Protein synthesis in sperm: Dialog between mitochondria and cytoplasm

Molecular and Cellular Endocrinology 282 (2008) 45–55

Review

Protein synthesis in sperm: Dialog between mitochondria and cytoplasm Yael Gur, Haim Breitbart ∗ The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Abstract Ejaculated sperm are capable of using mRNAs transcripts for protein translation during the final maturation steps before fertilization. In a capacitation-dependent process, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes while the cytoplasmic translation machinery is not involved. Our findings suggest that new proteins are synthesized to replace degraded proteins while swimming and waiting in the female reproductive tract before fertilization, or produced due to the specific needs of the capacitating spermatozoa. In addition, a growing number of articles have reported evidence for the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. It is known that all of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment is far out of proportion to the number of proteins involved, as there have been multiple movements and duplications of genes. However, the evolutionary retention (or secondary uptake) of the mitochondrial machinery for translation of nuclear-encoded mRNAs may shed light on this paradox. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Protein synthesis; Mitochondria; Sperm

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The sperm mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The targeting of mRNAs to mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Import of RNA into mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unexpected localization of mitochondrial rRNA in cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for translation of nuclear mRNAs to the mitochondrial ribosomes in mammalian spermatozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . Two possible mechanisms for protein synthesis in sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In this review, we will outline the scientific results that lead us to believe that protein translation in mature sperm is a general phenomenon in mammals. We address the evidence that this newly discovered process is within the midpiece mitochondria and involves the mitochondrial translation machinery. In addition, a significant number of articles have reported evidence for ∗

Corresponding author. Tel.: +972 3 5318201; fax: +972 3 6356041. E-mail address: [email protected] (H. Breitbart).

0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2007.11.015

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the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. According to the endosymbiont theory of the ␣-proteobacterial origins of mitochondria there have been significant and progressive moves of genetic machinery from the organelle to the nucleus. All of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment by the genome is out of proportion to the number of proteins involved to maintain mitochondrial functioning. A possible reason for this investment is to provide the proteins needed by the evolutionary retained (or secondary uptake)

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Fig. 1. Immunocytochemistry to detect the expression of the protein receptors during capacitation. Human (A–C) and bovine (D and E) sperm cells at zero time and at 1 h and 4 h of incubation in capacitation medium were dried on slides and exposed to anti-AT1-R antibody or anti-PR-R, followed by a second rhodamine-conjugated antibody using fluorescent microscopy.

mitochondrial machinery so it can translate nuclear-encoded mRNAs. Protein synthesis in sperm was first observed when the localization of major hormone receptors involved in fertilization was followed during capacitation. Both the Angiotensin II type I receptor (AT1-R) and the progesterone receptor (PR-R) were localized to the sperm midpiece at the beginning of capacitation and were dispersed to other cell regions during capacitation (Fig. 1). These differences of protein spreading throughout capacitation were at first believed to involve intracellular translocation. Unexpectedly, the changes in molecular localization were associated with changes in total protein content in the cell (as seen by western blotting). By incorporation of labeled amino acids, a general and wide occurrence of de novo synthesis in mature sperm was revealed. This involved the production of tens of new polypeptides in different species, a process that started at the beginning of capacitation and proceeded along the next few hours until fertilization (Gur and Breitbart, 2006). The complexity of protein synthesis in spermatozoa then started to become incontrovertible. Unpredictably, protein production in spermatozoa proved sensitive to the mitochondrial mRNA translation inhibitor dchloramphenicol (CP) (Gur and Breitbart, 2006). This was the first association of protein synthesis with the mitochondrial translation machinery. Later, mRNA translation was found to be sensitive to other mitochondrial translation inhibitors such as gentamycin and tetracycline and insensitive to the cytoplasmic 80S ribosomal inhibitor cycloheximide (Gur and

Breitbart, 2006). Because the mitochondrial genome encodes only for 13 polypeptides, producing five proteins altogether, it does not account for the tens of proteins synthesized during sperm capacitation. Furthermore, analysis of the specific proteins that were synthesized during capacitation demonstrated that nuclear-encoded proteins are indeed translated by mitochondrial-type ribosomes (Gur and Breitbart, 2006). Evidence from studies on somatic cells also revealed the existence of nuclear-encoded proteins inside the somatic mitochondria, which raises the question of whether their existence might be associated with mRNA translation in these mitochondria as well as in sperm. 2. The sperm mitochondria The mitochondria of mammalian spermatozoa are restricted to the midpiece of the flagellum. They wrap helically around the outer dense fiber axoneme complex in a species-specific manner during spermiogenesis to form a cylinder-shaped mitochondrial sheath. Within the sheath, adjacent mitochondria associate both end to end and along their lateral surfaces. This positioning of a concentrated array of mitochondria adjacent to the flagellum is believed to be an efficient way to provide at least some of the energy required for motility (Olson and Winfrey, 1992). The unique arrangement of sperm mitochondria around the sperm midpiece and their structural interactions with each other and with cytoskeletal elements raises the possibility that the mitochondrial membrane could be organized into domains

