Patterns, mechanisms, and functions of translation regulation in mammalian spermatogenic cells.

Translational regulation is a fundamental aspect of the atypical patterns of gene expression in mammalian meiotic and haploid spermatogenic cells. Every mRNA is at least partially translationally repressed in meiotic and haploid spermatogenic cells, but the extent of repression of individual mRNA species is regulated individually and varies greatly. Many mRNA species, such as protamine mRNAs, are stored in translationally repressed free-mRNPs in early haploid cells and translated actively in late haploid cells. However, translation does not regulate developmental expression of all mRNAs. Some mRNAs appear to be partially repressed for the entire period that the mRNA is expressed in meiotic and haploid cells, while other mRNAs, some of which are expressed at high levels, are almost totally inactivated in free-mRNPs and/or generate little or no protein. This distinctive phenomenon can be explained by the hypothesis that translational repression is used to prevent the potentionally deleterious effects of overproduction of proteins encoded by overexpressed mRNAs. Translational regulation also appears to be frequently altered by the widespread usage of alternative transcription start sites in spermatogenic cells. Many ubiquitously expressed genes generate novel transcripts in somatic spermatogenic cells containing elements, uORFs and secondary structure that are inhibitory to mRNA translation, while the ribosomal proten L32 mRNA lacks a repressive element that is present in somatic cells. Very little is known about the mechanisms that regulate mRNA translation in spermatogenic cells, largely because few labs have utilized in vivo genetic approaches, although there have been important insights into the repression and activation of protamine 1 mRNA, and the role of Y-box proteins and poly(A) lengthening in mRNA-specific translational activation mediated by the cytoplasmic poly(A) element binding protein and a testis-specific isoform of poly(A) polymerase. A very large literature by evolutionary biologists suggests that the atypical patterns of gene expression in spermatogenic cells are the consequence of the powerful and unusual selective pressures on male reproductive success.

A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells.

This review focuses on the striking differences in the patterns of transcription and translation in somatic and spermatogenic cells in mammals. In early haploid cells, mRNA translation evidently functions to restrict the synthesis of certain proteins, notably protamines, to transcriptionally inert late haploid cells. However, this does not explain why a substantial proportion of virtually all mRNA species are sequestered in translationally inactive free-messenger ribonucleoprotein particles (free-mRNPs) in meiotic cells, since most mRNAs undergo little or no increase in translational activity in transcriptionally active early haploid cells. In addition, most mRNAs in meiotic cells appear to be overexpressed because they are never fully loaded on polysomes and the levels of the corresponding protein are often much lower than the mRNA and are sometimes undetectable. A large number of genes are expressed at grossly higher levels in meiotic and/or early haploid spermatogenic cells than in somatic cells, yet they too are translated inefficiently. Many genes utilize alternative promoters in somatic and spermatogenic cells. Some of the resulting spermatogenic cell-altered transcripts (SCATs) encode proteins with novel functions, while others contain features in their 5'-UTRs, secondary structure or upstream reading frames, that are predicted to inhibit translation. This review proposes that the transcriptional machinery is modified to provide access to specific DNA sequences during meiosis, which leads to mRNA overexpression and creates a need for translational fine-tuning to prevent deleterious consequences of overproducing proteins.

Developmental expression of Y-box protein 1 mRNA and alternatively spliced Y-box protein 3 mRNAs in spermatogenic cells in mice.

Y-box proteins bind DNA and RNA and are characterized by a cold shock domain and a carboxyl-terminus containing clusters of aromatic and basic residues that alternate with clusters of acidic residues. Y-box proteins 1 and 3 in mouse testis were cloned here by 3' rapid amplification of cDNA ends (RACE) using a degenerate primer. Northern blots and reverse transcription-polymerase chain reaction (RT-PCR) established that the levels of Y-box protein 1 and 3 mRNAs are regulated individually: (i) Y-box protein 1 mRNA is strongly expressed in kidney, whereas Y-box protein 3 mRNA is strongly expressed in heart and muscle; (ii) Y-box protein 1 and 3 mRNAs are weakly expressed in early prepubertal testis and strongly expressed in pachytene spermatocytes, round spermatids, and elongated spermatids; and (iii) prepubertal testes and meiotic and haploid spermatogenic cells express two alternatively spliced Y-box protein 3 mRNAs encoding isoforms with different carboxyl termini, whereas somatic tissues primarily express one form. Sucrose gradients reveal that approximately 27% of both Y-box protein 3 mRNAs are translationally active in adult testis. In conclusion, spermatogenic cells in mice express five isoforms of Y-box proteins including Y-box protein 1, and two isoforms each of Y-box proteins 2 and 3. This multiplicity is intriguing because Y-box proteins are thought to activate transcription and repress translation in spermatogenic cells.

