Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos

Abstract

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 p < 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 ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 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 BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 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.

Keywords: C-type natriuretic peptide; NPR2; cattle; embryonic metabolism; transcript abundance.

Figure 1

NPR2 localization in bovine oocytes and pre-implantation-stage embryos. The green color indicates NPR2 staining, and the blue color indicates nuclear staining (DAPI). NPR2 protein was expressed in bovine oocytes and embryos at all stages. GV, germinal vesicle; M II, metaphase II; PZs, presumptive zygotes; Morula, morula stage; Blastocyst, blastocyst stage. Bar = 50 µm.

Figure 2

Box plot of fluorescence intensity of NPR2 in oocytes and pre-implantation-stage embryos. Results are presented as the median and 1° and 3° interquartile intervals of five replicates/stage using 8 structures in total. Different letters above each box represent significant differences (p ≤ 0.05). GV, germinal vesicle; M II, metaphase II; PZs, presumptive zygotes; Morula, morula stage; Blastocyst, blastocyst stage. AU, arbitrary units.

Figure 3

Effect of CNP treatment in IVC on differential gene expression in morula. Data represent the fold change in relative target abundance related to the reference gene. Downregulated transcription ACAT1 (p = 0.040), CASP3 (p = 0.082), and BMP15 (p = 0.013) and upregulated transcription ADCY6 (p = 0.057) with the addition of CNP (400 nM) on the D1 of the culture. Results are represented by least squares means ± SEM of four replicates/group. Different letters above each bar represent significant differences (p ≤ 0.08). Control (no treatment) and C-400 (400 nM of CNP).

Figure 4

Multivariate analysis plots of the abundance of transcripts derived from untreated (control) and CNP-treated morula. (A) Cluster analysis heatmap showing transcriptional profiles abundance in only 12 genes most impacted from morula treated with 400 nM CNP and the control group. (B) Two-dimensional PLS-DA discrimination score plot between groups (5 morulas/group in 4 replicates).

Figure 5

Multivariate analysis plots of the abundance of transcripts derived from untreated (control) and CNP-treated blastocyst. (A) Cluster analysis heatmap showing transcriptional profiles abundance in only 11 genes most impacted from blastocyst treated with 400 nM CNP and the control group. (B) Two-dimensional PLS-DA discrimination score plot between groups (3 embryos/group in 4 replicates).

Figure 6

Illustrative experimental design. IVC, in vitro culture; EXP, experiment; GV, germinal vesicle; MII, metaphase II; PZs, presumptive zygotes; MO, morula; BL, blastocyst; CTL, control group; CNP, group treated with C-type natriuretic peptide. © 2024 BioRender.