Viability and protein phosphorylation patterns of boar spermatozoa agglutinated by treatment with a cell-permeable cyclic adenosine 3',5'-monophosphate analog.
Viability and protein phosphorylation patterns of boar spermatozoa agglutinated by treatment with a cell-permeable cyclic adenosine 3',5'-monophosphate analog.
复制标题
用细胞可渗透的环腺苷 3,5-单磷酸类似物处理后凝集的公猪精子的活力和蛋白质磷酸化模式。
作者：
H. Harayama
当猪精子的细胞内环腺苷3',5'-单磷酸（cAMP）信号级联在头部被激活时，它们在头部相互凝集。本研究的目的是研究camp依赖性凝集猪精子的生存能力和蛋白质磷酸化模式。将射精后的精子清洗，然后在含有聚乙烯醇（mkhh - pva）和0.1 mM sp -5,6-二氯-1- β -d -核呋喃基-苯并咪唑-3',5'-单磷硫酸盐（cimps，一种细胞渗透性cAMP类似物）的改良的Krebs-Ringer HEPES培养基中在38.5℃下孵育180分钟。各孵育期后取等量精子悬浮液，用SYBR14-PI染色和活力法检测凝集状态，用Western blotting和间接免疫荧光法检测蛋白磷酸化状态。在不加cBiMPS孵育180分钟的对照样品中，不到30%的精子在头部相互凝集，超过70%的凝集精子为碘化丙啶（PI）阳性（死亡）。然而，用cBiMPS孵育30分钟内，头对头凝集精子的百分比迅速增加到约60%，但此后没有显著改变。此外，在cBiMPS孵育180分钟的样品中，凝集精子的pi阳性细胞百分比（约30%）显著低于对照样品（超过70%）。这一结果也得到了支持，在cBiMPS孵育180分钟的样品中，凝集精子的运动细胞百分比远高于未孵育的对照样品。Western blotting和间接免疫荧光显示，cbimps诱导的丝氨酸/苏氨酸磷酸化（如>220 kd、220 kd、180 kd、84 kd和54 kd）在30分钟内主要出现在凝集精子和游离精子的连接片段和主要片段，30分钟后在中间片段出现额外磷酸化。此外，在与cBiMPS孵育超过30分钟后，几乎一半的凝集精子的连接段和主段中酪氨酸磷酸化程度强烈，中间段中酪氨酸磷酸化程度中等，而在自由精子中则很少发生酪氨酸磷酸化（如>220 kd、190 kd、93 kd、59 kd、54 kd和32 kd）。这些发现与以下建议一致：激活camp信号级联导致活精子快速（30分钟内）头对头凝集；在camp依赖性凝集和游离精子的连接和主要片段中快速（30分钟内）蛋白丝氨酸/苏氨酸磷酸化，以及随后（30分钟后）在它们的中间片段中磷酸化；以及在camp依赖性凝集精子的连接、中间和主要片段中缓慢（超过30分钟）的蛋白酪氨酸磷酸化。基于这些建议，我们得出结论，许多依赖camp的凝集精子是活细胞，其中camp信号级联导致蛋白丝氨酸/苏氨酸和酪氨酸磷酸化在整个鞭毛中被激活。
Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in the head. The aim of the present study is to examine viability and protein phosphorylation patterns of cAMP-dependently agglutinated boar spermatozoa. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, a cell-permeable cAMP analog) at 38.5 degrees C up to 180 minutes. Aliquots of the sperm suspensions were recovered after various incubation periods and then used to examine the state of agglutination, the viability by SYBR14-PI staining and motility assay, and the state of protein phosphorylation by Western blotting and indirect immunofluorescence. In the control samples incubated without cBiMPS for 180 minutes, less than 30% of the total spermatozoa were agglutinated with one another at the heads, and more than 70% of the agglutinated spermatozoa were propidium iodide (PI)-positive (dead). However, the incubation with cBiMPS rapidly increased the percentages of head-to-head agglutinated spermatozoa to approximately 60% within 30 minutes, but did not significantly change them thereafter. In the samples incubated with cBiMPS for 180 minutes, moreover, the percentages of PI-positive cells of the agglutinated spermatozoa (approximately 30%) were significantly lower than those obtained in the control samples (more than 70%). This result was supported by the observation that the percentages of motile cells of the agglutinated spermatozoa were much higher in the samples incubated with cBiMPS for 180 minutes than in the control samples incubated without cBiMPS. As revealed by Western blotting and indirect immunofluorescence, cBiMPS-induced serine/threonine phosphorylation of the proteins (eg, >220 kd, 220 kd, 180 kd, 84 kd, and 54 kd) appeared mainly in the connecting and principal pieces of both agglutinated and free spermatozoa within 30 minutes, and additional phosphorylation occurred in the middle piece later than 30 minutes. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kd, 190 kd, 93 kd, 59 kd, 54 kd, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. These findings are consistent with the following suggestions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; and slow (later than 30 minutes) protein tyrosine phosphorylation in the connecting, middle, and principal pieces of the cAMP-dependently agglutinated spermatozoa. Based on these suggestions, we conclude that many of cAMP-dependently agglutinated spermatozoa are live cells in which cAMP-signaling cascades leading to protein serine/threonine and tyrosine phosphorylation are activated in the whole flagellum.
影响因子：
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作者：
P. Visconti;H. Galantino-Homer;Grace D. Moore;Janice L. Bailey;Xiaoping Ning;Miguel W. Fornés;G. S. Kopf
通讯作者：
P. Visconti;H. Galantino-Homer;Grace D. Moore;Janice L. Bailey;Xiaoping Ning;Miguel W. Fornés;G. S. Kopf
影响因子：
3.6
作者：
Naz, RK
通讯作者：
Naz, RK
Phorbol 12-myristate 13-acetate 抑制人因子依赖性造血细胞系中粒细胞-巨噬细胞集落刺激因子诱导的蛋白酪氨酸磷酸化。
DOI：
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发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Kanakura,Y;Druker,B;DiCarlo,J;Cannistra,SA;Griffin,JD
通讯作者：
Griffin,JD