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Center for Research on Reproduction and Women's Health,3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160
Department of Anatomy and Cell Biology,4 McGill University, Montreal, Quebec, Canada H3A 2B2
Department of Biology,5 University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
ABSTRACT
MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.
gene regulation, sperm, spermatid, spermatogenesis, testis
The DNA/RNA-binding protein MSY2 is a male and female germ cell-specific member of the Y-box family of proteins [1]. Y-box proteins are ubiquitously expressed from bacteria to humans [2], and the structure of bacterial Y-box proteins has been determined [3, 4]. Y-box proteins contain a highly conserved sequence element, the cold shock domain that is required for DNA binding. Within the cold shock domain are two RNA-binding motifs essential for mRNA binding [5]. Protein interactions with other proteins are mediated by the amino and carboxyl termini of Y-box proteins, which are variable in length and sequence and contain arginine-rich domains similar to the RNA-binding domains of the human immunodeficiency virus tat protein. In vitro RNA-binding studies suggest that at low concentrations, Y-box proteins stimulate translation, whereas high levels of Y-box proteins inhibit translation [6, 7].
In the testis, the germ cell-specific Y-box protein, MSY2, is abundant in meiotic and postmeiotic germ cells, where it binds to specific mRNAs in the nucleus and facilitates their storage/translational repression in the cytoplasm [8–10]. This process depends on MSY2 recognizing the sequence CTGATTGGC/TC/TAA, a DNA motif in the promoter of many genes specifically expressed in male germ cells [10]. After binding to its consensus promoter sequence, MSY2 binds to transcripts of the gene and stabilizes and represses their translation in cytoplasmic RNA-protein complexes.
Both male and female mice that lack MSY2 are phenotypically normal but are infertile [11]. In males, spermatogenic arrest occurs, and females contain defective oocytes. Because of the multiple sites in which MSY2 interacts with DNA or mRNA in the nucleus and cytoplasm, there are several cellular disruptions that could be the cause of the spermatogenic arrest in mice lacking MSY2. To discover the molecular basis of the infertility of male mice lacking MSY2, we have examined several transcriptional and posttranscriptional processes in Msy2-null male mice and their control littermates. We find that in the absence of MSY2, transcription, mRNA processing, and mRNA intracellular transport appear normal. However, a polysomal redistribution and decreases in the amount of many mRNAs normally expressed in meiotic and postmeiotic germ cells are seen in mice lacking MSY2. These decreases in mRNA levels occur before morphological defects are detected and are likely a cause of the spermatogenic arrest and male infertility.
The protocol of Hinoi et al. [12] was followed. In brief, an enriched germ cell preparation was prepared from the testes of 22-day-old wild-type and Msy2-null mice with collagenase to dissociate the cells of the seminiferous tubules. The mice were raised on the premises, and all investigations were conducted in accordance with the Guide for Care and Use of Laboratory Animals (1966); the Institutional Animal Care and Use Committee approved all procedures involving animals in advance. The dissociated cells (5 x 106) were lysed in 0.5 ml of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40 and incubated for 30 min at 4°C. After centrifugation at 500 x g for 1 min, nuclei were harvested and resuspended in 200 µl of ice-cold glycerol storage buffer (50 mM Tris-HCl [pH 8.3], 5 mM MgCl2, 0.1 mM EDTA [pH 8.0], and 40% glycerol). For the transcription assays, the nuclear suspension (200 µl) was mixed gently with 200 µl of 2x reaction buffer (10 mM Tris-HCl [pH 8.0], 5 mM MgCl2, 0.3 M KCl, 5 mM DTT, 1 mM ATP, 1 mM CTP, and 1 mM GTP) and 100 µCi of 
32P UTP. After incubation at 30°C for 30 min, the reaction mixtures were digested with RNase-free DNase I (25 µg/ml) at 37°C for 5 min, and RNA was purified with TriReagent (Sigma, St. Louis, MO). For hybridization, DNAs (1 µg), encoding the full-length coding region of target genes, were prepared by RT-PCR and spotted on nitrocellulose membranes by a DNA slot apparatus. Hybridizations were carried out for 24 h at 65°C in Quick hybridization solution (Stratagene, La Jolla, CA) with a hybridization solution of 5 x 106 cpm RNA/5 ml. After filter washing as described in the manufacturer's protocol, the blots were incubated at 37°C for 30 min with 2x saline sodium citrate (SSC; 0.15 M NaCl and 0.015 M sodium citrate) containing RNase A (10 µg/ml), washed twice in 2x SSC at 37°C for 30 min, and quantitated by PhosphorImager (Amersham Biosciences, Piscataway, NJ).