Y. Gur, H. Breitbart / Molecular and Cellular Endocrinology 282 (2008) 45–55

with unique structures and functions. Assuming that the mitochondrion is active in producing transmembrane proteins, it is possible that this organelle is useful for co-translation and translocation of certain proteins (Bibi, 1998; Herskovits and Bibi, 2000). The typical mammalian sperm midpiece contains approximately 50–75 mitochondria with one copy of mitochondrial DNA (mtDNA) in each (Hecht et al., 1984). However, sperm mtDNA shows a high rate of mutations compared with somatic mitochondria, possibly because of exposure to mutagens during the long free life of the cell. This poor quality of the mitochondrial genome may be associated with the need to degrade the sperm mtDNA in the embryo. Although parental mitochondria might enter the oocyte, they are destroyed shortly after fertilization. The sperm mitochondrial proteins are ubiquitinated during spermatogenesis and later subjected to specific proteolysis during preimplantation development (Sutovsky et al., 1999). Most cells in the body contain between 103 and 104 copies of mtDNA. This is usually a circular molecule of approximately 16.6 kb that is made up of two separate strands: the heavy strand (H-strand), which has a higher G + T content, and the light strand (L-strand). The mtDNA encodes 13 proteins, 13 mRNAs, 14 tRNAs and RNA primers for heavy chain replication, all of which are transcribed and translated in the mitochondrion (Cummins, 1998). Still, the mitochondrion needs more then 700 different nuclear-encoded proteins to operate (Neupert, 1997). These are targeted to the mitochondria, translocated through the mitochondrial membranes and sorted to the different organelle subcompartments. Separate translocases in the mitochondrial outer membrane (TOM complex) and inner membrane (TIM complex) facilitate recognition of preproteins and transport them across the two membranes (Pfanner and Wiedemann, 2002). The nuclear and mitochondrial genomes contribute to the synthesis and assembly of mitochondrial proteins and to their functions: respiration, regulation of energy production, mitochondrial gene expression and nuclear control of the mitochondrial assembly (Poyton, 1980; Poyton and McEwen, 1996). The mammalian mitochondrial ribosome has a smaller sedimentation coefficient (55S), compared with the bacterial ribosome (70S), and consists of both large (39S) and small (28S) subunits. The 39S subunits contain 16S and the 28S subunits contain 12S rRNAs. The mammalian mitoribosome has no counterpart to the bacterial 5S rRNA. The protein composition of the mitoribosome has been estimated to be about 75%, which indicates that large parts of bacterial rRNA domains have been replaced by protein components during mitochondrial evolution from a eubacteria-like endosymbiont in eukaryotic cell progenitors (Suzuki et al., 2001). Initiation of translation in mammalian mitochondria is a characteristic feature of mitochondrial protein synthesis. Mitochondrial mRNAs that are encoded by mtDNA do not have well-known leader sequences, such as a Shine–Dalgarno sequence, on the 5 side of their initiation codons nor do they have a cap structure at their 5 ends (Attardi, 1985; Ojala et al., 1980). Mitochondrial protein synthesis starts directly at the initiation codons that are located on the 5 ends of these mRNAs.

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The 28S small subunit of the mitoribosome is thought to play a key role in this peculiar initiation system. Another way to ensure the targeting of a nuclear-encoded protein to another cellular organelle is to transport the mRNA encoding that protein to the ribosome close to the relevant subcellular compartment. Mitochondrial precursor proteins carry amino terminal targeting sequences to insure that they will be imported into the mitochondria. mRNAs encoding mitochondrial proteins do not encounter mitochondria by chance, but use mRNA export machinery and “fast-track” the early stages of mitochondrial protein sorting (Lightowlers et al., 1996). Co-translational and post-translational protein import mechanisms exist in different cell types (Crowley and Payne, 1998; Herrmann and Neupert, 2000; Ni et al., 1999; Tokatlidis et al., 2000). Co-translational translocation of proteins into mitochondria is done by binding the ribosome to the mitochondria and to the required GTP and transit peptide (Alder and Theg, 2003; Crowley and Payne, 1998). This process could be facilitated by mRNA localization (Lightowlers et al., 1996). Both pathways require leader sequences that are resistant to protease degradation and the assistance of numerous cytosolic factors such as mitochondrial stimulating factors and heat shock proteins to lead the mRNA or preprotein to the mitochondria and protect it (Stan et al., 2003). However, a new theory claims that protein translocation into mitochondria is not tightly coupled with the translation reaction at the ribosome. In contrast to the transport of proteins into the endoplasmic reticulum, mitochondrial precursor proteins can accumulate in the cytosol and are imported after their synthesis is completed (Voos, 2003). The transport of preproteins into the mitochondria is mediated by chaperones. Molecular chaperones are generally thought to protect newly synthesized proteins and ensure that they fold correctly. For example, the chaperone proteins heat shock protein (Hsp) 70 and Hsp90 help target certain proteins to the mitochondria (reviewed by Ellis, 2003). In addition to the mitochondrial proteins that are needed for known mitochondrial functions, several studies revealed the localization of other nuclear-encoded proteins inside the mitochondria, such as phosphoproteins and protein kinases (including pyruvate dyhydrogenase kinase, protein kinase A, protein kinase C␦, stress activated kinase, A-Raf and unidentified kinases) (Arai et al., 1996; Thomson, 2002), and GTP-binding proteins (Thomson, 1998). The role of these proteins inside the mitochondria is not yet known. However, we can now hypothesize that those proteins are found inside the mitochondria while being translated there. There are also mechanisms in different cell types for the export of proteins from the mitochondria to other compartments. Many proteins that were originally characterized based on non-mitochondrial functions have been shown, unexpectedly, to be identical to mitochondrial matrix proteins (Soltys and Gupta, 1999). The subcellular distribution of these proteins cannot be explained by the conventional pathways for protein trafficking. This raises the possibility that there is specific trafficking from the mitochondria to other cellular compartments.

