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Translational regulation and deadenylation of a protamine mRNA during spermiogenesis in the mouse*1
Abstract
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.
... Primates and most rodents, as well as a subset of other placental mammals, express two PRMs: PRM1 and PRM2 [6]. Both corresponding genes are transcribed in round spermatids (steps 7-9 in mice) [22,23], and the mRNAs are stored in the form of cytoplasmic ribonucleoprotein particles until they are translated in elongating and elongated spermatids (steps 12-16) 6 Andrologia [24,25]. Following transcript storage, mouse Prm1 mRNA seems to be translated earlier (step 12) than Prm2 mRNA (steps 14 and 15) [26]. ...
... Following transcript storage, mouse Prm1 mRNA seems to be translated earlier (step 12) than Prm2 mRNA (steps 14 and 15) [26]. Therefore, our results suggest that RB2 specifically binds to PRMs expressed in steps 12-16 haploid spermatids in mouse seminiferous tubules [24][25][26]. ...
... Therefore, the negative charge of the three sulfate residues in RB2 could ionically bind to the guanidyl group of Arg at pH 10 but not to the neutral chemical components of Lys. The present study demonstrated that RB2 specifically stained steps 12-16 spermatids, in which most PRMs are translated, at pH 10, as previously reported [24][25][26], whereas the specificity of RB2 staining was lost at a neutral pH when both Lys and Arg are positively charged. This pH-dependent specificity could be due to differences in the Arg content among the basic proteins, histones, transition proteins, and PRMs. ...
Reactive blue 2 (RB2) dye specifically binds to the nuclei of human spermatozoa under weakly alkaline conditions, thereby providing a new method for assessing sperm quality. However, this technique has not yet been applied to other mammalian species, such as well-established rodent models, which would allow evaluation of the male reproductive toxicity of new drug candidates in nonclinical studies. We aimed to evaluate the usefulness of RB2 staining in assessing testicular and epididymal sperm toxicity in mice using a busulfan-induced infertility model. Male C57BL/6J mice were intraperitoneally administered 40 mg/kg of busulfan. After 28 days, the testes and epididymis were collected and stained with RB2 at pH 10. In vitro evaluations were conducted on uncoated glass slides with RB2 mixed with mouse synthetic protamines, protamines extracted from the human spermatozoa or intracellular protein components from somatic cells without protamines. Following peanut agglutinin lectin histochemistry, RB2-positive cells were observed in elongating and elongated spermatids at all stages except for stages IX–XI of the seminiferous epithelium. After busulfan administration, the proportion of RB2-positive germ cells in the seminiferous tubules was significantly decreased, and no RB2-positive spermatozoa were found in the caput epididymis of treated mice. Aggregates were observed in a mixture of RB2 dye (pH 10) and protamines but not in a mixture of intracellular protein components without protamines, and this specificity was lost at a neutral pH. Our study demonstrated that RB2 specifically stains steps 12–16 spermatids, indicating specific binding to the protamines expressed in these spermatids. The RB2 staining technique has potential as a biomarker for male reproductive toxicity, allowing for the rapid visualization of protamination in an animal model commonly used for the evaluation of male reproductive toxicity.
... The protamine genes Prm1 and Prm2 encode small basic proteins first transcribed in haploid round spermatids (Mali et al., 1989). The protamine mRNAs are stored in translationally inert mRNP particles for up to 10 days, until translational activation in elongated spermatids (Balhorn et al., 1984;Kleene et al., 1984;Kleene and Flynn, 1987). Translational regulation of the protamine mRNAs is mediated by sequences in their 3′ untranslated region (3′ UTR) (Braun et al., 1989;Fajardo et al., 1997;Zhong et al., 2001). ...
... ODF2 is one of three outer dense fiber proteins that assemble as nine fibers surrounding the axoneme of the sperm tail (reviewed by Kierszenbaum, 2002). All six of these proteins have been shown to be under translational control during murine spermatogenesis (Balhorn et al., 1984;Bunch et al., 1998;Kleene et al., 1984;Kleene and Flynn, 1987;Yelick et al., 1989). Translation of five of the six markers was dramatically reduced in PMH transgenic mice when compared with wild-type mice (Fig. 9). ...
In developing male germ cells, newly synthesized protamine mRNAs are stored for up to 7 days before translational activation. Translational repression of protamine 1 (Prm1) mRNA requires sequences present in its 3′ untranslated region (UTR) and substantial evidence suggests a role for the murine Y-box protein MSY4 in this process. To determine if MSY4 can mediate translational repression in vivo, we generated transgenic mice in which the temporal window of MSY4 expression was extended during spermatogenesis. Expression of MSY4 disrupted the normal completion of spermatogenesis and caused dominant sterility. Immunocytochemical analysis of several markers, including the protamines, indicated that MSY4 prevented normal activation of translation. mRNAs whose translation was inhibited contained at least one MSY4 RNA recognition site, suggesting sequence-dependent translational repression. Altered translational activation resulted in defective processing of protamine 2 and severe defects in sperm morphogenesis. These results suggest that MSY4 plays an active role in translational repression of several mRNAs in differentiating spermatids.
... Prm1 and Tnp2 mRNAs are first detected in round spermatids but are not translated until several days later in elongating spermatids [69,[86][87][88]. Interestingly, these mRNAs only exhibit a partial release from translational repression (for example, less than half of the total Prm1 and Prm2 mRNAs become polysome-associated in elongating spermatids). ...
... A striking observation from northern blot analysis of translationally regulated mRNAs in spermatids is a difference in the electrophoretic mobility of specific mRNA species in ribosome-free mRNP and polysome-associated sucrose gradient fractions from mouse testis [86,88,107]. In the mRNP fractions, mRNAs encoding Prm1 or Tnp1 for example, appear as homogenous transcripts. ...
Tissue development and homeostasis are dependent on highly regulated gene expression programs in which cell-specific combinations of regulatory factors determine which genes are expressed and the post-transcriptional fate of the resulting RNA transcripts. Post-transcriptional regulation of gene expression by RNA-binding proteins has critical roles in tissue development—allowing individual genes to generate multiple RNA and protein products, and the timing, location, and abundance of protein synthesis to be finely controlled. Extensive post-transcriptional regulation occurs during mammalian gametogenesis, including high levels of alternative mRNA expression, stage-specific expression of mRNA variants, broad translational repression, and stage-specific activation of mRNA translation. In this chapter, an overview of the roles of RNA-binding proteins and the importance of post-transcriptional regulation in male germ cell development in the mouse is presented.
... Accordingly, mRNAs encoding structural proteins required for cellular remodeling are transcribed in early haploid spermatids and stored as translationally inert messenger ribonucleoprotein particles (mRNPs). Subsequent translational activation of the repressed messages in late spermatids accompanies poly(A) shortening [8,9]. On the other hand, poly(A) tails of some mRNAs are known to be longer in early haploid spermatids than in meiotic spermatocytes [10][11][12][13][14]. Thus, the mRNA metabolism plays a critical role in spermiogenesis. ...
... Notably, the abundance of poly(A)shortened forms of Prm1, Prm2, and Tnp2 mRNAs of the sizes 0.4, 0.6, and 0.6 kb, respectively, in the Pabpc2 -/testes was comparable with that in the Pabpc2 +/+ and Pabpc2 +/testes. Consistent with the previous findings that poly(A) shortening accompanies translational activation of these three mRNAs [8,9], the PRM2 and TNP2 levels were unaffected by the loss of PABPC2 (Fig. 3B). The levels of PGK2 and AKAP4 were also similar among the Pabpc2 +/+ , Pabpc2 +/-, and Pabpc2 -/testes. ...
