Since the discovery and isolation of the pluripotent gene Nanog in mouse ESCs for the first time (Wang et al., 2003), its role in embryonic development and maintaining pluripotency in ESCs and the regulatory networks had been deeply studied (Chambers et al., 2003, Darr et al., 2006, Mitsui et al., 2003). From then on, Nanog had also been reported in birds (Canon et al., 2006, Lavial et al., 2007) and some model organisms, medaka (Camp et al., 2009, Wang et al., 2011) and zebrafish (Schuff et al., 2012, Theunissen et al., 2011). Among the non model fish, there were a few studies on Nanog in goldfish (Marandel et al., 2012), Paralichthys olivaceus (Gao et al., 2013) and blunt snout bream (Yu et al., 2017). However, little was known about Nanog gene of small sea angelfish. In this study, we cloned Nanog of C. vrolikii, an important marine ornamental fish, and analyzed its sequence characteristics and expression pattern, defined Cv-Nanog as the Nanog ortholog because of its conserved features that we hereby described, so as to lay a foundation for further study on the mechanism of cell differentiation and gonadal development of C. vrolikii.
In this study, a total of 2,136 bp cDNA fragment of Cv-Nanog was obtained with encoding 436 amino acids. Like medaka (Wang et al., 2011), zebrafish (Theunissen et al., 2011) and blunt snout bream (Yu et al., 2017), Cv-Nanog contains a conserved HOX domain, located at 222-284 (Fig. 2). In addition, the HOX region also contained a conserved YKQVKTWFQN motif that was speculated to be a nuclear localization signal, similar to that observed in human Nanog (Chang et al., 2009, Do et al., 2007, Pan and Pei, 2003). The C-terminal of Nanog in mammalian contained a tryptophan pentapeptide repetitive sequence (Chambers et al., 2003, Hart et al., 2004, Medvedev et al., 2009), some studies also pointed out that the WR region played an important role in the interaction with other pluripotent network regulatory proteins (Mullin et al., 2008, Wang et al., 2008), but a similar repetition of W was not found in Nanog of teleost fish and birds. Structurally, they may not have the conserved function as Nanog of mammalian. However, later studies confirmed that Nanog can play an effective role in the process of cell reprogramming in mammals, birds and teleost fish (Theunissen et al., 2011), indicating that it had a conserved function in vertebrates. As an important component structure of Nanog, HOX domain can recognize and bind specific DNA sequences to play its role (Jauch et al., 2008). Multiple sequence alignment found that except the conserved Hox domain, the conservation of other sequences was relatively low. The sequence identity of full-length Cv-Nanog protein was lower (16.5%) compared to that of the mouse Nanog (Table 1). In zebrafish, the sequence identity of Dr-Nanog was much lower (13%) compared to that in mouse whereas they were functionally conserved (Theunissen et al., 2011). It was speculated that Nanog gene had the characteristics of rapid evolution and rich diversity. The phylogenetic tree elucidated that the Cv-Nanog belonged to the Perciformes subgroup with the closest relationship to M. saxatilis in the Perciformes sub-clade. These showed that the HOX domain of Nanog was very conservative in evolution and very important in the function.
Gonadal development is crucial to the growth and reproduction of fish. An army of studies had demonstrated that Nanog gene played essential roles in embryogenesis, gonadal development and maintaining the pluripotency and self-renewal ability of ESCs in vertebrates (Ben-Porath et al., 2008, Clark et al., 2004, Silva et al., 2006, Wang et al., 2011). The expression of Cv-Nanog in tissues showed that in adult tissues, the transcript of Cv-Nanog was specifically expressed in gonads at different developmental stages with much higher in ovaries than that in testis, and the expression of Cv-Nanog was relatively low (or even almost not) in other adult tissues (Fig. 5). The expression of Nanog in the gonads and early developmental embryos was reported, too (Ambady et al., 2010, Ben-Porath et al., 2008, Beltrami et al., 2007, Chambers et al., 2007). In humans, Nanog was mainly expressed in ESCs, and also in testes and ovaries (Clark et al., 2004), and ovarian cancer cells (Hoei-Hansen et al., 2005, 2007). In mouse, the expression of Nanog was significantly higher than that in heart, kidney, spleen, liver, and differentiated cells (Hart et al., 2004, Ventea et al., 2012). In medaka, the expression of Nanog was exhibited in the gonads, predominantly in pluripotent cells (Camp et al., 2009, Wang et al., 2011). C. vrolikii is a naturally reversed fish, the mark of the beginning of the gonad reversal is the male germ cells beginning to appear in the ovary,named hermaphrodite, and then the male germ cells are further differentiated, finally the gonad was transformed into pure testis (Zhong et al., 2021). In this study, there were differences in the expression of Cv-Nanog in gonadal tissues at different developmental stages with the level OⅣ > OⅡ > OT > T in turn (Fig. 5). It is speculated that Nanog were likely to be indispensable in the process of the gonadal development, which may also be closely related to the maternal expression pattern of Nanog (Schuff et al., 2012, Wang et al., 2011, Yu et al., 2017).