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3. The targeting of mRNAs to mitochondria The import of tRNAs and rRNAs into the mitochondria has been described in several species, but there is no precedent for the import of mRNA into mammalian mitochondria. However, targeting of mRNA to the vicinity of the mitochondria was reported in several species and might be active in sperm as well. Clarification of the mitochondrial import process that has emerged in recent years should not obscure its complexity. Several lines of evidence suggest that the mitochondrial protein import process may begin before the proteins of the TOM machinery recognize the mitochondrial-targeting signal. Pioneering studies (Kellems et al., 1974, 1975) demonstrated that translationally active ribosomes loaded with mRNA molecules encoding mitochondrial precursor proteins accumulate on the surface of yeast mitochondria. Other studies have confirmed that translation and import are associated with each other. The translocation of specific mRNAs to the vicinity of mitochondria appears to require cis-acting signals in the RNA molecule. mRNA targeting does not require the translation of the 5 sequence, suggesting that targeting occurs independently of translation. Marc et al. (2002) found two main classes of mRNAs that encode mitochondrial proteins in yeast: mRNA molecules that are transcribed from genes with known homologues identified in bacteria and are present in mitochondrion-bound polysomes, and mRNA molecules that are transcribed from genes of eukaryotic origin and are present in free cytosolic polysomes. In our recent publication, we showed the presence of mRNA for AKAP110 in the polysomal fraction isolated from bovine sperm. The polysomal fractions were found to be based on mitochondrial ribosomes. This may indicate that AKAP110 is translated in spermatozoa (Gur and Breitbart, 2006). It is known that in somatic cells, mRNA targeting to the mitochondria is led by chaperon molecules as members of the AKAP family. These regulate cAMP-dependent PKA signaling pathways and other enzymes. Each protein contains a targeting domain that anchors it to a given cytoskeletal element. AKAPs carry a KH domain that participates in various RNA/protein-binding interactions, including associations with structural elements in the 3 -untranslated regions (3 -UTR) of mRNAs. These interactions affect the localization, stabilization and translational regulation of mRNAs (Ginsberg et al., 2003). AKAP121, derived from the mouse, tethers PKA to the mitochondrial outer surface. AKAP121 binds at least two distinct, nuclear-encoded mRNAs: the Fo-f subunit of the mitochondrial ATP synthase and the 3 UTR of the manganese superoxide dismutase. There is also the possibility that other mRNAs bind to AKAPs in vivo and are translocated to the mitochondria. Another protein from this family is the ATM1 yeast protein that also binds mRNAs in the 3 -UTR and leads molecules to the vicinity of the mitochondria (Corral-Debrinski et al., 2000). 4. Import of RNA into mitochondria Most of the evidence indicating that RNA is imported into the mitochondria involves tRNAs, of which at least 24 different

