Male germ cell proliferation and apoptosis in sexually immature meagre Argyrosomus regius (Asso, 1801) treated with recombinant follicle stimulating hormone

Abstract

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated.

In vivoeffect of recombinant FSH and LH administered to meagre (Argyrosomus regius) at the initial stages of sex differentiation

“…As previously mentioned the arrGTHs used in the present study have induced spermatogenesis (Zupa et al, 2023) and steroid production in 1 kg meagre (personal observation). In addition, rGTHs produced with the same expression system and at similar doses (12-18 µg kg -1 ) have successfully in adult fish induced spermatogenesis in Senegalese sole (Solea senegalensis) (Chauvigné et al, 2017(Chauvigné et al, , 2018, European eel (Anguilla anguilla) (Peñaranda et al, 2018), flathead grey mullet (Mugil cephalus) (Ramos-Júdez et al, 2021 and meagre (Zupa et al, 2023) and oogenesis in flathead grey mullet (Ramos-Júdez et al, 2021. These previous studies would indicate that the hormone and doses used were effective, although all these studies worked with fish at a more advanced stage of gametogenesis.…”

“…The arrGTHs used in this study have been demonstrated to be biologically active. The same arrFSH preparations induced full spermatogenesis in 17 month-old, 1 kg meagre (Zupa et al, 2023) and applied to the same fish both arrFSH and arrLH were shown to increase steroid levels and induce spermiation (personal observation Neil Duncan). This is typical of the rGTHs produced with the CHO expression system that produce glycosylated proteins with high biological activity in vivo (Molés et al, 2020).…”

“…This is typical of the rGTHs produced with the CHO expression system that produce glycosylated proteins with high biological activity in vivo (Molés et al, 2020). In adult fish rGTHs have induced increases in steroid levels and spermatogenesis in Senegalese sole ( Solea senegalensis ) (Chauvigné et al, 2017, 2018), European eel ( Anguilla anguilla ) (Peñaranda et al, 2018), flathead grey mullet ( Mugil cephalus ) (Ramos-Júdez et al, 2021, 2022) and meagre (Zupa et al, 2023) and oogenesis in flathead grey mullet (Ramos-Júdez et al, 2021, 2022). However, despite of the demonstrated biological activity of the arrGTHs used in the present study, there was no effect of the arrGTHs on E2 plasma levels and actually plasma E2 decreased significantly in all treatments including control groups from week 0 to weeks 3 and 6.…”

“…The mRNA sequences are available in the European Nucleotide Archive (ENA) with project accession number PRJEB57583. The arrFSH sequences were previously reported by Zupa et al, (2023) and the sequences were:…”

“…A wide range of expression systems have been used for rGTH production and the Chinese hamster ovary (CHO) cells have been found to produce rGTH glycosylated proteins with terminal sialic acids that have high biological activity in vivo (Molés et al, 2020). The rGTHs have been successfully used to overcome reproductive dysfunctions in adult cultured fish and rFSH has proved to stimulate spermatogenesis in Senegalese sole ( Solea senegalensis ) (Chauvigné et al, 2017, 2018), European eel ( Anguilla anguilla ) (Peñaranda et al, 2018), flathead grey mullet ( Mugil cephalus ) (Ramos-Júdez et al, 2021, 2022) and meagre (Zupa et al, 2023) and oogenesis in flathead grey mullet (Ramos-Júdez et al, 2021, 2022). The entire process of spermatogenesis from spermatogonia through to motile spermatozoids and the entire process of oogenesis from perinuclear oocytes through to viable ovulated ova was induced using a combination of rFSH and rLH administered to adult flathead grey mullet arrested in the early stages of gametogenesis (Ramos-Júdez et al, 2021, 2022).…”