Expression of Mannose 6-Phosphate Receptor Messenger ...

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Sertoli cells were isolated from 17-day-old mice as de- scribed previously ..... de Kretser DM (ed), The Molecular Biology of the Male Reproductive System. ... 245. 22. Escalier D, Gallo J-M, Albert M, Meduri G, Bermudez D, David G, Schrevel J.
BIOLOGY OF REPRODUCTION 50, 429-435 (1994)

Expression of Mannose 6-Phosphate Receptor Messenger Ribonucleic Acids in Mouse Spermatogenic and Sertoli Cellsl DEBORAH A. O'BRIEN, 2'3 JEFFREY E. WELCH,4 KERRY D. FULCHER, 4 and E.M. EDDY 4

The Laboratoriesfor Reproductive Biology, Department of Pediatrics,Department of Cell Biology and Anatomy, University of North Carolinaat Chapel Hill,3 Chapel Hill, NC 27599-7500 Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of EnvironmentalHealth Sciences, National Institutes of Health,4 Research Triangle Park, NC 27709 ABSTRACT Spermatogenic and Sertoli cells isolated from the mouse synthesize different proportions of the two mannose 6-phosphate receptors (MPR) during overnight culture periods (O'Brien et al., Endocrinology 1989; 125:2973). To determine the relative expression of MPR mRNAs in these cells, poly(A) + RNAs were examined by Northern blot analysis using cDNA probes specific for the cation-independent (CI) and cation-dependent (CD) MPRs. A single CI-MPR transcript, -10 kb in size, was present in all tissues and cell types examined. Like the CI-MPR protein, this transcript was more abundant in Sertoli cells than in spermatogenic cells isolated from adult testes. The CD-MPR is the predominant MPR synthesized by pachytene spermatocytes or round spermatids. Multiple CD-MPR transcripts were detected in these cells, including a 2.4-kb CD-MPR mRNA that was indistinguishable from CD-MPR transcripts in somatic tissues and Sertoli cells. Smaller CD-MPR mRNAs of -1.4 and 1.6 kb were prominent in pachytene spermatocytes and round spermatids, respectively, but were faint or undetectable in somatic tissues. These smaller CD-MPR mRNAs did not hybridize with an 0.9-kb restriction fragment derived from the CD-MPR 3' untranslated region (UTR), suggesting that alternate polyadenylation signals are used to produce multiple CD-MPR transcripts in spermatogenic cells. When poly(A) tracts were selectively removed from germ cell RNAs by ribonuclease H treatment, identical 1.3-kb CD-MPR mRNAs were detected in pachytene spermatocytes and round spermatids, indicating that the size difference between the 1.4- and 1.6kb transcripts is due to variations in poly(A) tail length. These alterations in the 3' UTR of the CD-MPR transcripts may affect mRNA stability or translation during spermatogenesis.

INTRODUCTION Mammalian cells generally contain two mannose 6-phosphate receptors (MPR) that are encoded by distinct genes and have diverse characteristics (reviewed in [1, 2]). Although both MPRs are capable of binding to the mannose 6-phosphate (M6P) recognition marker on acid hydrolases and directing these enzymes to lysosomes, the larger cation-independent (CI) MPR is a more efficient mediator of lysosomal targeting [3]. The CI-MPR has an apparent Mr of 275 000 and binds M6P-containing ligands independent of divalent cations, both within the cell and at the cell surface where it mediates endocytosis [1, 2]. Cloning of mammalian CI-MPR genes has revealed that this receptor is identical to the receptor for insulin-like growth factor II (IGF-II) [4, 5], with distinct binding sites for IGF-II and M6P [6]. Recent studies indicate that the CI-MPR is linked to signal transduction mechanisms that respond to IGF-II or M6P-containing growth factors [7, 8]. The smaller cation-dependent (CD) MPR has a subunit Mr of -46 000, requires divalent cations for optimal ligand binding in some species, does not participate in receptor-mediated endocytosis of M6PAccepted October 4, 1993. Received July 6, 1993. 'This work was supported by HD26485 (DA.O.) P30-HD18968 (Laboratories for Reproductive Biology), and CA16086 (UNC Lineberger Comprehensive Cancer Center) grants from the National Institutes of Health. Correspondence: Deborah A. O'Brien, Ph.D., Laboratories for Reproductive Biology, CB# 7500, MacNider Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7500. FAX: (919) 966-1856.

