Molecular Human Reproduction, Vol. 6, No. 9, 779-788,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Developmental expression of Y-box protein 1 mRNA and alternatively spliced Y-box protein 3 mRNAs in spermatogenic cells in mice
Department of Biology, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125–3393, USA
Abstract
Y-box proteins bind DNA and RNA and are characterized by a cold shock domain and a carboxyl-terminus containing clusters of aromatic and basic residues that alternate with clusters of acidic residues. Y-box proteins 1 and 3 in mouse testis were cloned here by 3' rapid amplification of cDNA ends (RACE) using a degenerate primer. Northern blots and reverse transcription–polymerase chain reaction (RT–PCR) established that the levels of Y-box protein 1 and 3 mRNAs are regulated individually: (i) Y-box protein 1 mRNA is strongly expressed in kidney, whereas Y-box protein 3 mRNA is strongly expressed in heart and muscle; (ii) Y-box protein 1 and 3 mRNAs are weakly expressed in early prepubertal testis and strongly expressed in pachytene spermatocytes, round spermatids, and elongated spermatids; and (iii) prepubertal testes and meiotic and haploid spermatogenic cells express two alternatively spliced Y-box protein 3 mRNAs encoding isoforms with different carboxyl termini, whereas somatic tissues primarily express one form. Sucrose gradients reveal that ~27% of both Y-box protein 3 mRNAs are translationally active in adult testis. In conclusion, spermatogenic cells in mice express five isoforms of Y-box proteins including Y-box protein 1, and two isoforms each of Y-box proteins 2 and 3. This multiplicity is intriguing because Y-box proteins are thought to activate transcription and repress translation in spermatogenic cells.
cold-shock domain/spermatogenesis/transcriptional regulation/translational regulation/Y-box proteins
Introduction
Spermatogenesis is the remarkable developmental process that produces male gametes, spermatozoa (described in Russell et al., 1990). Spermatogenesis begins with diploid cells, spermatogonia, which divide repeatedly generating large numbers of cells. After withdrawing from the mitotic cycle, the cells, (spermatocytes), enter meiosis. Meiosis ends with the reduction divisions, and the resulting haploid cells, (spermatids), undergo profound changes in the structure of their nuclei, Golgi, flagella and mitochondria as they differentiate into mature spermatozoa. The haploid phase lasts ~13 days in mice and can be subdivided into early haploid cells, round spermatids, (which are active in transcription), and late haploid cells, (elongated spermatids), which exhibit no detectable transcription due to changes in chromatin structure.
Analysis of the distribution of specific mRNAs in sucrose gradients reveals that virtually all mRNA species in meiotic and haploid spermatogenic cells in mammals exhibit high levels, 25–100%, of translationally inactive free-messenger ribonucleoprotein particles (free-mRNPs) indicating that the initiation of translation is at least partially repressed (reviewed in Kleene, 1996). Protamine mRNAs are the best known members of a potentially large group of mRNAs that are strongly translationally repressed in round spermatids and actively translated in elongated spermatids after the cessation of transcription (Kleene, 1989
). The observation that premature protamine 1 mRNA translation arrests spermatogenesis in round spermatids in transgenic mice indicates the importance of translational regulation in restricting the synthesis of this protein to elongated spermatids (Lee et al., 1995a).
Sucrose gradient analyses also indicate that the overwhelming majority of (but certainly not all) mRNAs exhibit little or no developmental change in translational activity in pachytene spermatocytes and round spermatids (Kleene, 1996). In addition, the proportions of polysomal proenkephalin and hemiferrin mRNAs are very similar in pachytene spermatocytes, round spermatids and elongated spermatids (Kew et al., 1989; Stallard et al., 1991). However, large differences in the proportions of various mRNA species in free-mRNPs also indicate that the extent of repression is mRNA-specific (Kleene, 1996; Cataldo et al., 1999). The purpose of storing mRNAs that are not developmentally activated is enigmatic, although it may be a mechanism of fine tuning protein accumulation, possibly to adjust protein synthesis during the intricate differentiation of spermatozoa or to prevent deleterious effects of overproducing proteins encoded by overexpressed mRNAs (Cataldo et al., 1999).
