RESUMO
TSP1 was reported to be involved in multiple biological processes including the activation of TGF-ß signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17ß-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia.
Assuntos
Proteínas de Peixes/genética , Folículo Ovariano/metabolismo , Trombospondina 1/genética , Tilápia/metabolismo , Animais , Aromatase/genética , Aromatase/metabolismo , Feminino , Proteínas de Peixes/metabolismo , Sistema de Sinalização das MAP Quinases , Folículo Ovariano/crescimento & desenvolvimento , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Trombospondina 1/metabolismo , Tilápia/fisiologiaRESUMO
A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3-/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3-/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.
Assuntos
Reprodução , Somatomedinas/metabolismo , Espermatogênese , Tilápia/fisiologia , Androgênios/farmacologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Camundongos Knockout , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Somatomedinas/genética , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Tilápia/genéticaRESUMO
In the present study, Amh was found to be abundantly expressed in the granulosa cells of the primary growth follicles, and Amhr2 in the granulosa cells, oogonia and phase I oocytes in tilapia by immunohistochemistry. In addition, Amh and Amhr2 were also found to be expressed in the brain and pituitary. Heterozygous mutation of either amh or amhr2 resulted in increased primary growth follicles and decreased fertility, and homozygous mutation resulted in hypertrophic ovaries with significantly increased primary follicles and failed transition from primary to vitellogenic follicles. Expression of gnrh3 in the brain, fsh and lh in the pituitary and serum E2 concentration were significantly decreased in both mutants. Significantly increased apoptosis of follicle cells was observed in both mutants. However, administration of E2 failed to rescue the folliculogenesis defects of the mutants. Our results suggested that Amh acts in a dose-dependent manner by binding Amhr2 in tilapia.