Mouse testes contain several isoforms of cytoplasmic poly(A)-binding proteins (PABPCs), including ubiquitous PABPC1 and testis-specific PABPC2/PABPt. PABPC2 is characterized by its absence from translationally active polyribosomes and elongating spermatids. To elucidate the function of PABPC2 in spermatogenesis, we produced mutant mice lacking PABPC2. The PABPC2-null mice showed normal fertility. The processes of spermatogenesis and sperm migration in the testes and epididymides, respectively, were normal in the mutant mice. When the involvement of PABPC2 in translational regulation of haploid-specific mRNAs was examined, these mRNAs were correctly transcribed in round spermatids and translated in elongating spermatids. Moreover, immunoblot analysis revealed low abundance of PABPC2 relative to PABPC1 in spermatogenic cells. These results suggest that PABPC2 may be either functionally redundant with other PABPCs (including PABPC1) or largely dispensable for translational regulation during spermiogenesis.
... During spermiogenesis, protamine genes are expressed soon after completion of meiosis, in round spermatids (Hecht et al., 1986a;. Subsequently, transcripts are stored as cytoplasmic ribonucleoprotein particles for several days until protein synthesis, which takes place in elongating spermatids (Kleene et al., 1984;Kleene, 1989). Most studies on protamines have focused on their interaction with nuclear chromatin and the relevance of chromatin reorganization and nuclear reshaping for sperm morphology and performance. ...
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.
... Perhaps the best described examples of translationally regulated spermiogenesis mRNAs encode the major genome compaction proteins, transition proteins 1 and 2 (TNP1 and 2) and protamine 1 and 2 (PRM1 and 2) which are expressed starting late in meiosis and undergo translation suppression until activation during mid-to late spermiogenesis [5][6][7][8]. Mutations that impact translation of these proteins are associated with severe differentiation defects. For example, mice with Prm1 3' UTR loss exhibit no translation repression and have early DNA compaction with a total arrest of spermatid differentiation [9]. ...
ADAD1 is a testis-specific RNA-binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm showed defective DNA compaction, abnormal head shaping, and reduced motility. Mutant testes demonstrated minimal transcriptome changes; however, ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Further, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Additional analyses demonstrated impaired subcellular localization of multiple proteins, suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC (linker of nucleoskeleton and cytoskeleton) complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1’s impact on the NPC (nuclear pore complex), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1's impact on translation is required for a NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1’s influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype.
... In contrast, from round to elongating spermatids, the poly(A) length gradually decreased (Phase II, Fig. 1D), suggesting that the shortening of the transcript poly(A) tails coincides with the delayed translation progressing. A good agreement was also found when comparing this result with those in the previous reports showing that shortening of the poly(A) tails correlates with translational activation in spermiogenesis (Kleene et al., 1984;Kleene, 1989). For example, transcript isoforms of Spata6 mRNAs start to be expressed in late pachytene spermatocytes, and more isoforms continue to be expressed through the entire haploid phase. ...
As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for up to hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoproteins (RNPs) for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNPs to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a novel mechanism underlying uncoupled translation and transcription in haploid male germ cells.
... Protamine mRNA accumulates in mouse round spermatids with a poly(A) tail length of approximately 150 nucleotides [110]. None of the protamine RNAs associates with ribosomes in round spermatids, while they split into two populations in the elongating spermatids. ...
Multiple RNA pathways are required to produce functional sperm. Here, we review RNA post-transcriptional regulation during spermatogenesis with particular emphasis on the role of 3' end modifications. From early studies in the 1970s, it became clear that spermiogenesis transcripts could be stored for days only to be translated at advanced stages of spermatid differentiation. The transition between the translationally repressed and active states was observed to correlate with the shortening of the transcripts' poly(A) tail, establishing a link between RNA 3' end metabolism and male germ cell differentiation. Since then, numerous RNA metabolic pathways have been implicated not only in the progression through spermatogenesis, but also in the maintenance of genomic integrity. Recent studies have characterized the elusive 3' biogenesis of Piwi-interacting RNAs (piRNAs), identified a critical role for messenger RNA (mRNA) 3' uridylation in meiotic progression, established the mechanisms that destabilize transcripts with long 3' untranslated regions (3'UTRs) in post-mitotic cells, and defined the physiological relevance of RNA exonucleases and deadenylases in male germ cells. In this review, we discuss RNA processing in the male germline in the light of the most recent findings. A brief recollection of different RNA-processing events will aid future studies exploring post-transcriptional regulation in spermatogenesis.
... D'autres études opteraient pour une traduction plus tardive pour la Prm1, c'est à dire à partir des stades 13-14 (Mali et al., 1988). Il a été montré que si les ARNm de Prm1 et Prm2 comprenant un signal de polyadénylation de 160 résidus sont inactifs, 80 à 95% des ARNm de Prm1 et Prm2 comprenant un signal de polyadénylation de 30 résidus sont transcriptionnellement actifs (Kleene et al., 1984 ;Heidaran et Kistler, 1987 ;Kleene, 1989Kleene, , 1993. Le rôle essentiel de ce signal de polyadénylation a également été mis en évidence chez la souris : si la séquence 3'UTR du gène codant pour la Prm1 est remplacée par la séquence 3'UTR de l'hormone de croissance humaine, la traduction a lieu de suite après transcription et dans ce cas, les souris sont stériles et la maturation de la protamine2 n'a pas lieu (Lee et al., 1995). ...
Le processus de maturation des spermatides, la spermiogénèse, est unique en de nombreux points et implique des réorganisations majeures des différents compartiments cellulaires. Pendant les stades de spermatides allongées et condensées, ce processus inclut notamment une compaction drastique du génome paternel qui sera transmis à la future descendance. Lors de ce processus la quasi-totalité des histones est enlevée et celles-ci sont remplacées par des protéines spécifiques, les protamines, qui s'associent au génome paternel et le compactent dans les spermatozoïdes matures. Ce processus de réorganisation est essentiel afin d’éviter des dommages à l’ADN avant et pendant la fécondation et afin qu’un épigénome paternel spécifique soit correctement transmis à la descendance. Les mécanismes de réorganisation de la chromatine font tout d’abord intervenir une vague d’hyperacétylation des histones qui induit ensuite leur enlèvement. L’incorporation de H2A.L.2, un variant spécifique du testicule, va permettre au nucléosome d’adopter une structure plus ouverte et permettre l’invasion de l’ADN par les protéines de transition qui contrôlent le recrutement des protamines et leur incorporation dans le génome paternel. La protamine-2 est synthétisée sous forme d’un précurseur immature qui nécessite d’être clivée pour être correctement intégrée à l’ADN. Cependant, le rôle fonctionnel de la maturation de la protamine-2 dans la spermatogenèse normale et son implication dans l’infertilité n’ont pas été explorées. Mon travail de thèse vise à caractériser cette maturation à l’aide d’un anticorps spécifique de la Pré-Prm2. Nous avons démontré qu’il y avait une accumulation de Pré-Prm2 dans plusieurs modèles de souris où il y avait un défaut dans le processus de transition des histones vers les protamines et que le processus de maturation était donc lié au processus d’enlèvement des histones. Nous avons également pu démontrer que l’action des protéines de transition et des protamines est complètement simultanée et que les deux protéines interagissent. En outre, l’anticorps spécifique de la Pré-PRM2 humaine montre qu’il y a une rétention de pré-PRM2 chez certains patients infertiles et que cette rétention est corrélé à des défauts de compaction de l’ADN, de formation de l’acrosome et de mobilité spermatique. Enfin, à partir d’un certain seuil d’accumulation aberrante de Pré-PRM2, le taux de fécondation est diminué. En conclusion, cette étude a montré qu’une mauvaise maturation de la PRM2 a des conséquences sur le génome mâle, et que cet outil pourrait apporter des informations essentielles en clinique humaine dans l’avenir.
... For ADAD1, this conclusion is buoyed by previous observations demonstrating it binds to the 3′ UTR of the protamine 1 (Prm1) mRNA 17 . Prm1 is among the classically defined translationally regulated transcripts in the testis 32 and proteins that bind to its 3′ UTR are often associated with translational regulation or RNA storage. However, mutation of Adad1 did not impact Prm1 translation or the translation of several other translationally regulated transcripts as reported by Connolly et al. 18 suggesting ADAD1 may post-transcriptionally regulate a different set of transcripts. ...
Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.
... Similarly, during spermatogenesis in the mouse, the transcription of mRNAs coding for protamine, an arginine-rich chromosome protein, occurs shortly after completion of meiosis and stops about a week later. During this time the mRNAs are stored, polyadenylated with some 160 A residues, in the cytoplasm as translationally inert ribonucleoprotein particles (Kleene et al, 1984). Activation of translation in elongated spermatids is coupled with a shortening of the poly A tail to about 30 residues, and since this occurs slightly after translational activation, deadenylation is thought unlikely to be a primary cause for activation. ...
In recent years the 3'untranslated regions (3'UTRs) of eukaryotic mRNAs have emerged as a repository of signals that determine mRNA localization, stability, translation, and cytoplasmic polyadenylation. Furthermore, these regions bind transacting factors to form specific complexes that control gene expression at the post-transcriptional level. This thesis describes the investigations of the functional role of the 3'untranslated region of four murine skeletal myosin heavy chain (MyHC) mRNAs. 3' RACE was used firstly to isolate and clone the slow type 1, and fast 2a, 2b, and 2x MyHC 3'UTRs. Sequence analysis revealed not only the isotypic conservation of the 3'UTRs but also two conserved motifs in the fast 3'UTRs which could be involved in regulation of gene expression or act as putative binding sites for proteins. The results of transfection experiments with hybrid CAT/MyHC 3'UTRs plasmids showed that, after taking into account the effect of deleting the CAT 3'UTR, the MyHC 3'UTRs had no major effects on translation and stability of reporter mRNA in transfected C2C12 cells. RNA-protein interactions of the MyHC 3'UTRs were investigated by bandshift analyses. Results demonstrated that the MyHC 3'UTR-protein complexes were muscle-specific. Competition assays to determine sequence specificity showed that binding could be competed out by unlabelled MyHC 3'UTR and polyadenylic acid but not rat growth hormone 3'UTR. Deletion analysis suggests that secondary structure is important for protein binding. Purification of the protein from shifted complexes identified a 33-kDa protein, aldolase A, as a MyHC 3'UTR-binding protein. These results and the role of aldolase A in the post-transcriptional regulation of MyHC are discussed.
... The morphological and functional development of male germ cells is a reflection of the changes in the testicular microenvironment during spermatogenesis when expression of specific mitochondrial marker proteins was evaluated: with attention to hsp60, hsp70, protamin-1, Lon-protease, sulfhydryl-oxidase, cytochrome C, cytochrome Ct, endopeptidases, chaperonin 60, transition proteins 1, 2, etc. [32,43]. The expression of several sperm mitochondrial proteins may be altered in asthenozoospermic patients [62]. ...
The aim of our review article is related to the sperm mitochondrial biology during living cycle-sper ma-togenesis, when the sperm mitochondria are independent biomarkers of male germ cell dif fe rentiation, sperm capacitation and motility, as well as of spermatozoa health and fertility. An increased understanding of sperm mitochondrial structure, functions, bioenergetics, remodeling/plasticity and especially studies of the own mitochondrial genome (mtDNA), may be useful in the routine andrological clinical practice and assisted reproduction.
... With prior knowledge that the trout protamine mRNA was under developmental translational regulation (Iatrou and Dixon 1977), I selected the recombinant DNA with the greatest number of clones. Sucrose gradient analysis demonstrated that this small mRNA, encoding an unknown protein, was stored in translationally repressed mRNPs in round spermatids (early haploid cells), and recruited onto polysomes in elongated spermatids (late haploid cells) (Kleene et al. 1984). I suspected that I had fortuitously cloned a protamine mRNA, because this mRNA reproduced Iatrou and Dixon (1977) unusual finding that poly(A) tails on repressed trout protamine mRNAs were longer than those on actively translated mRNAs. ...
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
... Posttranscriptional control plays a major role in the successful development of male gametes (Schafer, Nayernia et al. 1995, Laiho, Kotaja et al. 2013). In the case of chromatin compaction, the mRNAs encoding Prm1 and Prm2 are synthesized in round spermatids and stored as mRNPs for up to 7 days before being recruited for translation in elongating spermatids (Kleene, Distel et al. 1983, Kleene, Distel et al. 1984. Translational repression of Prm1 is mediated by sequences in its 3'UTR (Fajardo, Haugen et al. 1997) and temporal translational delay is essential for completion of spermatid differentiation (Lee, Haugen et al. 1995). ...
Micro RNAs (miRNAs), which are ~22 nucleotide (nt) long RNA molecules and several RNA binding proteins (RBPs) engage in an RNA dependent post-transcriptional gene silencing process known as RNA interference (RNAi). In the canonical miRNA biogenesis pathway, an enzyme known as DICER cleaves the ~70nt pre-miRNA to a ~22nt long miRNA that is loaded into the RNAi effector mechanism, the RNA induced silencing complex (RISC). Several in vitro studies provide suggestive evidence that mammalian double stranded RNA binding proteins (dsRBPs), such as TARBP2, act as DICER cofactors in miRNA processing and RISC loading to promote RNAi activity. A screen attempting to identify translational regulators of the murine Protamine1 gene identified TARBP2 as a potential translation regulator. It is unknown if TARBP2 has a role in miRNA biogenesis in vivo, or if the translation regulation of Prm1 during murine spermatogenesis is dependent on TARBP2 mediated miRNA biogenesis. Murine embryos with a constitutive null allele of Tarbp2 and adult mice with a germ cell-specific loss of TARBP2 were generated to lead to understanding of the role of TARBP2 in miRNA biogenesis and TARBP2 mediated post-transcriptional gene regulation during spermatogenesis. Here, I describe that TARBP2 regulate biogenesis of a sub-set of miRNAs during murine embryonic development and spermatogenesis. Also, the role of TARBP2 dependent miRNAs in post-transcriptional regulation of gene expression during murine spermatogenesis will be discussed.
... There are several precedents for a translational control during spermiogenesis. The protamime mRNA (43,44) contains elements at the 3Ј-end that controls the timing of translation during spermiogenesis; it is transcribed at steps 5-6 of spermiogenesis and translated later at steps 9 -10. Whether similar regulatory elements controlling translation are present in the PDE4D mRNA is not known. ...
The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and carboxyl-terminus domains. Multiple PDE proteins with heterogeneous amino termini are derived from each gene. To understand the significance of this heterogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase protection analysis with mRNA for testes at different ages of development showed that two transcripts (PDE4D1 and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcript appears at day 30 and increased during testid maturation. This latter transcript codes for a long variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was est...
... A particularly surprising outcome concerned the PRM and TNP. It has been long stated that their mRNAs in mouse, rat and human start to be transcribed in RS [81][82][83][84][85] and remain repressed until the stages of elongating spermatids, when they are translated [86,87]. This means that in mouse, for example, this repression mechanism would be operative for up to a week. ...
Background
Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.
Results
We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.
Conclusions
This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2618-1) contains supplementary material, which is available to authorized users.
... Spergen-4 protein was, however, detected only in elongated spermatids (step 16-19) in the seminiferous epithelium, but not seen in spermatocytes and round spermatids, suggesting delayed translation of Spergen-4 mRNA into protein. The delayed translation of transcript, as reported in the cases of nuclear proteins (Kleene et al., 1984;Iguchi et al., 2004) and flagella structural proteins (Morales et al., 1994;Carrera et al., 1994;Yang et al., 2003;Iida et al., 2004b), may be caused by translation repression and subsequent activation of translation at specific stages of spermiogenesis. Translation represion is a major form of gene regulation during spermatogenesis and is believed to provide a way for spermatogenic cells to overcome the transcriptional silencing associated with chromatin condensation in elongating spermatids (Iguchi et al., 2004). ...
We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.