types are required to read the universal genetic code. It has been postulated that tRNAs are imported into the mitochondria in plants (Huang and Jungmann, 1995), yeast (Entelis et al., 1998) and protozoa (reviewed in Kashikawa et al., 1999). Cytosolic and mitochondrial tRNAs in these species can be divided into four categories according to their localization and genetic origin. Most tRNAs in eukaryotes are either nuclear-encoded and function in the cytosol or are mitochondrially encoded and involved in organellar protein synthesis. There may be RNAs other than tRNAs that are targeted to mammalian mitochondria. One of these is the RNA component of RNase MRP, a site-specific endoribonuclease that is supposed to be present in the organelle in very low amounts and is involved in primer RNA cleavage during the replication of mitochondrial DNA (Chang and Clayton, 1987; Puranam and Attardi, 2001; Tarassov et al., 1995). The second candidate for import into mammalian mitochondria is the RNA component RNase P, an endoribonuclease that is involved in the processing of the 5 ends of tRNAs (Doersen et al., 1985). An important question is how a negatively charged molecule such as RNA can cross the hydrophobic environment of the mitochondrial double membrane in the countercurrent of a proton gradient. This could be accomplished with the assistance of pre-protein import machinery, ATP hydrolysis energy, membrane charge and outer membrane receptors (Entelis et al., 2001). 5. Unexpected localization of mitochondrial rRNA in cells In addition to the import of RNAs into the mitochondria, spermatozoa can also export mitochondrial-type ribosomes, particularly in embryos, to different unexpected locations in the cells. For example, Xenopus embryos express the mitochondrial ribosomal large RNA (Kobayashi et al., 1998), and the germ plasm of Drosophila embryos express the mitochondrial ribosomal large and small RNAs (Amikura et al., 2001a,b; Kashikawa et al., 1999). Mitochondrial-type ribosomes were also found outside of the mitochondria in a discrete region of the egg cytoplasm in ascidian (Ogawa et al., 1999), sea urchin (Oka et al., 1999) and planarian embryos (Sato et al., 2001). Ribosome-like particles are associated with spermatid acrosomal membranes (Mollenhauer and Morre, 1978). Because the origin of the acrosome is the Golgi apparatus, which in the liver contains functional polyribosomes (Elder and Morre, 1976), it is possible that the sperm acrosome also contains mitochondrialtype ribosomes. The nuclear localization of RNA in the mature sperm of rat and human has been demonstrated using the immunogold procedure for electron microscopy (Pessot et al., 1989). The U1 and U2 small nuclear RNA transcripts that are involved in premRNA splicing were confined to the rat sperm nucleus (Concha et al., 1993). Several mRNAs were identified in the nucleus of mature sperm of rodent and humans (Chiang et al., 1994; Kramer and Krawetz, 1997; Kumar et al., 1993; Miller et al., 1994, 1999; Rohwedder et al., 1996). Moreover, in plants, RNA is found in the mature pollen grain, which contains a store of presynthesized mRNAs (Mascarenhas, 1993).

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The 16S and 12S rRNAs were localized to the nucleus of mouse and human spermatozoa by in situ hybridization with a specific probe (Villegas et al., 2000, 2002). The possibility that the 16S rRNA in the nucleus is the result of nuclear transcription of a mitochondrial pseudogene was ruled out. These results suggest that the mitochondrial RNA is translocated from the organelle to the nucleus of the sperm by an unknown mechanism, and it was hypothesized that these rRNAs exit from mitochondria and are translocated to the nucleus (Villegas et al., 2002). 6. Evidence for translation of nuclear mRNAs to the mitochondrial ribosomes in mammalian spermatozoa The validity of our initial results was established by demonstrating that protein synthesis could be attributed unquestionably to spermatozoa and not to somatic cells (blood cells or epithelial cells), spermatogenic cells or microorganisms that may contaminate the ejaculates (Gur and Breitbart, 2006). Changes in the amounts of several proteins during capacitation led us to look for protein synthesis. Nascent proteins in sperm were labeled using [35 S] amino acid and BODIPY-lysine-tRNALys incorporation. [35 S] Amino acid autoradiography is not possible at the level of the sperm cell compared with tissue sections, because the separated cells are too small, so we used BODIPY-lysine-tRNALys incorporation to visualize the translation process. This was found to be time-dependent, capacitation-dependent and sensitive to mitochondrial translation inhibitors. However, neither BODIPYlysine-tRNALys nor [35 S] amino acid incorporation was affected by a eukaryotic cytoplasmic translation inhibitor. The origin of BODIPY-lysine-tRNALys incorporation is apparently the sperm midpiece (Gur and Breitbart, 2006). The incorporation of labeled amino acids began within 2 min of incubation in capacitating conditions, indicating that sperm cells need newly made proteins immediately at the beginning of capacitation (Gur and Breitbart, 2006). Thus, these data contradict the notion that a sperm cell contains all of the proteins that it needs for its journey to the oocyte and fertilization including residing in the sperm reservoir (Suarez, 2002), swimming in the reproductive tract and capacitating. It is possible that a new set of proteins – or more of the existing proteins – are required to initiate capacitation in sperm. Thus, the cell may need to translate a new set of proteins to continue with capacitation. This is consistent with an initial elevation of newly synthesized protein labeling in the first hour and then with a constant low level of protein labeling for several hours, presumably to replace degraded proteins. This possibility is supported by the observation that there was no further elevation in the kinetics of [35 S] amino acid incorporation for up to 4 h after the first hour of capacitation (Gur and Breitbart, 2006). One way we tried to link protein synthesis with sperm function was to examine the effect of CP on the sperm-specific proteins CatSper, PKA-Cs, ATPase ␣4 and AKAP 110. All of these proteins have a nuclear-encoded origin, and all of them are decreased during capacitation in the presence of CP (Gur and Breitbart, 2006). Other examined proteins belong to several different protein families, including protein kinases, hormone

