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Gordon Dixon's pioneering work on the replacement of histones by protamines during spermatogenesis inspired research as recombinant DNA became widely used to analyze gene expression in mammalian spermatogenic cells. The impact of recombinant DNA began immediately with the identification of mouse protamine 1 as a haploid-expressed mRNA, resolving a decades-long controversy whether gene expression in haploid spermatogenic cells distorts transmission of alleles to progeny. Numerous insights into the biology of spermatogenesis followed as the sequences of many mRNAs revealed that the patterns of gene expression in spermatogenic cells are astonishingly different from those in other cells in the mammalian body. Studies of these phenomena have generated fundamental insights across reproductive, molecular and evolutionary biology. Abbreviations: PRM1: protamine 1; PRM2: protamine 2; TCE: translation control element.

Y-box proteins are single-strand DNA- and RNA-binding proteins distinguished by a conserved cold shock domain (CSD) and a variable C-terminal domain organized into alternating short modules rich in basic or acidic amino acids. A huge literature depicts Y-box proteins as highly abundant, staggeringly versatile proteins that interact with all mRNAs and function in most forms of mRNA-specific regulation. The mechanisms by which Y-box proteins recognize mRNAs are unclear, because their CSDs bind a jumble of diverse elements, and the basic modules in the C-terminal domain are considered to bind nonspecifically to phosphates in the RNA backbone. A survey of vertebrate Y-box proteins clarifies the confusing names for Y-box proteins, their domains, and RNA-binding motifs, and identifies several novel conserved sequences: first, the CSD is flanked by linkers that extend its binding surface or regulate co-operative binding of the CSD and N-terminal and C-terminal domains to proteins and RNA. Second, the basic modules in the C-terminal domain are bona fide arginine-rich motifs (ARMs), because arginine is the predominant amino acid and comprises 99% of basic residues. Third, conserved differences in AA (amino acid) sequences between isoforms probably affect RNA-binding specificity. C-terminal ARMs connect with many studies, demonstrating that ARMs avidly bind sites containing specific RNA structures. ARMs crystallize insights into the under-appreciated contributions of the C-terminal domain to site-specific binding by Y-box proteins and difficulties in identifying site-specific binding by the C-terminal domain. Validated structural biology techniques are available to elucidate the mechanisms by which YBXprot (Y-box element-binding protein) CSDs and ARMs identify targets., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The protamine 1 (Prm1) and sperm mitochondria-associated, cysteine-rich protein (Smcp) mRNAs exemplify a widespread pattern of mRNA-specific regulation of mRNA translation in post-meiotic spermatogenic cells, spermatids. Both mRNAs are transcribed and initially stored in free-mRNPs in early spermatids, and translated on polysomes in late spermatids. In this study, we demonstrate that the 5' and 3'-UTRs and the 3' terminus of the Smcp 3'-UTR are required for normal repression of the Smcp mRNA in transgenic mice. RNA affinity chromatography and mass spectrometry sequencing identified Y-box protein 2 (YBX2/MSY2) as the major protein that interacts with the 3' terminus of the Smcp 3'-UTR and a Y-box recognition sequence, GCCACCU, in the translation control element that is necessary for Prm1 mRNA repression. Depletion of YBX2 in Ybx2-null mice prematurely activates Prm1 and Smcp mRNA translation in early spermatids. Fluorescent in situ hybridization reveals that the Smcp intron, the Smcp mRNA, and both Smcp-Gfp transgenic mRNAs are strongly concentrated in the chromatoid body, and that theYbx2-null mutation does not eliminate the Smcp mRNA from the chromatoid body. This and previous findings suggest that the Smcp pre-mRNA is spliced and associates with YBX2 in the chromatoid body, and that repressed free-mRNPs are stored in the general cytoplasm. As YBX2 is the predominant protein in testis free-mRNPs, it likely represses many mRNAs in early spermatids. The mechanisms by which YBX2 represses the Smcp and Prm1 mRNAs are relevant to reproductive medicine because mutations in the human YBX2 gene correlate with abnormal protamine expression and male infertility., (© 2015 Society for Reproduction and Fertility.)