Besides ubiquitous poly(A)-binding protein, cytoplasmic 1 (PABPC1), testis-specific PABPC2/PABPt (in humans, referred to as PABPC3), and female and male germline-specific PABPC1L/ePAB, have been reported in the mouse testis. Recent in silico analysis additionally identified testis-specific Pabpc6 in the mouse. In this study, we characterized PABPC6 and its mutant mice. PABPC6 was initially detectable in the cytoplasm of pachytene spermatocytes, increased in abundance in round spermatids, and decreased in elongating spermatids. PABPC6 was capable of binding to poly(A) tails of various mRNAs and interacting with translation-associated factors, including EIF4G, PAIP1, and PAIP2. Noteworthy was that PABPC6, unlike PABPC1, was barely associated with translationally active polysomes and enriched in chromatoid bodies of round spermatids. Despite these unique characteristics, neither synthesis of testicular proteins nor spermatogenesis was affected in the mutant mice lacking PABPC6, suggesting that PABPC6 is functionally redundant with other co-existing PABPC proteins during spermatogenesis.
Cell differentiation is associated with global changes in translational activity. Here, we characterize how mRNA poly(A) tail processing supports this dynamic. We observe that decreased translation during neuronal differentiation of P19 cells correlates with the downregulation of 5'-terminal oligopyrimidine (TOP) transcripts which encode the translational machinery. Despite their downregulation, TOP transcripts remain highly stable and show increased translation as cells differentiate. Changes in TOP mRNA metabolism are reflected by their accumulation with poly(A) tails ∼60-nucleotide (nt) long. The dynamic changes in poly(A) processing can be partially recapitulated by depleting LARP1 or activating the mTOR pathway in undifferentiated cells. Although mTOR-induced accumulation of TOP mRNAs with tails ∼60-nt long does not trigger differentiation, it is associated with reduced proliferation of neuronal progenitors. We propose that while TOP mRNAs are transcriptionally silenced, their post-transcriptional regulation mediated by a specific poly(A) processing ensures an adequate supply of ribosomes to complete differentiation.
Herein, we compare the different experimental regimes used to induce testicular heat stress and summarise their impact on sperm production and male fertility. Irrespective of the protocol used, scrotal heat stress causes loss of sperm production. This is first seen 1–2 weeks post heat stress, peaking 4–5 weeks thereafter. The higher the temperature, or the longer the duration of heat, the more pronounced germ cell loss becomes, within extreme cases this leads to azoospermia. The second, and often underappreciated impact of testicular hyperthermia is the production of poor‐quality spermatozoa. Typically, those cells that survive hyperthermia develop into morphologically abnormal and poorly motile spermatozoa. While both apoptotic and non‐apoptotic pathways are known to contribute to hyperthermic germ cell loss, the mechanisms leading to formation of poor‐quality sperm remain unclear.
Mechanistically, it is unlikely that testicular hyperthermia affects messenger RNA (mRNA) abundance, as a comparison of four different mammalian studies shows no consistent single gene changes. Using available evidence, we propose two novel models to explain how testicular hyperthermia impairs sperm formation. Our first model suggests aberrant alternative splicing, while the second model proposes a loss of RNA repression. Importantly, neither model requires consistent changes in RNA species.
In brief:
Protamines package and shield the paternal DNA in the sperm nucleus and have been studied in many mouse models over decades. This review recapitulates and updates our knowledge about protamines and reveals a surprising complexity in protamine function and their interactions with other sperm nuclear proteins.
Abstract:
The packaging and safeguarding of paternal DNA in the sperm cell nucleus is a critical feature of proper sperm function. Histones cannot mediate the necessary hypercondensation and shielding of chromatin required for motility and transit through the reproductive tracts. Paternal chromatin is therefore reorganized and ultimately packaged by protamines. In most mammalian species, one protamine is present in mature sperm (PRM1). In rodents and primates among others, however, mature sperm contain a second protamine (PRM2). Unlike PRM1, PRM2 is cleaved at its N-terminal end. Although protamines have been studied for decades due to their role in chromatin hypercondensation and involvement in male infertility, key aspects of their function are still unclear. This review updates and integrates our knowledge of protamines and their function based on lessons learned from mouse models and starts to answer open questions. The combined insights from recent work reveal that indeed both protamines are crucial for the production of functional sperm and indicate that the two protamines perform distinct functions beyond simple DNA compaction. Loss of one allele of PRM1 leads to subfertility whereas heterozygous loss of PRM2 does not. Unprocessed PRM2 seems to play a distinct role related to the eviction of intermediate DNA-bound proteins and the incorporation of both protamines into chromatin. For PRM1, on the other hand, heterozygous loss leads to strongly reduced sperm motility as the main phenotype, indicating that PRM1 might be important for processes ensuring correct motility, apart from DNA compaction.
Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell-specific RNA-binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mouse mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.
Introduction:
Histopathological characteristics of cytomegalovirus (CMV) lymphadenitis have been well described. Rare studies have reported the immune status and clinical features. Clinically, experts believed that CMV lymphadenitis develops in immunocompromised and immunocompetent patients. Infectious mononucleosis (IM)-like syndrome is the most well-known clinical presentation.
Methods:
We reviewed archived CMV immunohistochemical stains on lymphoid tissues. The clinicopathological features of CMV-positive cases were studied.
Results:
For lymph nodes, we detected CMV in 29% (5/17) allogeneic peripheral blood hematopoietic stem cell transplantation (PBSCT) recipients, 29% (4/14) post-autologous PBSCT patients, 13% (6/47) patients treated with intravenous chemotherapy, and 9% (9/96) immunocompetent patients. We detected CMV in 7% (2/24) of tonsils but not in the nasopharynx, tongue base, or spleen specimens. The patients with iatrogenic immunodeficiency ranged from 37 to 76 years old. CMV infections developed a few years after lymphoma treatment (median duration after allogeneic PBSCT, 932 days; after autologous PBSCT, 370 days; and after chemotherapy, 626 days). The most common clinical presentation was neck mass (13/25, 42%), followed by asymptomatic image finding (10/25, 40%). Positron emission tomography/computed tomography (PET/CT) scan showed increased uptake compared to the liver in all patients (11/11, 100%). Of 10 lymphoma patients, 8 (80%) had a Deauville score of 4-5; they accounted for 30% (8/27) of lymphoma patients with false-positive PET/CT scan results. All cases were self-limiting. 96% (23/25) cases had Epstein-Barr virus coinfection, and EBER-positive cells were predominantly in a few germinal centers.
Conclusions:
Cytomegalovirus (CMV) lymphadenitis and tonsillitis were subclinical infections, not primary CMV infection with IM-like syndrome. The lymphadenopathy typically developed a few years after lymphoma treatments in the middle-aged and the elderly. The lesions mimicked lymphoma relapse in PET scans. Therefore, recognizing CMV infection in lymphoid tissues is of clinical importance.
The development of gametes was of much interest as the meiotic events are quite different between the two sexes. Female gametogenesis occurred during embryonic development whereas male gametogenesis continued into adulthood and was more easily studied. A practical concern, of much importance for domestic animal breeding, was the separation of X- and Y-bearing sperm and much effort was expended trying to do so. Also, it had generally been assumed that there was no gene expression after meiosis such that the evolutionary selection would only occur on the zygote and not on the gametes. This turned out not to be true and other mechanisms, intracellular bridges for the sharing of postmeiotically synthesized gene products, were found to mostly maintain gamete neutrality. However, the much studied t-alleles provided a major exception to this neutrality and the elucidation of the transmission ratio distortion they produced was a major project for mouse geneticists.
Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.
Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.
The mouse H19 gene was identified as an abundant hepatic fetal-specific mRNA under the transcriptional control of a trans-acting locus termed raf. The protein this gene encoded was not apparent from an analysis of its nucleotide sequence, since the mRNA contained multiple translation termination signals in all three reading frames. As a means of assessing which of the 35 small open reading frames might be important to the function of the gene, the human H19 gene was cloned and sequenced. Comparison of the two homologs revealed no conserved open reading frame. Cellular fractionation showed that H19 RNA is cytoplasmic but not associated with the translational machinery. Instead, it is located in a particle with a sedimentation coefficient of approximately 28S. Despite the fact that it is transcribed by RNA polymerase II and is spliced and polyadenylated, we suggest that the H19 RNA is not a classical mRNA. Instead, the product of this unusual gene may be an RNA molecule.