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containing glycoproteins, and does not bind IGF-II [1, 2]. In addition, the CD-MPR mediates the secretion of M6P-containing glycoproteins from tissue culture cells [9]. Mouse genes for the CI-MPR and CD-MPR have been designated Igf2r and M6pr, respectively, in the Encyclopedia of the Mouse Genome maintained at The Jackson Laboratory. Distinct proportions of the CI-MPR and CD-MPR are synthesized by mouse spermatogenic and Sertoli cells, suggesting that these receptors may serve multiple functions in the seminiferous epithelium [10]. Pachytene spermatocytes and round spermatids synthesize mainly the CD-MPR and smaller amounts of the CI-MPR. Sertoli cells synthesize markedly higher levels of the CI-MPR and also secrete at least ten M6P-containing glycoproteins [11]. Both spermatogenic and Sertoli cells endocytose M6P-containing glycoproteins, presumably via the CI-MPR [10, 11]. We hypothesize that these receptors may have roles in mediating germ cell-Sertoli cell interactions and/or in targeting hydrolytic enzymes to the acrosome. A quantitative enzyme-linked immunosorbent assay has been used to determine the molar ratio of CD-MPR to CIMPR in several human cell lines and tissues [12]. Both the quantity of MPRs and the molar ratio were found to vary substantially between tissues and cells, suggesting that the steady-state concentrations of the two MPRs are regulated independently. In the human testis, the CD-MPR/CI-MPR molar ratio was 2.7 [12]; this is consistent with our findings that the CD-MPR is the predominant MPR synthesized by

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germ cells in the later part of meiosis and during spermiogenesis [10]. Our studies of mouse spermatogenic and Sertoli cells provide further evidence that expression of the MPRs is differentially regulated, with distinct cell types in the testis synthesizing markedly different proportions of the two receptors. Both transcriptional and translational control mechanisms have been documented for a variety of genes expressed during spermatogenesis (reviewed in [13,14]). In addition to germ cell-specific and/or stage-specific patterns of expression, unique transcripts that are shorter or longer than their somatic counterparts are common in spermatogenic cells [13-15]. Therefore, we have examined mRNAs for the CI-MPR and CD-MPR in pachytene spermatocytes, spermatids, and Sertoli cells to determine whether mRNA and protein levels are correlated in these cells and whether germ cells and somatic cells express identical transcripts for these receptors. The CI-MPR transcripts detected in these cells were of predicted size and abundance. In contrast, CDMPR mRNA levels were higher than anticipated in Sertoli cells, and spermatogenic cells expressed multiple CD-MPR transcripts that varied with respect to the length of the 3' untranslated region (UTR). Our examination of both the transcription and translation of MPRs in these cells should provide further insights into the regulation of MPR expression in the seminiferous epithelium. MATERIALS AND METHODS Animals Spermatogenic cells and most tissues for RNA preparations were isolated from adult CD-1 mice (Charles River Laboratories, Raleigh, NC). Sertoli cells, thymus, and skin were isolated from juvenile CD-1 mice from the National Institute of Environmental Health Sciences breeding colony, established with micefrom Charles River. Procedures involving animals were approved in advance by the appropriate Institutional Animal Care and Use Committee. Isolation of Spermatogenic and Sertoli Cells Cells from the seminiferous epithelium were isolated by sequential enzymatic dissociation of adult testes, yielding a single-cell suspension referred to here as "mixed germ cells" and consisting predominantly of pachytene spermatocytes and spermatids [16,17]. Pachytene spermatocytes, round spermatids, and condensing spermatids were purified from adult seminiferous cell suspensions by unit gravity sedimentation [16,17]. Purities of pachytene spermatocytes and round spermatids (steps 1-8) exceeded 90% in all experiments. Condensing spermatid populations contained 3040% nucleated spermatids (steps 9-16) plus cytoplasts derived primarily from these late haploid-phase cells. Sertoli cells were isolated from 17-day-old mice as described previously [10, 11]. Briefly, seminiferous cell sus-

