Quantitative analysis of mRNA translation in mammalian spermatogenic cells with sucrose and Nycodenz gradients

Abstract

Background: Developmental and global regulation of mRNA translation plays a major role in regulating gene expression in mammalian spermatogenic cells. Sucrose gradients are widely used to analyze mRNA translation. Unfortunately, the information from sucrose gradient experiments is often compromised by the absence of quantification and absorbance tracings, and confusion about the basic properties of sucrose gradients.

Methods: The Additional Materials contain detailed protocols for the preparation and analysis of sucrose and Nycodenz gradients, obtaining absorbance tracings of sucrose gradients, aligning tracings and fractions, and extraction of equal proportions of RNA from all fractions.

Results: The techniques described here have produced consistent measurements despite changes in personnel and minor variations in RNA extraction, gradient analysis, and mRNA quantification, and describes for the first time potential problems in using gradients to analyze mRNA translation in purified spermatogenic cells.

Conclusions: Accurate quantification of the proportion of polysomal mRNA is useful in comparing translational activity at different developmental stages, different mRNAs, different techniques and different laboratories. The techniques described here are sufficiently accurate to elucidate the contributions of multiple regulatory elements of variable strength in regulating translation of the sperm mitochondria associated cysteine-rich protein (Smcp) mRNA in transgenic mice.

Figure 1

Nycodenz gradient analysis of 18S ribosomal RNA, and the Ldhc and Smcp mRNAs in 26 dpp prepubertal mouse testis. A cytoplasmic extract of the testes of three 26 dpp mice was sedimented on a Nycodenz gradient, collected as 19 fractions from the bottom, RNAs were extracted from each fraction, and the levels of 18S rRNA, Ldhc and Smcp mRNAs were determined by sequential hybridization of each probe to a Northern blot and phosphorimage analysis. The methods and probes are described in the Additional file 1 and [5]. 18s rRNA, blue triangles; Ldhc mRNA, black squares; Smcp mRNA, red circles. The peaks of polysomes and free-mRNPs are indicated.

Figure 2

Analysis of mRNA translation in purified pachytene spermatocytes purified by sedimentation on bovine serum albumin gradients. Dissociated testicular cells from 16 dpp (A) and adult mice (B) were purified by sedimentation at 1XG on bovine serum albumin gradients, pachytene spermatocytes were collected, cultured for 1 hr at 32°C in RPMI 1040 medium in 5% CO₂ in air, cytoplasmic extracts were prepared and sedimented on sucrose gradients. The preparation in panel A contained 4.28 × 10⁶ pachytene spermatocytes. The gradients in panels A and B were sedimented at 35,000 rpm in the SW60Ti rotor for 100 and 80 min, respectively. The absorbance tracings of both gradients at 254 nm are shown. In addition, in Panel A the RNAs were extracted from each fraction, and the distribution of the Ldhc and Pgk2 mRNAs was analyzed by sequential hybridization of a single Northern blot. The fractions were collected from the bottom, and the fraction labeled P contains RNA extracted from the pellet on the bottom of the ultracentrifuge tube. The full scale absorbance of the UV analyzer was 0.32 using a flow cell with a 2 mm path length. The positions of the fractions in the absorbance tracing in panel B are numbered. Fraction 0 in the absorbance tracing is the void and does not contain RNA.

Figure 3

Recovery of [³²P]-labeled T7 bacteriophage RNA polymerase transcripts from the fractions of sucrose and Nycodenz gradients. Each fraction of sucrose and Nycodenz gradients was spiked with ~100,000 cpm of [³²P]-labeled T7 bacteriophage RNA polymerase transcript, RNA was extracted from each fraction using the procedures described in Additional file 1, and the amount of radiolabeled RNA was determined by Cherenkov counting in a scintillation counter. The results are depicted as the percentage of cpm in each fraction relative to the average cpm in all of the fractions in each gradient. Fraction 1 of the sucrose gradients contains RNA extracted from the pellet.