Transcriptional insights on the incomplete cytoplasmic maturation and developmental potential of oocytes cultured without granulosa cells in mice

Abstract

Background: Oocyte maturation is crucial for female fertility and embryonic development, encompassing nuclear and cytoplasmic maturation. Supportive cells of follicles, such as granulosa cells, are essential for oocyte growth and maturation. Oocytes can achieve nuclear maturation without granulosa cells during in vitro maturation (IVM). However, there is still a higher chance of incomplete cytoplasmic maturation for these oocytes with mature nuclei compared with oocytes cultured with granulosa cells. Oocytes with incomplete cytoplasmic maturation have lower fertilization rates and developmental potential than mature ones, although underlying mechanisms are poorly understood. Identifying key genes and signaling pathways associated with oocyte cytoplasmic maturation can help further elucidate the maturing process of oocytes and understand the impact of immature oocytes on embryonic development, throwing insights into the strategy to improve the success rate of assisted reproductive technologies.

Results: Our study investigated murine oocytes maturing with and without granulosa cells. IVM without granulosa cells yielded oocytes with lower nuclear maturation rates than IVM with granulosa cells and in vivo maturation (IVO). Even though oocytes could achieve nuclear maturation without granulosa cells, they showed incomplete cytoplasmic maturation featuring higher levels of reactive oxygen species, lower mitochondrial density, and higher proportions of cells with abnormal distributions of cortical granules. Of note, oocytes with immature and mature cytoplasm had distinct transcriptional profiles. In the immature oocytes, we observed a deficient mRNA restoration of genes in crucial regulatory pathways of cellular growth and division, potentially affecting embryonic development. Differentially expressed genes (DEGs) between immature and mature oocytes were identified to be highly expressed in different pre-implantation stages, such as the MII oocyte, the 8-cell stage, and the ICM stage. Identified DEGs were enriched in key regulatory pathways of fertilization and embryonic development, such as energy and metabolic pathways. These observations indicated that the impeded development potential of oocytes with immature cytoplasm might be the result of abnormal gene expressions during oocyte maturation.

Conclusions: We show that granulosa cells are important for both nuclear and cytoplasmic maturation of oocytes. Abnormal gene expression in oocytes with incomplete cytoplasmic maturation may be associated with potential defects in fertilization and embryonic development.

Keywords: Embryonic development; Fertilization; Granulosa cells; In vitro maturation; Oocyte cytoplasmic maturation.

Fig. 1

Differences in maturation of oocytes with different maturation methods. (A) The schematic diagram illustrates the workflow of oocyte collection and in vitro maturation (IVM) in mice, with the in vivo maturation (IVO) group serving as a control. Oocytes of the IVM group, including denuded oocytes (DO) and cumulus-oocyte complexes (COC), were collected from ovaries 48 h after PMSG treatment. Oocytes of the IVO group were collected 16 h after hCG treatment. (B) Representative bright field images show the morphology of premature and mature oocytes from the DO and COC groups after IVM, with mature oocytes exhibiting germinal vesical breakdown (GVBD) and polar body extrusion (PB1). (C) Bright field images display oocytes from IVO, COC, and DO groups after cumulus cell removal with hyaluronidase from groups of IVO and COC. (D) Statistical analysis of the germinal vesicle breakdown (GVBD) rates in oocytes from the IVO, COC, and DO groups (n = 5 per group). (E) Statistical analysis of the PB1 rates in oocytes from the IVO, COC, and DO groups (n = 5 for each group). The data in panels D and E are shown as the mean ± SD. Statistical significance: n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical analysis was performed by a two-tailed unpaired Student’s t-test. Scale bar in panel B: 15 μm. Scale bar in panel C: 100 μm

