Haploid expression of a mouse testis alpha-tubulin gene.

A complementary DNA clone for an alpha-tubulin has been isolated from a mouse testis complementary DNA library. The untranslated 3' end of this complementary DNA is homologous to two RNA transcripts present in postmeiotic cells of the testis but absent from meiotic cells and from several tissues including brain. The temporal expression of this alpha-tubulin complementary DNA provides evidence for the haploid expression of a mammalian structural gene.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1.

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455-464; Hecht et al. (1986) Exp. Cell Res. 164, 183-190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.

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.

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.

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.

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.

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.

Equations describing the effects of the transient stability of subpopulations of nascent hnRNAs on the kinetics of turnover of hnRNA.

The equations that have been used previously to analyse the rate of decay of hnRNA implicitly assume that nascent hnRNAs are degraded stochastically. This assumption is inconsistent with electron-microscopic studies of transcription cited here, which show that nascent hnRNAs are not degraded during transcription, implying that hnRNA degradation occurs only after termination of transcription and release of the hnRNA from chromatin. Equations are derived describing the accumulation of radioactivity hnRNA during continuous labelling assuming that nascent hnRNAs are stable and that hnRNAs decay with first-order kinetics only after completion of transcription. The effects of the transient stability of nascent hnRNAs on the kinetics of hnRNA turnover can become important when the half-life of the hnRNA is shorter than the time to transcribe an hnRNA from the point of initiation to the point of termination. These equations should prove useful in studies of hnRNA turnover that require a precise description of the labelling kinetics of nascent and completed subpopulations of hnRNA.

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.

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.

Translation of mouse testis poly(A)+ mRNAs for testis-specific protein, protamine 1, and the precursor for protamine 2.

Since previous studies have suggested that the mammalian protamine mRNAs are translated poorly in cell-free systems, we directly measured the efficiency of translation of mouse protamine 1 mRNA. We found that mouse testis poly(A)+ mRNA stimulates the synthesis in the wheat germ and reticulocyte cell-free systems of three prominant translation products which can be resolved by electrophoresis through acid urea polyacrylamide gels containing 8 M urea. These translation products have been identified as testis-specific protein, protamine 1, and the precursor to protamine 2 by several criteria, including labeling with amino acids, [35S]cysteine, and [3H]leucine, which are known to be specific to some of these proteins from the nucleotide sequences of recombinant DNAs. Surprisingly, the mobility of the testis-specific protein translation product is slightly reduced and the mobility of both protamine translation products is drastically reduced unless the extracts of cell-free translations are coelectrophoresed with the appropriate carrier. The fraction of [35S]cysteine- labeled protamine 1 translation product was compared with the fraction of testis poly(A)+ mRNA as protamine 1 mRNA which we measured in dot blots with the use of an SP6 RNA polymerase transcript for protamine 1. The results demonstrate that protamine 1 mRNA is translated only slightly less efficiently than the average testis poly(A)+ mRNA.

Transcription of similar sets of rare maternal RNAs and rare nuclear RNAs in sea urchin blastulae and adult coelomocytes.

We studied the sequences transcribed in the rare class of hnRNA and the rare maternal RNA set in blastula embryos and a tissue of adult sea urchins, coelomocytes. About 26% of labelled single-copy DNA formed hybrids which bound to hydroxyapatite after three cycles of hybridization with nuclear RNA from blastulae and coelomocytes. This corresponds to transcription of about 50% of the single-copy genome by both cell populations. To compare the rare hnRNA sequences synthesized by blastulae and coelomocytes directly, labelled single-copy DNA was hybridized with blastula nuclear RNA to high RNA C0t, fractionated into sequences complementary and non-complementary to blastula nuclear RNA by chromotography on hydroxyapatite, and then each fraction was rehybridized with nuclear RNA from blastulae and coelomocytes. About 62% of the labelled DNA complementary to blastula nuclear RNA and about 1.5% of the labelled DNA non-complementary to blastula nuclear RNA hybridized with nuclear RNA from both cell populations. Thus, coelomocytes and blastula embryos transcribe essentially the same single-copy sequences in the rare hnRNA class. A probe for the rare maternal RNA set was isolated by hybridizing single-copy DNA with total egg RNA to high RNA C0t. 65-67% of this probe hybridized with whole-cell RNA from eggs, blastulae, plutei and coelomocytes demonstrating that essentially all rare maternal RNAs are present, and presumably transcribed, in blastulae, plutei and coelomocytes.

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.

Characterization of a cDNA clone encoding a basic protein, TP2, involved in chromatin condensation during spermiogenesis in the mouse.

A cDNA clone encoding a small cysteine and serine-rich basic protein has been isolated from a mouse testis cDNA library. This cDNA clone encodes the mouse homologue of a protein involved in the initial phases of condensation of chromatin during spermiogenesis in rats, TP2, based on similarities in the sequence of the carboxyl terminus, composition, molecular weight, and electrophoretic mobility. Mouse TP2 can be divided into a highly basic domain comprising about one-third of the polypeptide chain at the carboxyl terminus and a much less basic domain comprising the remaining two-thirds at the amino terminus. The 5' end of the mouse TP2 mRNA contains two in-phase initiation codons both of which may be used generating two polypeptides which differ in length at the amino terminus. Southern blots demonstrate that there is a single copy of the TP2 gene in the mouse genome and Northern blots demonstrate that the polyadenylated TP2 mRNA is present at high and essentially equal levels in early and late haploid cells, and that it is virtually absent from meiotic cells.

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 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.

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.

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.