Guidobaldi Héctor Alejandro, Teves María Eugenia, Uñates Diego Rafael, Anastasía Agustín, Giojalas Laura Cecilia
Centro de Biología Celular y Molecular, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina.
PLoS One. 2008 Aug 22;3(8):e3040. doi: 10.1371/journal.pone.0003040.
Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.
在哺乳动物中，已确定精子对几种雌性来源具有趋化性，如卵泡液（FF）、输卵管液以及来自卵丘（CU）和卵母细胞（O）的条件培养基。尽管有几种物质被确认为精子化学引诱剂，但孕酮（P）似乎是最佳的化学引诱剂候选物，原因如下：1）精子表达细胞表面P受体；2）获能精子在体外会被低浓度P的梯度化学吸引；3）CU细胞在排卵后产生并分泌P；4）P的梯度可能在CU周围保持稳定；5）体内精子趋化最可能的部位可能在CU附近和/或内部。本研究的目的是验证在作为哺乳动物动物模型的兔子中，P是否是兔卵母细胞 - 卵丘复合体（OCC）分泌的精子化学引诱剂。通过视频显微镜和计算机图像分析，我们观察到只有CU是精子引诱剂的稳定来源。通过免疫细胞化学在这些细胞内定位该激素，并通过酶免疫测定在条件培养基中检测到，CU产生并分泌P。此外，通过蛋白质印迹检测到兔精子表达细胞表面P受体，并通过免疫细胞化学将其定位在顶体区域。为了证实P是CU分泌的精子化学引诱剂，采用三种不同方法抑制了精子对OCC条件培养基的趋化反应：用抗P抗体去除OCC条件培养基中的P，用P反向梯度破坏OCC条件培养基的引诱剂梯度，并用特异性抗体阻断精子P受体。我们得出结论，只有CU而非卵母细胞分泌P，并且后者通过细胞表面受体对精子产生化学吸引作用。我们的研究结果可能对人类、具有经济重要性的动物以及濒危物种的辅助生殖程序具有重要意义。
