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. 2023 Apr 28;13(1):7013.
doi: 10.1038/s41598-023-34102-1.

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

Rosa Zupa  1 , Neil Duncan  2 , Ignacio Giménez  3 , Constantinos C Mylonas  4 , Chrysovalentinos Pousis  1 , Letizia Passantino  5 , Rezart Cuko  1 , Aldo Corriero  6
Affiliations

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

Rosa Zupa et al. Sci Rep. .

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.

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Conflict of interest statement

Rosa Zupa, Neil Duncan, Constantinos C. Mylonas, Chrysovalentinos Pousis, Letizia Passantino, Rezart Cuko and Aldo Corriero declare no competing interest. Ignacio Ginenéz is associated with the biotech company Rara Avis Biotech, S. L., which produced the recombinant gonadotropin employed in this study.

Figures

Figure 1
Figure 1
Mean (± sd) gonadosomatic index (GSI) of untreated (CONTROL 0, n = 5), treated with saline solution for 6 weeks (CONTROL 6, n = 9) or treated with rFsh for 6 weeks (TREATED, n = 4) juvenile meagre males. Different letters represent statistically significant differences (ANOVA; P < 0.05).
Figure 2
Figure 2
Micrographs of testis sections from juvenile meagre. (a) Testis section of an untreated fish (CONTROL 0 group); (b) Testis section of a fish treated with saline solution for 6 weeks (CONTROL 6 group); (c) Testis section of a fish treated with rFsh for 6 weeks (TREATED group). H-E staining. Magnification bar = 1 mm. sz = spermatozoa.
Figure 3
Figure 3
Micrographs of testis sections from (a) a juvenile meagre treated with saline solution for 6 weeks (CONTROL 6 group) and (b) a juvenile fish treated with rFsh for 6 weeks (TREATED group). H–E staining. Magnification bars = 50 μm. Arrowhead = single type A spermatogonium; dashed arrow = type A spermatogonial cyst; curved arrow = type B spermatogonial cyst; scI = primary spermatocyte cyst; scII = secondary spermatocyte cyst; sd = spermatid cyst; sz = spermatozoa.
Figure 4
Figure 4
Mean (± sd) diameter of seminiferous tubules of untreated (CONTROL 0, n = 5), treated with saline solution for 6 weeks (CONTROL 6, n = 9) or treated with rFsh for 6 weeks (TREATED, n = 4) juvenile meagre males. Different letters represent statistically significant differences (ANOVA; P < 0.05).
Figure 5
Figure 5
Micrographs of testis sections of (a) a juvenile meagre treated with saline solution for 6 weeks (CONTROL 6 group) and (b) a juvenile fish treated with rFsh for 6 weeks (TREATED group), immunostained with antibodies against the proliferating cell nuclear antigen (PCNA), which stains brown the nuclei of proliferating cells. Magnification bars = 50 μm. Arrowhead = anti-PCNA–positive single type A spermatogonium; arrow = anti-PCNA–positive spermatogonial cyst; dashed arrow = anti-PCNA–positive primary spermatocyte cyst.
Figure 6
Figure 6
Changes in mean (± sd) anti-proliferating cell nuclear antigen (PCNA) positive germ cell density in untreated (CONTROL 0, n = 5), treated with saline solution for 6 weeks (CONTROL 6, n = 9) or treated with rFsh for 6 weeks (TREATED, n = 4) juvenile meagre males. (a) Anti-PCNA positive single A spermatogonia. (b) Anti-PCNA positive spermatocysts. Different letters represent statistically significant differences (ANOVA; P < 0.05).
Figure 7
Figure 7
Micrographs of testis sections from (a) a juvenile meagre treated with saline solution for 6 weeks (CONTROL 6 group) and (b) a juvenile meagre treated with rFsh for 6 weeks (TREATED group), stained with the terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labeling (TUNEL) method, with apoptotic cells appearing as dark blue dots. Magnification bars = 100 μm. Arrow = TUNEL–positive spermatogonial cyst; arrowhead = TUNEL–positive single type A spermatogonium.
Figure 8
Figure 8
Changes in mean (± sd) surface occupied by apoptotic germ cells in untreated (CONTROL 0, n = 5), treated with saline solution for 6 weeks (CONTROL 6, n = 9) and treated with rFsh for 6 weeks (TREATED, n = 4) juvenile meagre males. Different letters represent statistically significant differences (ANOVA; P < 0.05).
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References

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