Laboratorio de Biología de la Reproducción, Facultad de Ciencias de la Salud, Departamento Biomédico, Universidad de Antofagasta, Antofagasta 1240000, Chile.
Instituto Antofagasta, Universidad de Antofagasta, Antofagasta 1240000, Chile.
Cells. 2021 Dec 11;10(12):3501. doi: 10.3390/cells10123501.
The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.
蛋白酶体在精子获能开始时通过 SACY/PRKACA 途径增加其活性；这种增加对于获能的进展是必需的。PRKA 活性在获能过程中也增加并保持高水平。然而，在此过程中细胞内 cAMP 水平下降。我们的目标是评估蛋白酶体在精子获能开始后调节 PRKA 活性的作用。将有活力的人精子在存在和不存在环氧酶素或 0.1%DMSO 的情况下孵育。通过 Western blot 评估 PRKA 活性；PRKA 底物的磷酸化模式（pPRKAs）；PRKAR1、PRKAR2 和 AKAP3 的表达。通过免疫荧光评估 pPRKAs、PRKAR1、PRKAR2 和 AKAP3 的定位。环氧酶素处理改变了定位和磷酸化模式，并降低了 pPRKAs 阳性精子的百分比。PRKA 活性在获能 1 分钟时显着增加，并在整个孵育过程中保持高水平。然而，环氧酶素处理在 30 分钟后显着降低了 PRKA 活性。此外，蛋白酶体降解 PRKAR1 和 AKAP3，但具有不同的时变动力学。我们的结果表明，PRKAR1 是蛋白酶体调节 PRKA 的靶标。