Y-box proteins (YBP) are widely thought to be the proximal cause of the widespread translational repression in both male and female germ cells (reviewed in Matsumoto and Wolffe, 1998; Evdokmova and Ovchinnikov, 1999; Sommerville, 1999). YBPs are DNA and RNA binding proteins whose primary sequence is divided into a highly conserved cold shock domain, a variable amino terminus, and a carboxyl-terminus domain consisting of alternating clusters rich in aromatic and basic amino acids and clusters rich in acidic amino acids, known as islands. YBPs function as transcriptional activators or repressors in the nucleus, and as translational activators or repressors in the cytoplasm. The opposing effects of YBPs on translation depend on the ratio of protein to mRNA: low ratios facilitate translation by melting mRNA secondary structure, while slightly higher ratios promote the formation of multimers mediated by the carboxyl terminus domain that render mRNPs inaccessible to the translational apparatus (Matsumoto and Wolffe, 1998; Evdokmova and Ovchinnikov, 1999). The Xenopus laevis YBP, FRGY2, appears to be capable of inhibiting translation of all mRNAs, although RNA binding and translational masking by FRGY2 are enhanced by a hexanucleotide with consensus sequence, AACAUC (Bouvet et al., 1995; Matsumoto et al., 1996). The binding of YBPs to mRNA is further regulated by phosphorylation (Kick et al., 1987; Murray et al., 1991).
The expression of mouse YB protein 2 (MSY2) has been analysed with antibodies and cDNA probes (Kwon et al., 1993; Gu et al., 1998). MSY2 is expressed exclusively in male and female germ cells, binds to all mRNAs tested, and is associated with free-mRNPs in total testis (Gu et al., 1998; Herbert and Hecht, 1999). The levels of MSY2 are correlated with elevated free-mRNPs in pachytene spermatocytes and round spermatids, and decreases in the levels of MSY2 are correlated with increases in translational activity of selected mRNAs in elongated spermatids (Kwon et al., 1993; Oko et al., 1996). In addition, gel mobility supershifts implicate MSY2 in transcription of the protamine 2 and cytochrome ct genes (Nikolajczyk et al., 1995; Yiu et al., 1997).
In the present study, a strategy combining a degenerate primer and 3' rapid amplification of cDNA ends (RACE) was used to identify Ybp genes that are expressed in mouse testis. Ybp mRNAs are particularly amenable to cloning by degenerate PCR because the cold shock domain is highly conserved (Wolffe, 1994; Sapru et al., 1996; Gu et al., 1998). We isolated cDNAs encoding two Ybp mRNAs that are expressed in mouse testis. The first, Msy1, has previously been shown to be expressed in mouse testis (Tafuri et al., 1993), but the patterns of expression in spermatogenic cells have not been accurately described. The second gene, referred to here as Msy3, is very similar to a Ybp gene referred to as DBA in human and MY-1 in rat (Sakura et al., 1988; Sapru et al., 1996). In the present study, it was shown that Msy3 mRNA was expressed at high levels in spermatogenic cells, that the transcripts are alternatively spliced encoding two isoforms, and that the Msy1 and Msy3 mRNAs persist at high levels in elongated spermatids after the cessation of transcription.
Materials and methods
Cell separation, RNA purification and Northern blotting
CD-1 male mice 6, 8, 10, 12, 14, 16, 18, and >60 days old were obtained from Charles River Laboratories. Pachytene primary spermatocytes, round spermatids and elongated spermatids were purified by sedimenting single cell suspensions of adult seminiferous tubules at unit gravity on bovine serum albumin gradients as described by Romrell et al. (1976). RNAs were extracted from tissues and cells with the Trizol reagent (Gibco-BRL, Bethesda, MD, USA).
For Northern blot analysis, RNAs were denatured with formaldehyde and formamide, electrophoresed through 0.8% agarose gels containing 2.2 mol/l formaldehyde for 6–17 h at 35 V, and blotted to nitrocellulose (Kleene et al., 1994). The probes were as follows: Msy1, a 1052 nucleotide (nt) EcoRI cDNA fragment (Tafuri et al., 1993); Msy3, the 1720 nt cDNA displayed in Figure 1. Northern blots were hybridized and washed under stringent conditions as described previously (Cataldo et al., 1999). The amount of RNA in the various RNA samples was controlled by hybridization to a probe for rat 18S ribosomal RNA (Chan et al., 1984).