pensions were prepared by sequential enzymatic dissociation and cultured in TK medium, a serum-free, growth-factorsupplemented MEM [10]. Ovine FSH (100 ng/ml; NHPP, Baltimore, MD) was added to the medium in some experiments. After two days in culture, spermatogenic cells were removed by hypotonic lysis [18]. Sertoli cells prepared by this method have typical morphological features and secrete several of the same glycoproteins as rat Sertoli cells [10, 11]. RNA Isolation and Northern Blot Analysis Total RNA were isolated from cells or tissues quick-frozen in liquid nitrogen, and their integrity was verified as described previously [19]. Poly(A) + RNA was isolated from cells or tissues through use of the FastTrack oligo(dT)-cellulose kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions. RNA samples were separated by electrophoresis through denaturing gels containing formaldehyde and were transferred to nylon membranes (Nytran from Schleicher and Schuell, Keene, NH or GeneScreen Plus from Du Pont NEN Research Products, Boston, MA) [19]. Estimates of transcript size were made by comparison to an 0.24-9.5-kb RNA stan32 dard ladder (Life Technologies, Grand Island, NY). P-Labeled probes were prepared by random priming using a T7 DNA polymerase kit (Stratagene, La Jolla, CA or Pharmacia LKB Biotechnology, Piscataway, NJ). Northern blotting was performed essentially as described in previous studies [19], except that the final stringent washes of the membranes were conducted at 60 0C. In some experiments, the SDS concentration in the hybridization and wash solutions was increased from 0.1% to 1% and the stringent wash times were extended (two 45-min washes) as recommended by the manufacturer of GeneScreen Plus. Complementary DNA probes used in this study were kindly provided by Dr. Peter Lobel (Center for Advanced Biotechnology and Medicine, Piscataway, NJ). They include a 5-kb EcoRI fragment of the mouse CI-MPR cDNA; an 0.85kb HindIII-Pst I restriction fragment from the bovine CD-MPR cDNA; and 1.4- and 0.9-kb EcoRI restriction fragments from the mouse CD-MPR cDNA. Removal of Poly(A) Tails with Ribonuclease H Poly(A) + RNA samples (10 ig each) were hybridized with oligo(dT) and digested with ribonuclease H (RNase H; Life Technologies) as described previously [20]. Control RNA samples were incubated under identical conditions except that RNase H and oligo(dT) were omitted. RESULTS Spermatogenic and Sertoli Cells Synthesize a Single CI-MPR mRNA Northern blots containing poly(A) + RNA from mouse tissues and cells isolated from the testis were hybridized with

M6P RECEPTOR mRNAs IN SERTOLI AND GERM CELLS

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FIG. 2. Northern blot analysis of CD-MPR mRNA expression in several mouse tissues. When Northern blots were hybridized with a 1.4-kb mouse CD-MPR cDNA probe containing the coding region, a 2.4-kb mRNA (arrows) was the predominant transcript detected in brain (1), skin (2), thymus (3), stomach (4), skeletal muscle (5), spleen (6), liver (7), ovary (8), uterus (9), seminal vesicle (10), and epididymis (11). Fifteen micrograms of total RNA was loaded per lane and the Northern blot was exposed for two days.

Poly(A) + RNA was isolated from Sertoli cells cultured for 5-6 days with or without 100 ng/ml FSH. The inclusion of FSH in the culture medium did not alter the steady-state levels of the CI-MPR transcript detected in these cells (Fig. 1).

FIG. 1. Expression of the CI-MPR mRNA in cells isolated from the mouse seminiferous epithelium. When Northern blots were hybridized with a 5-kb mouse CI-MPR cDNA probe, a single transcript (-10 kb, arrows) was detected in poly(A)+ RNAs (9 iLg/lane) from testis (T), pachytene spermatocytes (P), round spermatids (R), condensing spermatids (C), Sertoli cells (S), and brain (B). Similar levels of the CI-MPR transcript were present in Sertoli cells cultured 100 ng/ml FSH. Messenger RNAs were visualized by autoradiography after a three-day exposure of the Northern blot.

Pachytene Spermatocytes and Round Spermatids Synthesize Multiple CD-MPR mRNAs

a 5-kb mouse CI-MPR cDNA probe; results are shown in Figure 1. A single CI-MPR transcript, comparable in size to the 9.5-kb transcript reported for calf liver [5], was detected in testis, isolated pachytene spermatocytes, round spermatids, and condensing spermatids. The CI-MPR mRNA in spermatogenic cells was indistinguishable from the more abundant CI-MPR mRNA in somatic cells and tissues including Sertoli cells, brain (Fig. 1), and liver (not shown). This transcript was not detected on Northern blots containing 10-15 ig total RNA from spermatogenic cells; this is consistent with the low steady-state levels of CI-MPR protein in pachytene spermatocytes and round spermatids [10]. Similar proportions of the CI-MPR transcript were observed on three different Northern blots prepared with poly(A) + RNAs from spermatogenic and Sertoli cells isolated in three separate experiments.