Isolation of Highly Purified Nuclear and Cytoplasmic RNA from Testes
Nuclear and cytoplasmic extracts were prepared from enriched germ cell preparations of 22-day-old wild-type and Msy2-null mice following the protocol of Dignam et al. [13], with the modification that all buffers contained a protease inhibitor cocktail (Roche, Mannheim, Germany). Testicular cells were dissociated as previously described [14] and homogenized 20 times twice with a 10-min incubation interval on ice in a Teflon glass homogenizer. Cell breakage was monitored by phase-contrast microscopy. Nuclei were pelleted by centrifugation at 12 000 x g for 30 min at 4°C in an Eppendorf Centrifuge (model 5415D). Total RNA was purified from the nuclear and cytoplasmic extracts with TriReagent, and aliquots (1 µg) were reverse transcribed and used for quantitative PCR (Q-PCR) analysis.
Total testis RNA from 22-day-old and adult wild-type and Msy2-null mice was prepared with TriReagent, treated with DNase I (amplification grade; Invitrogen, Carlsbad, CA), and reverse transcribed for PCR analysis. Primers were designed to cross introns to distinguish mature mRNAs from precursor mRNAs containing introns. The primers for Msy4, 5'-attggagaggctgaagacaaa-3' and 5'-ttttgccttgtttttgaatggt-3', would detect a precursor mRNA of 767 nt and a mature mRNA of 280 nt; the primers for acrosin: 5'-cagcctcctgaactcccact-3' and 5'-ccctccgtcactacgttgtatt-3', would detect a precursor mRNA of 407 nt and a mature mRNA of 195 nt; the primers for protamine 1, 5'-atggccagataccgatgctgccgc-3' and 5'-ggacttgctattctgtgcatc-3', would detect a precursor of 249 nt mRNA and a mature mRNA of 176 nt; and the primers for protamine 2, 5'-atggttcgctaccgaatgaggagc-3' and 5'-ttagtgatggtgcctcctacatttcc-3', would detect a precursor mRNA of 427 nt and a mature mRNA of 314 nt. Genomic DNA from adult wild-type mice was used as a positive control template to demonstrate that the larger RNAs could be detected in our assays.
Polysomal Gradient Fractionation and Analysis
Testes from two adult wild-type and three MSY2-null mice were decapsulated, homogenized on ice in 20 mM Hepes, pH 7.6, 40 mM KCl, and 1.5 mM MgCl2, and centrifuged at 2300 x g for 10 min at 4°C. The supernatants (0.3 ml) were layered over 10%–30% sucrose gradients (5 ml) on a 60% sucrose cushion (0.25 ml). Following centrifugation at 100 000 x g rpm for 1.5 h at 4°C in an SW28 rotor, the gradients were fractionated into 17 tubes. Each fraction was extracted with TriReagent (Sigma), and the purified RNAs were used for Northern blot analysis. The distribution of protamine 2 mRNA in ribonucleoprotein particles (RNPs) and polysomes was used to calibrate the sucrose gradient. The following probes were prepared for each mRNA as follows: acrosin (BC103579, 290–485 nt), Msy4 (AF246224, 917-1197 nt), Pgk2 (BC061054, 1275–1530), glyceraldehyde phosphate dehydrogenase (Gapdh; BC085275, 223–379 nt), and clusterin (Clu; NM_013492, 92–1281). The Northern blots were quantitated with a PhosphorImager (Amersham), and the expression level for each fraction was represented by the percentage of the total.
Quantification of mRNAs by Q-PCR
Total testis RNA from 17-day-old, 22-day-old, and adult wild-type and MSY2-null mice was purified with TriReagent. One microgram of each preparation was assayed by Q-PCR. The expression level of each mRNA was normalized to 18S rRNA, and the Delta-Delta Ct method was used for quantification. To compare the data from the three developmental stages, the expression levels of the 17- and 22-day-old mice were normalized to those of the adult samples.