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Isolation of Ybp cDNAs by RT–PCR using a degenerate primer to the cold shock domain.
Ybp cDNAs were isolated using a 3'-RACE strategy in which the mouse testis RNA was reverse transcribed using an oligo(dT) primer, and then PCR-amplified using a degenerate upstream primer encoding a highly conserved sequence in the cold shock domain, EFDVVEGEK (Tafuri et al., 1993
; Sapru et al., 1996; Gu et al., 1999). 1 µg RNA in 5 µl DH2O was denatured for 10 min, chilled on ice, and copied in a 50 µl reaction containing 50 mmol/l KCl, 100 pmol of oligo dT primer (primer 1, Table I), 1.5 mmol/l MgCl2, 1.0 mmol/l dNTPs, 10 mmol/l Tris–HCl (pH 7.9) and 100 IU Superscript reverse transcriptase (Gibco-BRL) at 42°C for 60 min followed by heat inactivation at 95° for 10 min. 3 µl of the reverse transcriptase reaction was amplified in a 50 µl PCR reaction containing 50 mmol/l KCl, 0.1% Triton X-100, 1.5 mmol/l MgCl2, 10 mmol/l Tris–HCl (pH 9.0), 20 pmol of the upstream degenerate and oligo(dT) primers (primers 1 and 2, Table I), 0.4 mmol/l dNTPs and 0.2 IU Taq DNA Polymerase (Promega, Madison, WI, USA). PCR reactions were overlaid with mineral oil and amplified using an MJ-Research thermocycler: 5 min denaturation at 95°C, followed by 30 cycles, 94°C for 1 min, 60°C for 45 s, 72°C for 1 min. A final extension at 72°C for 5 min completed the programme.
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Cloning and sequencing
5 µl of the RT–PCR reactions were separated by electrophoresis through 2% agarose gels and stained with ethidium bromide. Individual bands were excised, purified with GeneClean (Bio101, La Jolla, CA, USA), ligated into pGEM-T (Promega-Biotec), and electroporated into Escherichia coli DH5
. Msy1 and Msy3 cDNAs were isolated from a mouse testis DNA library (Stratagene Cloning Systems, La Jolla, CA, USA) using cloned Msy1 and Msy3 PCR products that had been labeled by the random primer method. Plasmid DNAs were purified with a miniplasmid kit (Qiagen, Valencia, CA, USA). The sequences reported here were determined on both strands using an Applied Biosystems automatic sequencer, and have been deposited in GenBank under accession numbers: AF248546 and AF248547.
RT–PCR analysis of the patterns of expression of alternatively spliced Msy3 mRNAs
RT–PCR using purified cell type RNAs from adult and prepubertal testis and tissues was performed as described above except that a random hexamer was used to prime the reverse transcriptase reaction and the PCR annealing temperatures varied from 60–62°C. The primers used to amplify specific segments of the Msy3 mRNA are listed in Table I
and their positions are indicated in Figure 1.
Sucrose gradient analysis
The distribution of mRNAs in the polysomal and non-polysomal compartments of testis was analysed by sedimenting cytoplasmic extracts of total adult testes on sucrose gradients and extracting the RNAs from gradient fractions under conditions that avoid differential losses of RNA as described previously (Cataldo et al., 1999). The distribution of mRNAs for the two isoforms of Mys3, Msy3S and Msy3L, in the sucrose gradient fractions were analyzed by hybridizing the Msy3L cDNA displayed in Figure 1, and a 149 nt specific probe for the Msy3L mRNA prepared by PCR amplification of Msy3L cDNA with primers 5 and 8 (Table I) to Slot Blots. The relative amounts of mRNA in each fraction were quantified with a Storm Model 840 phosphorimager.
