Sequence-independent assembly of spermatid mRNAs into messenger ribonucleoprotein particles

Abstract

During mammalian spermatogenesis, meiosis is followed by a brief period of high transcriptional activity. At this time a large amount of mRNA is stored as messenger ribonucleoprotein (mRNP) particles. All subsequent processes of sperm maturation occur in the complete absence of transcription, primarily using proteins which are newly synthesized from these stored mRNAs. By expressing transgene mRNAs in the early haploid spermatids of mice, we have investigated the sequence requirements for determining whether specific mRNAs in these cells will be stored as mRNP particles or be assembled into polysomes. The results suggest that mRNAs which are transcribed in spermatids are assembled into mRNP particles by a mechanism that acts independently of mRNA sequence. Our findings reveal a fundamental similarity between the mechanisms of translational control used in spermatogenesis and oogenesis.

FIG. 1

Transgene design and mRNA expression. (A) Transgene design. Colors refer to sequences derived from the sources indicated. Positions of translation initiation and termination codons are indicated. Fusion sites for the hgh 3′-UTR are indicated above the transgenes. Transcription initiation sites are denoted by bent arrows. Introns are shown as constrictions in the colored boxes in transgene mP1-hGH-hGH-3′ and transgene 4. (B) Relative GFP mRNA and Prm1 mRNA expression. At the top are indicated the samples in each lane. Mouse lines are designated by the transgene number followed by a unique letter designation for each founder carrying that transgene; “wt” denotes wild-type mice. RNase protection assays were performed with the indicated samples and the Prm1-GFP probe. Lane p contains a roughly 1:300 dilution of nondigested probe; lane c is a control lane containing probe hybridized to yeast RNA. Comparison to wild-type testis confirmed the identity of the transgene-specific signals. GFP-specific and Prm1-specific bands were excised from gels and were quantitated by liquid scintillation counting. Specific activities were corrected for differences in the radiolabeled UTP content of each protected fragment, and the ratios are presented below the autoradiogram. (C) GFP mRNA levels. The internal GFP probe, which does not hybridize to endogenous Prm1 mRNA and which gives an identical protected fragment with all of the GFP transgene mRNAs and with the synthetic control mRNA, was used to quantitate transgene mRNA levels. Assays were performed on RNA samples from each GFP transgenic mouse line and were quantitated by liquid scintillation counting of excised gel bands; data are presented in Table 1.

FIG. 2

Expression of GFP protein. (A) Confocal fluorescent microscopy with bright-field back-lighting on whole seminiferous tubules from wild-type or line 1a mice. (B) Confocal fluorescent and bright-field microscopy on 100-μm vibratome sections and on 10-μm cryosections of mouse line 1a testes. Yellow arrows indicate tubules with round spermatids; red arrows indicate tubules containing elongating spermatids; the pink arrow denotes a tubule with only prehaploid germ cells. (C) Whole live seminiferous tubules from mouse line 1a observed by confocal fluorescence microscopy showing regions with germ cells in the round spermatid stage (yellow arrow), the elongating spermatid stage (red arrow), and regions with only prehaploid germ cells (pink arrow). (D) Confocal fluorescent microscopy of seminiferous tubules from mouse lines 2a and 3b. Left, panels were photographed with a 6× objective and conditions used for panels A to C; right, panels photographed with a 16× objective and 30-fold-higher laser excitation energy. Arrows are as in other panels.