Morphological Analyses and Quantitation
Testes and epididymides from a 5-mo-old Msy2-null mouse and its wild-type littermate were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer. After fixation, the tissue was impregnated with osmium tetroxide (OsO4), dehydrated in ethanol, cleared in propylene oxide, and embedded in Epon. Semithin sections (1 µm thick) were cut with an ultramicrotome, stained with toluidine blue, and examined by light microscopy.
Micrographs of cross sections of seminiferous epithelium were taken at a magnification of 400x and printed by a factor of 1.5x (final magnification 600x). Tubular and luminal diameters were measured in millimeters and converted to micrometers by the following equation: diameter in millimeters x 1000 ÷ 600. Because of the absence of late spermatids in the null mice, the precise stages of the cycle of the seminiferous epithelium of Msy2-null testes were determined on the basis of the morphology of the acrosomic system in round spermatids. In total, 20 micrographs were taken from cross sections of Msy2-null and wild-type seminiferous tubules at stages VIII and I. The mean and SEM were obtained for each of these groups. Statistical comparisons between Msy2-null and wild-type seminiferous tubules were performed with an impaired two-tailed Student t-test. Differences were considered significant if P < 0.05.
Following transcription, mRNAs undergo several posttranscriptional processing and transport events before becoming available for protein synthesis in the cytoplasm. Because MSY2 functions in both the nucleus and cytoplasm, the infertility seen in male mice lacking MSY2 could be a result of a deficiency in any or all of the steps of mRNA synthesis, maturation, stability, or translation where MSY2 participates. Therefore, to start to define the molecular cause(s) of the spermatogenic arrest leading to infertility, we have compared transcriptional and posttranscriptional processes in mice lacking MSY2 and in their wild-type littermates. For all the assays, selected mRNAs that are bound or not bound by MSY2 will be examined [10]. This will allow us to assess the direct effect of MSY2 for the bound subset of mRNAs as well as the secondary effects on nonbound mRNAs as cell deterioration and germ cell loss occur.
Transcription Is Normal in Male Germ Cells of Msy2-Null Mice
To measure transcription, nuclear run-on assays were performed. Because the testes of 22-day-old Msy2-null and control mice (but not adult mice) contain the same populations of cells and spermatogenesis appears morphologically normal, the testes of 22-day-old mice were used for transcriptional analyses. In the first wave of spermatogenesis, both meiotic and postmeiotic cell types were present in the testes of 22-day-old mice. Because MSY2 is expressed in only meiotic and postmeiotic male germ cells, transcription was assayed for two postmeiotically expressed mRNAs, i.e., transition protein 2 (Tnp2) and activator of Crem-tau transcription (Act); three mRNAs expressed during meiosis, i.e., acrosin, Msy4, and cyclin A1; and three control mRNAs, i.e., Clu, Gapdh, and ß-actin. Similar levels of mRNA transcription in wild-type and Msy2-null mice were detected for all eight mRNAs (Fig. 1). Quantitative PCR assays of purified mRNA from the isolated nuclei showed similar amounts of mRNAs for these genes in the wild-type and Msy2-null mice, confirming the nuclear run-on assays (data not shown). We conclude that germ cell transcription is normal in the absence of MSY2.
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Intron Removal and Subcellular mRNA Transport Are Normal in Msy2-Null Mice
Following transcription, mRNAs undergo intron removal before being transported from the nucleus into the cytoplasm. To determine if a deficiency in intron removal exists in Msy2-null mice, primers that bridged introns of Msy4, Acr, Prm1, and Prm2 were used to assay for nonspliced and mature mRNAs. Following nuclear isolation [13], assays for the nuclear RNA U6snRNA established that more than 70% of the U6snRNA was in the nuclear fraction. Gel electrophoresis of PCR products of RNAs of Msy4, Acr, Prm1, and Prm2 isolated from the nuclear and cytoplasmic fractions of adult mice showed identical sizes in the mRNAs encoding the meiotically/postmeiotically expressed mRNAs, Msy4 and Acr, and the two postmeiotically expressed mRNAs, protamines 1 and 2 (Fig. 2). Identical results were obtained from total nuclear RNA from 22-day-old-mice (data not shown). The detection of only mature processed mRNAs confirmed that normal intron excision can occur in the absence of MSY2 in male germ cells.
