Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg

Abstract

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple-shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister-like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10-15 microns from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

“…However, studies on the molecular mechanisms of fish sperm decondensation process has been difficult because it is hard to develop a nice cell-free system based on fish eggs for the performance of in vitro biochemical studies. Most of the studies on early developmental events of sperm nuclei after fertilization in fish were based on the methods of microinjection together with cytological and ultrastructural observation [12][13][14]. It is necessary to develop a nice cell-free system to study the sperm decondensation and pronucleus formation in fish.…”

“…It has been demonstrated in bisexual reproduction fish that sperm nucleus undergoes nuclear envelope breakdown, nucleus swelling, and pronuclear formation with a new nuclear envelope after fertilization [12,15]. In contrast to common bisexual reproduction fish, within the fertilized eggs of gibel carp (Carassius auratus gibelio), the heterologous sperm nucleus keeps in condensation and is failed to form male pronucleus, while some of the homologous sperm nuclei can decondense and transform into male pronucleus.…”

Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish

“…So, the donor nucleus will be more likely to migrate to the microvillae cluster in animal pole, the place where the fertilization occurs (Wolenski and Hart, 1987), through the cytoplasmic flows together with the pronuclei at the time of the activation.…”

“…In this way, the animal pole is segregated through the place where the female nucleus will be located (Wolenski and Hart, 1987). So, this enabled detection of the incipient animal pole to deposit the somatic nucleus in the female perinuclear region.…”

“…The micromanipulation area was not cooled (room temperature). The donor nucleus was injected in the incipient animal pole, just where zygote nucleus was being constituted (Wolenski and Hart, 1987). The reconstructed embryos were incubated under the same described conditions.…”