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receptors, ion channels and others. Thus, sperm cells synthesize a wide range of proteins during capacitation depending both on the half-life of the protein and its role in capacitation. Measuring the effect of CP on the quantity of proteins is an indirect method to detect protein synthesis, as this actually inhibits the resynthesis of degraded proteins. Gel electrophoresis may not be sensitive enough to identify a small reduction for proteins, and proteins with a longer half-life probably required a longer incubation period with CP before its inhibitory effect on the translation of the protein could be observed. On the other hand, by immunoprecipitation of [35 S] amino acid-incorporated proteins we can follow a specific protein. AT1-R, PR-R, and PKC␣ (Breitbart et al., 1992), which are involved in acrosome reaction and capacitation, were immunoprecipitated and the incorporation of [35 S] amino acid was completely inhibited by CP and unaffected by cycloheximide (Gur and Breitbart, 2006). This was the first confirmation that specific nuclearencoded mRNA transcripts may be translated by CP-sensitive ribosomes. The immunoprecipitated proteins were tested for intracellular localization. AT1-R was initially localized to the tail midpiece at zero time (Fig. 1A), and translocated to the head and tail at one (Fig. 1B) and at 4 h of capacitation (Fig. 1C), supporting the role of AT1-R in acrosome reaction mediating (Gur et al., 1998) and motility needed for fertilization (Vinson et al., 1996). PR-R was designated primarily to the tail midpiece (Fig. 1D), and during capacitation translocated to the rest of the tail and head (Fig. 1E). This observation is consistent with the known physiological role of PR-R, which is needed first in the tail for motility (Uhler et al., 1992) and later in the head for the acrosome reaction (Osman et al., 1989). It seems that inhibition of protein translation with CP did not inhibit translocation of the existing molecule to their target site (Fig. 2). A weak signal could still be observed in the tail and the margins of the head, implying that proteins that had been translated earlier had been translocated there to exert their effect on cellular functions. The remaining AT1-R molecules were dispersed along the tail midpiece and in the post-acrosome region (Fig. 2B), while the PR-R had a different dispersion: most of these molecules were located in the post acrosome region and along the tail, but no staining could be seen in the midpiece region (Fig. 2D). We conclude that the midpiece region is a translation site for both receptors, but serves as an active site only for the AT1-R. Recent studies have reported that spermatozoa contain a complex yet specific repertoire of mRNA transcripts that are expressed during spermatogenesis. This wider screenings were done by Ostermeier et al. (2002) and Zhao et al. (2006), using a microarray assay and serial analysis of gene expression (SAGE), to define the molecular fingerprint of human spermatozoa. The microarrays identified ∼5000 transcripts in ejaculated spermatozoa from normal fertile men (Ostermeier et al., 2002). Using the SAGE technique, between 2459 and 2712 unique transcripts were identified (Zhao et al., 2006). Most of the sperm RNAs that were identified correspond to mitochondrial transcription and respiration proteins, and to the nuclear and plasma membrane proteins that participate in signal transduction, oncogenesis and cell proliferation.

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Fig. 2. Immunocytochemical detection of AT1-R and PR-R proteins in the presence of d-chloramphenicol. Human and bovine spermatozoa were incubated in capacitation medium in the presence of d-chloramphenicol (CP; 100 ␮g/ml) for 4 h. Proteins were probed with a specific antibody against either AT1-R (A and B) or PR-R (C and D) and with a rhodamine-conjugated secondary antibody.

Proteins produced via gene translation in the sperm prove the existence of mRNA transcripts in the cell. We first looked for the mRNA transcripts of the synthesized proteins: mouse and bovine PKA-Cs, human PKC␣ and PKC␤I, bovine 16S rRNA, mouse and human CatSper, mouse CatSper2, human and rat ATPase ␣4, AKAP 110, and human and bovine AT1 receptor (Gur and Breitbart, 2006). No studies in the literature have examined the transcription of these mRNA transcripts in ejaculated sperm, although some articles from 30 years ago showed that there is RNA synthesis in mature spermatozoa (Hecht and Williams, 1978; MacLaughlin and Terner, 1973; Premkumar and Bhargava, 1973). In our work, actinomycin D, which is a transcription inhibitor, did not influence protein synthesis (Gur and Breitbart, 2006). This indicates that translation is not transcription-dependent. Thus, sperm cells may use stable, long-term mRNA transcripts to synthesize novel proteins (Miller and Ostermeier, 2006;

