Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

Poly(A) shortening accompanies the activation of translation of five mRNAs during spermiogenesis in the mouse

I have compared the quantity and the length of the poly(A) tracts of five haploid-expressed mRNAs in the polysomal and nonpolysomal fractions of round and elongating spermatids in mice: transition proteins 1 and 2, protamines 1 and 2, and an unidentified mRNA of about 1050 bases. Postmitochondrial supernatants of highly enriched populations of round and elongating spermatids (early and late haploid spermatogenic cells) were sedimented on sucrose gradients, and the size and amount of each mRNA in gradient fractions were analyzed in Northern blots. In round spermatids, all five mRNAs are restricted to the postpolysomal fractions, but in elongating spermatids about 30-40% of each mRNA is associated with the polysomes. The distribution of these mRNAs in sucrose gradients suggests that all five mRNAs are stored in a translationally repressed state in round and early elongating spermatids, and that they become translationally active in middle and late elongating spermatids. The translationally repressed forms of all five mRNAs are long and homogenous in size, whereas the polysomal forms are shorter and more heterogenous due to shortening of their poly(A) tracts. The relationship between translational activity and poly(A) size exemplified by these five mRNAs may be typical of mRNAs which are translationally repressed in round spermatids and translationally active in elongating spermatids.     

Target organ regulation of substance P in sympathetic neurons in culture

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.     

Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: proteins associated with the cytoplasmic polysomal and non-polysomal mRNAs (messenger ribonucleoprotein complex)

Cytoplasmic polysomal and non-polysomal mRNA-associated proteincomplexes were isolated from, and characterized in, developingsomatic and zygotic embryos of alfalfa (Medicago sativa L.).³⁵S-methionine-labelled intact embryos were irradiated withultraviolet light (UV) in situ to cross-link mRNA and proteinsoccurring within one bond length, and the polysomal and non-polysomalfractions were extracted. Then the mRNA-protein complexes wereisolated from the fractions and separated using two cycles ofaffinity chromatography on an oligo(dT)-cellulose column. Followingdigestion with RNase A and T1 and micrococcal nuclease, mRNA-associatedproteins were separated by SDS-PAGE. Several polypeptides of 15–150 kDa were associated withthe polysomal and non-polysomal (ribonucleoprotein, mRNP) fractionsof alfalfa embryos after UV-cross-linking. Many of the polypeptidesassociated with the polysomal and non-polysomal mRNAs were qualitativelysimilar, although their concentration in the two fractions wasdifferent. However, some developmentally stage-specific polypeptideswere found to be associated with the non-polysomal mRNA fractionduring the early stages of embryogenesis (precotyledonary) ofsomatic embryos. Thus the presence of mRNPs during embryogenesishas been demonstrated, and proteins intimately associated withthe mRNAs identified. Key words: Embryogenesis, translational control, protein synthesis, messenger ribonucleoproteins, alfalfa (Medicago sativa L.)     

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

DNA polymerase-beta and poly(ADP)ribose polymerase mRNAs are differentially expressed during the development of male germinal cells.

We have examined the steady-state mRNA levels in spermatogenic cells of two nuclear enzymes that appear to be involved in DNA repair, DNA polymerase-beta (pol-beta) and poly(ADP)ribose polymerase (PADPRP). Two pol-beta mRNAs of 1.3 kb and 1.4 kb were detected in extracts from mouse testes. In leptotene/zygotene spermatocytes a low level of the 1.4-kb mRNA was observed. Both pol-beta mRNAs were found in meiotic pachytene spermatocytes, with the 1.3-kb form being more abundant. In contrast, the 1.4-kb form was more abundant in haploid round spermatids. Polysome gradient analyses indicated that the two pol-beta mRNAs were predominantly present in the nonpolysomal fractions of spermatocytes. In round spermatids, a larger fraction of the 1.4-kb pol-beta mRNA was associated with polysomes, correlating well with the higher levels of pol-beta enzyme detected during spermiogenesis. The pattern of PADPRP mRNA expression differed from the expression of pol-beta mRNA. The two PADPRP mRNAs of 3.7 and 3.8 kb were present in type A and type B spermatogonia, reached their highest levels in pachytene spermatocytes, and were greatly reduced in haploid round and elongating spermatids. Most of the pachytene spermatocyte PADPRP and mRNAs were present in polysomes, whereas a greater percentage of PADPRP mRNAs in round spermatids were detected in the nonpolysomal fractions. This finding correlates with the immunocytochemical nuclear localization of this enzyme in pachytene spermatocytes. These data demonstrate that different developmental patterns of mRNA expression and translational regulation exist for the pol-beta and PADPRP mRNAs during differentiation of male germinal cells.     

Peptide coding capacity of polysomal and non-polysomal messenger RNA during growth of animal cells.

The major peptides encoded by cytoplasmic RNA preparation translated in vitro in extracts of wheat germ were displayed by two-dimensional electrophoresis on polyacrylamide gels. With this assay system polysomal and non-polysomal RNA preparations were found to differ in coding capacity. These differences tended to be greater in RNA preparations from stationary phase cells than in those from exponential phase cells. These differences were maintained when the concentrations of potassium and magnesium were above or below the optimal concentrations for incorporation. Most of the messenger RNA activities preferentially in the post-polysomal region could be driven into the polysomal region in the presence of cycloheximide. We conclude that these measurements are valid measurements of concentrations of individual functional mRNA species in these RNA preparations.     

Cross-linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr = 49 000 and 52 500 were the major components. In contrast polypeptides of Mr = 43 000 and 57 000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions.     

The effect of age on the properties of poly(A)-containing messenger RNA in Physarum polycephalum

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.     

Ultrastructural distribution of poly (A)+ RNA during Trypanosoma cruzi-cardiomyocyte interaction in vitro: a quantitative analysis of the total mRNA content by in situ hybridization

Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A)+ RNA remained about the same up to 24 h post-infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.     

Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Selective gene expression during sporulation of Physarum polycephalum.

The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.