SPERMATOGONIAL PROLIFERATION AND APOPTOSIS IN PREPUBERTAL MEAGRE Argryrosomus regius TREATED WITH RECOMBINANT FOLLICLE STIMULATING HORMONE, AND COMPARISON WITH ADULTS

Introduction The meagre Argyrosomun regius (Asso, 1801) is a promising aquaculture species and advancing puberty using recombinant gonadotropins could shorten the generation time for selective breeding programs (Zupa et al., 2023). The aim of this study was to assess the effects of recombinant follicle stimulating hormone (rFsh) administration on spermatogonial proliferation and apoptosis in prepubertal meagre reared in indoor tanks through a comparison with adult fish reared in sea cages. Material and Methods Prepubertal meagre males (18-months old) reared in indoor tanks at IRTA (La Ràpita, Spain) underwent a six-weeks treatment with increasing doses of rFsh (week 0: 6 µg/kg; week 1: 9 µg/kg; week 2 to week 6: 12 µg/kg); control prepubertal males were injected weekly with 1 mL of saline solution. Prepubertal fish samplings took place before the treatment (week 0; control fish, N = 6) and after 6 weeks of treatment (week 6; control fish, N = 9 and rFsh-treated fish, N = 4). Adult males (6-years old) belonging to a commercial stock reared in sea cages in the Gulf of Taranto (Ionian Sea, Italy) were sacrificed during early (March-April 2021; N = 7) and advanced (June 2021; N = 4) phases of spermatogenesis. Testis samples were fixed in Bouin's solution and embedded in paraffin wax. Deparaffinized sections were stained with hematoxylin-eosin; proliferating spermatogonia were identified through the immunohistochemical detection of the proliferating cell nuclear antigen (PCNA); apoptotic germ cells were identified through the TUNEL method. Results and Discussion The rFsh-treated fish had larger testes compared to both control groups, had larger seminiferous tubules that contained all stages of spermatogenesis and had more abundant luminal spermatozoa (Fig. 1a, b). The testes of adult fish sampled in March-April were in active spermatogenesis with all germ cell types in the germinal epithelium and luminal spermatozoa (Fig. 1c); while in June, all adults were fully mature, showing residual spermatogenetic activity in a thin germinal epithelium and plenty of luminal spermatozoa (Fig. 1d). Anti-PCNA immunostaining was observed in the nuclei of single spermatogonia, spermatogonia in cysts and primary spermatocytes (Fig. 1e), but only single spermatogonia were considered for quantitative analysis. The TUNEL reaction labelled spermatogonia and spermatocytes (Fig. 1f). Fish treated with rFsh showed a significant decrease of proliferating and apoptotic single spermatogonia. In adults, spermatogonial proliferation was significantly higher during the early phase of spermatogenesis compared with the advanced phase in June, and apoptosis significantly increased from the early to the advanced phase of spermatogenesis (Table 1). The treatment with rFsh stimulated spermatogenesis advancement in prepubertal meagre and induced a significant reduction in spermatogonial proliferation and apoptosis. In adult fish, germ cell apoptosis was low during the early spermatogenesis phase and increased during the advanced spermatogenesis phase. This observation confirms that apoptosis plays a major role in regulating germ cells/Sertoli cells ratio and in preventing aberrant germ cell development during spermatogenesis in adult fish (Prisco et al., 2003; Zupa et al., 2013). Moreover, the present data support our previous hypothesis that in prepubertal meagre apoptosis is involved in the inhibition of spermatogonial survival and progress towards meiosis (Zupa et al., 2023).