Gramajo-Bühler M C, Zelarayán L, Sánchez-Toranzo G
Department of Developmental Biology, Higher Institute for Biological Research, San Miguel de Tucumán, Argentina.
Faculty of biochemistry, chemistry and Pharmacy, Institute of Biology 'Dr. Francisco Barbieri', National University of Tucumán, San Miguel de Tucumán, Argentina.
Reprod Domest Anim. 2016 Feb;51(1):150-7. doi: 10.1111/rda.12659. Epub 2015 Dec 23.
The mechanisms involved in fertilization are the centre of attention in order to determine the conditions required to reproduce in vitro the events that take place in vivo, with special interest in endangered species. Previous data from mouse sperm, where acrosome reaction (AR) occurs more often in the interstitium of the cumulus oophorus, contribute to strengthen the use of progesterone as a physiological inducer of this process. We studied the participation of protein kinase A (PKA), phospholipases A2 and C (PLA2 , PLC) in the AR induced by progesterone from Chinchilla epididymal spermatozoa. The addition of db-cAMP to the incubation medium caused an increase of 58% in the AR, while the use of H89 (30 μm), a PKA inhibitor, reflected a decrease of 40% in the percentage of reacted gametes. The assays conducted with arachidonic acid showed a maximum increase of 23% in the AR. When gametes were pre-incubated with PLA2 inhibitors, a dose-dependent inhibitory effect was observed. The addition of phorbol12-myristate13-acetate (10 μm) revealed higher percentages of AR induction (60%). When PLC was inhibited with neomycin and U73122, a dose-dependent decrease in AR percentages was observed. Combined inhibition of PKA, PLA2 and PLC, AR values similar to control were obtained. This work shows evidence, for the first time in Chinchilla, that progesterone activates the AC/cAMP/PKA system as well as sperm phospholipases and that these signalling pathways participate jointly and cooperatively in AR. These results contribute to the understanding of the complex regulation that is triggered in sperm after the effect of progesterone.
为了确定体外重现体内发生的事件所需的条件，尤其是对濒危物种的研究，受精过程所涉及的机制成为了关注的焦点。先前来自小鼠精子的数据表明，顶体反应（AR）在卵丘间质中更常发生，这有助于强化将孕酮用作该过程生理诱导剂的应用。我们研究了蛋白激酶A（PKA）、磷脂酶A2和C（PLA2、PLC）在龙猫附睾精子中由孕酮诱导的AR中的作用。向孵育培养基中添加二丁酰环磷腺苷（db - cAMP）导致AR增加了58%，而使用PKA抑制剂H89（30μm）则使反应配子的百分比降低了40%。用花生四烯酸进行的试验显示AR最大增加了23%。当配子与PLA2抑制剂预孵育时，观察到剂量依赖性抑制作用。添加佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（10μm）显示出更高的AR诱导百分比（60%）。当用新霉素和U73122抑制PLC时，观察到AR百分比呈剂量依赖性下降。联合抑制PKA、PLA2和PLC时，获得了与对照相似的AR值。这项工作首次在龙猫中证明，孕酮激活了AC/cAMP/PKA系统以及精子磷脂酶，并且这些信号通路在AR中共同且协同发挥作用。这些结果有助于理解孕酮作用后精子中触发的复杂调节机制。