FIG. 3

Velocity sedimentation analysis of the distribution of mRNAs between mRNP particles and polysomes. Mouse testis or mouse Hepa cell cytoplasms were sedimented through exponential 10 to 85% sucrose gradients, and total RNA was purified from each fraction. At the top of panel A is shown an ethidium bromide-stained agarose gel of RNAs from a typical gradient (mouse line 1b). Gradient fractions (numbered from the top of the tube) are indicated. Below are representative autoradiograms of RNase protection assays on gradient fractions. The mouse line represented in each assay is listed at the left; adjacent to this is indicated the mRNA species being assayed. The positions of fractions containing mRNP particles and polysomes are indicated at the top. The Prm1 mRNA is much shorter than the other mRNAs and therefore assembles into smaller mRNP particles and smaller polysomes. For this reason, all Prm1 signals are shifted one to two fractions toward the top of the gradient (toward the left on the autoradiogram). As controls, GFP-hGH mRNA, either with (A, bottom) or without (not shown; both mRNAs gave similar results) the 91-base prm1 leader sequence, was expressed in mouse Hepa cells from the CMV promoter. The asterisk in lane 8 of the Cre sample in panel B (line 5a) denotes a gradient fraction for which the RNA pellet was lost during purification.

FIG. 4

Expression and mRNP association of transgene mRNA from mouse line 6a. (A) Confocal fluorescent and bright-field microscopy of whole live seminiferous tubules. Due to the high expression of GFP protein in this line, very low excitation energy was used (about 3% of that used for line 1a in Fig. 2). Arrows are as in Fig. 2. (B) Relative levels of nascent GFP transcripts in nuclei from lines 1c and 6a. RNase protection assays were performed as described above on the indicated amounts of RNA isolated from whole testis (total) or purified nuclei (nuclear), using the internal GFP probe (upper two autoradiograms) or the GAPD-s probe (below). At the left are given the mouse line used and the identity of each protected fragment. The schematic at the bottom shows that GAPD-s pre-mRNA retaining intron 1 hybridizes to a 147-base region of the probe (dark hatched line below the RNAs); mRNA with exon 1 spliced onto exon 2 hybridizes to a 177-base region of the probe. To compare relative levels of spliced and unspliced transcripts, the radioactivity of each band was determined by liquid scintillation counting and was corrected for differences in the number of radiolabeled UTP residues in each protected fragment (31 and 49 for nonspliced and spliced, respectively). By assuming equal hybridization efficiency, we calculate that 43% of the GAPD-s transcripts in testis nuclei have not yet removed intron 1. (C) Velocity sedimentation analysis of GFP transgene mRNA and endogenous Prm1 mRNA. Assays were as in Fig. 3 except that for detecting GFP mRNA, the internal probe was used.

FIG. 5

Transgene mRNA, GAPDH mRNA, and Prm1 mRNA levels in explanted mouse line 4b testis cell populations purified by FACS. (A) Bright-field and fluorescent microscopy of trypan blue-stained cell explants. The yellow arrow denotes a trypan blue-stained (dead) cell. Red arrows indicate large multinucleate cells. Testis cells show extreme size variation; however, phase-contrast microscopy (not shown) revealed that most of the large cells in the micrographs are multinuclear (see Materials and Methods). (B) Preparative FACS on explanted cells. Left, distribution of cell types seen using forward and side scatter criteria; center, distribution of fluorescence intensities observed; right, gating parameters used in this study. Cells gated as GFP⁺ are green, cells gated as GFP⁻ are red, and other cells (which were discarded) are gray. The colors in the left panel correspond to those in the right panel and thus give an indication of differences in the forward and side scatter properties of the various GFP⁺ and GFP⁻ cell subpopulations. (C) RNase protection analyses of GFP mRNA from the transgene and the endogenous Prm1 and GAPDH mRNAs in the sorted GFP⁺ and GFP⁻ cell populations and in the nonsorted explant. GFP and Prm1 mRNAs were detected with the Prm1-GFP probe, which maps the cap sites of both transcripts. Each lane contained 2 μg of total RNA from the indicated cell sample. The GFP⁺ sample contained 25% as much GAPDH mRNA as the GFP⁻ sample, whereas FACS reanalysis of the GFP⁺ cell sample indicated that it contained 17.2% contamination with GFP⁻ cells. By assuming that these were expressing the same amount of GAPDH mRNA per cell as cells in the pure GFP⁻ population, we estimate that 69% (17.2% ÷ 25%) of the total GAPDH signal in the GFP⁺ sample arose from the contaminating GFP⁻ cells (see text).