Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT‐PCR

Abstract

Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT‐PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.

Global decay of mRNA is a hallmark of apoptosis in agingXenopuseggs

“…In the present study, we performed transcript profiling in individual living Xenopus oocytes using multiple collections of nanoliter amounts of the oocyte cytoplasm and RT-qPCR. This technique was shown to exert a minimal effect on oocyte viability 12 . The expression profiling of 18 genes, classified into different functional types, such as house-keeping, regulatory, apoptosis-related, metabolic, etc., revealed a great difference of their expression levels (Fig.…”

“…The hormone promotes oocyte transition from prophase I to metaphase II, producing fertilization-competent metaphase-arrested eggs. Previously, we have demonstrated that Xenopus oocytes can withstand multiple cytoplasmic collections, remaining intact for at least 48 hours 12 . In contrast, matured meiotically arrested eggs are easily activated or hyperactivated by pricking with a collection needle.…”

“…In detail, the technique of multiple cytoplasmic collections from Xenopus oocytes was described previously 12 . Briefly, capillaries for cytopasmic collections were prepared using a dual-stage glass micropipette puller from Narishige (Tokyo, Japan).…”

Multienzyme activity profiling for evaluation of cell‐to‐cell variability of metabolic state

“… 17 Interestingly, a study of 16 individual stage VI oocytes revealed low cell‐to‐cell variability of GAPDH mRNA expression compared to several other genes that have not been assigned housekeeping functions (e.g., stat3 and pdk3 ). 47 Post‐transcriptional mechanisms may therefore underlie the heterogeneity of GAPDH protein expression revealed by our analysis.…”

“…This possibility is strengthened by the fact that the cytoplasmic pools of GAPDH and MDH1 account for 96 and 94%, respectively, of the total cellular amount of these enzymes (calculations based on published ensemble protein concentration data and compartment volume estimates) 17 . Interestingly, a study of 16 individual stage VI oocytes revealed low cell‐to‐cell variability of GAPDH mRNA expression compared to several other genes that have not been assigned housekeeping functions (e.g., stat3 and pdk3 ) 47 . Post‐transcriptional mechanisms may therefore underlie the heterogeneity of GAPDH protein expression revealed by our analysis.…”