Ostermeier et al., 2002). Long-term mRNA might be used to synthesize proteins in the female reproductive tract during storage in the sperm reservoir (Suarez, 2002) and during capacitation. In addition, the mRNA might be transported to the egg and participate in the zygote development. Such mRNA transcripts may have elements that stabilize them and increase their halflife (AT1-R mRNA, Guhaniyogi and Brewer, 2001: for example lactate dehydrogenase mRNA stabilization by PKA and PKC, Huang and Jungmann, 1995). The localization of sperm-specific mRNAs was therefore critical for understanding and elucidating the translation mechanism. CatSper mRNA was localized in permeabilized cells to the midpiece area of mouse spermatozoa (Fig. 3). Comparing the signal at zero time with 3 h of capacitation demonstrated a significant reduction in mRNA content. The reduction in signal in the mitochondria area (Fig. 3) compared with elevation of protein level from the mitochondria area to other cell compartments

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Fig. 3. In situ hybridization of mRNA in mouse sperm. Mouse sperm were incubated in capacitating medium for 3 h. Samples of cells at (A) 0 h and (B) 3 h were smeared on slides and permeabilized. In situ hybridization was performed by hybridizing DIG-conjugated mouse-CatSper antisense on the slides, probing with anti-DIG FITC antibody. The cells were visualized using confocal microscopy.

also indicates that the site of translation is the mitochondria. Further examination of mouse CatSper, mouse PKA-Cs and bovine AT1-R transcripts and the related proteins at an intraorganelle level, confirmed the localization of transcripts and their translated products to the mitochondria themselves (Gur and Breitbart, 2006). 7. Two possible mechanisms for protein synthesis in sperm The mechanism by which nuclear-encoded mRNA transcripts are translated by mitoribosomes is presently unclear. Two possible mechanisms have been hypothesized, based on the data that had been gathered to this point (Gur and Breitbart, 2007). According to the first mechanism, mRNA transcripts might be translated by CP-sensitive ribosomes that are located outside the mitochondria but attached to its surface. Alternatively, the nuclear mRNA might be imported into the mitochondria to be translated by CP-sensitive ribosomes that are located inside the mitochondria. Although the import of tRNAs and rRNAs into the mitochondria has been described in several species, there is no precedent for the import of mRNA into mammalian mitochondria. The first hypothesis is supported by several studies that found that mitochondrial proteins are translated near the mitochondria. Co-translational translocation of proteins is achieved by binding of the ribosome to the mitochondria. The ribosome contains a number of proteins that are attached to the mitochondria or located nearby (Crowley and Payne, 1998; Marc et al., 2002; Ni et al., 1999). One of the ways to ensure the targeting of a nuclearencoded protein to another cellular organelle is to transport the mRNA that encodes the protein to the ribosome that is near the correct subcellular compartment. Mitochondrial precursor proteins carry amino terminal targeting sequences to ensure that they are imported into the mitochondrion. mRNA transcripts that

encode mitochondrial proteins do not encounter mitochondria by chance: they use the mRNA export machinery to “fast-track” the early stages of mitochondrial protein sorting (Lithgow et al., 1997). The AKAP family of proteins is involved in these mechanisms. These proteins have a pivotal role in the localization, stabilization and translation of mitochondrially directed proteins (Corral-Debrinski et al., 2000; Ginsberg et al., 2003). Interestingly, the major mRNA that was identified while being translated on mitochondrial ribosome in sperm is AKAP110, a protein that belongs to the AKAP family (Gur and Breitbart, 2006). It is also possible that nuclear mRNAs, which are translated by mitochondrial-type ribosomes outside the mitochondria, are imported into the mitochondria for further processing. Other studies have found that mitochondrial translation components are localized outside the mitochondria. For example, mitochondrial-type ribosomes in the germ plasm of Drosophila embryos (Clermont et al., 1994) and mitochondrial rRNAs in discrete regions of egg cytoplasm in ascidian, sea urchin and planarian embryos (Ogawa et al., 1999; Oka et al., 1999; Sato et al., 2001) were found outside the mitochondria. Further, particles resembling ribosomes that were associated with spermatid acrosomal membranes have also been described (Mollenhauer and Morre, 1978). In addition, it has been reported that mitochondrial rRNA is localized to cellular locations outside the mitochondria. Although several reports described these findings in embryos (Amikura et al., 2001a,b; Kashikawa et al., 1999; Oka et al., 1999), 11 other reports described the localization of mitochondrial rRNA to different areas of spermatozoa in several species (see above for citations). Another article reported that there are 12S and 16S rRNAs (components of the small and large subunits of the mitochondrial ribosome, respectively) in the nucleus of mouse spermatogenic cells and human spermatozoa. The possibility that the rRNAs in the nucleus are the result of nuclear

