Sperm-carried IGF2: towards the discovery of a spark contributing to embryo growth and development

Abstract

Spermatozoa have been shown to carry key RNAs which, according to animal evidence, seem to play a role in early embryo development. In this context, a potential key growth regulator is insulin-like growth factor 2 (IGF2), a highly conserved paternally expressed imprinted gene involved in cell growth and proliferation which, recent observations indicate, is expressed in human spermatozoa. We herein hypothesized that sperm IGF2 gene expression and transmission at fertilization is required to support early embryo development. To test this hypothesis, we analyzed sperm IGF2 mRNA levels in the same semen aliquot used for homologous assisted reproductive technique (ART) in infertile couples and correlated these levels with embryo morphokinetics. To find a mechanistic explanation for the observed results, the transcriptomes of blastocysts obtained after injection of Igf2 mRNA in mouse parthenotes were analyzed. Sperm IGF2 mRNA negatively correlated with time of 2-cell stage (t2), t3, t4, t5, and time of expanded blastocyst (tEB), independently of maternal age, body mass index, anti-Müllerian hormone levels, and oocyte quality. An IGF2 mRNA index >4.9 predicted the ability of the embryos to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; P < 0.001). In the animal study, transcriptome analysis demonstrated that 65 and 36 genes were, respectively, up- and down-regulated in the experimental group compared to the control group. These genes belong to pathways that regulate early embryo development, thus supporting the findings found in humans. This study has the potential to challenge the longstanding tenet that spermatozoa are simply vehicles carrying paternal DNA. Instead, it suggests that IGF2 mRNA in healthy spermatozoa provides critical support for early embryo development. Pre-ART sperm-carried IGF2 mRNA levels may be used as a marker to predict the chances of obtaining blastocysts to be transferred for infertile couples undergoing ART.

Keywords: IGF2; blastocyst; embryo development; embryo kinetics; embryo morphology; sperm IGF2.

Graphical abstract

Study hypothesis. Low levels of sperm-carried IGF2 levels interfere with early embryo development, while high levels support early embryo mitosis and allow for the development of a viable blastocyst. IGF1R, insulin-like growth factor 1 receptor; IGF2, insulin-like growth factor 2.

Figure 1.

Animal experimental protocol. One Shot™ TOP10 Chemically Competent E. coli colonies were transfected with a plasmid carrying the Igf2 gene, which served as gene reservoir. Plasmids were used for gene transcription and Igf2 mRNA synthesis. Oocytes were retrieved from 8 to 12-week-old superovulated FVB/NJ mice. They were separated from the cumulus and incubated in a parthenogenic medium. Three hours after incubation, parthenotes were injected with a fluorescent Gfp mRNA (control group, n = 45) or with Igf2 plus Gfp mRNAs (experimental group, n = 45). After reaching the blastocyst stage, 96 h after injection, embryos were harvested and sequenced. Gfp, Green fluorescent protein; Igf2, insulin-like growth factor 2.

Figure 2.

Unadjusted correlation analysis between IGF2 mRNA and tPN_fading (n = 106 embryos), t2 (n = 106 embryos), t4 (n = 96 embryos), and t6 (n = 77 embryos). Only the times with significant results are shown. Sperm IGF2 expression (calculated as the IGF2 mRNA/β-actin mRNA ratio) negatively correlated with embryo development times: tPN_fading, t2, t4, and t6. t, time; PN, pronuclei.

Figure 3.

Sperm IGF2 expression: predictive analysis for embryo capability to reach the blastocyst stage on Day 5. An IGF2 mRNA index of >4.9 predicted the capability of the embryo to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; p < 0.001) The sample size of the analysis was 131 embryos. Sperm IGF2 expression was calculated as the IGF2 mRNA/β-actin mRNA ratio.

Figure 4.

Distribution of sperm IGF2 mRNA according to embryo morphology from Day 2 to Day 4. Panel A shows the quartile distribution of sperm IGF2 mRNA in the two groups of morphologically good (n = 36) and poor (n = 22) embryos. Significant differences in quartile distribution were found between the two groups, with a higher prevalence of low IGF2 levels (I quartile) and a lower prevalence of IGF2 levels falling into the II and III quartiles in morphologically poor compared to morphologically good embryos. *P < 0.05, Chi-squared test. Panel B shows the violin plots of the distribution of sperm IGF2 mRNA values in the groups of morphologically good (n = 36) and poor (n = 22) embryos. The distributions of the two groups were not significantly different. Sperm IGF2 expression was calculated as the IGF2 mRNA/β-actin mRNA ratio.

Figure 5.

Heatmap showing differently expressed genes in parthenotes injected with Igf2 mRNA (n = 45) compared to controls (n = 45). An adjusted P-value <0.05 and a fold change >2 indicate a statistically significant change in differential gene expression. A total of 65 genes were found to be up-regulated and 36 were down-regulated. Four replicates for un-injected (controls) and for Igf2 mRNA injected samples were used for the analysis, with several animals used for each replicate.

Figure 6.

Overview of enrichment analysis results obtained with g:Profiler. Upper panel. The Manhattan interactive plot illustrates the results of the enrichment analysis. The x-axis represents functional terms grouped and color-coded according to data sources [Molecular Function from GO is red; Biological Process from GO is orange, Cellular Component from GO is green, KEGG from biological pathways is fucsia, Reactome from biological pathways is electric blue, Wiki Pathways from biological pathways is sky blue, TRANSFAC from regulatory motifs in DNA is blue, mirTarBase from regulatory motifs in DNA is water green, CORUM from protein databases is green, HP from Human phenotype ontology is violet]. The y-axis shows the P-values of enrichment adjusted to the negative log10 scale. Light circles represent not significant terms (if present). Bottom panel. Source, Term ID, Term Name, and adjusted P-values of significant terms are shown. The query can be found at: https://biit.cs.ut.ee/gplink/l/9vD3nuvtTh. Abbreviations. BP, biological process; CC, cellular component; GO, gene ontology; HP, human phenotype; MIRNA, miRTarBase; MF, molecular function; REAC, Reactome; TF, TRANSFAC; WP, WikiPathways.