Northern blots containing total RNA from several mouse tissues were hybridized with bovine (not shown) or mouse CD-MPR cDNA probes (Fig. 2). A 2.4-kb CD-MPR transcript was detected with either probe in brain, skin, thymus, stomach, skeletal muscle, spleen, liver, ovary, uterus, seminal vesicle, and epididymis. Relatively small amounts of this mRNA were apparent on multiple Northern blots using two different preparations of skeletal muscle RNA, suggesting that the CD-MPR transcript is not abundant in this tissue. The 2.4-kb CD-MPR mRNA also was prominent on Northern blots of poly(A) + RNA from Sertoli cells cultured with or without 100 ng/ml FSH (Fig. 3), even though these cells apparently synthesize very little CD-MPR protein [10]. Spermatogenic cells displayed multiple CD-MPR mRNAs when Northern blots of total or poly(A)+ RA were hybridized with the 0.85-kb bovine CD-MPR probe (Fig. 3). The 2.4-kb CD-MPR mRNA and smaller transcripts (-1.41.6 kb) were equally prominent in poly(A) + RNA from the testis, mixed germ cells, pachytene spermatocytes, and round

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FIG. 3. CD-MPR mRNA expression in cells isolated from the mouse + seminiferous epithelium. This Northern blot of poly(A) RNAs (10 ig/lane) probe. As in other cDNA was hybridized with an 0.85-kb bovine CD-MPR tissues (Fig. 2), a 2.4-kb mRNA was present in testis (T), mixed germ cells (G), pachytene spermatocytes (P), round spermatids (R), and Sertoli cells (S) cultured ± 100 ng/ml FSH. CD-MPR transcripts were faint or undetectable in condensing spermatids (C). In addition to the 2.4-kb transcript, smaller CD-MPR mRNAs were equally prominent in testis, mixed germ cells, pachytene spermatocytes, and round spermatids. The 1.4-kb mRNA detected in pachytene spermatocytes was consistently smaller than the 1.6-kb mRNA seen in round spermatids. This Northern blot was exposed for seven days.

spermatids. Although transcripts for the CI-MPR (Fig. 1) and for actin (not shown) were present in condensing spermatids, CD-MPR mRNA were faint or not detected in these cells. The smaller CD-MPR transcripts were resolved as two distinct mRNAs in separated spermatogenic cells. Pachytene spermatocytes had a 1.4-kb transcript, while round spermatids had a slightly longer 1.6-kb transcript (Fig. 3). Smaller CD-MPR mRNAs in Germ Cells Lack Part of the 3' UntranslatedRegion A mouse CD-MPR cDNA was digested with EcoRI to produce restriction fragments of 1.4 and 0.9 kb, as diagrammed in Figure 4a. Northern blots containing total RNA from mixed germ cells, pachytene spermatocytes, and round spermatids 32 were hybridized with these two P-labeled restriction frag-

scripts detected with the bovine probe (see Fig. 3) in mixed germ cells (G), pachytene spermatocytes (P), and round spermatids (R). Only the 2.4-kb mRNA hybridized with the 0.9-kb fragment from the 3' UTR (c). The Northern blots contained 15 jg/lane total RNA. Blot b was exposed for two days and blot c was exposed for ten days.

ments. The 1.4-kb mouse CD-MPR fragment, which contains the coding region, hybridized to the same mRNAs detected in poly(A) + RNAs with the bovine probe (Fig. 4b; compare with Fig. 3). In contrast, the 0.9-kb fragment derived from the 3' UTR hybridized only to the 2.4-kb mRNA (Fig. 4c). Germ Cell CD-MPR mRNAs Have Stage-Specific Variations in Poly(A) + Tails The smaller CD-MPR mRNAs detected in pachytene spermatocytes (1.4 kb) and round spermatids (1.6 kb) differed

