RESUMEN
To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.
Asunto(s)
Animales , Masculino , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Ratones Noqueados , Espermatogénesis/genética , Espermatogonias/metabolismo , Testículo/metabolismoRESUMEN
<p><b>OBJECTIVE</b>To construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.</p><p><b>METHODS</b>PIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.</p><p><b>RESULTS</b>We successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.</p><p><b>CONCLUSION</b>The construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.</p>
Asunto(s)
Animales , Masculino , Ratones , Línea Celular , Vectores Genéticos , Lentivirus , Genética , Plásmidos , Proteínas Inhibidoras de STAT Activados , Genética , Espermatocitos , Biología Celular , TransfecciónRESUMEN
<p><b>OBJECTIVE</b>To screen and identify PIAS2-interacting proteins from the mouse spermatogonial cDNA library using the yeast two-hybrid system, and to investigate the action mechanism of PIAS2 in spermatogenesis.</p><p><b>METHODS</b>With pGBKT7-PIAS2 as a bait plasmid, the positive clones interacting with pGBKT7-PIAS2 were screened from the mouse spermatogonial cDNA library, the inserted fragments were sequenced and underwent bioinformatic analysis, and their interaction was verified using the yeast two-hybrid system.</p><p><b>RESULTS</b>Through screening, sequencing, homology analysis and yeast two-hybrid verification, we obtained 8 different candidate proteins interacting with PIAS2, including Cyfip2, Psmb3, Nmel, nischarin, Ints10, Nsun5, Gnb211 and Ndufaf3.</p><p><b>CONCLUSION</b>Eight different genes were successfully obtained using the yeast two-hybrid system, and their encoding proteins interacted with PIAS2, which might be related with male fertility regulation. Our findings have offered some new clues to the action mechanism of PIAS2 in spermatogenesis.</p>