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MSY2 binds to a subset of mRNAs in the nucleus and stabilizes them in the cytoplasm as part of an mRNA-protein complex. To determine whether a buildup of nuclear transcripts in Msy2-null mice could contribute to the arrest of spermatogenesis, Q-PCR was used to quantitate the relative amounts of mRNA encoding 12 genes in highly purified nuclear and cytoplasmic fractions from 22-day-old wild-type and Msy2-null mice (Table 1). Five of the genes, Prm1, Tnp2, Acr, Msy1, and Msy4, encode mRNAs that are bound by MSY2, and six of the genes, Ldh3, Ccna1, Ccna2, Hdac1, Hspa2 (also known as Hsp70.2), and ß-actin, encode mRNAs not bound by MSY2 [10]. As a control, testicular mRNA that is not bound by MSY2, the Sertoli cell transcript, Clu, was assayed. None of the Msy2-bound or nonbound mRNAs accumulated in the nuclei of mice lacking MSY2, suggesting that mRNA movement to the cytoplasm is not dependent on MSY2 (Table 1).
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Meiotic and Postmeiotic Germ Cell mRNAs Are Markedly Reduced in Msy2-Null Mice
Although the testes of 17- and 22-day-old mice appear morphologically normal in Msy2-null mice, spermatogenesis arrests in the haploid phase of germ cell development in adult mice lacking MSY2 [11]. To determine whether biochemical changes can be detected before morphological changes are seen, we used Q-PCR to quantitate the amounts of 17 different mRNAs in the testes of prepubertal and adult wild-type and MSY2-lacking mice. As expected for testes lacking late-stage spermatids, marked reductions in the mRNAs normally present in the late-stage postmeiotic male germ cells were seen (Fig. 3). Indeed, the mRNAs encoding five postmeiotically expressed genes, Prm1, Prm2, Tnp1, Tnp2, and Act, were decreased to as low as 10% of controls. More significantly and likely contributing to the spermatogenic arrest, 12 mRNAs expressed during meiosis, i.e., Pgk2, Acr, Msy1, Msy4, Tsn, Ldh3, Papb, Hspa2, Ccna1, Ccna2, Hdac1, and Caml2, were also markedly decreased in the testes of adult Msy2-null mice compared to their littermate controls. Decreases were also detected for many of the 17 mRNAs in testes from 22-day-old Msy2-null mice, despite normal testicular morphology at the light microscopic level. No decreases were seen for these mRNAs in the testes of 17-day-old mice, nor were decreases evident in the mRNA amounts for three control mRNAs, i.e., Clu, Gapdh, or ß-actin, when 18S rRNA was used as a loading control in the testes of 17-day-old, 22-day-old, or adult Msy2-null mice. Thus, in the absence of MSY2, we detected substantial decreases in the amounts of mRNAs present in spermatogenic cells before morphological changes could be detected by light microscopy. In total, our assays of transcriptional and posttranscriptional processes suggest that MSY2 is essential to maintain the stability of specific mRNAs in male germ cells.
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In the Absence of MSY2, There Is an Enrichment of Msy2-Bound mRNAs on Polysomes
Because many genes are posttranscriptionally regulated in the mammalian testis [15, 16], many germ cell mRNAs are predominantly found in RNPs, often in association with MSY2. Because protein synthesis occurs on polysomes and because stored mRNAs are present in RNPs, movement of mRNAs between these fractions is often used as an indicator of translation or mRNA storage [17]. To determine if there were changes in the RNP/polysomal mRNA distribution in Msy2-null mice, we compared the mRNA amounts in the RNPs and polysomes of two distinct populations of germ cell mRNAs, those that are bound by MSY2 and those that are not bound, in adult Msy2-deficient mice and their littermate wild-type controls (Fig. 4). The mRNA distribution of acrosin and Msy4, two mRNAs normally bound by MSY2, showed different redistributions in sucrose gradients of total mRNA from wild-type and Msy2-null mice (Fig. 4, A and B). Both acrosin and MSY4 showed a decrease in the RNP fraction and an increase in the polysomes in Msy2-null mice compared to wild-type controls. No changes in mRNA distribution were seen for Pgk2 or Gapdh mRNAs, two mRNAs not bound by MSY2 (Fig. 4, C and D). The presence of Clu mRNA solely in polysomes established the integrity of the polysomes in the extracts and sucrose gradients (Fig. 4E). The redistribution of Msy2-bound but not nonbound mRNAs suggests that the destabilization of the mRNAs in RNPs leading to the loss of a subset of mRNAs occurs following mRNA translation as a result of precocious translation in the absence of MSY2.