M6P RECEPTOR mRNAs IN SERTOLI AND GERM CELLS

FIG. 5. Northern blot analysis of CD-MPR mRNAs following RNase H treatment to remove poly(A) tails. The Northern blot containing both control and RNase H-treated poly(A)+ RNAs (10 pg/sample) from testis (T), pachytene spermatocytes (P), and round spermatids (R) was hybridized with the 1.4-kb mouse CD-MPR probe. After RNase digestion, the 2.4-kb transcript was converted to -2.3 kb, and both the 1.4-kb mRNA in pachytene spermatocytes and the 1.6-kb mRNA in round spermatids were reduced in size to -1.3 kb. The blot was exposed for seven days.

in size by -200 nucleotides. To determine whether this difference resulted from variations in poly(A) tail length, mRNAs from testes, pachytene spermatocytes, and round spermatids were hybridized with oligo(dT) and digested with RNase H to remove poly(A) tracts. The treated mRNA were compared to untreated poly(~)+RNAs on Northern blots hybridized with the 1.4kb mouse CD-MPR cDNA probe (Fig. 5). After RNase H treatment, transcripts of identical size (-2.3 and 1.3 kb) were detected in pachytene spermatocytes and round spermatids. Therefore, the 1.4- and 1.6-kb CD-MPR transcripts present in these spermatogenic cells have poly(~) tails of different lengths. DISCUSSION

Recent studies in this laboratory have examined MPRs as potential mediators of germ cell-Sertoli cell interactions and/ or acrosomal targeting. These roles would require both temporal and spatial regulation of MPR expression in the seminiferous epithelium. Receptors used selectively for acrosomal targeting are likely to accumulate during late meiotic or early post-meiotic phases, when the synthesis of acrosin

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and other acrosomal constituents is initiated [21-231. Similarly, signalling between germ cells and ~ertolicells may be modulated by stage-specific variations in cell surface receptors or their effectors. To assess the differential expression of the CI-MPR and CD-MPR in the seminiferous epithelium, we have examined MPR mRNAs and proteins [lo] in Sertoli cells and germ cells at defined stages of spermatogenesis. The MPR are not abundant proteins in tissues isolated from adult animals [I, 121 or in cells isolated from the adult mouse testis [lo]. Consequently, our studies have employed up to 10-pg samples of PO~Y(A)+ RNA to ensure detection of the MPR mRNAs. When MPR were isolated by affinity chromatography from metabolically labeled mouse testis cells in an earlier study, the IGF-II/CI-MPRwas the predominant MPR synthesized by Sertoli cells. Smaller amounts of this receptor were synthesized by pachytene spermatocytes and round spermatids [lo]. The CI-MPR mRNAs detected in the present study reflect a similar distribution, with higher transcript levels in Sertoli cells compared to pachytene spermatocytes or round spermatids. In addition, CI-MPR transcripts persist in condensing spermatids, suggesting that synthesis of this receptor may continue during the late haploid phase of spermatogenesis when transcription no longer occurs. A single -10-kb mRNA for the CI-MPR was identified in all cell types and tissues examined. Examination of CD-MPR mRNAs in germ cells and Sertoli cells revealed several features that were not predicted from previous studies of MPR proteins. Although very little CDMPR protein was isolated from Sertoli cells [lo], a 2.4-kb CD-MPR mRNA, comparable to the predominant CD-MPR transcript in several somatic tissues, was relatively abundant in these cells. It is unlikely that this difference between protein and mRNA levels is an artifact of cell culture, since the Sertoli cells used for each study were isolated and cultured under identical conditions. The inclusion of FSH (100 ng/ ml) in the Sertoli cell culture medium did not alter transcript levels for either the CD- or CI-MPR. Recently, a CDMPR pseudogene has been identified in the mouse with 5' sequences that confer promoter activity in vitro [24].In preliminary studies using an oligonucleotide probe specific to the pseudogene sequence, evidence of a transcript derived from the pseudogene was not detected in mouse Sertoli cells. Because two CD-MPR isoforms with different affinities for M6P have been isolated from bovine testis [25],we also have considered the possibility that CD-MPR in Sertoli cells were missed in our earlier study using phosphomannan affinity chromatography to isolate MPR proteins. However, comparison of our isolation methods [lo] with those used for bovine testis MPRs [25] suggests that receptors with lower &nity for M6P would still have been detected in our mouse Sertoli cell preparations. Further studies will be required to clarlfy the apparent post-transcriptional regulation of CDMPR expression in Sertoli cells.

