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Spawning Induction in Sterlet Sturgeon Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: Analysis of hormone profiles and spawning indices Sedigheh Mohammadzadeh, Sakineh Yeganeh, Fatemeh Moradian, Sylvain Milla, Bahram Falahatkar To cite this version: Sedigheh Mohammadzadeh, Sakineh Yeganeh, Fatemeh Moradian, Sylvain Milla, Bahram Fala- hatkar. Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: Analysis of hormone profiles and spawning indices. Aquaculture, Elsevier, 2020, 166, pp.736108. 10.1016/j.aquaculture.2020.736108. hal-03070192 HAL Id: hal-03070192 https://hal.archives-ouvertes.fr/hal-03070192 Submitted on 15 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Spawning induction in Sterlet sturgeon (Acipenser ruthenus) with recombinant GnRH: 2 Analysis of hormone profiles and spawning indices 3 4 Sedigheh Mohammadzadeha, Sakineh Yeganeh*a, Fatemeh Moradianb, Sylvain Millac and 5 Bahram Falahatkard 6 a Fisheries Department, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and 7 Natural Resources University, Sari, Iran 8 b Department of Basic Sciences, Sari Agricultural Sciences and Natural Resources University, 9 Sari, Iran 10 c University of Lorraine, Unit of Animal Research and Functionalities of Animal Products, USC 11 INRA 340, F-54505 Vandoeuvre-lès-Nancy, France 12 d Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, 13 Guilan, Iran 14 15 Running head title: Spawning induction in Sterlet sturgeon with rGnRH 16 17 * Corresponding author 18 Sakineh Yeganeh 19 Fisheries Department, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and 20 Natural Resources University, Sari, P. O. Box 578, Mazanadaran, Iran. 21 Phone number: +98 1133687574 22 Email: [email protected]; [email protected] 23 1 24 Highlights 25 1. rGnRH had a positive effect in final maturation in Sterlet sturgeon. 26 2. rGnRH at the dose of 10 µg kg body weight-1 was efficient in spawning induction. 27 3. rGnRH can stimulate the secretion of sex-steroids in vivo. 28 4. rGnRH can be used as a specific homologue to treat reproductive disorders in fish. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 2 48 Abstract 49 In the present study, the biological function of recombinant gonadotropin-releasing hormone 50 associate peptide (rGnRH) was tested for the induction of ovulation and spawning in Sterlet 51 sturgeon (Acipenser ruthenus) broodstock. For this purpose, Sterlet sturgeon broodstock were 52 divided into four treatments and treated with following preparing procedure: 0.9% NaCl (C); 2.5 53 μg kg body weight-1 (BW) LHRH-A2 (luteinizing hormone-releasing hormone-A2, L2.5); 2.5 μg 54 kg BW-1 rGnRH (rGn2.5); 10 μg kg BW-1 rGnRH (rGn10). The capability of the rGnRH for 55 eliciting biological response was studied in vivo by evaluating the changes of 17β estradiol (E2), 56 progesterone (P) and testosterone (T) and by its ability for inducing final maturation. Blood 57 samples were collected at the time of first injection, 10 h after the first injection and at ovulation 58 time. The L2.5 or rGn10 treatments led to significantly lower E2 concentration 10 h after the first 59 injection and at ovulation time compared to the baseline measures. E2 levels increased in the 60 rGn2.5 treatment 10 h after the first injection and by ovulation time compared to the time of first 61 injection. T levels were significantly increased in the L2.5 or rGn10 treatments 10 h after the first 62 injection compared to the time of first injection and it further decreased at ovulation time. No 63 significant differences were found in C and rGn2.5 treatments between the different sampling 64 times. P secretion was significantly higher in the rGn10 or L2.5 treatments compared to C and 65 rGn2.5 treatments 10 h after the first injection and at ovulation time. No significant difference 66 was found in P secretion in the C and rGn2.5 treatments at different sampling times. The results 67 showed that the rGnRH at the highest dose (10µg kg BW-1) was more efficient than the other 68 treatments in the induction of spawning and this dose caused P secretion and spawning in vivo. 69 Our results present the possibility of using rGnRH in artificial reproduction of sturgeons and it 70 can be introduced as a specific homologue to treat reproductive disorders in fish. 3 71 Keywords: Recombinant GnRH; Acipenser ruthenus; Sex steroids; Ovulation; Spawning 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 4 94 Introduction 95 Sterlet sturgeons (Acipenser ruthenus) are one of the 27 sturgeon species that are endangered 96 worldwide (Muller et al., 2018). They are promising candidates for freshwater aquaculture 97 production and they have several indisputable advantages