mRNA-specific regulation of translational activity plays major roles in directing the development of meiotic and haploid spermatogenic cells in mammals. Although many RNA-binding proteins (RBPs) have been implicated in normal translational control and sperm development, little is known about the keystone of the mechanisms: the interactions of RBPs and microRNAs with cis-elements in mRNA targets. The problems in connecting factors and elements with translational control originate in the enormous complexity of post-transcriptional regulation in mammalian cells. This creates confusion as to whether factors have direct or indirect and large or small effects on the translation of specific mRNAs. This review argues that gene knockouts, heterologous systems, and overexpression of factors cannot provide convincing answers to these questions. As a result, the mechanisms involving well-studied mRNAs (Ddx4/Mvh, Prm1, Prm2, and Sycp3) and factors (DICER1, CPEB1, DAZL, DDX4/MVH, DDX25/GRTH, translin, and ELAV1/HuR) are incompletely understood. By comparison, mutations in elements can be used to define the importance of specific pathways in regulating individual mRNAs. However, few elements have been studied, because the only reliable system to analyze mutations in elements, transgenic mice, is considered impractical. This review describes advances that may facilitate identification of the direct targets of RBPs and analysis of mutations in cis-elements. The importance of upstream reading frames in the developmental regulation of mRNA translation in spermatogenic cells is also documented.

To facilitate identifying translational control elements by studies of mutations in transgenic mice, a database of orthologous 5' and 3' ends of 12 messenger RNA (mRNA) species from 13 to 23 mammals that undergo delayed translational activation in spermatids was constructed for the Acev2, Akap3, Akap4v2, Gapdhs, Odf1, Prm1, Prm2, Prm3, Smcp, Spata18, Tnp1, and Tnp2 mRNAs. This database, available here, was searched for conserved sequences in conserved positions and known translational control elements. Numerous potential mRNA-specific elements were identified, including upstream open reading frames, conserved sequences upstream and downstream of the poly(A) signal, and noncanonical and multiple poly(A) signals. RNA electrophoresis mobility shift assays demonstrate that Y-box proteins bind 30 of the 36 permutations of the degenerate Y-box recognition sequence (YRS), [UAC][CA]CA[UC]C[ACU], and this information was used to identify hundreds of YRSs in the untranslated region (UTR) database. Collectively, these findings suggest that the distal ends of both UTRs are particularly well conserved, implying that translation of each mRNA is regulated by mechanisms involving the poly(A) binding protein and the closed loop. In addition, the 5' flanking regions of all 12 genes have conserved, gene-specific sequences and configurations of elements that resemble the binding site of the testis-specific isoform of cyclic AMP response element modulator, and all 12 genes lack retrogene paralogues, demonstrating the efficacy of mechanisms that limit the proliferation of retroposons in the male germ line. This study illustrates the power of comparative genomics in identifying novel hypothetical regulatory elements for analysis with biochemical and in vivo genetic approaches.

The chromatoid body is a dynamic organelle that is thought to coordinate the cytoplasmic regulation of mRNA translation and degradation in mammalian spermatids. The chromatoid body is also postulated to function in repression of mRNA translation by sequestering dormant mRNAs where they are inaccessible to the translational apparatus. This review finds no convincing evidence that dormant mRNAs are localized exclusively in the chromatoid body. This discrepancy can be explained by two hypotheses. First, experimental artifacts, possibly related to peculiarities of the structure and function of the chromatoid body, preclude obtaining an accurate indication of mRNA localization. Second, mRNA is not stored in the chromatoid body, because, like perinuclear P granules in Caenorhabditis elegans, the chromatoid body functions as a center for mRNP remodeling and export to other cytoplasmic sites.

Background: Developmental and global regulation of mRNA translation plays a major role in regulating gene expression in mammalian spermatogenic cells. Sucrose gradients are widely used to analyze mRNA translation. Unfortunately, the information from sucrose gradient experiments is often compromised by the absence of quantification and absorbance tracings, and confusion about the basic properties of sucrose gradients., Methods: The Additional Materials contain detailed protocols for the preparation and analysis of sucrose and Nycodenz gradients, obtaining absorbance tracings of sucrose gradients, aligning tracings and fractions, and extraction of equal proportions of RNA from all fractions., Results: The techniques described here have produced consistent measurements despite changes in personnel and minor variations in RNA extraction, gradient analysis, and mRNA quantification, and describes for the first time potential problems in using gradients to analyze mRNA translation in purified spermatogenic cells., Conclusions: Accurate quantification of the proportion of polysomal mRNA is useful in comparing translational activity at different developmental stages, different mRNAs, different techniques and different laboratories. The techniques described here are sufficiently accurate to elucidate the contributions of multiple regulatory elements of variable strength in regulating translation of the sperm mitochondria associated cysteine-rich protein (Smcp) mRNA in transgenic mice.

