Complete depletion of primordial germ cells in an All-female fish leads to Sex-biased gene expression alteration and sterile All-male occurrence

Abstract

BackgroundGynogenesis is one of unisexual reproduction modes in vertebrates, and produces all-female individuals with identical genetic background. In sexual reproduction vertebrates, the roles of primordial germ cells on sexual dimorphism and gonadal differentiation have been largely studied, and two distinct functional models have been proposed. However, the role of primordial germ cells remains unknown in unisexual animals, and it is also unclear whether the functional models in sexual reproduction animals are common in unisexual animals.ResultsTo solve these puzzles, we attempt to utilize the gynogenetic superiority of polyploid Carassius gibelio to create a complete germ cell-depleted gonad model by a similar morpholino-mediated knockdown approach used in other examined sexual reproduction fishes. Through the germ cell-depleted gonad model, we have performed comprehensive and comparative transcriptome analysis, and revealed a complete alteration of sex-biased gene expression. Moreover, the expression alteration leads to up-regulation of testis-biased genes and down-regulation of ovary-biased genes, and results in the occurrence of sterile all-males with testis-like gonads and secondary sex characteristics in the germ cell-depleted gynogenetic Carassius gibelio.ConclusionsOur current results have demonstrated that unisexual gynogenetic embryos remain keeping male sex determination information in the genome, and the complete depletion of primordial germ cells in the all-female fish leads to sex-biased gene expression alteration and sterile all-male occurrence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2130-z) contains supplementary material, which is available to authorized users.

Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio)

“…The cDNA library was constructed as previously described (Li et al, 2014a). The real-time quantitative PCR (RT-qPCR) was performed as previously (Liu et al, 2015a). The PCR cycling conditions were: 94°C (2 min) for heat denaturing, followed by 40 cycles of 94°C (15 s), 57°C (15 s), 72°C (20 s), and additional 72°C (2 min).…”

“…β-actin was used as an internal control. All the samples were analyzed in triplicates, and relative expression level of target gene was calculated with the 2 − ΔCT methods (Liu et al, 2015a;Yue et al, 2013).…”

“…However, unlike most unisexual vertebrates (Avise, 2008), gibel carp has been revealed to have multiple reproduction modes including unisexual gynogenesis and sexual reproduction (Zhou et al, 2000;Gui and Zhou, 2010;Li et al, 2014a;Mei and Gui, 2015;Zhang et al, 2015), and a high male incidence has been also observed in many natural populations (Jiang et al, 2013;Li et al, 2016). Utilizing it as an evolutional development biology model for screening reproduction-related genes, many important genes, such as oocytespecific and ovarian development-associated genes C-type lection (Dong et al, 2004), C1q-like (Mei et al, 2008(Mei et al, , 2014 and spindlin (Sun et al, 2010), oocyte-specific H2A variant h2af1o (Wu et al, 2009;Yue et al, 2013) and stem-loop binding protein-encoded gene slbp2 (Liu et al, 2015a), testis determination-related gene Dmrt1 (Li et al, 2014a), and germ cell marker genes Vasa and Dazl (Xu et al, 2005;Peng et al, 2009), have been identified and functionally characterized in gibel carp. In a previous study, we have utilized the superiority of unisexual gynogenesis in gibel carp to create a complete germ cell-depleted gonad model by a dnd-pecific morpholino-mediated knockdown approach, and revealed that the complete depletion of primordial germ cells in the gynogenetic all-female fish leads to sex-biased gene expression alteration and sterile all-male occurrence (Liu et al, 2015a).…”

“…Utilizing it as an evolutional development biology model for screening reproduction-related genes, many important genes, such as oocytespecific and ovarian development-associated genes C-type lection (Dong et al, 2004), C1q-like (Mei et al, 2008(Mei et al, , 2014 and spindlin (Sun et al, 2010), oocyte-specific H2A variant h2af1o (Wu et al, 2009;Yue et al, 2013) and stem-loop binding protein-encoded gene slbp2 (Liu et al, 2015a), testis determination-related gene Dmrt1 (Li et al, 2014a), and germ cell marker genes Vasa and Dazl (Xu et al, 2005;Peng et al, 2009), have been identified and functionally characterized in gibel carp. In a previous study, we have utilized the superiority of unisexual gynogenesis in gibel carp to create a complete germ cell-depleted gonad model by a dnd-pecific morpholino-mediated knockdown approach, and revealed that the complete depletion of primordial germ cells in the gynogenetic all-female fish leads to sex-biased gene expression alteration and sterile all-male occurrence (Liu et al, 2015a). However, molecular characterization of the dnd has been not reported and its expression pattern is still unclear in gibel carp.…”

A novel allotetraploid gibel carp strain with maternal body type and growth superiority

“…As an evolutionary development biology model, gibel carp may be in a transitional phase of unisexual to sexual reproduction, and may have relative stability and flexibility (Gui, 1997;Gui and Zhou, 2010;Liu et al, 2015). Cytological observation indicated that heterologous sperm were condensed in the inseminated gibel carp eggs, whereas the inseminated eggs were treated with trypsin or extremely cold shock, the heterologous sperm nucleus could be decondensed to thereby form pronucleus.…”