A role of Pumilio 1 in mammalian oocyte maturation and maternal phase of embryogenesis

Abstract

Background: RNA binding proteins play a pivotal role during the oocyte-to-embryo transition and maternal phase of embryogenesis in invertebrates, but their function in these processes in mammalian systems remain largely understudied.

Results: Here we report that a member of the Pumilio/FBF family of RNA binding proteins in mice, Pumilio 1 (Pum1), is a maternal effect gene. The absence of maternal PUM1 in the oocyte does not affect meiotic maturation but leads to abnormal preimplantation development. Furthermore, genome-wide transcriptome analysis of oocytes and embryos revealed that there is a concomitant perturbation of the mRNA milieu. Of note, putative PUM1 mRNA targets were equally perturbed as non-direct targets, which indicates that PUM1 regulates the stability of maternal mRNAs both directly and indirectly. We show Cdk1 mRNA, a known PUM1 target essential for meiosis and preimplantation development, is not degraded appropriately during meiosis, leading to an increase in CDK1 protein in mature oocytes, which indicates that PUM1 post-transcriptionally regulates Cdk1 mRNA; this could partially explain the observed abnormal preimplantation development. Furthermore, our results show that maternal and zygotic PUM1 are required for postnatal survival.

Conclusions: These findings indicate that PUM1 is essential in the process of cytoplasmic maturation and developmental competence of the oocyte. These results reveal an important function of maternal PUM1 as a post-transcriptional regulator during mammalian embryogenesis.

Keywords: Oocyte maturation; Post-transcriptional regulation; Preimplantation embryo.

Fig. 1

Depletion of maternal Pum1 leads to abnormal preimplantation development. a The schematic of the four crosses used. Of note, zygotic Pum1 is not expressed maximally until four-cell stage, therefore maternal PUM1 protein is the only source of PUM1 protein during early preimplantation development. Cross I: m+z+ indicates presence of maternal and zygotic PUM1 protein, progeny are Pum1^+/+; Cross II: m−z+ indicates maternal PUM1 absent and zygotic PUM1 present, progeny are Pum1^+/−; Cross III: m−z− indicates absence of both maternal and zygotic PUM1, progeny are Pum1^−/−; Cross IV: m+z+ indicates maternal and zygotic PUM1 present from maternal Pum1 allele, progeny are Pum1^−/+. b, c Left panels show the percentages of embryos observed at each developmental stage (after excluding unfertilized oocytes). Right upper panels are representative light microscopy images of embryos collected at different time points. Scale bar (white): 500 µm. Right lower panel shows the number of matings for each cross and the mean number (± SD) of each type of embryo seen. Dpc: days postcoitum; 2C and 4C: two-cell and four-cell, embryos, respectively; 8C/M: eight-cell stage embryo and morula; B: blastocyst; abnormal: presumed fragmented embryos; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

PUM1 is dispensable for oocyte maturation. a Oocyte in vitro maturation using WT (n = 49) and Pum1^−/− (n = 53), three females of each genotype were used. b The GVBD time course for Pum1^+/+ (WT) (n = 29) and Pum1^−/− (KO) (n = 23) oocytes. Two females of each genotype were used

Fig. 3

Pum1^−/− MII oocytes show significant changes in transcriptome compared to Pum1^+/+ MII oocytes, and lack of PUM1 protein during oocyte maturation leads to a reduction in the dynamics of the mRNA pool. a Heatmap of Spearman correlation coefficient of Pum1^+/+ (WT) and Pum1^−/− (KO) GV and MII oocytes. b, c Scatterplots of the unchanged and differentially expressed (DE) genes for WT GV vs. KO GV and WT MII vs. KO MII. d Gene ontology analysis using Gene Ontology Consortium website was performed on the genes up/downregulated in KO compared to WT MII oocytes. e, f Scatterplots of the DE and unchanged genes during Pum1^+/+ (WT) and Pum1^−/− (KO) oocyte maturation

Fig. 4

Maternal PUM1 regulates mRNA degradation by direct and indirect mechanisms. a The comparison of the mRNA transcripts normally found lower in WT MII (degraded) and the mRNA transcripts found lower in KO MII. b The genes were classified according to Class type as described in the table. c The percentage of genes found in Class 1–3. d GO analysis of the genes in Class 2. e, f The genes found in b were further classified into whether they had a Pumilio-response element (PRE) or without a PRE and the percentages of each class is presented

Fig. 5

Absence of maternal PUM1 affects transcripts typically stabilized in Pum1^+/+ oocyte maturation to a greater extent than those that are typically degraded. a The comparison of the mRNA transcripts usually found higher in WT MII (stabilized) and the mRNA transcripts found higher in KO MII. b The genes were classified according to Class type as described in the table. c The percentage of genes found in Class 4–7. d GO analysis of the genes in Class 5. e, f The genes found in b were further classified into those with PRE and without PRE

Fig. 6

The maternal effect of PUM1 on mRNA milieu continues into early preimplantation development. a The scatterplots for comparison of the transcriptome changes during MII to two-cell transition between the different matings. b The comparison of the transcripts lower (degraded) in WT MII to m+z+ two-cell embryos transition and KO MII to m−z+ two-cell embryo transition. c The comparison of the transcripts higher (stabilized) in WT MII to m+z+ two-cell embryos transition and KO MII to m−z+ two-cell embryo transition. d, e GO analysis of the transcripts dysregulated in KO MII to m−z+ two-cell embryo transition

Fig. 7

Lack of maternal PUM1 in oocytes causes an increase in CDK1 protein levels. a Representative images of the immunostaining of WT (Pum1^+/+) and KO (Pum1^−/−) MII oocytes with the anti-CDK1 antibody. Scale bar (white): 50 μm. b The quantification of the fluorescence signal. n = total number of oocytes scored. CTCF, corrected total cellular fluorescence (units) per oocyte (mean ± SD); WT, Pum1^+/+ MII oocytes; KO, Pum1^−/− MII oocytes. * p < 0.05