The sperm mitochondria-associated cysteine-rich protein (Smcp) mRNA is transcribed in step 3 spermatids, and is stored in free mRNPs until translation begins ∼6 days later in step 11. To identify sequences that control the timing of Smcp mRNA translation, mutations in both UTRs were analyzed in transgenic mice using green fluorescent protein (GFP), squashes of seminiferous tubules, and quantification of polysomal loading in adult and 21 dpp testes in sucrose and Nycodenz gradients. GFP fluorescence is first detected in step 9 spermatids in lines harboring a transgene containing the Gfp 5' UTR and Smcp 3' UTR. Unexpectedly, this mRNA is stored in large, inactive mRNPs in early spermatids that sediment with polysomes in sucrose gradients, but equilibrate with the density of free mRNPs in Nycodenz gradients. Randomization of the segment 6-38 nt upstream of the first Smcp poly(A) signal results in early detection of GFP, a small increase in polysomal loading in 21 dpp testis, inactivation of the formation of heavy mRNPs, and loss of binding of a Y-box protein. GFP is first detected in step 5 spermatids in a transgene containing the Smcp 5' UTR and Gfp 3' UTR. Mutations in the start codons in the upstream reading frames eliminate translational delay by the Smcp 5' UTR. Collectively, these findings demonstrate that Smcp mRNA translation is regulated by multiple elements in the 5' UTR and 3' UTR. In addition, differences in regulation between Smcp-Gfp mRNAs containing one Smcp UTR and the natural Smcp mRNA suggest that interactions between the Smcp 5' UTR and 3' UTR may be required for regulation of the Smcp mRNA.

The comparative genomics of the Odf1, Prm1, Prm2, Tnp1, and Tnp2 genes in 13-21 diverse mammalian species reveals striking similarities and differences in the sequences that probably function in the transcriptional and translational regulation of gene expression in haploid spermatogenic cells, spermatids. The 5' flanking regions contain putative TATA boxes and cAMP-response elements (CREs), but the TATA boxes and CREs exhibit gene-specific sequences, and an overwhelming majority of CREs differ from the consensus sequence. The 5' and 3' UTRs contain highly conserved gene-specific sequences including canonical and noncanonical poly(A) signals and a suboptimal context for the Tnp2 translation initiation codon. The conservation of the 5' UTR is unexpected because mRNA translation in spermatids is thought to be regulated primarily by the 3' UTR. Finally, all of the genes contain a single intron, implying that retroposons are rarely created from mRNAs that are expressed in spermatids.

The protamine gene cluster containing the Prm1, Prm2, Prm3, and Tnp2 genes is present in humans, mice, and rats. The Prm1, Prm2, and Tnp2 genes have been extensively studied, but almost nothing is known about the function and regulation of the Prm3 gene. Here we demonstrate that an intronless Prm3 gene encoding a distinctive small acidic protein is present in 13 species from seven orders of mammals. We also demonstrate that the Prm3 gene has not generated retroposons, which supports the contention that genes that are expressed in meiotic and haploid spermatogenic cells do not generate retroposons. The Prm3 mRNA is first detected in early round spermatids, while the PRM3 protein is first detected in late spermatids. Thus, translation of the Prm3 mRNA is developmentally delayed similar to the Prm1, Prm2, and Tnp2 mRNAs. In contrast to PRM1, PRM2, and TNP2, PRM3 is an acidic protein that is localized in the cytoplasm of elongated spermatids and transfected NIH-3T3 cells. To elucidate the function of PRM3, the Prm3 gene was disrupted by homologous recombination. Sperm from Prm3(-/-) males exhibited reductions in motility, but the fertility of Prm3(-/-) and Prm3(+/+) males was similar in matings of one male and one female. We have developed a competition test in which a mutant male has to compete with a rival wild-type male to fertilize a female; the implications of these results are also discussed.

The Smcp mRNA encoding the sperm mitochondria-associated cysteine-rich protein is translationally repressed in round spermatids and translationally active in elongated spermatids. The patterns of transcription and translation of fusions of the Smcp promoter, the green fluorescent protein coding region (Gfp) and various combination of the Smcp and Gfp 5' UTR and 3' UTR have been studied in transgenic mice. 518 nt of Smcp 5' flanking region and 8 nt of 5' UTR drive transcription of mRNAs containing the Gfp coding region in early round spermatids at the same transcription start site as the natural Smcp gene. Transcripts containing both the Gfp 5' and 3' UTRs are translationally active in step 2 spermatids as detected by GFP fluorescence in squashes of living seminiferous tubules from adult testes, and the presence of polysomal mRNAs in sucrose gradient analyses of testes from 21-day-old prepubertal mice, which contain early round spermatids and lack elongated spermatids. By comparison, expression of GFP is delayed until steps 5 and 10, respectively, in transcripts containing the Smcp 5' UTR and Gfp 3' UTR and the Gfp 5' UTR and the Smcp 3' UTR. Sucrose gradient analysis of 21-day-old testes demonstrates that transcripts containing the Smcp 3' UTR exhibit a bimodal distribution in free-mRNPs and polysomes, indicating that the 3' UTR blocks the expression of GFP after the transcripts have entered the elongation phase, a mechanism that may involve microRNAs. The Smcp 5' UTR reduces the levels and size of polysomes in adult testis. In addition, the natural Smcp mRNA contains a positive control element that counteracts the inhibition of translation by the Smcp 5' UTR in adult testis, and the Smcp 3' UTR strongly localizes GFP fluorescence in step 10 spermatids.

