We investigated the distribution of
The microtubule cytoskeleton is essential for a variety of cellular processes, including cell movement, organelle transport, and cell division. Moreover, in oocytes and early embryos, microtubules have been implicated in localization of important embryonic determinants such as
In most animal cells, centrosomes are composed of a pair of centrioles, surrounded by an amorphous cloud of electron-dense material, the pericentriolar material (PCM) [
The zebrafish (
Early embryonic, maternally regulated vertebrate cell cycles (i.e., cleavage) differ from somatic cell cycles. Early embryonic cells have rapid synchronous cell cycles with alternating M (mitosis) and S (DNA synthesis) phases and lack G (gap/growth) phases. The rapid repeated cleavages result in cells with successively smaller volumes. Zebrafish embryogenesis begins with 10 metasynchronous mitotic cycles [
To visualize the cellular distribution of
Figure
Cellular distribution of
In cleavage and blastula staged embryos, the
During cleavage,
The results support our previous hypothesis [
Since embryonic centrosomes were undetectable in the 1-cell stage then suddenly appeared during the first mitotic division, we wanted to further investigate their cellular distribution in dividing blastomeres in later embryonic stages. The image data demonstrated that the centrosome arrays alternated from curvilinear or arc-shaped to punctate in one direction, then from punctate to arc-shaped or curvilinear in another direction, while the nuclear division orientation alternated after each cell division (Figure
Summary of the alternating orientation of centrosomes and nuclei during zebrafish early embryogenesis using epifluorescence microscopy. Schematic cartoons of simplified nuclei (n; red dots) and curvilinear arc-shaped/ring (A/R) or punctate (p) centrosomes (green lines or dots, resp.) are depicted above each representative micrograph doubly labeled with DAPI (red) and GTU-88 (green). (a) First mitosis. (b) 2-cell stage with each cell in anaphase/telophase. (c) 4-cell stage with cells in prometaphase/metaphase. (d) 4-cell stage with each cell in early anaphase. (e) 4-cell stage with cells in telophase. (f) 8-cell stage, (g) 16-cell stage. (h) 32-cell stage.
The results presented here, demonstrating a lack of typical
Centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development [
In unfertilized mouse oocytes, meiosis is arrested in the second meiotic metaphase.
Besides
Centrosomes are assembled at fertilization, in which the centriole pair, inherited from the male gamete, binds nucleating components from the female gamete [
In
In the mouse model, after fertilization,
In unfertilized bovine oocytes,
When the
In summary, the distribution of
The colocalization of
In newt embryos, cyclin B1 begins to accumulate in the nucleus during interphase in synchronous cleavage, and its greatest expression is in the centrosomes and the nucleus at prometaphase. The centrosomes of the principle sperm nucleus and the zygote nucleus have greater accumulation of both
Establishment of polarity in
The cycles of centriole and centrosome duplication are intimately coupled. Centrioles initiate duplication at the onset of S phase in a process that requires Cdk2 (reviewed in [
Wild-type zebrafish were obtained from a local distributor and housed in a dedicated, temperature (28°C) and photoperiod (14L) controlled fish room in racks of food-quality plastic containers (6 and 12 l) with flow-through dechlorinated water. Fish were fed 1-2 times daily a diet of TetraMin and dried brine shrimp flakes. This research was carried out under an IACUC approved protocol.
Gravid adult female zebrafish were decapitated and the spinal cords pithed, the whole ovary was removed and placed in a Petri dish half filled with Cortland’s solution (NaCl 7.25 g, CaCl2-2H2O 0.23 g, KCl 0.38 g, MgSO4-7H2O 0.23 g, NaHCO3 1.0 g, penicillin 30 mg, and streptomycin 50 mg made up to 1000 mL with double distilled water). Then the opaque fully grown oocytes were dissected from the ovary with watchmaker forceps and pooled into a 1.5 mL microcentrifuge tube.
The opaque fully grown oocytes were separated from the others following the above procedure, pooled into a 60 × 15 mm Petri dish with 10 mL fresh Cortland’s solution with 1
Male and female fish were placed in separate compartments within spawning tanks, and the separation barriers were removed just prior to the desired mating time. This allowed collection of embryos at precise times after fertilization. Crosses were set up in the afternoon using 3 females and 4-5 males per breeding tank. After mating, males and females were separated again and allowed to rest for at least one week before the next cross. Embryos were collected next morning and were washed to remove any debris [
Anti-
Before incubation in primary antibody, the fixed oocytes or embryos were rehydrated into 100% PBS in a graded series. The chorions of embryos were manually removed with watchmaker forceps after rehydration. Then the sample oocytes or embryos were incubated in 5% nonfat milk (Carnation) in PBS solution for 4–6 hours at room temperature with gentle rotation. After the samples were washed several times with PBS, they were incubated with 1 mL 1: 200 primary antibody GTU-88 overnight at 4°C. Then the oocytes or embryos were washed in PBS for 8 hours at room temperature, changing buffer at 1-hour intervals. The embryos were then incubated with 750
Embryos, oocytes, and eggs were examined on a Nikon C1 laser confocal microscope equipped with Nikon triple-laser scanning assembly using Argon (ex.488 nm), green helium-neon (ex.543 nm), and red helium-neon (ex.633 nm) lasers. Objectives of 10X and 20X were used. Some images presented are projections of up to three optical sections, providing increased depth of focus.
Microtubule
17
Microtubule organizing center
4′,6-Diamidino-2-phenylindole
Germinal vesicle
Blastodisc.
The authors have no financial or other conflict of interests regarding this work.
This work was supported in part by the Department of Biology, the University of Memphis Graduate School, a Faculty Research Grant from the University of Memphis, and the American Society for Cell Biology Minority Affairs Council. The authors thank the Integrated Microscopy Center, the University of Memphis, for their support in confocal microscopy analysis.