Comparative Transcriptomic Analysis of In Vitro Matured Oocytes From Cyclic and Prepubertal Gilts Reveals Distinct Molecular Patterns

ABSTRACT

In vitro embryo production is a key tool in reproductive biotechnology, enabling genetic improvement, basic research on early development, and the generation of biomedical models. Despite high rates of nuclear maturation achieved during in vitro maturation (IVM) of porcine oocytes, inadequate cytoplasmic maturation continues to limit oocyte competence and subsequent development, particularly when using oocytes from prepubertal gilts (PG). The molecular basis underlying differences in oocytes from cyclic females remains incompletely understood. In this study, we performed a comparative transcriptomic analysis of immature (IMO) and in vitro matured oocytes (MO) from cyclic (CG) and PG using RNA sequencing (n = 20/group in 6 replicates). Differential expression analyses (FDR < 0.05) revealed extensive transcriptional changes between groups. The CG group showed enrichment of genes related to MAPK and PI3K–Akt signalling, and extracellular matrix interactions in MO vs. IMO, consistent with a more coordinated and developmentally competent maturation program. In contrast, oocytes from the PG group were enriched in genes related to cell cycle regulation, DNA damage response and homologous recombination, indicating a likely impairment in cytoplasmic maturation. Overall, our results provide complementary insight into the differences in the biological processes underlying IVM of oocytes retrieved from PG or CG in the porcine species.

1. Introduction

Porcine embryo in vitro production (IVP) provides an essential platform in porcine reproduction, with relevance for livestock production, developmental biology and biomedical research. Despite major advances in in vitro maturation (IVM) protocols that reliably achieve nuclear maturation, the acquisition of full oocyte competence remains suboptimal. It represents a key limitation to embryo developmental potential (Lin et al. 2015). Numerous studies across mammals have demonstrated that oocytes derived from prepubertal females exhibit reduced developmental potential compared with those from sexually mature cyclic females (Braga et al. 2019; Pawlak et al. 2011). Despite the widespread use of prepubertal gilts (PG) as oocyte donors for embryo IVP, the molecular mechanisms underlying their reduced oocyte competence remain incompletely understood. Although previous studies have shown that meiotic progression and nuclear maturation are largely comparable between PG and cyclic gilts (CG) (Pawlak et al. 2011), these parameters alone fail to predict developmental competence, suggesting that deficiencies in cytoplasmic maturation are critical (Appeltant et al. 2016). Recent studies of the oocyte transcriptome have begun to reveal differences in gene expression between oocytes of varying developmental competence (Braga et al. 2019). However, most of these investigations relied on targeted approaches or microarray technologies (Paczkowski et al. 2011). A comprehensive RNA‐seq analysis comparing maturation‐associated transcriptional dynamics between PG and CG has yet to be performed. Therefore, in the present study, we performed RNA‐seq analysis of immature (IMO) and in vitro‐matured (MO) oocytes from PG and CG.

2. Material and Methods

2.1. Experimental Design

Ovaries were collected from gilts slaughtered at a local abattoir (Comarán S.L. Aranjuez, Madrid), classed as PG or CG based on the absence or presence of corpora lutea, respectively. A subset of cumulus‐oocyte complexes (COCs) was sampled immediately at collection (IMO), while the remaining COCs were cultured for 44 h to collect MO. For each group, six independent biological replicates were analysed, each comprising pooled samples of 20 oocytes. Samples were snap‐frozen and subjected to RNA‐seq analysis.

2.2. IVM

COCs with homogeneous cytoplasm and at least two intact layers of cumulus cells were washed and cultured in pre‐equilibrated IVM medium, consisting of TCM‐199 supplemented with 0.55 mM glucose, 0.9 mM sodium pyruvate, antibiotics, 0.1 mg/mL PVA, 0.57 mM cysteine and 10 ng/mL epidermal growth factor. IVM medium was supplemented with 10 IU eCG and 10 IU hCG for 22 h, followed by an additional 22 h culture in hormone‐free IVM medium. IVM was performed at 38.5°C, in a humidified atmosphere and 5% CO₂ in air. After IVM, COCs were vortexed at 1660 rpm for 2 min in TL‐HEPES‐PVA supplemented with 0.1 mg/mL hyaluronidase to remove cumulus cells. Oocyte nuclear maturation was then assessed by Hoechst staining.

