As the first allotetraploid fish
As the first allotetraploid fish in the world, our lab selectively breed the allotetraploid hybrid fish (4nAT, 4n = 200) from the distant hybridization offspring F3 of female Red Crucian Carp (Carassius auratus red var., abbreviated as RCC, 2n = 100) and male Common Carp (Cyprinius carpio L., abbreviated as CC, 2n = 100). On this basis, the allotriploid fish (abbreviated as 3n, 3n = 150) is generated by interploidy hybridization with diploid RCC as the maternal parent and 4nAT as the paternal parent (Chen et al., 2009). This 3n fish is characterized by stable reproductive sterility. Firstly, histological analysis of gonad cross-sections showed ovary of 3n fish stayed at stage I (Tao et al., 2008). In agreement with this result, the hypothalamic-pituitary-gonadal (HPG) axis in 3n fish is abnormal (Long et al., 2006, Long et al., 2009a, Long et al., 2009b). Moreover, gene expression of the meiotic gene dmc1 is lower in the ovaries of 3n fish than in diploid and allotetraploid fish (Tao et al., 2008). In the testis, chromosome spreading studies showed that 50 bivalents are formed in diploid fish, whereas 50 bivalents and 50 univalent are formed in allotriploid fish, which indicated that there is abnormal pairing between homologous chromosomes during meiosis (Zhang et al., 2004). Sterility of this 3n fish might be caused by pachytene impairment, which provided a perfect research material for studying the mechanism under pachytene checkpoint in fish. De novo DNA methyltransferases (Dnmts) are a kind of enzyme to precisely mark DNA methylation to new positions without methylated DNA template and involve in regulating multiple important biological processes (Chen and Riggs, 2011). In mammals, de novo Dnmts contains Dnmt3A, Dnmt3B, and Dnmt3L with similar constitution of domains, except that Dnmt3L lacks a catalytic domain (Chedin, 2011). However, Dnmt3L can bind both Dnmt3A and Dnmt3B, acting as a cofactor to promote their catalytic activities (Suetake et al., 2004). Previous study has reported that de novo Dnmts regulated female germ cell development by silencing retrotransposons (Lucifero et al., 2007). Moreover, another conditional knockout mouse study reported that, during oocyte growth, Dnmt3A established DNA methylation patterns in germ angiotensin receptor blockers and Dnmt3B regulated early development (Kaneda et al., 2010). Furthermore, meiotic catastrophe, such as forming aberrant branching and anastomosing synaptonemal complexes, were observed in Dnmt3L-deficient spermatocytes of male mice due to changing DNA methylation patterns of the repetitive elements (Bourchis and Bestor, 2004). What is more, all female oocytes from knockout mice of lymphoid specific helicase (Lsh), another epigenetic regulator essential for DNA methylation, also exhibited incomplete synapsis (La Fuente et al., 2006; Zhu et al., 2006). All these studies proved the important role of Dnmts in oocyte development, especially the pachytene checkpoint in mammals. In teleost fish, six de novo Dnmts (Dnmt3–8) have been identified in zebrafish (Goll and Halpern, 2011), whereas homologue of Dnmt3L is absent from the fish genome. Most studies focused on investigating the roles of Dnmts in the effects of toxins from the environment and embryonic development (Aluru et al., 2015; Dorts et al., 2016; Seritrakul and Gross, 2014; Takayama et al., 2014). Till now, there are few data reported the correlation between de novo Dnmts and reproductive sterility in fish. Among the six Dnmts identified in fish, dnmt3 was reported to be mainly expressed in zebrafish gonads (Smith et al., 2011). Present study intends to elucidate the roles of the de novo DNA methyltransferase dnmt3 in regulating sterility in 3n fish. Firstly, we identified the sequence of dnmt3 in different ploidy fish and analyzed its gene expression, which showed that dnmt3 was mainly expressed in gonads and exhibited lower expression in ovaries of sterile 3n fish when compared to the fertile RCC and 4nAT. Then, to explore the relationship between HPG axis and DNA methylation, we used two kinds of important hormones of HPG axis, human chorionic gonadotrophin (hCG) and estradiol (E2) to treat 3n fish in vivo. On one hand, the results revealed that dnmt3 is upregulated in E2 treatment group, while no obvious change in hCG treatment group. On the other hand, consistent with variation of dnmt3 gene expression, msh4, a meiosis-specific gene which stabilizes double Holliday junctions thus plays an important role in homologue chromosome synapsis, decreased and exhibited lower DNA methylation level of CpG island after E2 treatment, and also have no significant change after hCG treatment. All the results indicated that dnmt3 is regulated by E2, and its downstream influence on meiotic progression might be correlated with msh4 gene. Taken together, our findings provide new insights on the epigenetic regulation of sterility in female cyprinid fish. These results may be useful in the fields of reproductive biology and genetics as well as in practical aquatic organism breeding.