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The CD-MPR is the predominant MPR synthesized by pachytene spermatocytes and round spermatids in culture [10]. Unlike CI-MPR transcripts, CD-MPR mRNA were sufficiently abundant in these cells to be detected in total RNA samples. However, CD-MPR transcripts were undetected in condensing spermatids, even though the less abundant CIMPR mRNAs were retained in these late haploid cells. Both pachytene spermatocytes and round spermatids contained approximately equal amounts of two CD-MPR mRNAs. The larger CD-MPR transcript in spermatogenic cells was indistinguishable from the 2.4-kb mRNA detected in somatic cells and tissues. Since this transcript is more abundant in Sertoli cells, it is possible that minor Sertoli cell contamination (< 10%) contributed to the 2.4-kb CD-MPR mRNA detected in pachytene spermatocytes. However, the 2.4-kb transcript was equally prominent in mixed germ cells (< 2% Sertoli cells) and in round spermatids, a germ cell population that generally does not contain Sertoli cells. The smaller 1.4- and 1.6-kb mRNAs in pachytene spermatocytes and round spermatids, respectively, contain the CD-MPR coding region but not the full 3' UTR. Minor transcripts of similar size have been detected in bovine liver [26] and in several mouse tissues when Northern blots contained 20 g total RNA [24] or were exposed for longer periods. As in the current study, the smaller CD-MPR transcripts in bovine liver did not hybridize with a probe derived from the 3' end of the cDNA [26]. It is likely that the the two CD-MPR mRNAs result from the use of alternate polyadenylation signals that have been identified in the nucleotide sequence of both the bovine [26] and mouse [24] CD-MPR. In the mouse sequence, the polyadenylation signals are located 217 and 1210 bp from the termination codon; this would account for the -1-kb difference seen between the germ cell transcripts. Other truncated mRNAs that result from the use of alternate polyadenylation signals have been identified in spermatogenic cells. An unusual upstream polyadenylation signal is used to produce the shortened transcript for the c-abl proto-oncogene that appears in round spermatids [2729]. This shortened c-abl mRNA persists in condensing spermatids and appears to be more stable than the longer transcript found in somatic cells [27]. Similarly, shorter mRNAs for 31 ,4-galactosyltransferase that appear in round spermatids have been attributed to the preferential use of an upstream polyadenylation signal [30]. Further studies are needed to determine the regulation and consequences of use of alternate polyadenylation signals in spermatogenic cells. An additional variation in CD-MPR transcript size is apparent when pachytene spermatocyte and round spermatid mRNAs are compared. The 1.6-kb mRNA present in round spermatids has a poly(A) tail that is -200 nucleotides longer than the 1.4-kb mRNA in pachytene spermatocytes. Transcripts for cytochrome cT and LDH-C, which both encode germ cell-specific isoforms, exhibit similar variations in

poly(A) tail length [31-33]. In both cases, the longer transcripts are preferentially associated with polysomes [31, 33], suggesting that poly(A) tail length may regulate translation. It has not yet been determined whether specific CD-MPR mRNAs in spermatogenic cells have different stabilities or are associated with polysomes. In both Xenopus and mouse oocytes, AU-rich sequences upstream from the polyadenylation signal have been identified as cytoplasmic polyadenylation elements (CPE) [3436]. These sequences direct the cytoplasmic elongation of the poly(A) tract, which is both necessary and sufficient to stimulate the translation of specific mRNAs during meiotic maturation. Interestingly, a CPE sequence (UUUUUAU) is present in the mouse CD-MPR transcript 30 nucleotides upstream from the first polyadenylation signal [24]. Similar AUrich sequences are also found near the poly(A) addition signals in cytochrome CT [31] and LDH-C transcripts [37, 38]. Further studies of spermatogenic cell mRNAs with longer poly(A) tails are needed to determine whether these mRNAs result from new transcription or from cytoplasmic polyadenylation of existing mRNA. Our studies have provided evidence for the differential expression of CI-MPR and CD-MPR mRNAs and proteins in Sertoli cells and germ cells at different stages of spermatogenesis. Findings from Northern analysis and the structural features of the CD-MPR mRNAs, in particular, suggest that expression of these receptors may be regulated by multiple mechanisms in the seminiferous epithelium. We are currently extending our studies of the MPR to earlier stages of spermatogenesis, using both isolated cells and testicular sections in which the cellular associations between germ cells and Sertoli cells are retained. These studies, together with functional assays, should provide further insights into the regulation of MPR expression during spermatogenesis and the roles of these receptors in the testis. ACKNOWLEDGMENTS The authors thank Drs. P. Lobel and J.E. Pintar for providing cDNA probes for the mannose 6-phosphate receptors; Dr. C.A Gabel for his advice and his support of this project; A. Taylor, Jr. for excellent technical assistance; M.R. Hollowbush for assistance in manuscript preparation; and NIDDK and the National Hormone and Pituitary Program, University of Maryland, Baltimore, MD for rat growth hormone and ovine FSH.

