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

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

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

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

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.     

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.     

A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.     

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.     

Stimulation of DNA repair by the spermatidal TP1 protein

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.     

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1–6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)⁺ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1–6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley-Liss, Inc.     

Role of follitropin receptor signaling in nuclear protein transitions and chromatin condensation during spermatogenesis

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.     

Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain

Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.     

Stage-specific expression of rat transition protein 2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe.

The present study has used methoxyacetic acid (MAA)-induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7-12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis.     

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

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

Nuclear basic protein transition during sperm differentiation. Primary structure of the spermatid-specific protein S2 from the dog-fish Scylliorhinus caniculus

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.     

Target organ regulation of substance P in sympathetic neurons in culture

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

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.     

Isolation of a cDNA clone for transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.