Glial cell derived neurotrophic factor (GDNF) was first purified and cloned in rat glial cell line cultures and is a distant relative of the TGF-β superfamily [1]. In mammals, a vast number of studies suggest that in addition to being a neurotrophic factor, GDNF plays a critical role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) in testis and oogenesis in ovary. In testis, GDNF is expressed in Sertoli cells and peritubular myoid cells [2,3]. Its deficiency leads to reduced SSCs and progressive germ cells loss, whilst its overexpression leads to SSCs accumulation and failure to respond appropriately to differentiation signals [4,5]. In vitro cell culture, the addition of GDNF can promote the sustained proliferation and maintenance of SSCs in mouse and the other mammals such as human, pig and sheep [[6], [7], [8]]. In ovary, GDNF is expressed in oocytes and somatic cells such as cumulus cells and granulosa cells in mouse and human [9,10]. In cultured immature oocytes, treatment with GDNF can up-regulate the level of cyclin D1, and promote the extrusion of the first polar body and the maturation of oocytes [11,12].

Retinoic acid (RA) is a crucial factor in the early differentiation of SSCs. Conditional knockdown of the key genes of RA synthesis in Sertoli cells, including Aldh1a1, Aldh1a2, Aldh1a3 or Rdh10 [13,14], or treatment of neonatal mice with the Aldh1a inhibitor WIN 18446, can lead to the accumulation of SSCs [15]. Studies show that RA is negatively correlated with the expression level of Gdnf in mouse testis [16], and can transcriptionally down-regulate its expression in Sertoli cells [17]. These data imply that the regulation of GDNF by RA is involved in the balance of self-renewal and differentiation of SSCs.

Reports on fish GDNF homologs are limited to some fishes, including rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes), zebrafish (Danio rerio) and dogfish (Scyliorhinus canicula) [[18], [19], [20], [21], [22]]. Unlike mammals, there are two or more GDNF replicate genes in fishes due to the third round of genome replication unique to teleost or fish genus-specific genome replication [23]. In rainbow trout, three gdnf homologs have been reported, namely gdnfa1, gdnfa2, and gdnfb. Rainbow trout gdnfb is mainly expressed in type An undifferentiated spermatogonia, and its expression level is downregulated during the rapid proliferation of spermatogonia, but dramatically upregulated during the late stage of spermatogenesis [21,22]. In medaka, two gdnf homologs, gdnfa and gdnfb, have been reported and play a role in the proliferation of a spermatogonial cell line (SG3) derived from medaka testis [18]. In zebrafish, two gdnf (gdnfa and gdnfb), and their family receptor alpha-1 homologs (gfrα1a and gfrα1b) have been identified [19,24], respectively. Zebrafish gdnfa is expressed in Sertoli cells and Leydig cells, and its homozygous mutation leads to abnormal spermatogenesis and male sterility [19]. Besides, recombinant human GDNF can increase the mitotic index of types A spermatogonia in zebrafish [24]. Meanwhile, it is reported that feeder cells which were genetically modified to express zebrafish gdnfa can promote the proliferation and maintenance of female zebrafish germ line stem cells in vitro [25], implying that it is involved in oogenesis. In Nile tilapia, using anti-human GFRα1 antibody (Abcam, ab84106), obvious signals were observed in single type An undifferentiated spermatogonia [26]. In dogfish, gdnf/gfrα1 homologs have been identified and recombinant human GDNF can promote the proliferation and maintenance of dogfish spermatogonia with stem characteristics for at least 5 months [27,28]. Collectively, these studies suggest that Gdnf/Gfrα1 signaling might have conserved role in fish SSCs and oogenesis just like that in mammals. Nonetheless, our knowledge about the two GDNF homologs in fish is limited and far from to be elucidated.

Nile tilapia is an important aquaculture fish in the world [29]. It has attributes of strong adaptability, strong disease resistance, fast growth rate, short spawning cycle (14 days), availability of all-XX and all-XY genetic fish through sex linked marker assisted breeding, high-quality genome sequences and feasibility of gene editing technology [[30], [31], [32], [33]]. Therefore, it is considered an excellent model for studying fish reproduction. In the present study, the full length of two paralogous gdnf open reading frame (ORF) sequences from Nile tilapia (named as Ongdnfa and Ongdnfb hereafter) were cloned and characterized. Furthermore, their expression profiles during spermatogenesis and oogenesis and their responsiveness to RA treatment was investigated.