Results
Isolation of Ybp cDNAs
A 3' RACE strategy was used to isolate recombinant DNAs encoding novel Ybps in mouse testis. Total mouse testis RNA was reverse transcribed using an oligo(dT) primer, and then amplified by PCR using the oligo (dT) primer and a degenerate primer encoding a perfectly conserved amino acid sequence in the cold shock domain of amphibian and mammalian Ybps. The PCR products were subcloned and 15 clones were sequenced. The sequences of six clones displayed no similarity to sequences in GenBank (data not shown), while nine clones described below were similar to two known Ybp genes.
The sequences of eight clones exhibited high similarity to mouse ovary Msy1 cDNA (Tafuri et al., 1993). In addition, several Msy1 cDNAs were isolated from a mouse testis cDNA library using Msy1 5' RACE products as a probe. The sequences of these cDNAs were identical to that of Msy1 cDNA reported previously (Tafuri et al., 1993).
The sequence of another clone encoded another YBP, referred to as DBA in human and MY-1 in rat (Sakura et al., 1988; Sapru et al., 1996). This PCR product was used to screen a mouse testis cDNA library resulting in the identification of two groups of cDNAs clones, referred to below as Msy3S and Msy3L, that differed by the presence or absence of 207 nt, corresponding to 69 amino acids in the carboxyl terminus domain encoding proteins of 292 and 361 amino acids shown in Figure 1. The observation that the sequences of the Msy3S and Msy3L mRNAs are identical outside the 207 base insertion/deletion implies that the two forms of Msy3 mRNA are generated by alternative splicing. MSY3S and MSY3L are identical at 86 and 98% of amino acid positions to human DBA and rat MY-1 respectively (Sakura et al., 1988; Sapru et al., 1996). Although the 3' UTR of the Msy3 mRNA contains two consensus polyadenylation signals, AAUAAA, and one example of the most common non-consensus signal, AUUAAA (Wickens, 1990), the 3' proximal signal was used exclusively in the cDNAs we characterized.
It is important to note that screening cDNA libraries with Msy1 and Msy3 cDNA probes always detected cDNAs whose sequences were identical to the probe, and never detected heterologous Ybp cDNAs, indicating that probes are specific for each Ybp mRNA.
Multiple alignment of mammalian Ybps
A CLUSTALW multiple alignment of the deduced amino acid sequences of MSY1 (Tafuri et al., 1993), MSY2 and MSY2A (Gu et al., 1998), and MSY3S and MSY3L is presented in Figure 2. The cold shock domain is the most conserved segment of Ybps and exhibits a high proportion of identical amino acids, but differs by several conservative and non-conservative amino acid substitutions in MSY1, MSY2 and MSY3. In contrast, the amino and carboxyl terminus domains exhibit few conserved amino acid sites, although they are organized similarly. The amino termini vary in length from 56–92 amino acids, except for MSY2A which is 17 amino acids. The four longer amino termini and are rich in alanine (25–32%), glycine (11–18%), hydroxyl amino acids (13–21%) and proline (9–18%). The carboxyl terminus domains of MSY1, MSY2 and MSY3L contain four basic/aromatic islands and four acidic islands. However, owing to the 69 amino acid deletion, the carboxyl terminus domain of MSY3S contains only three islands of each kind.
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Northern blot analysis of the expression of Msy1 and Msy3 mRNAs
The levels of Msy1 and Msy3 mRNAs were compared in Northern blots of 10 µg total cellular RNA from several somatic tissues and testis (Figure 3a
). The Msy1 probe hybridizes to a transcript of ~1500 nt, which is expressed at low levels in brain, spleen, thigh muscle and liver, and at high levels in kidney and heart. Quantification by phosphorimaging indicated that the Msy1 mRNA levels were slightly higher in testis (~10%) than in any somatic tissue studied here, and not many-fold higher as previously reported (Tafuri et al., 1993). In contrast, the Msy3 probe hybridizes to a transcript of about ~1650 bp that is expressed at low levels in brain, heart, spleen and liver, and at high levels in thigh muscle. The Msy3 mRNA was expressed at ~3-fold higher levels in testis than in somatic tissues, and the mRNA was slightly larger and more heterogenous in size, ~1650–1850 bp, presumably due to high levels of the Msy3L mRNA whose presence is demonstrated with RT–PCR below. That the probes for Msy1 and Msy3 hybridize with different sized mRNAs was clearly established by hybidizing Northern blots to first one probe, then rehybridizing the same filter to a second probe.