2.3. RNA Isolation and Sequencing

Total RNA was isolated using the miRNeasy Tissue/Cells Advanced Micro Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. RNA quantity and integrity were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Madrid, Spain) and RNA 6000 Pico Kit on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA), respectively. RIN values were higher than 8 for all samples. RNA‐seq libraries were prepared using the Stranded Total RNA Prep with Ribo‐Zero Plus (Illumina), and library size distribution and quality were evaluated using the High Sensitivity DNA assay on a 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced on an Illumina NextSeq 550 platform using paired‐end 2 × 75 bp reads under BioProject accession PRJNA1414671.

2.4. Statistics and Bioinformatics

Differences in IVM rates between CG and PG were analysed using Student's t‐test (Welch correction), and results are expressed as mean ± SEM. A value of p < 0.05 was considered statistically significant. Raw sequencing reads were processed and analysed using Partek Flow software (Partek Inc., St. Louis, MO, USA). Reads were aligned to the porcine genome ( Sus scrofa 11.1, Ensembl) (Dobin et al. 2013). Differential gene expression was assessed using the DESeq2 median‐of‐ratios method algorithm implemented in Partek. Genes were differentially expressed when FDR < 0.05.

3. Results

3.1. IVM Rates

Both groups displayed similar nuclear maturation rates (CG: 84.60% ± 0.91% and PG: 89.45% ± 2.79%) (p > 0.05).

3.2. Gene Expression Patterns

When comparing IMO vs. MO, we observed a total of 2248 differentially expressed genes (DEGs) (1128 downregulated and 1120 upregulated) in the CG group and 2499 (1121 downregulated and 1378 upregulated) in the PG group (Figure 1A,B). Venn diagrams revealed substantial overlap of genes between the IMO vs. MO groups, with 1924 genes commonly altered in PG and CG. In contrast, 324 genes in CG, respectively, 575 genes in PG were uniquely altered (Figure 1C).

FIGURE 1.

Comparative transcriptomic and functional profiling of immature (IMO) and in vitro‐matured (MO) oocytes from cyclic (CG) and prepubertal gilts (PG). (A, B) Volcano plots of gene expression changes in CG (A) and PG (B) groups (IMO vs. MO), showing log₂ fold change versus −log₁₀(FDR) with thresholds of |log₂FC| = 1 and FDR < 0.05. (C) Venn diagram showing shared and unique differentially expressed genes (DEGs; FDR < 0.05) between CG and PG. (D) Mirror bar plot of enriched Gene Ontology (GO) terms in CG (left) and PG (right) groups. (E, F) Bubble plots of enriched pathways in CG (E) and PG (F) groups, where bubble size reflects the number of DEGs and colour indicates −log₁₀ (p‐value).

3.3. Enrichment Analysis

In CG, enriched GO terms in IMO vs. MO were mainly associated with extracellular and membrane‐associated components, calcium ion binding, oxidoreductase activity, endoplasmic reticulum function and cellular responses to hypoxia. In contrast, PG showed enrichment for GO terms related to cell cycle regulation, chromosome organization and segregation, DNA damage response and repair and nuclear‐related processes (Figure 1D–F).

