. b This particular zygote was recovered from the preparation, fixed, and serial semi-thick
sectioned. Shown is a cross-section of a centriole in one of the centrosomes. The
other centriole was found in a different section. Scale bar 0.1 ?m

Practical considerations

1. Sea urchins and other echinoderms are a cost-effective experimental system. Large
quantities of eggs and sperm can be collected from one or a few organisms for organelle
isolation and biochemical studies. The gametes and zygotes live in sea water; they
function under atmospheric conditions; and they do not require sterile conditions.
One must culture the zygotes at a temperature similar to that at which that particular
species lives—typically this is below normal room temperatures.

2. The eggs are large (~70–140 ?m in diameter depending on the organism). During the
early mitoses the spindles and asters are large compared to somatic cells. The eggs
of some species are optically clear and thus well suited to in vivo imaging using
polarization or differential interference contrast optics. Importantly, one can follow
the behavior of spindles and centrosomes in vivo without fluorescent probes using
a polarization microscope (Figs. 2a, 3). Since centrioles organize the pericentriolar material that nucleates the asters,
the pattern of aster doubling can be used as a measure of the number and duplication
of the centrioles. For example, a centrosome containing the normal two centrioles
precisely doubles from one cell cycle to the next. A centrosome containing one centriole
does not double in the first cell cycle but then doubles between all subsequent cycles
(see 2]).

Fig. 3. Second mitosis in a Lytechinus pictus zygote. This image shows use of the polarizing microscope to image spindles and asters
in living zygotes of a sea urchin that has optically clear eggs. The spindles are
negatively compensated and appear dark. Quadrants of the asters are bright because
the microtubules in those quadrants are oriented at right angles to those of the central
spindle. Scale divisions are 10 ?m apart

3. One can follow individual zygotes in vivo and then recover each for correlative
fixed cell light microscopy or electron microscopy (Fig. 2a, b). To precisely characterize the number and spatial arrangement of centrioles
in zygotes, one needs to use serial semi-thick section analysis (see 10]). A method for recovering and fixing for electron microscopy individual zygotes previously
followed in vivo in microinjection preparations is described in 7]. For specifics on sealed or open-faced imaging preparations for microinjection/micromanipulation
see 3].

4. The zygote cell cycle is rapid and roughly synchronous for a population of eggs
fertilized at the same time. For the commonly used echinoderms with native temperatures
ranging from 13 to 22 ?, cell cycle duration ranges from 40 min to 1.5 h depending
upon the temperature. Since the investigation of centrosome/centriole duplication
often requires following the cells for multiple cell cycles, echinoderm zygotes allow
one to do an experiment in a morning or afternoon, not the several days needed for
somatic cells. Since experiments usually last a few hours, the investigator can be
continuously present at the microscope to follow/image in detail 5 or more zygotes
per run. For the use of polarization or differential interference optics, a rotating
stage allows one to align each zygote to optimize contrast. Simple time lapse microscope
systems, without computer programmed focus and stage position, allow one to follow
only a few cells at a time, often without proper orientation in the field.

5. The sea urchin zygote is robust and will tolerate experimental insults that would
arrest kill untransformed somatic cells. Zygotes can be mechanically fragmented and
better survive micromanipulation than cultured cells. Also, zygotes have a finite
but much greater tolerance for exposure to blue and near UV light than untransformed
somatic cells. For example, exposure of untransformed human cells (RPE1) to 488 nm
light delivered through a conventional epifluorescence pathway leads to dose-dependent
cell cycle arrest or cell death (Douthwright and Sluder, submitted). Sea urchin zygotes
tolerate equivalent exposures to 366 nm light even though it is more energetic. In
this regard, a note of caution: when imaging Hoechst-stained metaphase sea urchin
chromosomes in the DAPI channel of a fluorescence microscope, one must be careful
to limit exposures to the excitation light. Longer exposures can cause the sister
chromosomes to stick together and when the cell enters anaphase, there is chromosome
bridging and non-disjunction.

6. Even though eggs and zygotes are not amenable to transfection, there are well-established
methods for microinjecting eggs and zygotes prior to following their behavior in vivo.

7. The genomes of S. purpuratus and L. variegatus have been sequenced giving the investigator access to the specific DNA sequences
of centriolar proteins. New expression of specific proteins in zygotes can be inhibited
by the injection of engineered antisense Morpholino oligonucleotides (reviewed in
11]). Recently, genome editing in individual sea urchin zygotes with the CRISPR/Cas9
system has been reported 12].