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Northern blots were used to determine the testicular cell types that express high levels of Ybp mRNAs in staged prepubertal mice in comparison with the appearance of various stages of spermatogenic cells in prepubertal mice of known ages (Bellve et al., 1977
). In these experiments, the testes from 10 prepubertal mice at each age were used to average developmental differences in individual mice. As shown in Figure 3b, the levels of Msy1 and Msy3 mRNAs are low in testes of prepubertal mice of 6–10 days old which contain somatic cells and spermatogonia, and in the testes of 12 day old mice which contain somatic cells, spermatogonia and leptotene and zygotene spermatocytes. The levels of Msy1 and Msy3 mRNAs increase sharply in the testes of 14 day-old mice, corresponding to the initial appearance of pachytene spermatocytes, and increase progressively with age, consistent with findings described below that the transcripts of both genes are expressed at high levels in pachytene spermatocytes, round spermatids and elongated spermatids.
The levels of Msy1 and Msy3 mRNAs were also analysed in Northern blots of total cytoplasmic RNA extracted from middle and late pachytene spermatocytes, round spermatids, and elongated spermatids that had been purified by sedimentation on bovine serum albumin gradients. All three cell types were ~90% pure. Figure 3c reveals that the Msy1 and Msy3 mRNAs are present at high and similar levels in pachytene spermatocytes, round spermatids and elongated spermatids. It is unclear whether the slightly lower mobility of Msy mRNAs in round spermatids is caused by a process of poly(A) lengthening and shortening that occurs on several, and possibly all, mRNAs during these stages of spermatogenesis (Fujimoto et al., 1988; Kleene, 1989), or an electrophoresis artefact.
RT–PCR analysis of the patterns of expression of alternatively spliced Msy3 mRNAs
Since we were unable to resolve the Msy3S and Msy3L mRNAs as two bands in Northern blots, the expression of these mRNAs was analysed by RT–PCR using primers that flank the alternatively spliced regions. The sequences of these primers are shown in Table I and their positions in the Msy3 mRNA are shown in Figure 1. The ethidium bromide-stained gel shown in Figure 4A reveals that a single band is present in somatic tissues corresponding to Msy3S mRNA, while each of the three major bands (332, ~300, and 125 bp) were observed in testes from adult, all stages of prepubertal mice, and purified pachytene spermatocytes, round spermatids and elongated spermatids. The sequences of the 332 and 125 bp bands were identical to the sequences of the Msy3S and Msy3L mRNAs shown in Figure 1. The fourth minor band slightly larger than the 332 bp band was not observed with other sets of primers and was not characterized.
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The observation that several additional sets of primers designed to amplify the alternatively spliced region also generated three bands (not shown) argued that the middle band (~300 bp) was not generated by spurious amplification of an unrelated mRNA and might, therefore, correspond to a third alternatively spliced form of Msy3 mRNA. However, three primer sets consisting of one primer inside the alternatively spliced region and a second primer downstream or upstream of the alternatively spliced region generated one band, instead of the expected two bands (Figures 4B–D
). In addition, we sequenced 13 clones of the middle band. The sequences of eight clones matched MsyL, while the sequences of five clones matched Msy3S. Thus, the ~300 bp RT–PCR product appears to be an artefact described previously (Zacharias et al., 1994), a heteroduplex between the Msy3S and Msy3L PCR products, and does not correspond to a bona fide alternatively spliced mRNA.
We also wish to point out that the absence of the 332 nt band in ethidium bromide stained agarose gels of RT–PCR reactions of somatic tissue RNAs in Figure 4A also suggests incorrectly that the Msy3L mRNA is not expressed in somatic tissues. In reality, the Msy3L mRNA is present and can be detected in all of the somatic tissues studied here by Southern blots, owing to the greater sensitivity of hybridization (data not shown). In addition, the Msy3L mRNA was detected in somatic cells by RT–PCR with three primer sets designed to selectively amplify the Msy3L mRNA as shown in Figure 3B–D. We conclude that the levels of Msy3L and Msy3S mRNAs are fairly similar in spermatogenic cells and in unidentified cell types in prepubertal testis, while the Msy3S mRNA is much more abundant than the Msy3L mRNA in somatic tissues.