The promoter of the Poly(A) binding protein 2 (Pabp2) retroposon is derived from the 5'-untranslated region of the Pabp1 progenitor gene.

The mouse Pabp2 retroposon encodes an isoform of poly(A) binding protein that is expressed in meiotic and early haploid spermatogenic cells. In the present study, we have determined the transcription start site of the Pabp2 gene to clarify the source of its promoter, a prerequisite for expression of retroposons and preservation of their function by natural selection. The 5' end of the mouse Pabp2 retroposon exhibits extensive similarity to the entire 5' UTR of the human PABP1 mRNA, but there is no similarity upstream of the transcription start site of the human PABP1 mRNA, indicating that the Pabp2 gene lacks 5' flanking sequences of the parental PABP1 gene. Oligonucleotide-directed RNase H cleavage and 5' rapid amplification of cDNA ends both indicate that the transcription start site of the mouse Pabp2 gene is located approximately 330 bases downstream of the capsite of the PABP1 mRNA, indicating that the Pabp2 promoter is derived from the PABP1 5' UTR.

A quantitative sucrose gradient analysis of the translational activity of 18 mRNA species in testes from adult mice.

Sucrose gradients have been widely used to study the translational activity of mRNA species in meiotic and haploid spermatogenic cells in mammals. Unfortunately, the results of these studies have been very inconsistent. The purpose of the present study was to obtain accurate and reproducible measurements of the translational activity of a large number of testicular mRNA in sucrose gradients. Extracts of adult testes and cultured seminiferous tubules were sedimented on sucrose gradients, and the distribution of 18 mRNA species was quantified by phosphoimaging. The proportions of various mRNA species sedimenting with polysomes in meiotic and haploid cells (approximately 6-74%) is less than typical of efficiently translated mRNAs (85-90%), demonstrating that the initiation of translation of virtually all mRNA species is at least partially inhibited and that the extent of inhibition is mRNA-specific. Most mRNA species in meiotic and early haploid spermatogenic cells are translated on polysomes in which the ribosome spacing is somewhat wider than in somatic cells, 100-150 verses 80-100 bases. However, the ribosome spacing on protamine mRNAs is unusually close (40-50 bases), and the spacing on poly(A) binding protein mRNA is unusually wide (212-272 bases), thus suggesting that the rate of translational initiation, termination and/or elongation is regulated on translationally active forms of certain mRNA.

cDNA copies of the testis-specific lactate dehydrogenase (LDH-C) mRNA are present in spermatogenic cells in mice, but processed pseudogenes are not derived from mRNAs that are expressed in haploid and late meiotic spermatogenic cells.

Retroposons are a class of genes created by reverse transcribing a processed mRNA and inserting the DNA copy into genomic DNA in germ-line cells. The present study concerns the question: Are retroposons created in meiotic and haploid spermatogenic cells? We demonstrate that polymerase chain reaction amplifies cytoplasmic DNAs with the expected intronless-structure of endogenous reverse transcriptase copies of the processed lactate dehydrogenase C mRNA encoding the testis-specific isoform of lactate dehydrogenase. Quantification of cytoplasmic LDH-C mRNA and endogenous cDNA by competitive RT-PCR and PCR, respectively, indicates that the level of LDH-C cDNA is lower by a factor of about 10(7) than the level of LDH-C mRNA in the cytoplasmic nucleic acids extracted from the testes of 14-day-old mice, and that about 1 in 10(5) meiotic cells contains an endogenous cDNA copy of LDH-C mRNA. A review of the literature reveals that a large number of genes including the LDH-C gene, whose expression is restricted to spermatogenic cells, are always single copy. Collectively, these observations suggest that reverse transcriptase cDNA copies of mRNAs are present in meiotic and haploid spermatogenic cells, but these cDNAs are not integrated into genomic DNA.