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In the Absence of MSY2, Spermatids Show Incomplete Nuclear Condensation and Degeneration
To obtain an improved assessment in Msy2-null mice of any visible morphological changes that occurred before spermatogenesis arrests, we analyzed 50 sections of seminiferous tubules from Msy2-null mice and 50 sections from wild-type littermate control mice. In Msy2-null testes, an occasional step 14 spermatid was seen in stage IV of the seminiferous cycle. Between stages V and VIII, round spermatids, but not later-stage-specific condensing spermatids, were detected (Fig. 5). Between late-stage VIII and early-stage IX of the cycle, a massive degeneration of step 8 and 9 spermatids was observed. Approximately 50% of the round spermatids formed multinucleated bodies and exhibited vacuolated cytoplasm (Fig. 5, A and B). Surviving step 9 spermatids initiated nuclear condensation at stage IX of the cycle. Prominent in the cells, nuclear condensation was incomplete, and the abnormal spermatids degenerated (Fig. 5, C–F). At stage IV of the cycle, complete degeneration of condensing spermatids was seen, often with dark cytoplasm and multinucleated bodies. As previously reported, epididymides of Msy2-deficient mice were virtually empty and contained few, if any, degenerating round spermatids [11].
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Consistent with the loss of late-stage germ cells in the absence of MSY2, there were decreases in the tubular and luminal diameters of seminiferous tubules in Msy2-null mice. The average diameter of wild-type seminiferous tubules was 235 µm (SD ± 20) compared to 200 µm (SD ± 15) at stage I and 245 µm (SD ± 15) vs. 205 (SD ± 13) at stage VIII of the cycle of the seminiferous epithelium for wild-type and Msy2-null mice, respectively. Similarly, compared to the average luminal diameters of 96 µm (SD ± 12) at stage I and 100 µm (SD ± 10) at stage VIII for wild-type mice, the average luminal diameters for Msy2-null mice were 33 and 35 µm (SD ± 17) at stages I and VIII. The decreases in seminiferous tubule luminal diameter in the Msy2-null mice were highly variable, ranging from 0 to 70 µm. All differences were statistically significant (P < 0.05).
In in vitro studies, many investigators have proposed that Y-box proteins stabilize mRNAs and suppress translation [5–7, 18–20]. In the present study, we show in an in vivo system that the lack of MSY2 in the testes leads to selective mRNA redistribution from RNPs to polysomes and a precocious loss of specific mRNAs. In contrast, other putative functions of MSY2, involving transcription, intron removal, and mRNA transport, appear normal. In light of its abundance in the cytoplasm of male germ cells, we propose that MSY2 plays a major role in mRNA storage and stabilization in male germ cells. This may also be true in the female, in which MSY2 constitutes 2% of total oocyte protein [21], and following fertilization, MSY2 markedly decreases until it, along with stored maternal mRNAs, is no longer detectable at the two-cell stage [1].