Sucrose gradient analysis of translational activity of Msy3S and Msy3L mRNAs
To assess the translational activity of Msy3S and Msy3L mRNAs, cytoplasmic extracts of adult testes were sedimented on sucrose gradients, and the proportions of translationally inactive and active mRNAs were quantified by phosphorimage analysis of Slot Blots of RNAs extracted from gradient fractions using procedures that recover equivalent proportions of RNAs from all fractions (Cataldo et al., 1999). The distribution of Msy3 mRNAs was analysed using two probes, one that hybridizes to both the Msy3S and the Msy3L mRNAs, and a second that hybridizes specifically to the Msy3L mRNA. The translational activity of the Msy3 mRNAs was analysed in three gradients, all of which displayed absorbance tracings at 254 nm (not shown) that were virtually identical to those reported previously (Cataldo et al., 1999). Quantification by phosphorimage analysis of Slot Blots using both probes show that ~26–29% of the Msy3L and Msy3S mRNAs sediment with polysomes (fractions 5–13 and the pellet) and the remainder, 71–74%, sediments with free-mRNPs and single ribosomes (fraction 1–4) (Figure 5A). When the cytoplasmic extract were treated with EDTA, a procedure that dissociates polysomes into mRNPs and ribosomal subunits, the proportion of Msy3 mRNAs sedimenting in the polysomal region of the gradient was drastically reduced. RT–PCR reveals that the Msy3S and Msy3L mRNAs exhibit similar distributions in sucrose gradients implying that both forms of Msy3 mRNA are translated with similar efficiencies (Figure 5B,C). The proportions of translationally active Msy3 mRNAs, ~26–29%, can be placed in perspective by comparison with the proportions of polysomal mRNAs of 18 species that have been quantified in total testis (Cataldo et al., 1999). The proportion of polysomal Msy1 mRNA, ~6%, is the lowest in this group; ~33% of total poly(A)+ mRNA is associated with polysomes in total testis; the testis-specific isoform of histone H1, H1t, exhibits the highest proportion of polysomal mRNA yet quantified in meiotic and haploid spermatogenic cells, ~72%; and 85–90% of actively translated mRNA species are associated with polysomes in somatic cells in testis and somatic tissues.
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= cytoplasmic extracts prepared and sedimented in buffer containing Mg2+ to preserve polysomes; = cytoplasmic extracts treated with EDTA to dissociate polysomes into mRNPs and ribosomal subunits. (B, C and D) reverse transcription–polymerase chain reaction (RT–PCR) analyses in which 1% of the RNA extracted from each gradient fraction was copied with reveres transcriptase using a random primer and then amplified with several pairs of primers: (B) primers 4 and 6; (C) primers 4 and 5; (D) primers 6 and 7. The specificities of the primers are described in the legend to Figure 4Discussion
The YBP family is distinguished by the highly conserved cold domain and a characteristic carboxyl terminus consisting of alternating aromatic/basic and acidic islands (Wolffe, 1994; Graumann and Marahiel, 1998). cDNAs derived from three genes, Msy1, Msy2, and Msy3, encoding five isoforms of Ybp proteins have been isolated in mice and other mammals (Sakura et al., 1988; Tafuri et al., 1993; Sapru et al., 1996; Gu et al., 1998). Two forms of Msy3 mRNA, Msy3S and Msy3L, are produced by alternative splicing encoding YBPs of 292 and 361 amino acids differing by the presence or absence of 69 amino acids in the carboxyl terminal domain. In addition, two transcripts of the Msy2 gene, denoted Msy2 and Msy2A, encode proteins of 282 and 360 amino acids differing in their amino terminal domains (Gu et al., 1998), but it is unclear whether the Msy2 and Msy2A mRNAs are produced by alternative promoters or alternative splicing. The five YBPs include four different amino termini, three very similar but non-identical cold-shock domains, and four different carboxyl termini.