The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.
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.
In the testis, germ cells which are separated from the serum by the blood-testis barrier rely primarily on the Sertoli cell to obtain nutrients. For example, transferrin synthesized by the Sertoli cell is important in delivering iron from the serum to the developing germ cells. Because of its role in the testis, Sertoli cell transferrin protein and mRNA have been extensively studied. By using RNA blot analysis of rat testicular tissue, we detected a transcript of 2.6 kb which is attributed to transferrin. In addition, we detected a novel mRNA of 0.9 kb which had sequence similarity to the 3' end of transferrin. This 0.9-kb mRNA was present in germ cells, but not Sertoli cells, liver, or brain. The primary source of this mRNA in the testis was round spermatids. Sequence analysis of a cDNA clone showed that this mRNA encoded a protein with sequence similarity to the carboxy terminus of transferrin. Polysome analysis indicated that this transcript was translated and may therefore have importance in the iron metabolism of germ cells. The evolutionary implications between the transferrinlike mRNA germ cells and the gene duplication event which resulted in the diferric binding of transferrin are discussed.
We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.
The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.
RNAs for proteins required in elongated spermatids are considered to be transcribed at an early stage and stored in cytoplasm, presumably in chromatoid body (CB), one type of nuage component (a unique structure that appears and disappears during spermatogenesis), because transcription of genes does not occur at late stages. In elongated spermatids, a large amount of tubulin molecules is required to form microtubules of manchette and flagellum. To investigate the possible role of CB in translation of tubulin mRNA, we performed immunofluorescence and immunoelectron microscopic localization studies of α- and β-tubulin in rat spermatogenic cells. β-tubulin was detected in CB, but α-tubulin was not. Other nuage components present in pachytene spermatocytes (ISPG, IMC, SB) were negative for both α- and β-tubulin. Our findings suggest that: (i) β-tubulin in round spermatids is translated within the CB, whereas α-tubulin is not; (ii) αβ-heterodimers are formed outside CB and incorporated into microtubules of manchette and flagellum.Japan Society for the Promotion of Science This article is protected by copyright. All rights reserved.
The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme's adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLB(D114A)), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLB(D114A) failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity.
As outlined in other chapters of this volume, opioid pep tides have been associated with numerous neuromodulatory and hormonal actions. In addition, recent studies have suggested an additional role as local paracrine/autocrine regulators in several peripheral organs and tissues. Instead of attempting to cover opioids in all peripheral tissues, this chapter will focus on selected examples of peripheral opioid gene expression that suggest a local function distinct from that of a neuromodulator or circulating hormone (with regard to the gastrointestinal tract, see Chap. 37).
The classic studies of Monesi (1,2) in the 1960s revealed an intricate pattern of RNA synthesis during spermatogenesis, characterized by transient transcriptional activity. During the early stages of meiotic prophase, a relatively low rate of [3H]uridine incorporation was detected; however, RNA synthesis increases at the mid-pachytene stage followed by diminution during the meiotic divisions. RNA synthesis once again increases in the round and elongating spermatids (up to step 8), followed by a decline in later stages where nuclear condensation occurs.
DNA-associated proteins of spermatozoa are different from somatic cell histones. In mammals, arginine-rich protamines replace histones and transition proteins in spermatids during compaction of the chromatin (Poccia, 1986). The mouse protamine mRNAs are expressed in early spermatids by a haploid genome (Kleene et al., 1983). To find the exact stages of differentiation where the expression of spermatidal nucleoprotein mRNAs occur, we used three different cDNA-hybridization techniques to measure protamine 1, protamine 2 and transition protein (TP1) mRNA levels.
Murine spermatogenesis initiates a few days after birth and continues for the duration of the sexual life of the animal. Spermatogenesis takes approximately 35 days and consists of the mitotic proliferation of spermatogonial cells, meiosis, and spermiogenesis, the haploid spermatid differentiation stage (Fig. 11.1). Transcription is ongoing throughout spermatogonial proliferation, meiosis, and the early spermiogenesis. In fact, unlike spermatogenesis in Drosophila where there is no postmeiotic transcription, in the mouse there is considerable elevation in the transcriptional apparatus shortly after meiosis (1). Transcription continues until the transition from the round to the elongating spermatid, and then ceases several days before to the completion of spermiogenesis (2, 3). The study of transcriptional activity has relied primarily on metabolic labeling studies, which are limited in their sensitivity. The available data do not allow us to distinguish between transcriptional silencing at the transition from round spermatid to elongating spermatid (about step 9), or the elongating spermatid to elongated spermatid transition (about step 13). Distinguishing between these two possibilities is important if one is to understand the reason for transcriptional silencing. Cessation of transcription at step 9 would likely involve developmental changes in the transcriptional apparatus, whereas transcriptional arrest at step 13 could be attributed to changes in chromatin structure that occur as chromosome condensation commences.
Several lines of evidence indicate that highly specialized transcriptional mechanisms ensure stringent stage-specific gene expression in the germ cells. Specific checkpoints correspond to the activation of transcription factors; these regulate gene promoters with a restricted pattern of activity, in a germ cell-specific fashion. There is also evidence that general transcription factors may be differentially regulated in germ cells. For example, TBP (TATA-binding protein) accumulates in early haploid germ cells at much higher levels than in any other somatic cell type. It has been calculated that adult spleen and liver cells contain 0.7 and 2.3 molecules of TBP mRNA per haploid genome-equivalent, respectively, while adult testis contain 80–200 molecules of TBP transcript per haploid genome-equivalent (1). In addition to TBP, TFIIB and RNA polymerase II also were found to be overexpressed in the testis (1). These remarkable features are consistent with the potent transcriptional activity that occurs in a coordinated manner during the germ cell differentiation. Here we discuss the characteristics of cAMP-responsive element modulator (CREM) (2), a transcription factor responsive to the cAMP signaling pathway and whose function is crucial for a normal germ cell differentiation program.
Spermatogenesis is a complex differentiative process, highly conserved in the course of evolution, represented by the ordered sequence of mitotic, meiotic and differentiative stages. The pioneering work of Oakberg (1956), Monesi (1965) and Clermont (1972) have clearly shown that these stages are characterized by a complex series of biosynthetic processes which make spermatogenesis an excellent model system for the study of molecular events related to cell differentiation. DNA, RNA and protein synthesis were initially studied by autoradiographic and histochemical techniques, and only later with biochemical analysis after the introduction of cell fractionation techniques (Lam et al., 1970; Meistrich, 1973) which made available germ cells at defined stage of differentiation. Further insight into the genetic events and the mechanisms which control germ cell differentiation seems now possible with the use of powerful tools like molecular biology and transgenic techniques.
Germ cell nuclear factor (GCNF), an orphan receptor in the nuclear receptor superfamily, is expressed predominantly in developing germ cells in the adult mouse. Two Gcnf transcripts (7.4 and 2.1 kilobase [kb]) encoded by a single copy gene are expressed in the testis of several mammalian species. To identify features that regulate Gcnf expression, we characterized the structure and sequence of the mouse gene and its two transcripts and determined the expression profile of the GCNF protein during spermatogenesis. Genomic fragments spanning part of the 5'-untranslated region (UTR), the coding sequence, and the complete 3'-UTR (similar to80 kb) were isolated and sequenced. The 3'-UTRs of the two transcripts are quite distinct. The 7.4 kb transcript, which appears earlier in spermatogenesis, has a very long 3'-UTR of 4451 nucleotides. In contrast, the 2.1 kb transcript, which is expressed predominantly during the haploid phase of spermatogenesis, has a 3'-UTR that is only 202 nucleotides in length. Additional analyses indicate that both transcripts share the same coding region and are associated with polysomes. A single GCNF protein band was detected in testis extracts by Western blotting with a specific antiserum. Immunohistochemical analysis showed that GCNF is localized in the nuclei of pachytene spermatocytes and round spermatids. GCNF is first detectable in early pachytene spermatocytes (stage II) and is continuously expressed until spermatids begin to elongate in stage IX. Although GCNF is generally distributed throughout the nucleus, it is particularly prominent in heterochromatic regions at some stages and in condensed chromosomes undergoing the meiotic divisions. This expression profile suggests that GCNF plays a role in transcriptional regulation during meiosis and the early haploid phase of spermatogenesis.