Fig. 2

Differences in cytoplasmic maturation of oocytes with different maturation methods. (A) Reactive oxygen species (ROS) levels (green) were detected in oocytes with nuclear maturation from the IVO, COC, and DO groups. BF, bright field. (B) Fluorescence intensity quantification of ROS was measured in oocytes from the IVO, COC, and DO groups after maturation (n = 10–16 oocytes per group). (C) Mitochondrial distribution (green) was detected in oocytes with nuclear maturation from the IVO, COC, and DO groups staining with MitoTracker. BF, bright field. (D) Fluorescence intensity and statistical analysis of mitochondrial signals were performed in oocytes from the IVO, COC, and DO groups after maturation (n = 22–33 oocytes per group). (E) Analysis of abnormal mitochondrial distribution rates in oocytes from the IVO, COC, and DO groups after maturation (n = 22–33 oocytes per group). (F) Distribution of cortical granules (CGs, green) was detected in oocytes with nuclear maturation from the IVO, COC, and DO groups. HOE, Hoechst. (G-H) Analysis of fluorescence intensity quantification and mislocalization rates of CGs were detected in oocytes from the IVO, COC, and DO groups after maturation (n = 15–25 oocytes per group). The data in panels B, D, E, G, and H are shown as the mean ± SD. Statistical significance: n.s., not significant; *, p < 0.05; ***, p < 0.001. Statistical analysis was performed by a two-tailed unpaired Student’s t-test or Fisher’s exact test. Scale bar: 15 μm

Fig. 3

Differentially expressed genes (DEGs) and gene ontology (GO) enrichment analysis in mature and immature mouse oocytes. (A) Schematic diagram of sample collection and Smart-seq2 single-oocyte sequencing of IVO, COC, and DO oocytes in mice. Oocytes were collected from 6–8 week-old mice (IVO, n = 7; COC, n = 7; DO, n = 6). All obtained oocytes were with extrusion of PB1. (B) Principal component analysis (PCA) plot of single-oocyte RNA transcriptomes from mature (IVO2, IVO3, IVO4, COC2, COC7) and immature oocytes (DO6, DO4, DO3, DO2, DO1). (C) Volcano plot of differentially expressed genes (DEGs) showing the downregulated genes (DOWN), upregulated genes (UP), and unchanged genes (NOT) between mature and immature oocytes. DEGs were defined as genes with an adjusted p-value < 0.05 and |log₂FC| > 1. (D) Heatmap of the top 50 DEGs (25 upregulated genes and 25 downregulated genes) between mature and immature mouse oocytes. DEGs were defined as genes with an adjusted p-value < 0.05 and |log₂FC| > 1, ranked by ascending p-value and descending |log₂FC|. (E) Enrichment analysis of DEGs and representative Gene Ontology (GO) terms in mature and immature mouse oocytes. Enriched pathways were significantly related to oocyte maturation, embryonic development, metabolism, and organelles

Fig. 4

Gene set enrichment analysis (GSEA) reveals important roles of nucleic acid stability and spindle pole in cytoplasmic maturation and early embryonic development. (A-C) GSEA enrichment showing significantly downregulated of the pathways: “Mitotic DNA replication”, “tRNA methylation” and “Mitotic spindle pole” in immature oocytes

Fig. 5

Incomplete maturation of oocytes affects the fertilization and pre-implantation embryonic development process in mice. (A) Boxplots showing gene expression of DEGs that are highly expressed in the MII oocyte stage (n = 130 genes) across different early embryonic stages. (B) Chord plot displaying selected GO terms enriched of DEGs highly expressed in the MII oocyte stage. Genes are ordered by log₂FC, with green indicating upregulation. (C) Boxplots showing the differential expression of Ppp3ca between mature and immature oocytes. (D) Chord plot displaying selected GO terms enriched of DEGs highly expressed in the 8-cell stage. Genes are ordered by log₂FC, with blue indicating upregulation. (E) Chord plot displaying selected GO terms enriched of DEGs highly expressed in the ICM stage. Genes are ordered by log₂FC, with orange indicating upregulation. Statistical significance: **, p < 0.01. P-value was calculated using a Wald test based on a negative binomial distribution