Fertilization of Eggs of Zebrafish, Danio rerio, by Intracytoplasmic Sperm Injection1

Abstract

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

“…Techniques that requires embryo (i.e. embryology, chimerism) or gamete manipulation (intracytoplasmic sperm injection, cryopreservation, nuclear transplantation, transgenesis) must also be performed in an adequate time postfertilization (Lin et al, 1992;Poleo et al, 2001;Lee et al, 2002;Liu et al, 2002;Sun et al, 2005). In this regard, sperm sampling is the first critical stage to develop such biotechniques.…”

Electroactivation of zebrafish (Danio rerio) eggs

“…In a same way it could be of interest its coupling to other techniques such as ICSI (Poleo et al, 2001).…”

Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish

“…This represented a critical point due to the technical difficulty in fixing the egg with the holding pipette because the egg rotated inside the chorion while this space was increasing. With the activation, the micropyle can be more easier detected but, a few seconds after the fertilization, the chorion hardened and the micropyle sealed (Poleo et al, 2001b). This made it very difficult to insert the donor nucleus through this point even if the microinjection pipette was fire polished, beveled and sharpened, as in our case.…”

“…In fish nuclear transplant, when the oocyte is activated but not enucleated, the donor nucleus is usually inserted in the perinuclear region of the oocyte, the closest as possible to the female nucleus, which is located in the cytoplasm subjacent to the plasma membrane under the micropyle (Amance and Iyengar, 1990). In various teleost species, such as medaka, catfish or tilapia, the animal pole position can easily be detected in pre-activated oocytes because the micropyle can be visualized at this stage, although, unfortunately, this is not the case in zebrafish (Poleo et al, 2001b).…”

“…In the few nuclear transplant (NT) studies reported to date in zebrafish (Li et al, 2000;Hu et al, 2002;Lee et al, 2002;Luo et al, 2009), the location of the donor nucleus injection place, the animal pole, requires previous oocyte activation because of the micropyle cannot be easily detected in this species (Poleo et al, 2001b).…”