A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture,
as well as the extent of sequence homology between related RNA or DNA species.
Nucleated erythroid cells isolated from the spleens of anemic mice were used to investigate the processing of the polyadenylic acid region of globin mRNA. Cells were labeled in media containing [3H] adenosine and transferred to media containing no radioactive precursor and incubated further in the presence or absence of actinomycin D. After various times following the transfer of the cells, globin mRNA was isolated using a combination of oligo(dT)-cellulose affinity chromatography, sucrose density centrifugation, and globin cDNA (the complementary DNA copy of globin mRNA)-cellulose affinity chromatography. The size of the poly(A) region was determined by polyacrylamide gel electrophoresis of the T1 and pancreatic RNase-resistant fragments. The prelabeled poly(A) region which initially comprises approximately 150 adenylate residues was found to become shorter with time, both in cells incubated in medium containing no radioactive precursor and in the presence of actinomycin D. After 9 h of incubation in the presence of actinomycin D, two major size classes of poly(A) were observed, one containing 35 to 45 adenylic acid residues and the other containing 55 to 65 residues. These two size classes are similar to those found in circulating reticulocytes suggesting that the poly(A) shortening observed in these cell incubation studies is similar to that which occurs in vivo. Two protein synthesis inhibitors, emetine and cycloheximide, were investigated with respect to their effect on poly(A) shortening. Neither drug inhibited the shortening of the poly(A) region of globin mRNA, suggesting that protein synthesis is not required for this process to occur.
Treatment of silkmoth chorion mRNAs with calf thymus RNase H (EC 3.1.4.34; RNA-DNA-hybrid ribonucleotidohydrolase) in the presence of oligo(dT) specifically and effectively removes the 3'-terminal poly(A) sequences. Excision of non-poly(A) fragments cannot be detected. Under these conditions, RNase H leads to increased electrophoretic homogeneity of rabbit globin mRNA, presumably as a result of removal of poly(A) sequences that are inherently variable in length. Treatment with RNase H converts the three diffuse zones of messages for the several chorion proteins into multiple sharp bands.
A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.
Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimum of 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.
Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimum of 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.
A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 x 10⁷ pachytene spermatocytes (98% purity), 1.1 x 10⁸ early spermatids (93% purity), and 1.1 x 10⁸ cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have retained their biochemical integrity.
Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.
The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [³H] arginine and [³H] lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididymides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [³H] arginine was seen in a number of spermatid basic proteins, including both the "testis-specific" protein (TP) and the protamines. As expected from previous studies, only one class of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [³H] lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [³H] lysine was initially incorporated into elongating spermatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nuclei of step 14.
The size of the poly(A) segment present at the 3'-end of mRNA molecules decreases gradually after entry of the mRNA into the cytoplasm. The size reduction seems to occur equally in free and polysome-bound mRNA. It is not affected by conditions that inhibit protein synthesis, such as treatment with cycloheximide and amino acid deprivation. The latter condition leads to a substantial increase in the size of nuclear poly(A). The rate of poly(A) cleavage could determine the length of time a mRNA molecule remains functional in the cytoplasm.
A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.
The ribosomes in polysomes increase about 30-fold from 0.75% to 20% of the total ribosomes during the first 2 hr after fertilization of sea urchin eggs. The amount of messenger RNA necessary for this increase in polysomes was calculated to be 6 × 10−12 gm or about 0.4% of the total RNA of the egg. The amount of newly synthesized RNA entering the polysomes right after fertilization was measured with radioactive precursor techniques and was no more than 15% of the total messenger RNA entering the polysomes over the same period. These direct measurements confirm that most of the newly active messenger RNA is not synthesized after fertilization but is apparently present in the egg beforehand.The polysomes do not change in size over the 2 hrs that more messenger RNA is becoming active, indicating that the messenger RNA molecules are released individually at various times over the 2-hr period. This suggests that the translational control mechanism in the egg acts directly at the level of the messenger RNA molecules.
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.
The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.
A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.
We have examined the relationship between the newly synthesized mRNA that enters polysomes in sea urchin embryos and the messengerlike RNA that enters the pool of ribosome-free ribonucleoprotein particles (free RNPs or informosomes). Although the RNA in the free RNPs turns over 25% more rapidly than in the polysomes, labeling kinetics indicate that the RNA containing poly(A) [poly(A)(+)RNA] and the RNA not containing poly(A) [poly(A)(-)RNA] within each cytoplasmic compartment have very similar half-lives. The poly(A)(+)RNA from both free RNPs and polysomes binds ribosomes almost equally well in a reticulocyte lysate, and this binding is sensitive to inhibitors of initiation. The poly(A)(-)RNA from polysomes initiates as well as poly(A)(+)RNA; however, poly(A)(-)RNA from free RNPs is only half as efficient in binding to ribosomes, and by this criterion is only 50% mRNA.
We have also examined the size and dynamics of shortening of the poly(A) tails of poly(A)(+)RNA from free RNPs and polysomes. Pulse-labeled poly(A) from both free RNPs and polysomes is about 180 nucleotides in length. Poly(A) shortening is very rapid in polysomes; steady-state labeled polysomal RNA is largely devoid of the 180-nucleotide-long poly(A) segments. Poly(A) shortening in free RNPs is slower; half of the poly(A) derived from steady-state free RNPs is still 180 nucleotides long. Despite this difference in the rates of poly(A) shortening, polysomes and free RNPs have very similar half-lives. There is, then, no obvious relationship between poly(A) shortening and turnover of mRNA in these embryos. The data are interpreted to mean that poly(A)(+)RNA from free RNPs is enriched for a class of mRNA that initiates less frequently in vivo than the bulk of the cellular mRNA.
Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and without degradation. The DNA is free of agarose and remains susceptible to digestion by restriction enzymes. The analytical technique uses selective precipitation of DNA with acetone and has been adapted to molecular hybridization scans of sequences in agarose gels. The sequence-monitoring system is quantitative, directly measuring the proportion of the probe complementary to a given DNA fragment and vice versa. It is especially suitable for analyzing restriction enzyme digests of DNA in mapping experiments.
We have used thermal elution from poly(U)-Sepharose to separate RNA from Dictyostelium discoideum into several fractions which differ in their respective poly(A) sizes. We have shown that most newly synthesized poly(A)-containing RNA from vegetative cells of this organism contains long poly(A) tracts which shorten with age, and that these new transcripts can be purified at least tenfold, and perhaps as much as 60-fold, from other cellular messenger RNAs by this technique. We have isolated newly synthesized mRNA and mRNAs of different poly(A) contents and analyzed their translation activities and translation products using mRNA-dependent wheat germ and reticulocyte lysates and two-dimensional gel electrophoresis. Our results demonstrate that translatable RNAs are not distributed equally amongst RNAs of different poly(A) contents; some appear to be relatively more abundant in newly synthesized mRNA than in RNAs containing shorter poly(A) tracts, while, others appear to be less abundant. A comparison of the translation products of newly synthesized poly(A)-containing RNA with those of other RNA fractions has led us to suggest that mRNA synthesis is pre-eminent in establishing the frequency distribution of mRNAs in vegetative cells of this organism, and that additional minor adjustments are made by differential stabilities. We also have shown that poly(A)-minus RNA from vegetative cells of this organism codes for only a small number of major proteins. Since shortening of poly(A) with age is a common occurrence in cells of higher organisms, thermal elution from poly(U)-Sepharose could be a generally applicable technique to use for enriching for mRNAs induced by alterations in developmental or metabolic states as well as for studying eukaryotic mRNA metabolism.
Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.
The rabbit beta-globin DNA insertion of the hybrid plasmid PbetaG1 (Maniatis et al., 1976) was sequenced by the method of Maxam and Gilbert (1977). A sequence of 576 nucleotides was determined and verified by pyrimidine tract analysis of double-stranded DNA, synthesized in vitro starting from beta-globin mRNA. The derived sequence is in complete agreement with previously reported partial mRNA sequencing data and with the predictions from the primary structure of the protein. Moreover, the globin DNA insertion is missing only 13 nucleotides corresponding to the 5' terminal sequence of the mRNA. The rabbit beta-globin mRNA consists of a coding region of 438 nucleotides, flanked by a 5' noncoding region of 56 nucleotides (including the initiation codon AUG but not the 7-methyl-guanine of the "cap structure") and by a 3' noncoding region of 95 nucleotides (including a UGA termination codon). The features of the mRNA sequence are discussed with specific attention to the selective use of particular codons, the probable existence extensively base-paired segments at the 5' terminal region and the ribosome binding site. The faithful representation of beta-globin mRNA in the PbetaG1 DNA insertion establishes the validity of used cloned DNA, initially derived from double-stranded DNA transcripts of mRNA, for studying the structure of eucaryotic genes.
THE 3'-OH poly(A) segment of globin mRNA ensures the stability of the message when injected into Xenopus oocytes1-4. We recently developed5 a specific method for the removal of the poly(A) tail from globin mRNA, based on synchronous processive phosphorolysis of mRNA using molar excess of E. coli polynucleotide phosphorylase at 0 °C. When injected into Xenopus oocytes, deadenylated globin mRNA is translated for a relatively short period and then rapidly degraded1,2. Native poly(A)-containing mRNA, however, is considerably more stable in the same conditions and is translated for extended periods of time1,2,7. The poly (A) segment itself is responsible for the stability of native globin mRNA in Xenopus oocytes since poly(A) re-addition to previously deadenylated mRNA restores the functional stability of the message3. We do not yet know, however, how poly(A) exerts its protective function; furthermore, the mechanism of degradation of poly(A)-free mRNA is not known. We report here the use of the enhancing effects of haemin on the translation of alpha-globin mRNA in frog oocytes6 to establish that, in these cells, the degradation of injected poly(A)-free alpha-globin mRNA is linked to its translation.
The amount of translatable histone mRNA in Xenopus oocytes, eggs, and embryos was found to be roughly similar, as judged by a cell-free translation assay. This observation provides further evidence that posttranscriptional control mechanisms bring about the 50-fold increase in the rate of histone synthesis during oocyte maturation. About three-quarters of the oocyte histone mRNA was found to be polyadenylated. However, polyadenylated histone mRNA was scarcely detectable in the egg and embryo. Our data are consistent with the speculation presented here that the disappearance of the poly(A)+ histone mRNA component at the time of conversion of oocyte to egg (maturation) is due to deadenylation.
Protamine messenger RNA was isolated in a very pure form from trout testes and used as a template for the synthesis of labeled complementary DNA (cDNA) of high specifiv activity. The cDNA was found to be a full-length transcript of protamine messenger RNA and was used as a probe for hybridization reactions with RNA preparations isolated from three subcellular compartments of differentiating trout testis cells. The RNA populations from the nuclei, polysomes, and postribosomal supernatant of these cells were fractionated into poly(A)-containing [poly(A)+] and poly(A)-free [poly(A)-] RNA to determine the distribution of these two forms of protamine mRNA in these cell compartments. At the early protamine stage of testis development, polysomal and postribosomal supernatant fractions contain almost equal quantities of poly(A)+ protamine mRNA, but poly(A)- protamine mRNA was found almost entirely in the polysomes.
Dramatic transitions occur in the nuclear proteins during spermiogenesis in rats. In order to determine more precisely when these transitions occur, we have employed centrifugal elutriation, velocity sedimentation at unit gravity, centrifugation in metrizamide gradients, and sonication to obtain relatively homogeneous populations of testis cells and nuclei. The results indicate that histones are present in step 1–8 spermatid nuclei but are not detectable after step 12. Nuclear proteins designated TP and TP2 are not detectable in step 1–8 spermatids but are present and actively synthesized in step 13–15 spermatids. These two proteins are turned over within 5 days after synthesis. A spermatidal basic nuclear protein, designated TP3, and the sperm basic nuclear protein, S1, are present in step 16–19 spermatids. Biochemical characterization of TP2 and TP3 are presented.
Whereas the original size of poly(A) in HeLa cells is about 200 nucleotides, at steady state most of the poly(A) in mRNA contains less than 50 nucleotides. An endonucleolytic attack on poly(A) is suggested as the most likely method to accumulate short pieces of poly(A). Both poly(A) shortening and mRNA turnover appear to be inhibited by emetine, a drug that stops translation. It is possible that a random endonucleolytic attack leads to scission of poly(A) to a size below which the mRNA is unstable.
Using an ATP:RNA adenyltransferase from Escherichia coli, a polyadenylic sequence was resynthesized onto rabbit globin mRNA from which the poly (A) segment had been previously removed. Conditions for obtaining a homogenous reconstituted globin mRNA preparation containing 30 adenylic residues per message molecule were determined. The reconstituted globin mRNA was microinjected into Xenopus laevis oocytes. Its stability was very similar to that of native mRNA.
The poly(A) sequences at the 3' end of mRNA and nuclear RNA molecules of mouse sarcoma and Chinese hamster cells are subject to an elongation process distinct from de novo synthesis. This process continues in cells treated with a high level of actinomycin D to block transcription. This results in the labeling of the steady-state poly(A) population in the cytoplasm and of unusually long poly(A) segments in the nucleus. In cells incubated with 3H-adenosine in the absence of drug treatment, cytoplasmic steady-state poly(A) segments with short labeled sequences at the 3' end can be detected by their heterogenous size distribution and by measurements of adenosine and AMP released by alkaline hydrolysis. These measurements indicate an average size of 8 residues for the labeled sequences. In the nucleus, a slow elongation of preexisting poly(A) chains can also be detected through measurements of AMP and adenosine. The cytoplasmic elongation process leads to turnover of the 3' end of the poly(A) sequence on mRNA, because of concomitant removal of AMP residues. It is apparently not linked to mRNA translation. The Chinese hamster and mouse sarcoma cells appear to differ markedly with respect to relative extents of poly(A) chain extension and de novo synthesis.
The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography.The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP·AE1 and SCP·AE2, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47–54.4%) and cysteine (7.7–12.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of [3H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.
An increase in the synthesis of polyadenylic acid, following fertilization, was examined in sea urchin, Strongylocentrotus purpuratus, and the marine worm Urechis caupo embryos. It was found that in both organisms there is a large increase in the incorporation of [3H]adenosine into the poly(A) region, of poly(A)-containing RNA, in the phase preceding the 2-cell stage. In sea urchin this rise was localized to the G2 period. Similar findings were made in Urechis indicating that polyadenylation of RNA may be a common postfertilization event in many developing organisms.
The experiments described demonstrate that once mRNA molecules reach the cytoplasm in HeLa cells the attached poly (A) sequences get continually shorter over a period during which the mRNA might be used for protein synthesis many hundreds of times. This time dependent shortening does not seem to be hindered by preventing the mRNA from engaging in protein synthesis.