The mouse gene encoding the testis-specific isoform of Poly(A) binding protein (Pabp2) is an expressed retroposon: intimations that gene expression in spermatogenic cells facilitates the creation of new genes.

The gene encoding the testis-specific isoform of mouse poly(A) binding protein (Pabp2) has been isolated and sequenced. Unexpectedly, comparison of the sequence of genomic and cDNAs demonstrated that the Pabp2 gene lacks introns, whereas all other functional Pabp genes in plants, amphibians, and mammals contain introns. Thus, the mouse Pabp2 gene is a retroposon, created by synthesizing a reverse transcriptase copy of a processed mRNA and inserting the copy into the genome. The Pabp2 retroposon is unusual because it is functional: previous work demonstrates that its promoter drives the accumulation of Pabp2 mRNA in meiotic and early haploid spermatogenic cells, and the Pabp2 mRNA encodes a protein whose size and RNA-binding specificities are characteristic of PABP in plants, yeast, and mammals (Kleene et al. 1994). Two novel factors can be implicated in the retention of function of the Pabp2 retroposon. First, the promoter of the Pabp2 gene is not derived from its intron-containing progenitor, Pabp1. Second, mRNAs encoding somatic PABP isoform, PABP1, are present at high levels in meiotic and haploid spermatogenic cells. Both features contrast with the phosphoglycerate kinase 2 retroposon, which is believed to compensate for the depletion of the somatic isoform due to X-chromosome inactivation in meiotic spermatogenic cells. We also document that more functional retroposons are expressed in meiotic and haploid spermatogenic cells than in any other tissue and speculate that transcriptional derepression in spermatogenic cells favors the creation of expressed retroposons.

Differential effects of heat shock on translation of normal mRNAs in primary spermatocytes, elongated spermatids, and Sertoli cells in seminiferous tubule culture.

Male germ cells in mice develop normally at 32 degrees C and spermatogenesis is severely inhibited by higher temperatures, including abdominal temperature, 37 degrees C. To examine the effects of heat stress on protein synthesis in various testicular cell types, seminiferous tubules were cultured at 32 degrees or 37 degrees C for 70 min or 42.5 degrees or 44 degrees C for 10 min followed by incubation for 60 min at 32 degrees C. Cultures were labeled with [35S]methionine, and the proteins that are soluble in 4% trichloroacetic acid were analyzed by acid-urea polyacrylamide gel electrophoresis. This culture system preserves the cytoarchitecture of the seminiferous epithelium and avoids breaking late haploid cells (elongated spermatids) during tissue dissociation. Incorporation of [35S]methionine into histone H1t, the testis-specific subtype of histone H1, in pachytene primary spermatocytes (meiotic cells) was reduced by about 33-50% following incubation at 37 degrees and 42.5 degrees C and by >/=90% after incubation at 44 degrees C. In contrast, exposure to 37 degrees, 42.5 degrees, and 44 degrees C had minimal effects on incorporation into transition proteins 1 and 2 in elongated spermatids. To determine whether heat stress inhibits translational initiation, the distribution of several mRNAs in cytoplasmic extracts of cultured tubules was analyzed by sucrose gradients and Northern blots. Exposure to 37 degrees and 44 degrees C produces incremental reductions in the size of polysomes translating H1t mRNA in pachytene spermatocytes and the sulfated glycoprotein 2 mRNA in Sertoli cells, the somatic cell type in the germinal epithelium. Neither 37 degrees nor 44 degrees C reduces the size or proportion of polysomal protamine 2 mRNA in elongated spermatids. These results demonstrate that the initiation of translation in pachytene spermatocytes and Sertoli cells is inhibited by exposure to abdominal temperature and that elongated spermatids are much more resistant to thermal stress.

Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

Patterns of translational regulation in the mammalian testis.

