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μ-阿片受体(MOR)在马卵丘-卵母细胞复合体中的表达与定位及其在卵母细胞减数分裂能力季节性调节中的作用

Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence.

作者信息

Dell'Aquila Maria Elena, Albrizio Maria, Guaricci Antonio Ciro, De Santis Teresa, Maritato Filippo, Tremoleda Jordi L, Colenbrander Ben, Guerra Lorenzo, Casavola Valeria, Minoia Paolo

机构信息

Department of Animal Production, University of Bari, Valenzano-Bari, Italy.

出版信息

Mol Reprod Dev. 2008 Aug;75(8):1229-46. doi: 10.1002/mrd.20869.

Abstract

The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 x 10(-8) M beta-endorphin in medium without additional hormones. Inhibitory effects of 10(-3) M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10(-8) M Naloxone. The exposure to 10(-3) M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. beta-Endorphin 3 x 10(-8) M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10(-8) M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus-oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes.

摘要

在马的卵母细胞、卵丘细胞和颗粒细胞中鉴定出了微阿片受体(MOR)。通过逆转录聚合酶链反应(RT-PCR),观察到一个441bp的片段。通过免疫印迹法,在冬季乏情期的样本中检测到一条65kDa的条带,而在繁殖季节回收的细胞中,发现了两条条带,分别为65kDa和50kDa。65kDa的条带在冬季乏情期标本中明显更强。在繁殖季节回收的样本中,随着卵泡大小的增加,这条带显著减少,并且在紧密的卵母细胞和卵丘细胞中更重。该蛋白定位于卵母细胞膜和卵母细胞及卵丘细胞的细胞质内。在无额外激素的培养基中添加3×10⁻⁸M的β-内啡肽后,通过共聚焦显微镜分析核染色质、微丝和微管,体外卵母细胞成熟率(MR)降低。在添加或不添加培养基中激素的情况下,均观察到10⁻³M纳洛酮对乏情期和春季过渡期采集的卵母细胞有抑制作用。在乏情期采集并在10⁻⁸M纳洛酮存在下培养的卵母细胞中观察到成熟率增加。全年回收的卵丘细胞暴露于10⁻³M纳洛酮会导致细胞内钙显著增加。仅在秋季过渡期和乏情期回收的卵丘细胞中,3×10⁻⁸M的β-内啡肽会导致钙显著增加。10⁻⁸M的纳洛酮不会引起细胞内钙的变化。我们得出结论,MOR在一年中的不同季节在马的卵丘-卵母细胞复合体中差异表达,并在马卵母细胞减数分裂能力的季节性调节中发挥作用。

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