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

Abstract

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

“…The high expression of Hira gene before fertilization is presumed to prepare for the sperm nucleus decondensation and the formation of male pronucleus. However, the heterologous sperm nucleus is not decondensed during fertilization of gyno-carp (Ge et al 1992;Li and Gui 2003). Hence Hira mRNA may not be required to translate to protein in early embryogenesis in gynocarp.…”

“…The demembranated heterologous sperm nucleus incubated in gibel carp egg extracts could not decondense at all (Li and Gui 2003), indicating that the mechanism of the gynogenesis in gibel carp is not due to the failure of sperm nuclear envelope breakdown, which is the case in Carassius auratus langsdorfii (Yamashita et al 1990). At present, the molecular mechanisms underlying the reproductive strategies of gibel carp are poorly understood, making this fish a promising study model for developmental biology (Gui 1996;Zhou et al 2000b;Xie et al 2001Xie et al , 2003.…”

“…Considering that the gibel carp has similar a phenotype of abnormal male pronucleus to that in Hira-mutated Drosophila (Ge et al 1992;Li and Gui 2003;Loppin et al 2000Loppin et al , 2005, gynogenetic reproduction may also involve the Hira gene. In order to further analyze the function of the Hira gene in fish development and in gynogenesis, we isolated Hira genes from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp Carassius auratus (gono-carp).…”

Molecular Characterization and Functional Commonality of Nucleophosmin/Nucleoplasmin in Two Cyprinid Fish

“…The egg extract preparation followed the protocol published previously (Li and Gui 2003). Immunodepletion was carried out according to the method described by Philpott et al (1991), with partial improvements.…”

“…Complete immunodepletion was confirmed by a Western blot. The demembranated sperm nucleus preparation and in vitro sperm decondensation experiment were performed according to the methods described previously (Li and Gui 2003).…”