ORIGINAL ARTICLE Induction of Gonadal Development in Protogynous Grouper with Orally Delivered FSH DNA Peter Palma 1,2 & Josephine Nocillado 1 & Joshua Superio 2 & Evelyn Grace de Jesus-Ayson 2 & Felix Ayson 2 & Akihiro Takemura 3 & Ming Wei Lu 4 & Abigail Elizur 1 Received: 8 May 2019 /Accepted: 17 July 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle- stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH β-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17β-estradiol (E 2 ), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin. Keywords Follicle-stimulating hormone . Oral delivery . Plasmid . Grouper . Protogynous . Sex change Introduction Hormonal manipulation has been an integral tool in aquacul- ture, particularly in controlling reproduction of cultured fish species (Mylonas et al. 2010). Early hormonal intervention by aquaculturists involved the administration of pituitary homog- enate derived from mature fish to overcome reproductive dysfunction (i.e., failure to spawn) among captive breeders (Donaldson and Hunter 1983). These pituitary preparations contain gonadotropins, particularly luteinizing hormone (LH), which promotes final gamete maturation through stim- ulation of gonadal synthesis of maturation-inducing steroid (MIS; 17,20β-dihydroxy-4-pregnen-3-one) (Levavi-Sivan et al. 2010). Commercially synthesized reproductive hor- mones of clinical grade were later applied in aquaculture such as analogs of gonadotropin-releasing hormone (GnRHa), a hypothalamic neuropeptide that stimulates pituitary release of LH (Peter et al. 1988; Zohar et al. 2010). hCG has also been a popular choice in aquaculture due to its strong LH receptor binding activity, capable of stimulating gonadal se- cretion of MIS. Enhanced in vivo stability and receptor bind- ing ability of GnRHa were achieved by modifying position 6 of the amino acid residue in the decapeptide (Peter and Yu 1997). Slow-release delivery systems, such as cholesterol pel- lets, ethylene-vinyl acetate implants, and biodegradable mi- crospheres, were also developed for species that would * Abigail Elizur AElizur@usc.edu.au 1 Genecology Research Centre, University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia 2 Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan, 5021 Iloilo, Philippines 3 Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan 4 Department of Aquaculture, National Taiwan Ocean University, Keelung City 202, Taiwan Marine Biotechnology https://doi.org/10.1007/s10126-019-09914-w