compared with the other sturgeon 98 species. They require smaller space in captivity due to smaller size and they display a quicker 99 sexual development (Akhavan et al., 2015; Abdollahpour et al., 2018). In comparison with 100 lengthy gonadal cycle in the large migratory species, they are an ideal model to study the 101 reproductive physiology of sturgeon due to earliest maturation (Akhavan et al., 2015). However, 102 the broodstock reproduction and the larval rearing in captivity are two major bottlenecks for its 103 aquaculture development (Abdollahpour et al., 2018). In captivity, sturgeons do not spawn and 104 this dysfunction may result from the combination of captivity-induced stress and absence of 105 suitable natural spawning condition (Alavi et al., 2012). Intramuscular administrations of 106 pituitary extracts (Linhart et al., 2003; Piros et al. 2002) and GnRHa (Linhart et al., 2000; Williot 107 et al. 2002; Van Eenennaam et al., 2008; Nazari et al., 2009; Roosta et al., 2018) have been used 108 to induce final maturation in both sexes. Various GnRH analogues with different half-lives exist 109 and they are used with differential efficiency for inducing final sexual development in sturgeon 110 broodstock. 111 GnRH is the main hypothalamic hormone regulating the synthesis and release of gonadotropins 112 (Nagahama and Yamashita, 2008; Gaillard et al., 2018(. At the N-terminal extremity of the 113 precursor structure of the GnRH protein, there is a signal peptide, followed by the decapeptide, 114 which is a bioactive site of the protein and is almost entirely preserved in all fish species. Next, 115 there is a proteolytic site, which is conserved in all vertebrates. Ultimately, at the C-terminal 116 extremity, there is a GnRH-associated peptide (GAP) that it is composed of 60 amino acids 5 117 approximately, which is less preserved in fish than other regions of the GnRH protein (Zmora et 118 al., 2002). Previous studies indicated that GAP is involved in the stabilization of GnRH structure 119 and that it is not related to the biological function of the protein (Andersen and Klungland, 120 1993). 121 In synthetic GnRH analogues, the biological active site (decapeptide region) is used for the 122 production of a hormone, because GAP region would not be involved in the biological activity of 123 GnRH. In addition, the amino acid sequences of GAP are long, so it is not economical to produce 124 by synthetic method. Apparently, a reliable, more effective, available and cheap product, useful 125 to induce the final maturation, is required (Aizen et al., 2017). Therefore, recombinant protein 126 methods are used instead of synthetic production for managing the costs (Mohammadzadeh et 127 al., 2020). The potential for the generation of recombinant hormones has been achieved in recent 128 years due to recent advances in construction cDNA encoding target protein. Different studies 129 have shown in vivo and in vitro effects of GnRH analogues or recombinant proteins to treat the 130 reproduction disorders in fish. In tench (Tinca tinca), treatment with GnRHa with or without a 131 dopamine antagonist increased luteinizing hormone levels and induced spawning (Podhorec et 132 al., 2016). Intramuscular administrations with different synthetic GnRH analogues and their 133 combinations with an anti-dopaminergic compound were also used to induce final maturation in 134 sterlet sturgeon (Ronyai, 2009). In the orange-spotted grouper (Epinephelus coioides) or 135 common carp (Cyprinus carpio), treatments with recombinant luteinizing hormone (LH) altered 136 the hormonal levels along the hypothalamus-pituitary-gonad axis (Cui et al. 2007; Aizen et al. 137 2017). An injection of rGnRH recently increased the plasma levels of 17α, 20β- dihydroxy-4- 138 pregnen-3-one, which led to final maturation induction in goldfish (Carassius auratus) 139 (Mohammadzadeh et al., 2020). Seemingly, producing rGnRH with suitable biological function 6 140 could pave the way for introducing an effective hormone therapy in aquaculture and could be 141 applied as specific analogues to treat reproductive disorders in some related species for 142 developing their aquaculture production. Due to the positive features regarding rGnRH for 143 inducing final maturation in breeding female fish (Mohammadzadeh et al., 2020), this study 144 tested rGnRH as an effective agent to stimulate sex steroids, inducing final maturation and 145 spawning in Sterlet sturgeon. 146 147 2. Material and methods 148 2.1. Production of recombinant GnRH 149 The amino acid sequence of pre pro cGnRH Beluga (Huso huso) (GenBank: EF534706.2) was 150 selected without its signal peptide.
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