Kisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E₂) and progesterone (P₄) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6–10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E₂ and P₄ was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3β-HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal-regulated kinase 1/2, ERK1/2 and p-ERK1/2) was studied in the GCs by qPCR. Significant E₂ production was found in the Kp 50 and 100 nM groups (p < .05), whereas P₄ production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p-ERK1/2 was observed in the Kp-treated GCs. It was concluded that Kp at 100 nM concentration stimulated E₂ production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp-mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

中文翻译：

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Kisspeptin 通过上调体外 Bubaline 颗粒细胞中的类固醇生成转录物和增殖标记来刺激雌二醇生物合成

Kisspeptin (Kp) 是 GnRH 释放的上游调节因子，对于生殖轴的发育和功能至关重要。之前，我们证明了 Kp 及其受体 (Kiss1r) 在 bubaline 卵巢活跃卵泡中的定位。本研究旨在确定 Kp 对水牛颗粒细胞 (GC) 功能的影响，特别是雌二醇 (E ₂ ) 和孕酮 (P ₄ ) 的产生，以及调节水牛增殖、凋亡和类固醇生成的基因的差异表达。屠宰后的循环水牛的卵巢具有6-10毫米大小的卵泡，用于分离GC用于体外研究。用 Kp（0、10、50 和 100 nM）处理原代 GC 培养物并孵育 48 小时。通过ELISA估计培养物上清液中E ₂和P ₄的产生。促性腺激素受体（FSHR和LHR）、类固醇生成基因（STAR、3β-HSD、CYP19A1）、增殖标志物（PCNA）、凋亡因子（CASP3和BCL2）和Kp信号分子（细胞外信号调节激酶1/2、通过 qPCR 在 GC 中研究了 ERK1/2 和 p-ERK1/2)。在 Kp 50 和 100 nM 组中发现显着的 E _{2产量 (} p  < .05)，而在 Kp 100 nM 组中 P ₄产量减少 ( p  < .05)。Kp 100 nM 处理的 GC 中 FSHR、ERK1/2、STAR 和 CYP19A1 伴随上调。此外，100 nM Kp 通过上调 BCL2（5.0 倍）和 PCNA（94.9 倍）的表达来刺激 GC 增殖。此外，在 Kp 处理的 GC 中观察到 p-ERK1/2 的高免疫反应性。结论是，100 nM 浓度的 Kp通过 ERK1/2、STAR 和 CYP19A1 上调类固醇生成途径并调节 GC 中的 PCNA 和 BCL2 表达来刺激 E _2产生。有必要使用不同组合的 Kp 拮抗剂进行进一步的实验，以建立 GC 中 Kp 介导的类固醇生成的信号通路，从而制定控制卵巢功能的策略。