4. Discussion

In the present study, nuclear maturation rates did not differ between groups. However, it is well recognized that PG oocytes can achieve nuclear maturation rates comparable to those of adult counterparts while still exhibiting deficiencies in cytoplasmic maturation, including molecular preparedness (Paczkowski et al. 2011; Pawlak et al. 2011), which is associated with reduced embryo development (Paczkowski et al. 2011). Therefore, we investigated transcriptomic changes associated with IVM of oocytes collected from PG and CG. Functional characterization of the unique DEGs within groups highlighted differences in key biological processes. In CG, we identified several DEGs associated with metabolic activity, extracellular matrix remodelling, and growth factor‐mediated signalling when comparing IMO vs. MO. Notably, the KIT ligand (KITLG), platelet‐derived growth factor receptor beta (PDGFRB), transforming growth factor alpha (TGFA), and dual specificity phosphatase 4 (DUSP4) genes, showed higher expression in MO compared to IMO. KITLG, PDGFRB and TGFA, act as mediators of oocyte somatic cell communication and signalling responsiveness (Appeltant et al. 2016; Zheng et al. 2023), whereas DUSP4 inhibits MAPK activity through phosphorylation dynamics (Kyriakis and Avruch 2012; Salhab et al. 2013). On the other hand, mitogen‐activated protein kinase kinase kinase 5 and 6 (MAP3K5 and MAP3K6), and dual specificity phosphatase 9 (DUSP9) were downregulated in MO vs. IMO. MAP3K5 and MAP3K6 function as upstream regulators of MAPK signalling cascades, whereas DUSP9 is a well‐known regulator of MAPK activity (Salhab et al. 2013). Together, these changes highlight a dual regulatory mechanism: a high capacity for oocyte‐somatic cell communication, coupled with a progressive attenuation of MAPK signalling as maturation advances, ensuring stronger activation during early meiotic resumption and tighter regulation in MO.

Furthermore, genes with key roles in glycolytic metabolism (Zhang et al. 2020), such as lactate dehydrogenase A (LDHA), phosphoglycerate kinase 1 (PGK1), and triosephosphate isomerase 1 (TPI1) were downregulated in MO vs. IMO. In contrast, genes ruling mitochondrial oxidative metabolism (malate dehydrogenase 2 (MDH2)) were upregulated in MO vs. IMO. This suggests that IMO maintains higher glycolytic activity, whereas MO suppresses glycolysis and increases mitochondrial oxidative capacity to secure the energy required for fertilization and early embryogenesis. This transcriptional profile is consistent with a more advanced cytoplasmic maturation state in CG compared with PG oocytes.

Additionally, several CG‐DEGs involved in protein processing were dysregulated in MO vs. IMO. For instance, mannosidase alpha class 1A member 1 (MAN1A1), defender against cell death 1 (DAD1), and SEC63 homologue (SEC63) genes were overexpressed in MO vs. IMO. These genes act as key components of the endoplasmic reticulum machinery responsible for protein folding and glycosylation (Lin et al. 2019), suggesting an enhanced capacity to process newly synthesized proteins after maturation correctly. This may help establish a cytoplasmic environment better prepared to sustain fertilization and early embryo development in CG compared to PG oocytes.

By contrast, PG displayed a markedly different functional landscape, predominantly related to cell cycle regulation, DNA damage response, and genome stability. Key downregulated DEGs in MO vs. IMO included Cyclin B1 (CCNB1), Checkpoint kinase 1 (CHEK1), BRCA1 DNA repair‐associated (BRCA1), and RAD51 recombinase (RAD51), all central regulators of meiotic cell cycle progression and DNA damage surveillance (Fang et al. 2022; Paczkowski et al. 2011). CCNB1 and CHEK1 are critical for meiotic cell cycle control, whereas BRCA1 and RAD51 mediate homologous recombination and genome integrity (Paczkowski et al. 2011). Our results suggest that PG oocytes rely more strongly on internal surveillance mechanisms that pause meiotic progression to allow DNA repair. This might reflect a greater need to ensure genomic stability before maturation is complete.

Overall, our results provide complementary insight into the differences in the biological processes underlying oocyte IVM of oocytes retrieved from CG or PG in the porcine species.

Author Contributions

C.A.M.‐S. and H.R.‐M.: conceptualization. C.G.C.‐G., P.B.‐C., D.G.‐C., C.A.M.‐S. and B.P.: methodology. C.G.C.‐G.: writing – original draft preparation. P.B.‐C., D.G.‐C., B.P., C.A.M.‐S., and H.R.‐M.: writing – review and editing. H.R.‐M. and C.A.M.‐S.: supervision. C.A.M.‐S. and H.R.‐M.: project administration. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by MICIU/AEI/10.13039/501100011033 and ERDF/EU (Project PID2023‐150622OA‐100), and by the LiU‐Agropor research agreement (86037‐293205).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are openly available in NCBI Sequence Read Archive (SRA) at https://www.ncbi.nlm.nih.gov/sra, reference number PRJNA1414671.