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transcription of a mitochondrial pseudogene was ruled out. The author suggested that the mitochondrial RNA might be translocated from the mitochondria to the nucleus of the sperm cell by an unknown mechanism of RNA translocation (Villegas et al., 2002). If protein translation takes place outside the mitochondria, which is supported by these articles (Amikura et al., 2001a,b; Kashikawa et al., 1999; Oka et al., 1999; Sato et al., 2001; Villegas et al., 2002), the translation mechanism would be simplified, because the nuclear-encoded mRNA transcripts would not need to enter the mitochondria to be translated. This leads us to the second hypothesis; suggesting that protein translation takes place inside the mitochondria. Our studies (Gur and Breitbart, 2006, 2007) localized RNA translation in mature spermatozoa convincingly to the mitochondrial translation machinery, but to further localize the site of translation we looked for mRNA at the in organello level. In situ hybridization was performed on thin sections of mouse and bovine spermatozoa, and an intramitochondrial hybridization of bovineAT1-R, and mouse CatSper and PKA-Cs was observed (Gur and Breitbart, 2006). The unexpected discovery of several nuclearencoded mRNA transcripts and their translated proteins, inside the mitochondria, strongly supports the second proposed model, in which mRNA is imported into the mitochondria to be translated. It is known that 16S and 12S ribosomal RNAs are present in the nucleus of mouse and human spermatozoa (Villegas et al., 2000, 2002). The possible presence of active ribosomes in the nucleus could explain the intranuclear localization of bovine AT1-R and PKA-Cs [1]. However, the presence of 16S and 12S ribosomal RNAs in bovine sperm has not been studied. It was recently shown that protein translation can take place in the nucleus of the HeLa cell (Iborra et al., 2001). Thus, it is pos-

sible that protein translation occurs in the sperm nucleus via ribosomes that are sensitive to CP. These publications together with our results may suggest that specific cellular loci may control atypical translation processes. The combination of evidences supports both extra and intramitochondrial translation by mitochondrial-type ribosomes is the basis for the current suggested model for protein translation in spermatozoa (Fig. 4). Assuming that the translation is intramitochondrial, further research is required to understand the special cellular mechanisms involved in the importing of mRNA into the mitochondria, in the translation of nuclear mRNA on mitochondrial-type ribosomes, and the exporting of the synthesized proteins to their target sites outside of the mitochondria. Other studies reported similar results regarding the localization of nuclear-encoded proteins. Carrey et al. (2002) researched the localization of enzymes involved in de novo biosynthesis of pyrimidine nucleotides in fruitfly and mammalian spermatozoa, and found glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase and UMP synthase in the nucleus and mitochondria of ram spermatozoa using immunogold electron microscopy. The authors claimed that these proteins were localized to the outer membrane of the mitochondria, but re-evaluation of the representative sections that were published indicates that the proteins may be intramitochondrial. Other studies similarly reported that their protein of interest was localized inside the mitochondria. The first of these is another member of cAMP-dependent protein kinase family: type II (PKA-II). This protein was found to be tightly associated with the sperm flagellum via the regulatory subunit (RII). The RII protein was immunogold-localized intramitochondrially in bovine and rat sperm (Lieberman et al., 1988). The

Fig. 4. A suggested model for extra and intramitochondrial protein translation in sperm. In mammalian spermatozoa, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes in the cytoplasm and in the mitochondria and then the translated proteins are translocated to their active.

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authors claimed staining to localize to the edges of the mitochondrion, but most of the gold grains illustrated are seen inside the mitochondria and even appear gathered in different areas of the mitochondrion, resembling polysome active sites. As we saw with PKA-Cs, labeling was found for RII in the fibrous sheath, although the labeling was much sparser than the labeling in the mitochondria. Several other articles revealed the localization of more proteins in the mitochondria by immunoelectron microscopy, including PKA-RI and PKA-RII␣ (Reinton et al., 1998), Akinase anchoring proteins, hAKAP220 and S-AKAP84 (Chen et al., 1997; Reinton et al., 1998). Thus, the sperm midpiece, which includes the mitochondria, appears to have all of the components needed for a functional PKA signaling system. The authors suggest that PKA in the mitochondria and its vicinity may have a role in the regulation of respiratory function, in the control of mitochondrial uptake of intracellular calcium and in the direct control of sperm motility. We suggest that the mitochondria may serve as a translation station for PKA proteins in spermatozoa on their way to their target sites. Peters et al. (1996) demonstrated the presence of components of the rennin–angiotensin system (RAS) pathway within the mitochondria of rat adrenal cortex by immunogold electron microscopy. The authors hypothesized that the intramitochondrial RAS components might play a role in adrenal steroid regulation. AT1-R is the major receptor mediating the hormonal responses of Angiotensin II in somatic and sperm cells. The localization of AT1-R and other components of RAS in mitochondria of somatic cells suggest a physiological role for AT1-R other than being translated in sperm mitochondria. In addition to the mitochondrial proteins that are needed for known mitochondrial functions, a few recent studies revealed that other nuclear-encoded proteins such as phosphoproteins, protein kinases (including pyruvate dyhydrogenase, protein kinase A, protein kinase C␦, stress activated kinase, A-Raf and unidentified kinases) (Thomson, 2002) and GTP-binding protein (Thomson, 1998) were localized inside of mitochondria. The role of these proteins inside mitochondria is not yet known. We may now speculate that these proteins were found in the mitochondria during their translation. Supporting this suggestion, Santangelo et al. (2005) showed that nuclear mRNAs colocalize with mitochondria in live somatic cells, using in situ hybridization. Furthermore, the K-ras and GAPDH mRNAs colocalized with mitochondrial 28S rRNA, the small subunit of the mitochondrial ribosome. These experiments were done on live cells, preventing the possibility for intra-organelle in situ hybridization. Therefore, based on our findings in sperm and on the existence of other nuclear originated proteins inside somatic mitochondria, it would be valuable to test for their intramitochondrial appearance in somatic cells. 8. Summary Translation of nuclear-encoded mRNAs by the mitochondrial ribosomes is a phenomenon that occurs in mature spermatozoa, and opens up the possibility for an alternative translation pathway in other cells. If the translation is indeed intramito-