The translational activity of more than 40 different mRNAs in rodent testes has been analyzed by determining the proportions of inactive free-mRNPs and active polysomal mRNAs in sucrose gradients. These mRNAs can be sorted into several groups comprising mRNAs with similar patterns of translational activity in particular cell types. mRNAs in testicular somatic cells sediment primarily with polysomes, indicating that they are translated efficiently, whereas the vast majority of mRNAs in late meiotic and haploid spermatogenic cells display high levels of free-mRNAPs, indicative of a block to the initiation of translation. Protamine mRNAs exemplify a group of mRNAs that is transcribed in round spermatids, stored as free-mRNPs for several days, and translated in elongated spermatids after the cessation of transcription. The extent to which the free-mRNPs in primary spermatocytes and round spermatids are due to developmental changes in translational activity is unclear. mRNAs at these stages can often be detected earlier than the corresponding protein, implicating either a delay in translational activation or difficulties in detecting the protein. In contrast, sucrose gradients consistently indicate little difference in the proportions of various mRNAs in free-mRNPs in primary spermatocytes and round spermatids, implying that the proportions of translationally active mRNAs remain essentially constant. Since the levels of some mRNAs appear to greatly exceed the amount that is translated, the biological significance of some free-mRNPs in meiotic and early haploid cells in unclear. There are numerous examples of controls over the translation of individual mRNAs in meiotic and haploid cells; the proportions of various mRNAs in free-mRNPs range from virtually none to virtually all, and individual mRNAs are activated at specific stages in elongated spermatids. Existing evidence is contradictory whether the mRNAs in the protamine/transition protein gene family are repressed by mRNP proteins of sequestration.

Developmental expression of poly(A) binding protein mRNAs during spermatogenesis in the mouse.

The poly(A) binding protein (PABP), a conserved protein that binds to the 3' poly(A) tail on mRNAs in eukaryotic cells, has been implicated in the regulation of mRNA stability and translation. Two PABP cDNAs with different sequences were isolated from mouse testis cDNA libraries. The predicted amino acid sequence of one, PABP1, is nearly identical (98.9%) to human liver PABP, while 80% of the amino acids of the second, PABPt, are identical to mouse and human PABPs. Northern blots reveal that there is one major PABP mRNA species in liver, muscle, kidney, and brain, two in spleen, and at least four in testis. The levels of PABP mRNA in testis are 5-10-fold higher than in these somatic tissues, but surprisingly the vast majority of all PABP mRNA size variants sediment more slowly than single ribosomes, indicating strong translational repression. Reverse transcriptase-polymerase chain reaction assays demonstrate that PABPt mRNAs are abundant only in testis. Northern blots of RNAs purified from highly enriched spermatogenic cells show that the high levels, multiple sizes of PABP mRNAs, and the PABPt mRNA are present in meiotic and early haploid spermatogenic cells, and are sharply reduced in late haploid cells. Comparison of the binding of PABP1 and PABPt to poly(A) Sepharose in vitro revealed subtle differences, even though PABPt contains substitutions for highly conserved aromatic amino acids that are thought to be necessary for binding to poly(A). The existence of two PABP isoforms in mouse spermatogenic cells could influence cytoplasmic gene expression during spermatogenesis.

Translational activity of mouse protamine 1 messenger ribonucleoprotein particles in the reticulocyte and wheat germ cell-free translation systems.

Protamine 1 mRNAs are inactivated by a block to the initiation of translation in early spermatids and are translationally active in late spermatids in mice. To determine whether translation of protamine 1 mRNAs is inhibited by a protein repressor, the translational activity of ribonucleoprotein particles and deproteinized RNAs were compared in the reticulocyte and wheat germ cell-free translation lysates. To isolate RNPs, cytoplasmic extracts of total testes were fractionated by large-pore gel filtration chromatography. Ribonucleoprotein particles in the excluded fractions stimulated synthesis of radiolabeled translation products for protamine 1 about twofold less effectively than deproteinized RNAs in the reticulocyte lysate, but were inactive in the wheat germ lysate. The ability of translationally repressed protamine 1 ribonucleoprotein particles to form initiation complexes with 80S ribosomes in the reticulocyte lysate was also measured. Protamine 1 ribonucleoprotein particles isolated by gel filtration and in unfractionated cytoplasmic extracts of early spermatids were nearly as active in forming initiation complexes as deproteinized mRNAs. The isolation of ribonucleoprotein particles in buffers of varying ionic strength, protease inhibitors, and several other variables had no major effect on the ability of protamine 1 ribonucleoprotein particles to form initiation complexes in the reticulocyte lysate. These results can be explained by artifacts in the isolation or assay of ribonucleoprotein particles or by postulating that protamine 1 mRNAs are inactivated by a mechanism that does not involve protein repressors, such as sequestration.

