RESUMEN
Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Espermatogénesis/genética , Proteínas de Anclaje a la Quinasa A/genética , Animales , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Motilidad Espermática/genética , Motilidad Espermática/fisiología , Cola del Espermatozoide/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
The effects of ethylene on tension wood formation were studied in 3-year-old Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings in two separate experiments. In experiment 1, ethylene evolution of buds and stems was measured using gas chromatography after 0, 2, 4, 7, 14, and 21 d of treatment; in experiment 2, both aminoethoxyvinylglycine (AVG) and AgNO3 were applied to the horizontally-placed stems, and the cell numbers on sites of applications were measured after 40 d. Ethylene evolution from buds was found to be much greater in tilted seedlings than in upright ones. The cell numbers of wood fibers in shoots and 1-year-old stems were reduced in treatments with 12.5 x 10(-7)micromol/L AVG, 12.5 x 10(-8)micromol/L AVG, and 11.8 x 10(-8)micromol/L AgNO3; whereas the horizontal and vertical diameters were reduced by treatment of 12.5 x 10(-7)micromol/L AVG. Ethylene evolutions of shoots and 1-year-old stems were inhibited greatly in comparison with the control by applying 12.5 x 10(-7)micromol/L AVG. The formation of a gelatinous layer of wood fibers was affected by neither AVG nor AgNO3 application. These results suggest that ethylene regulates the quantity of wood production, but does not affect G-layer formation in F. mandshurica Rupr. var. japonica Maxim. seedlings.