The loss of MSY2 in the testis following its gene targeting induces a massive loss of late step 8 and early step 9 spermatids (Fig. 5). About 50% of the entire population of round spermatids degenerate during stages VIII and IX, a time in the spermatogenic wave coincident with the initiation of nuclear condensation. In the Msy2-null mice, the nuclei of the surviving spermatids do not fully condense. The loss of spermatids due to the lack of MSY2 correlates with a decrease in seminiferous tubular and luminal diameters. Degenerating round spermatids form multinucleated bodies and exhibit darker vacuolated cytoplasm. Many spermatids degenerate at the onset of step 9, and surviving spermatids are unable to undergo nuclear condensation. The nuclei of surviving spermatids appear abnormal, being partially condensed or not condensed at all. Such incomplete condensation is consistent with deficiencies in nuclear protein composition and suggests that in the absence of MSY2, the synthesis of proteins required for DNA rearrangement and nuclear condensation is affected. Indeed, Q–PCR assays of mRNAs encoding the two transition proteins and two protamines detect substantial reductions in mRNA levels in the testes of 22-day-old mice, a time of germ cell differentiation when the mRNAs encoding these nuclear proteins are synthesized and stored (Fig. 3). In the maturing germ cell, the histones are replaced by transition proteins 1 and 2, which, in turn, are replaced by protamines 1 and 2. Gene targeting has demonstrated severe deficiencies in chromatin packaging in the absence of the transition proteins [22]. Deficiencies of either protamine 1 or 2 also lead to infertility and defective DNA compaction [23, 24]. Decreased amounts of the mRNAs of these nuclear proteins would lead to substantial protein deficiencies and incomplete condensation of the maturing germ cell nuclei. Although it is impossible to determine the initiating cause of the spermatogenic arrest because many other mRNAs show similar reductions in the testes of 22-day-old mice (Fig. 3), the impaired nuclear condensation is a morphologically obvious and dramatic change readily seen at the light microscope level.
To assess if the degenerating spermatids were eliminated via luminal sloughing, we examined the lumen of the epididymides. Because the lumen epididymides from Msy2-null mice rarely contained spermatids, it is likely that the elimination of these cells occurs via Sertoli cell phagocytosis. In fact, retention and degradation of spermatids within the cytoplasm of Sertoli cells were often observed.
Many different functions have been proposed for Y-box proteins [2, 5, 20, 21, 25–27]. The Xenopus ortholog of MSY2, FRGY2, has been proposed to exert both positive and negative effects on transcription and translation [28]. In oocytes, FRGY2 is a major component of RNPs, where it masks maternal mRNAs. It has also been linked to a coupling of transcription and translational activity of intron-less mRNAs in oocytes [29]. In mice, another coordination of nuclear and cytoplasmic functions for the Y-box protein, MSY2, exists. MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage in male germ cells, linking transcription and translational events [10]. In mouse oocytes, MSY2 has been shown to be essential for cytoplasmic mRNA localization and retention [30] and, in in vitro studies, has been demonstrated to repress translation [31]. Cell-free translation studies with other Y-box proteins expressed in somatic cells have indicated that Y-box proteins can modulate translation, stimulating translation at low concentrations and inhibiting translation at higher concentrations [5–7, 25]. Although our studies indicate that MSY2 plays a major role in stabilizing mRNAs, we cannot exclude that the spermatogenic arrests occur because MSY2 also exerts a stimulatory effect on translation. The movement of mRNAs from RNPs onto polysomes in Msy2-null mice suggests there is a precocious expression of mRNAs normally masked by MSY2 that leads to disruptions in the temporal expression of specific proteins. However, the effects of such early translation would be very transitory because of the instability of mRNAs in the absence of MSY2. This mRNA redistribution may lead to mRNA degradation, as mRNA turnover is often associated with translation. Interestingly, both Msy2-bound and nonbound mRNAs are lost in the male germ cells of mice lacking MSY2. This is likely due, in part, to the increased germ cell loss by apoptosis that occurs as spermatogenesis arrests [11] and to the interconnecting pathways among the many RNA-binding proteins present in the diverse RNA-protein regulatory particles of mammalian cells [32]. On the basis of its abundance and importance in both males and females [11], MSY2 plays a prominent role in posttranscriptional regulation in mammalian germ cells.
ACKNOWLEDGMENTS
We thank Dr. Y. Clermont for his insightful morphological assessments.
FOOTNOTES
1Supported by NIH grant HD 44449 (to N.B.H. and R.M.S.). 
Correspondence: 2 Norman B. Hecht, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, 1310 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160. FAX: 215 573 5408; e-mail: nhecht{at}mail.med.upenn.edu
Received: 27 June 2006.
First decision: 21 July 2006.
Accepted: 5 October 2006.
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