In the present study, we indicate that in fish, Cv-Nanog was expressed in both male and female gonads, further confirming that Cv-Nanog was the functional homolog of mammalian Nanog. However, it was not clear whether Nanog actually exerts a biological function in gamete maturation. In ovaries, Cv-Nanog RNA expression was localized in the cytoplasm of the oocytes at stage Ⅱ (Fig. 2 A1-A3), and it was only localized in the membrane of the oocytes with no positive signal in yolk granules at stage Ⅳ (Fig. 2 B1-B3). In OT stage, the Nanog RNA expression was distributed in both oocytes and sperm (Fig.6 C1-C3). On the other hand, in testis, Cv-Nanog RNA was highly expressed in the nucleus of cells in the periphery where spermatogonia was located and weakly expressed in sertoli cells (Fig. 2 D1-D3). The expression of Cv-Nanog in the gonadal germ cells was comparable to mouse and chicken (Canon et al., 2006, Lavial et al., 2011, Mitsui et al., 2003, Yamaguchi et al., 2005). Inconsistently, the Nanog RNA expression was present in spermatogonia or oogonia in medaka (Wang et al., 2011), in oogonia and early stage of oocytes in ovary, or in spermatogonia, spermatocytes, and spermatids in testis in blunt-snout bream (Yu et al., 2017). According to the current study of western blot, Cv-Nanog protein was most strongly expressed in OⅣ, then gradually decreased in OⅡ, OT and T (Fig. 5 C). The results of western blot analysis were compatible with those of qRT-PCR, suggesting that Nanog gene was expressed in gonads with highly in ovaries. Thus, the potentiating presence of Cv-Nanog mRNA and protein in ovary could be argued as its maternal inheritance, which was also agreement with the observations in teleosts (Camp et al., 2009, Gao et al., 2013, Marandel et al., 2012).
The studies demonstrated that the Nanog overexpression was indeed involved in regulating pluripotency reprogramming in mouse ESCs (Silva et al., 2006) and it can increase the proliferation rate and regulate the cell cycle in human ESCs (Zhang et al., 2009). The transient overexpression Nanog could activate the expression of Oct4 (5-fold), cMyc (2-fold), and Sall4 (5-fold) in somatic cells in porcine fetal fibroblast (PFF) (Zhang et al., 2011) and it could significantly increase the expression Oct4, cMyc and other genes in the gonads cell lines (LYCO and LYCT) of large yellow croaker (Larimichthys crocea) (Xu et al., 2021). Thereby, the overexpression of Nanog can recover the pluripotency of cells to improve the self-renewal ability of cells. Similarly, in this study, the growth rate of cells after Nanog overexpression was significantly accelerated (Fig. 3), this may be related to the activation of Oct4, Sox2, Klf4, and other pluripotency factors (Fig. 4), which can recover the pluripotency of cells to improve the self-renewal ability of cells. What’s more, the expression of sex-related genes of C. vrolikii was also different after Nanog overexpression (Fig. 4). In the core transcriptional network (Fig. 5), Cv-Nanog interacts with Sox2 and Oct4 to activate the self-renewal ability of cells, which will activate the TGF-beta signaling pathway and promote the gonadal development. Then, the Follicle-Stimulating Hormone (FSH) was promoted, the cAMP increased, and the expression of Cyp19a and Foxl2 was up-regulated the expression of Sox9 and Dmrt1 was down-regulated and eventually the ovarian development was promoted. The heightened abundance of the sex-related genes in the gonads suggested its participation in maternal inheritance and the gonadal development. Some studies had represented that Lr-Nanog may be involved in the development of undifferentiated germ cells in Labeo rohita (Patra et al., 2018). It would be of interest to clarify this particular aspect in the future of Nanog, it would also be fascinating to carry out the studies of Nanog in the regulatory mechanisms and pathways involved in gonadal development and maintenance of germ cells.