Gervasi Maria Gracia, Marczylo Timothy H, Lam Patricia M, Rana Shashi, Franchi Ana M, Konje Justin C, Perez-Martinez Silvina
Laboratory de Biología de la Reproducción en Mamíferos, Centro de Estudios Farmacológicos y Botánicos, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, Buenos Aires, Argentina.
PLoS One. 2013 Aug 16;8(8):e72521. doi: 10.1371/journal.pone.0072521. eCollection 2013.
Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.
哺乳动物的输卵管是精子的储存库，并提供了一个环境，使精子能够在此竞争使卵母细胞受精的机会。精子在输卵管中时会经历受精所必需的获能过程。精子与输卵管的相互作用对于精子获能至关重要，并且是一个受局部微环境严格调控的过程。此前我们报道过，内源性大麻素花生四烯酸乙醇胺（AEA）通过促进精子获能来调节精子从输卵管上皮细胞的释放。这项工作的目的是测量牛输卵管中AEA的含量，表征主要的AEA代谢途径，并确定这些在发情周期中如何变化。在本研究中，通过超高效液相色谱串联质谱法测量了在发情周期不同阶段采集的牛输卵管中AEA以及另外两种N-酰基乙醇胺（N-油酰乙醇胺和N-棕榈酰乙醇胺）的水平。结果表明，在发情周期中，所有三种N-酰基乙醇胺在输卵管上皮细胞内的水平都会波动。输卵管液中的花生四烯酸乙醇胺在发情周期中也有所变化，在排卵前后检测到的值最高。同侧输卵管至排卵侧的内源性大麻素水平高于对侧检测到的水平，这表明输卵管AEA的水平可能受卵巢激素调节。还通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法和免疫组织化学法测定了牛输卵管中N-酰基乙醇胺代谢酶的表达和定位，但在发情周期中未发现变化。此外，在卵泡液中检测到了纳摩尔水平的AEA，这表明在排卵期间，成熟卵泡可能会影响输卵管AEA水平，以形成有利于调节精子功能从而实现成功受精的内源性大麻素梯度。
