Graduate Program in Pharmacology and Biotechnology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-970, SP, Brazil.
Department of Biological Sciences, School of Sciences, Humanities and Languages, São Paulo State University (UNESP), Assis 19806-900, SP, Brazil.
Int J Mol Sci. 2024 Oct 11;25(20):10938. doi: 10.3390/ijms252010938.
The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ( = 0.057) and downregulated transcripts in ( = 0.013), ( = 0.040), and ( = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in , , , and transcripts and downregulation in , , , , , and transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.
C 型利钠肽(CNP)在与卵母细胞的相互作用以及暂时推迟自发减数分裂恢复中的作用已经得到了很好的描述。然而,其在胚胎着床前发育阶段的作用尚不清楚。因此,我们的研究旨在检测在生殖泡(GV)期、中期 II(MII)期、假定受精卵(PZ)期、桑椹胚(MO)期和囊胚(BL)期胚胎中存在经典 CNP 受体(利钠肽受体,NPR2),并随后观察在与 CNP 共培养时胚胎可能发生的变化。在实验 I 中,我们检测并定量了上述胚胎阶段的 NPR2。此外,在实验 II 中,我们旨在测试不同浓度(100、200 或 400 nM 的 CNP)在体外培养(IVC)中不同时间点(从第一天开始,即第 1 天,或从第 5 天开始)的纳入。在实验 III 中,在 IVC 中于第 1 天(D1)使用 400 nM 的 CNP,结果显示其对胚胎没有毒性作用,并且与对照组相比,在囊胚产生率方面显示出有潜在的良好结果。因此,我们分析了牛胚胎的胚胎发育率(D7)和孵化动力学(D7、D8 和 D9)。随后,收集桑椹胚和囊胚,并评估其能力和质量(细胞凋亡、氧化应激、增殖和分化)以及脂质代谢的转录丰度。差异概率 < 0.05,和/或倍数变化(FC)> 1.5 被认为具有统计学意义。我们证明了 NPR2 存在于囊胚发育阶段,此时膜受体显著减少。与对照组相比,共培养 400 nM CNP 后,囊胚的产生率没有统计学差异。然而,当我们评估桑椹胚转录物的丰度时,发现一些转录物的转录上调,而其他转录物的转录下调,如 ( = 0.057)和 ( = 0.013)、 ( = 0.040)和 ( = 0.082)。此外,在桑椹胚中,共有 12 个转录物的转录变化 FC > 1.5。在囊胚中,CNP 的处理诱导 ( = 0.007)、 ( = 0.022)、 ( = 0.022)和 ( = 0.035)转录物的上调以及 ( = 0.014)、 ( = 0.017)、 ( = 0.020)、 ( = 0.033)、 ( = 0.033)和 ( = 0.033)转录物的下调(FC > 1.5)。因此,我们的研究在牛胚胎着床前阶段鉴定和定量了 NPR2 的存在。此外,在 IVC 中使用 400 nM 的 CNP 是一种以前文献中未描述的浓度,调节了一些与胚胎代谢相关的转录物,且形态上对胚胎没有毒性。