The levels of the five Ybp mRNAs are regulated independently in adult tissues. The Msy1 and Msy3S mRNAs are expressed at low levels in several somatic tissues (Tafuri et al., 1993; Sapru et al., 1996), but the Msy1 mRNA is expressed at high levels in kidney and heart, and the Msy3S mRNA is expressed at high levels in skeletal muscle. In addition, the relative proportions of the alternatively spliced Msy3 transcripts are regulated: the Msy3S mRNA is the predominant transcript in most somatic tissues, while Msy3S and Msy3L and their homologues are present at similar levels in prepubertal and adult testis and in meiotic and haploid spermatogenic cells in mice and retina in rats (Sapru et al., 1996). In contrast, the Msy2 mRNA is expressed only in male and female germ cells (Gu et al., 1998). It is notable that the levels of all five Ybp mRNAs are higher in testis than in somatic tissues, although our results disagree with earlier findings that the Msy1 mRNA is grossly overexpressed in testis (Tafuri et al., 1993).
The findings that five Ybp mRNAs are expressed in pachytene spermatocytes, round spermatids and elongated spermatids raise questions concerning the patterns of expression of these proteins. Unfortunately, the expression of Ybps in spermatogenic cells in mice has only been studied with antibodies to amphibian Ybps, the specificity of which have not been rigorously characterized with recombinant proteins. A potential problem is illustrated by reports that antibodies to Xenopus laevis FRGY2 and mRNP3/mRNP4, a mixture of FRGY2 and a closely related protein, have been assumed to react specifically with both MSY1 and MSY2 (Tafuri et al., 1993; Gu et al., 1999). The fact that the predicted sizes of MSY2 and MSY3L are virtually identical, 360 and 361 amino acids, and that the levels of MSY1 and MSY3 cannot be predicted accurately from the relative levels and translational activity of their mRNAs create additional uncertainties. In addition, it is unclear whether the MSY1 is the predominant Ybp in all somatic mammalian cells (Evdokimova and Ovchinnikov, 1999), because the Msy3S mRNA appears to be much more abundant in skeletal muscle than the Msy1 mRNA.
Our finding that the Msy1 and Msy3 mRNAs are expressed at high, similar levels in pachytene spermatocytes, round spermatids and elongated spermatids is unexpected in view of the putative functions of YBPs in transcriptional activation and the widespread repression of mRNA translation in spermatogenic cells (Kwon et al., 1993; Tafuri et al., 1993; Nikolajczyk et al., 1995; Oko et al., 1995; Yiu et al., 1997). In particular, the levels of the Msy1 and Msy3 mRNAs and the levels of MSY2 protein (Oko et al., 1996) increase sharply in pachytene spermatocytes, while high levels of free-mRNPs begin earlier, in leptotene/zygotene spermatocytes (Kleene, 1996). Additional work will be required to determine whether the discrepancy lies in errors in the analysis of translational activity in early meiotic cells, the failure to detect a small but biologically significant increase in Ybps or Ybp mRNAs, or because translational repression is mediated by other mechanisms, such as the activities of translation initiation factors that have important roles in global translation repression in somatic cells (reviewed in Preiss and Hentze, 1999).
Another puzzling observation is the persistence of Msy1, Msy2 and Msy3 mRNAs after the cessation of transcription in elongated spermatids (Gu et al., 1998). This is unusual among RNA binding protein mRNAs that are expressed in spermatogenic cells such as the Pabp1, Pabp2, Prbp, Spnr, and Tenr mRNAs, all of which are undetectable in elongated spermatids, presumably because synthesis of the proteins is coupled to transcription (Kleene et al., 1994; Lee et al., 1995b; Schumacher et al, 1995a,b). It is reasonable to suppose that the high levels of Msy1, Msy2 and Msy3 mRNAs in transcriptionally active spermatogenic cells, pachytene spermatocytes and round spermatids, are utilized to produce YBPs that function as transcription factors and in packaging newly synthesized mRNAs into mRNPs. However, these ideas do not explain the persistence of high levels of Msy1, Msy2, and Msy3 mRNAs in transcriptionally inactive elongated spermatids. The persistence of Msy2 mRNA in elongated spermatids is especially puzzling (Gu et al., 1998), because MSY2 levels have been reported to decline sharply in elongated spermatids in the rat and mouse (Kwon et al., 1993; Oko et al., 1996). Apparently, the decreased translation of Ybp mRNAs in elongated spermatids is not accompanied by mRNA degradation, or YBPs are synthesized in elongated spermatids, but the steady state levels are low due to rapid turnover.