Multiple controls over the efficiency of translation of the mRNAs encoding transition proteins, protamines, and the mitochondrial capsule selenoprotein in late spermatids in mice.

The mRNAs encoding protamines 1 and 2, transition proteins 1 and 2, and the mitochondrial capsule selenoprotein are translationally repressed with long poly(A) tracts in early spermatids and translationally active with heterogenous shortened poly(A) tracts in late spermatids (Kleene, 1989). In the present study, the spacing of ribosomes on the translationally active forms of each mRNA was calculated from the length of the coding region and the polysome size determined by sucrose gradient and Northern blot analysis. In addition, the rate of initiation of these five mRNAs was compared in the reticulocyte cell-free translation lysate. Our results reveal at least four additional forms of translational control over these mRNAs: (1) The vast majority of the active forms of the transition protein 1 mRNA and both protamine mRNAs sediment with polysomes in which the ribosomes are spaced closer than is typical of mammalian mRNAs (31-38 vs 80-100 bases apart). This implies that the rate of initiation is unusually fast and/or that the rate of elongation is unusually slow. (2) The mitochondrial capsule selenoprotein mRNA also initiates efficiently in vivo and in vitro, but sediments with polysomes in which the ribosomes are spaced wider than on the protamine mRNAs. The small size of these polysomes can be explained by inefficient insertion of selenocysteine residues at UGA codons. (3) The transition protein 2 mRNA is translated on small polysomes and a relatively large fraction sediments as free mRNPs in vivo. In addition, the transition protein 2 mRNA initiates translation inefficiently at high mRNA concentration and efficiently at low mRNA concentration in vitro. These observations suggest that the transition protein 2 mRNA may be translated inefficiently because it is a weak competitor for a limiting initiation factor. (4) Since low levels of cycloheximide fail to increase the polysome loading of transition protein 2 mRNA in culture, active single ribosomes may also be limited in late spermatids.

Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of > 20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.

Nucleotide sequence of the gene encoding mouse transition protein 2.

The gene encoding the testis-specific basic chromosomal protein, mouse transition protein 2, is split by a single small intron that falls between the first and second nucleotides of a codon. Since the genes encoding protamines 1 and 2 and transition protein 1 in mammals contain a single intron in the same position, protamines and transition proteins appear to be evolutionarily related.

Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse.

We have characterized cDNA clones encoding the selenium-containing polypeptide of the keratinous mitochondrial capsule in mouse sperm. The longest open reading frame encodes a polypeptide 143 amino acids long which contains 21% cysteine and 27% proline and closely resembles the size and amino acid composition of bull mitochondrial capsule seleno-protein (V. Pallini, B. Baccetti, and A. G. Burrini, 1979, in "The Spermatozoon," D. W. Fawcett and J. M. Bedford, Eds., pp. 141-151, Urban & Schwartzenberg, Baltimore/Munich). The reading frame encoding the mitochondrial capsule seleno-protein ends with an amber stop codon suggesting that selenium is not incorporated cotranslationally into the protein by an opal suppressor selenocysteyl-tRNA as has been found for several eukaryotic and bacterial proteins. Northern blots using RNA extracted from purified spermatogenic cells and staged prepuberal mice suggest that the mitochondrial capsule seleno-protein mRNA is first transcribed in late meiotic cells and that the levels of the mRNA increase after meiosis in early haploid cells. Southern blots demonstrate that there is one copy of the gene in the mouse genome. The identification of this cDNA clone, in combination with previous work (K. C. Kleene, 1989, Development 106, 367-373) demonstrates that the mRNA for the mitochondrial capsule seleno-protein is translationally repressed with long homogenous poly(A) tracts in round spermatids and translationally active with shortened heterogenous poly(A) tracts in elongating spermatids.

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.

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.