There is evidence that the segregation distortion controlled by t alleles (t refers to the tailless condition that occurs in the compound heterozygote with T, Brachyury) in mice involves post insemination functional differences. Delayed mating (which results in fertilization after a shorter sojourn by spermatozoa in the female reproductive tract) partially nullifies the segregation distortion with several t alleles. Delayed mating decreased the segregation ratio for t6 from 1.6:1 to 1:1. The fact that one can alter the t allele effect by a post insemination variable implies two classes of spermatozoa with unequal physiological characteristics, possibly as the result of haploid gene expression. The quasi syncytial character of spermatozoal differentiation in mice may provide an explanation of the successful fertilization by gametes deficient in genes which are possibly expressed in the haploid state. As spermatozoa are highly differentiated cells with many distinctive organelles and isoenzymes, it is obvious that a large number of genes must be involved in their formation. Even if all these genes are transcribed before meiosis, unequal division of cytoplasm could lead to problems of regulation during spermatozoan differentiation. The high frequency of intercellular cytoplasmic bridges between spermatocytes and between spermatids would facilitate equal sharing of messenger RNAs and proteins while allowing aneuploid spermatozoa to receive gene products necessary for their development.
1.1. Ribonucleic acid and protein synthesis in germ cells and Sertoli cells of the mouse testis was studied using tritium autoradiography with 3H-uridine and 3H-amino acids, and cytochemical methods.2.2. Spermatogonia.—The rate of RNA and protein syntheses is much higher in the immature type A spermatogonia than in the mature type B spermatogonia. This difference in the synthetic rates is possibly related to a difference in the degree of DNA condensation between the two types of cells. Nuclear and cytoplasmic protein synthesis occurs at all stages of the division cycle, whereas chromosomal RNA synthesis stops during metaphase and anaphase.3.3. Meiosis.—In the autosomes, RNA synthesis ceases or falls to an insignificant level during two periods of the meiotic cycle, early prophase (leptotene to early pachytene) and late diakinesis to metaphase and anaphase I and II. Between these two minima it undergoes a stepwise increase during middle prophase to a peak in middle pachytene, followed by a drop during late pachytene to early diakinesis. In contrast to the autosomes, the heteropycnotic XY bivalent is invariably unlabeled with 3H-uridine throughout meiotic prophase. Protein synthesis, on the contrary, continues during the periods of arrest or depression of RNA synthesis, and is also present in the sex chromosomes.4.4. Spermiogenesis.—RNA synthesis stops very soon after second meiotic division, in very early spermiogenesis. The ribonucleic acids synthesized during meiosis and early spermiogenesis are completely lost from the nucleus both by breakdown and by transfer to the cytoplasm during the course of spermiogenesis, so that middle and late spermatids (from stage 9 to stage 15) do not show any detectable amount of RNA within the nucleus, whereas the cytoplasm exhibits fair amounts of RNA that had been synthesized at least a week earlier during meiotic prophase. The protein synthesis seen to occur in the cytoplasm of spermatid stages 9 to 15 is, then, probably sustained by “templates” produced during meiosis and conserved in the cell to become available during spermiogenesis.5.5. Histone transition during spermiogenesis.—Protein labeling over the nucleus is either absent or very low in spermatids, except in late, elongated, spermatids (stages 11 to 14) which show a marked nuclear labeling as early as 15 min after injection of 3H-arginine. With other amino acids, the nuclear labeling in elongated spermatids is either low or absent. The nuclear incorporation of 3H-arginine is accompanied by changes in the staining properties of nuclear histone (persistance of alkaline fast green stainability after acetylation) which indicate that elongated spermatids synthesize a new nuclear histone very rich in arginine that replaces the previous histone which is a typical histone rich in lysine. A number of evidences suggest that this “arginine-rich” spermatid histone is effectively synthesized within the nucleus, although the alternative interpretation of a cytoplasmic synthesis followed by transfer of the newly synthesized histone into the nucleus cannot be actually rejected.6.6. Sertoli cells.—Nucleoplasm and nucleolus of the supporting Sertoli cells incorporate very rapidly 3H-uridine and 3H-amino acids. The satellite karyosomes (or heteropycnotic bodies) flanking the nucleolus are, however, unlabeled with 3H-uridine and scarcely labeled with 3H-amino acids.7.7. The evidence that the heteropycnotic sex chromosomes during male meiosis and the heteropycnotic karyosomes in Sertoli cells do not incorporate RNA precursors is interpreted to indicate that only dispersed chromatin is active in ribonucleic acid synthesis.
Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.
A rapid decrease in the translational efficiency of mRNA synthesized during vegetative growth is associated with the initiation of development in Dictyostelium discoideum. In contrast, newly synthesized mRNA associates with polysomes with high efficiency. Discrimination between these two mRNA populations correlates with a rapid shortening of the poly(A) tract on the preexisting mRNA. A model is proposed in which a critical poly(A) length regulates the pattern of protein synthesis by affecting the efficiency with which mRNAs can interact with the translational machinery. The model suggests that transcriptional and translational controls can be coupled by altering the state of adenylation of the preexisting mRNA population. The model allows radical changes in the pattern of protein synthesis without wholesale destruction of preexisting mRNA.
A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
Messenger ribonucleic acid (mRNA) from fractionated mouse testis has been used as a template in the wheat germ and reticulocyte lysate cell-free translation systems. Cell-free translation products of deproteinized RNA from testis polysomes and from a nonpolysomal fraction (< 80 S) have been compared by one- and two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Wheat germ and reticulocyte ribosomes translate both polysomal and nonpolysomal RNA from testis with high efficiency. Analysis of the polypeptide products of these cell-free translation systems indicates a compartmentalization between polysome-bound and nonpolysomal mRNA in testis. With the assumption of equal template efficiency for those RNAs tested, three classes of radiolabeled polypeptide products have been distinguished: (1) polypeptide bands which represent an equal abundance of mRNA in each cell compartment; (2) polypeptide bands which represent a higher abundance of mRNA in the polysomal than in the nonpolysomal compartment; (3) polypeptide bands which represent a higher abundance of mRNA in the nonpolysomal than in the polysomal compartment. Compared with liver, the testis contained a larger proportion of ribosomes present as monosomes. Further, more poly(A)+ RNA and an equal or greater template activity were found to be associated with the nonpolysomal portion of testis cytoplasm than the polysomal fraction, suggesting that testis does not have as large a proportion of its messenger RNAs actively involved in protein synthesis as does liver. A comparison of cell-free translation products of deproteinized and nondeproteinized RNAs obtained from total testis cytoplasm had revealed similar polypeptide profiles with a few minor differences. These data suggest that some form of selective mRNA masking or sequestration in a subcellular compartment may be regulating the loading of specific nonpolysomal mRNAs onto polysomes.
Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.
When Drosophila tissue culture cells are shifted from 25 to 36 degrees C (heat shocked) the pre-existing mRNAs (25 degrees C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25 degrees C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding alpha-tubulin, beta-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25 degrees C. Increased temperature should lead to increased output of protein per ribosome. However, the 25 degrees C proteins are actually synthesized at less than 10% of 25 degrees C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25 degrees C mRNAs than they are on heat shock mRNAs in heat-shocked cells.
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.
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.
A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.
Total RNA, prepared from immature or mature mouse testes or from spermatogenic cells separated on the basis of sedimentation velocity, was translated in vitro. Mouse protamine-like histone could be identified as an in vitro translational product when [3H]arginine was used as the label. The mRNA for protamine-like histone was detected only after meiosis; the appearance of a peak of radioactivity comigrating with protamine-like histone occurred only when RNA from mature testes or late spermatid cell fractions was translated. Phosphoglycerate kinase-2 (ATP:3-phospho-D-glycerate I-phosphotransferase, EC 2.7.2.3) was identified as an in vitro translational product by affinity chromatography followed by two-dimensional gel electrophoresis or by specific immunoprecipitation when [35S]methionine was the label. The mRNA for phosphoglycerate kinase-2 was detected only in mature testes or late spermatid cell fractions. These translational assays for protamine-like histone and phosphoglycerate kinase-2 mRNAs suggest that these messages are transcribed after meiosis.
Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.
The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [3H]arginine [3H]lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididymides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [3H]arginine was seen in a number of spermatid basic proteins, including both the 'testis-specific'protein (TP) and the protamines. As expected from previous studies, only one case of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [3H]lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [3H]lysine was initially incorporated into elongating spermatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nuclei of step 14.