The special functions of the five isoforms of mammalian YBPs are likely to be interesting because YBPs have important functions as transcriptional and translational activators and repressors. Unfortunately, the diversity of the functions of YBPs and the limited information about the precise activities of the three domains leave open a myriad of possibilities. DNA binding is restricted to the cold shock domain, the cold shock and carboxyl terminus domains both bind RNA (Ladomery and Sommerville, 1994; Murray, 1994; Bouvet et al., 1995), and the carboxyl terminus domain enhances the formation of multimers (Matsumoto and Wolffe, 1998). In addition, the cold shock domain of FRGY2 binds strongly to the hexaribonucleotide AACUAC, while the cold shock and carboxyl terminus domains preferentially bind AG and UC homopolymers respectively (Ladomery and Sommerville, 1994; Bouvet et al., 1995). The observation that the various isoforms differ considerably in the carboxyl terminus domains, and in a small number of non-conservative substitutions in and around the cold shock domain suggests that MSY1, MSY2 and MSY3 may exhibit subtle differences in RNA binding specificity. This possibility is intriguing because the conventional view of YBPs as non-specific translational repressors does not account for the widely varying proportions of various mRNA species in free-mRNPs in meiotic and haploid spermatogenic cells (Kleene, 1996). Alternatively, the differences in the carboxyl terminus domain may produce differences in the ratio of protein to mRNA that determine whether YBPs facilitate or inhibit mRNA translation, and these ratios may be influenced by the ability, or inability, of various YBP isoforms to form multimers with each other. It is also relevant to point out that MSY2 has been reported to be associated exclusively with free-mRNPs in spermatogenic cells (Kwon et al., 1993) suggesting that MSY2 functions only as a translational repressor, and that translational activation is accomplished by MSY1 and/or MSY3. Finally, interactions between the various isoforms of YBPs and other transcription factors and mRNA binding proteins provide another level of modulating the effects of YBPs on transcription and translation.
As transcriptional activators and translational repressors, YBPs could have a profound impact on the patterns of gene expression in spermatogenic cells. Gene expression in spermatogenic cells is characterized by a phenomenon, quantitative transcriptional promiscuity, in which many genes are expressed at higher levels in spermatogenic cells than in somatic cells, but the mRNAs are translationally repressed. The Msy1, Msy3, poly(A) binding protein 1, TATA-binding protein and arylsulphatase A mRNAs are a few of the members of this group (Kleene et al., 1994; Kreysing et al., 1994; Persengiev and Kilpatrick, 1997; Schmidt and Schibler, 1997). It is tempting to speculate that YBPs, which possess activities as transcriptional activators and translational repressors, play a key role in this phenomenon by promoting mRNA transcription directly or by augmenting expression of other transcriptional activators, while simultaneously preventing deleterious effects of protein overproduction. Unfortunately, nothing is known about the effects of YBPs on differentiation of spermatogenic cells in vivo. Targeted disruption of the Msy1, Msy2 or Msy3 genes might lead to changes in both the patterns of mRNA transcription and translation in spermatogenic cells.
Note added in proof
After this manuscript was accepted for publication, Davies et al. (2000) reported a Y-box protein that is expressed at high levels in spermatogenic cells, MSY4. MSY4 is highly similar to the MSY3L in our article, and MSY4 and MSY2 form a complex that binds to the 3' UTR of the mouse protamine 1 mRNA.
Acknowledgments
We are grateful to A.P.Wolffe for generously providing the mouse ovary Msy1 plasmid. This work was supported by NSF Grants IBN-9418285 and MCB-9874491.
Notes
1 To whom correspondence should be addressed at: Department of Biology, University of Massachusetts Boston100 Morrissey Boulevard, Boston, MA 02125–3393, USA. E-mail:kenneth.kleene{at}umb.edu 
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Submitted on April 3, 2000; accepted on June 14, 2000.
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