Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo. We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6-38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA. All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation. In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

中文翻译：

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雌性大鼠垂体前叶体内促性腺激素释放激素-铜复合物 (Cu-GnRH) 的转录活性。

与以十肽一级结构中的氨基酸置换为特征的促性腺激素释放激素 (GnRH) 类似物不同，Cu-GnRH 分子保留了天然序列，但包含一个与氮原子稳定结合的 Cu2+ 离子，包括 His2 的咪唑环的氮原子。Cu-GnRH 可以通过 cAMP/PKA 信号在垂体前叶细胞中起作用，这表明它可能影响体内选定的促性腺网络基因转录。我们根据 Egr-1、Nr5a1 和 Lhb 在促黄体生成素 (LH) 合成中的作用分析了垂体 mRNA 的表达；和 Nos1、Adcyap1 和 Prkaca，因为它们依赖于 cAMP/PKA 活性。在两个独立的实验中，去卵巢的大鼠接受了 2 nM Cu-GnRH 的脑室内搏动（一个脉冲/小时或超过 5 小时的两个脉冲/小时）显微注射；2 nM antide（GnRH 拮抗剂）+ 2 nM Cu-GnRH；100 nM PACAP6-38（PACAP 受体拮抗剂）+ 2 nM Cu-GnRH。通过 qRT-PCR 确定所选 mRNA 的相对表达。根据 RIA 检查 LH 血清浓度。所有检查的基因都以依赖于脉冲频率模式的方式响应 Cu-GnRH 刺激并增加转录活性。Nr5a1、Lhb、Nos1、Adcyap1 和 Prkaca mRNA 的表达增加仅在接受复合物的大鼠中观察到，在 5 小时内以两次脉冲/小时的频率。对于两种应用的 Cu-GnRH 脉动模式，Egr-1 转录均上调。Cu-GnRH 对基因转录的刺激作用取决于 GnRH 受体和 PAC-1 的激活。总之，获得的结果表明，Cu-GnRH 复合物是一种 GnRH 类似物，能够在体内诱导 IP3/PKC 和 cAMP/PKA 依赖性促性腺激素网络基因转录。