Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish

Abstract

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of unstable amino acids with those that were more stable against proteolytic digestion as well as had a longer halflife, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Comparison of milt and blood parameters including testosterone and gonadotropins levels of sex‐reversed female rainbow trout ( Oncorhynchus mykiss ) by different steroid hormones

“…These results agree with those reported for sex‐reversed Nile tilapia ( Oreochromis niloticus ) (Phumyu et al., 2012). Additionally, applications of steroid hormones can trigger increases in red blood cell, haematocrit and haemoglobin values (Ma et al., 2020; Mohammadzadeh et al., 2020). The current study also reported the serum biochemical values of sex‐reversed female rainbow trout.…”

Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: Analysis of hormone profiles and spawning indices

“…Then, the column was washed with 10 bed volumes by washing buffer (50 mM Tris, 300 mM NaCl, 10 mM Imidazole and 0.05% Triton x-100 at pH 8.0). Finally, the His-tagged protein was eluted with 5 bed volumes elution buffer (50 mM Tris, 150 mM Imidazole and 1% (v/v) Triton x-100, pH 7.5) at a flow rate of 1 mL min -1 and before molecular characterization (Mohammadzadeh et al, 2020). rGnRH quantity was determined by Bradford's method (1976) with bovine serum albumin as a standard.…”

“…Apparently, a reliable, more effective, available and cheap product, useful to induce the final maturation, is required (Aizen et al, 2017). Therefore, recombinant protein methods are used instead of synthetic production for managing the costs (Mohammadzadeh et al, 2020). The potential for the generation of recombinant hormones has been achieved in recent years due to recent advances in construction cDNA encoding target protein.…”

“…In the orange-spotted grouper (Epinephelus coioides) or common carp (Cyprinus carpio), treatments with recombinant luteinizing hormone (LH) altered the hormonal levels along the hypothalamus-pituitary-gonad axis (Cui et al 2007;Aizen et al 2017). An injection of rGnRH recently increased the plasma levels of 17α, 20βdihydroxy-4pregnen-3-one, which led to final maturation induction in goldfish (Carassius auratus) (Mohammadzadeh et al, 2020). Seemingly, producing rGnRH with suitable biological function could pave the way for introducing an effective hormone therapy in aquaculture and could be applied as specific analogues to treat reproductive disorders in some related species for developing their aquaculture production.…”

“…Seemingly, producing rGnRH with suitable biological function could pave the way for introducing an effective hormone therapy in aquaculture and could be applied as specific analogues to treat reproductive disorders in some related species for developing their aquaculture production. Due to the positive features regarding rGnRH for inducing final maturation in breeding female fish (Mohammadzadeh et al, 2020), this study tested rGnRH as an effective agent to stimulate sex steroids, inducing final maturation and spawning in Sterlet sturgeon.…”