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chondrial, further research is required to understand the involved cellular mechanisms. Moreover, studies on a variety of somatic cells indicate that nuclear-encoded proteins are present inside the mitochondria and are coupled to mitochondrial ribosomes: their roles are yet to be clarified. Although our findings strongly support the intramitochondrial translation, the possibility for extramitochondrial translation cannot be excluded. Supporting this possibility are the growing number of articles on somatic cells and spermatozoa, indicating mitochondrial translation machinery components outside the mitochondria. This may offer a clue to the existence of a hitherto unknown mechanism of crosstalk between the mitochondria and the cytoplasm. Thus, mitochondrial translation of nuclear-encoded mRNAs might exist in all living cells. Acknowledgment We gratefully acknowledge J. Cummins for his helpful discussions. References Alder, N.N., Theg, S.M., 2003. Energy use by biological protein transport pathways. Trends Biochem. Sci. 28, 442–451. Amikura, R., Hanyu, K., Kashikawa, M., Kobayashi, S., 2001a. Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. Mech. Dev. 107, 97–104. Amikura, R., Kashikawa, M., Nakamura, A., Kobayashi, S., 2001b. Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos. Proc. Natl. Acad. Sci. U.S.A. 98, 9133–9138. Arai, M., Imai, H., Sumi, D., Imanaka, T., Takano, T., Chiba, N., Nakagawa, Y., 1996. Import into mitochondria of phospholipid hydroperoxide glutathione peroxidase requires a leader sequence. Biochem. Biophys. Res. Commun. 227, 433–439. Attardi, G., 1985. Animal mitochondrial DNA: an extreme example of genetic economy. Int. Rev. Cytol. 93, 93–145. Bibi, E., 1998. The role of the ribosome–translocon complex in translation and assembly of polytopic membrane proteins. Trends Biochem. Sci. 23, 51–55. Breitbart, H., Lax, Y., Rotem, R., Naor, Z., 1992. Role of protein kinase C in the acrosome reaction of mammalian spermatozoa. Biochem. J. 281, 473–476. Carrey, E.A., Dietz, C., Glubb, D.M., Loffler, M., Lucocq, J.M., Watson, P.F., 2002. Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa. Reproduction 123, 757–768. Chang, D.D., Clayton, D.A., 1987. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science 235, 1178–1184. Chen, Q., Lin, R.Y., Rubin, C.S., 1997. Organelle-specific targeting of protein kinase AII (PKAII). Molecular and in situ characterization of murine A kinase anchor proteins that recruit regulatory subunits of PKAII to the cytoplasmic surface of mitochondria. J. Biol. Chem. 272, 15247–15257. Chiang, M.H., Steuerwald, N., Lambert, H., Main, E.K., Steinleitner, A., 1994. Detection of human leukocyte antigen class I messenger ribonucleic acid transcripts in human spermatozoa via reverse transcription-polymerase chain reaction. Fertil. Steril. 61, 276–280. Clermont, Y., Rambourg, A., Hermo, L., 1994. Connections between the various elements of the cis- and mid-compartments of the Golgi apparatus of early rat spermatids. Anat. Rec. 240, 469–480. Concha, I.I., Urzua, U., Yanez, A., Schroeder, R., Pessot, C., Burzio, L.O., 1993. U1 and U2 snRNA are localized in the sperm nucleus. Exp. Cell Res. 204, 378–381. Corral-Debrinski, M., Blugeon, C., Jacq, C., 2000. In yeast, the 3 untranslated region or the presequence of ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria. Mol. Cell Biol. 20, 7881–7892. Crowley, K.S., Payne, R.M., 1998. Ribosome binding to mitochondria is regulated by GTP and the transit peptide. J. Biol. Chem. 273, 17278–17285.

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