The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron-exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5' UTR and 3' UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success.

Triple knockout mice were used to investigate the interactions of five genes that were expressed in meiotic and haploid spermatogenic cells in mice, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t), and sperm mitochondria-associated cysteine-rich protein (Smcp). TNP2 functions in the replacement of histones and the initial condensation of the spermatid nucleus. The linker histone subtypes H1.1 and H1t are expressed at high levels in meiotic and early haploid cells. ACR, a protease that is stored as a proenzyme in the acrosome, is activated during the acrosome reaction and functions in binding of sperm to the zona pellucida. SMCP is a structural protein in the outer membranes of sperm mitochondria that functions in motility. Previous work demonstrates that homozygous knockout mice lacking each of these proteins individually exhibit no defect in fertility on mixed genetic backgrounds. In contrast, the present study demonstrates that five triple knockout lines, Acr/H1.1/Smcp, Acr/Tnp2/Smcp, Tnp2/H1.1/Smcp, Acr/H1t/Smcp, Tnp2/H1t/Smcp, exhibit drastic reductions in fertility on mixed genetic backgrounds. Analysis of fertility parameters reveal that the decreased fertility is due to line-dependent defects in sperm motility in vitro correlated with reduced migration in the female reproductive tract, and decreased fertilization due to defects in adhesion of sperm to the zona pellucida, the membrane surrounding the egg. It was also found that triple knockout males, that are hemizygous for one locus and homozygous for two other loci, are as subfertile as homozygous triple knockout males, a phenomenon known as haploinsufficiency. These findings demonstrate that male fertility involves synergistic interactions of genes that function in sperm motility and sperm-egg adhesion during fertilization., (2005 Wiley-Liss, Inc.)

This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.

The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.

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., (Copyright 2003 S. Karger AG, Basel)

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.

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 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., (Copyright 1999 Academic Press.)

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.

Round spermatids and elongating spermatids were purified from a suspension of mouse testicular cells by sedimentation at unit gravity coupled with density gradient centrifugation through Percoll. Following separation, the two cell types were fractionated into polysomal and non-polysomal compartments. By comparison with round spermatids, elongating spermatids contain about one-half as much cytoplasmic RNA per cell, one sixth as much poly(A)+ RNA per cell and one-half the concentration of poly (A)+ mRNA in their cytoplasm. About two-thirds of the poly(A)+ messenger RNA (mRNA) was in the non-polysomal fraction in both cell types. Polypeptides whose synthesis was directed by cell-free translation of purified mRNA from each cell fraction were analyzed by two-dimensional gel electrophoresis. At the level of detection provided by the electrophoretic methods used, the majority of peptides from the polysomal and non-polysomal compartments for each cell type were similar. However, between the two cell types, approx. 5-10% of the polypeptides in the polysomal and non-polysomal fractions differed markedly in abundance. When the polypeptides encoded by the polysomal and non-polysomal mRNA from round spermatids were compared to the polypeptides encoded in the equivalent fractions from elongating spermatids, a significant reduction in number of polypeptides from elongating spermatids was seen. The presence of specific mRNAs in the non-polysomal fraction of round spermatids and in the polysomal fraction of elongating spermatids suggests that storage of mRNA in the cytoplasm and subsequent utilization provides a source of mRNA for proteins expressed at a time during spermiogenesis when transcription has terminated.

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 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.

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.

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.

We have analyzed the expression, through spermiogenesis, of a series of testicular cell polypeptides encoded by genes within the mouse t complex. Two of these polypeptides, TCP-3 and TCP-7, are synthesized in a testes-specific manner with highest levels of expression during haploid stages of spermatogenesis. A third, TCP-1, is also expressed at highest levels in haploid cells, and expression of this polypeptide continues until the last residual body stage of spermiogenesis. The genes that encode these polypeptides have been correlated with the t phenotype of transmission ratio distortion.

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.

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.

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.

The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.

Mouse spermatozoa contain two protamines with different amino acid sequences. By hybridizing Southern blots of a series of mouse-hamster somatic cell hybrids containing subsets of mouse chromosomes and a complete set of hamster chromosomes with 32P-labeled cDNAs for each mouse protamine, we assign the two mouse protamine genes to chromosome 16. This report presents the first evidence for chromosomal linkage of two sperm-specific, haploid regulated gene products.

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.

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.