The improvement of cryopreserved oocyte survival is imperative for the preservation of female fertility. In this study, we investigate whether P2Y2 receptors (P2Y2R) can be directly implicated in calcium (Ca²⁺) homeostasis misbalances observed during the cryopreservation process of cumulus oocyte complexes (COC). Firstly, RNA was extracted from bovine immature and mature oocytes and cumulus cells and real-time PCR performed to identify P2Y2R transcripts (experiment 1). Changes in intracellular calcium concentration [Ca2+]i of mature COC and oocytes (experiment 2) were measured upon exposure to cryoprotectants (CPA), UTP (P2Y2R stimulator, 100 μM), and/or suramin (P2Y2R inhibitor, 100 and 300 μM). The functional role of P2Y2R was investigated by analyzing the effect on oocyte viability of its modulation prior and during oocyte exposure to CPA (experiment 3). Mature COC were randomly divided into groups, and exposed to CPA and different P2Y2 modulators. Oocytes’ viability, cortical granules location, and competence for development were assessed. Results showed that P2Y2R mRNAs are expressed in both oocytes and cumulus cells. Stimulation with UTP and CPA led to [Ca2+]i increase, and this effect was totally or partially blocked by suramin (P2Y2R inhibitor). Oocyte exposure to CPA and UTP reduced embryo rates compared with control and suramin100μM (P ≤ 0.04). The observed enhanced premature zona hardening in oocytes exposed to CPA (P = 0.04) and UTP (P = 0.005) stimulus was inhibited by suramin 100 μM. In conclusion, inhibition of P2Y2R during cryoprotectant exposure reduces premature intracellular Ca²⁺ release and significantly improves the developmental competence of exposed bovine oocytes.

中文翻译：

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牛卵丘卵母细胞复合物中P2Y2受体的调节：对细胞内钙，透明带硬化和发育能力的影响。

冷冻保存的卵母细胞存活率的提高对于保持女性的生育能力至关重要。在这项研究中，我们调查P2Y2受体（P2Y2R）是否可以直接涉及在卵卵母细胞复合物（COC）的冷冻保存过程中观察到的钙（Ca ²⁺）稳态失衡。首先，从牛未成熟和成熟的卵母细胞和卵丘细胞中提取RNA，并进行实时PCR鉴定P2Y2R成绩单（实验1）。在暴露于冷冻保护剂（CPA），UTP（P2Y2R刺激剂，100μM）和/或苏拉明（P2Y2R抑制剂，100和300μM）后，测量成熟COC和卵母细胞的细胞内钙浓度[Ca2 +] i的变化（实验2）。 。通过分析卵母细胞暴露于CPA之前和期间，P2Y2R的功能对卵母细胞生存力的影响，对其功能进行了研究（实验3）。将成熟的COC随机分组，并暴露于CPA和不同的P2Y2调节剂中。评估了卵母细胞的生存力，皮质颗粒的位置和发育能力。结果表明，P2Y2R mRNA在卵母细胞和卵丘细胞中均表达。用UTP和CPA刺激导致[Ca2 +] i升高，这种作用被苏拉明（P2Y2R抑制剂）完全或部分阻断。P  ≤0.04）。 苏拉明100μM抑制了暴露于CPA（P  = 0.04）和UTP（P = 0.005）刺激的卵母细胞中增强的过早的透明带硬化。总之，在冷冻保护剂暴露期间抑制P2Y2R会减少细胞内Ca ^2+的过早释放，并显着提高暴露的牛卵母细胞的发育能力。