REFERENCES 1. Komfeld S. Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors. Annu Rev Biochem 1992; 61:307-330. 2. von Figura K. Molecular recognition and targeting of lysosomal proteins. Curr Opin Cell Biol 1991; 3:642-646. 3. Watanabe H, Grubb JH, Sly WS. The overexpressed human 46-kDa mannose 6phosphate receptor mediates endocytosis and sorting of 3-glucuronidase. Proc Natl Acad Sci USA 1990; 87:8036-8040. 4. Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WJ. Insulin-like growth factor II receptor as a multifunctional binding protein. Nature 1989; 329:301-307. 5. Lobel P, Dahms NM, BreitmeyerJ, ChirgwinJM, Kornfeld S. Cloning of the bovine 215-kDa cation-independent mannose 6-phosphate receptor. Proc Natl Acad Sci USA 1987; 84:2233-2237.

M6P RECEPTOR mRNAs IN SERTOLI AND GERM CELLS 6. MacDonald RG, Pfeffer SR, Coussens L, Tepper MA, Brocklebank CM, Mole JE, Anderson JK, Chen E, Czech MP, Ullrich A. A single receptor binds both insulinlike growth factor II and mannose-6-phosphate. Science 1988; 239:1134-1139. 7. Okamoto T, Nishimoto I, Murayama Y, Ohkuni Y, Ogata E. Insulin-like growth factor-ll/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II. Biochem Biophys Res Commun 1990; 168:1201-1210. 8. Rogers SA, Purchio AF, Hammerman MR Mannose 6-phosphate-containing peptides activate phospholipase C in proximal tubular basolateral membranes from canine kidney. J Biol Chem 1990; 265:9722-9727. 9. Chao H H-J, Waheed A,Pohlmann R, Hille A, von Figura K. Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes. EMBO J 1990; 9:3507-3513. 10. O'Brien DA, Gabel CA, Rockett DL, Eddy EM. Receptor-mediated endocytosis and differential synthesis of mannose 6-phosphate receptors in isolated spermatogenic and Sertoli cells. Endocrinology 1989; 125:2973-2984. 11. O'Brien DA, Gabel CA, Eddy EM. Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells. Biol Reprod; 49:1055-1065. 12. WenkJ, Hille A, von Figura K. Quantitation of M, 46000 and Mr 300 000 mannose 6-phosphate receptors in human cells and tissues. Biochem Int 1991; 23:723732. 13. Eddy EM, Welch JE, O'Brien DA. Gene expression during spermatogenesis. In: de Kretser DM (ed), The Molecular Biology of the Male Reproductive System. New York: Academic Press Inc.; 1993: 181-232. 14. Hecht NB. Gene expression during male germ cell development. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. Oxford: Oxford University Press; 1992: 464-503. 15. Wolgemuth DJ, Watrin F. List of cloned mouse genes with unique expression patterns during spermatogenesis. Mammalian Genome 1991; 1:283-288. 16. Romrell LJ, Bellv AR, Fawcett DW. Separation of mouse spermatogenic cells by sedimentation velocity. Dev Biol 1976; 49:119-131. 17. Bellve AR, Millette CF, Bhatnagar YM, O'Brien DA. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem 1977; 25:480-494. 18. Galdieri M, Ziparo E, Palombi F, Russo MA, Stefanini M. Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 1981; 5:249254. 19. Welch JE, Schatte EC, O'Brien DA, Eddy EM. Expression of a glyceraldehyde 3phosphate dehydrogenase gene specific to mouse spermatogenic cells. Biol Reprod 1992; 46:869-878. 20. Fulcher KD, Welch JE, Davis CM, O'Brien DA, Eddy EM. Characterization of laminin receptor messenger ribonucleic acid and protein expression in mouse spermatogenic cells. Biol Reprod 1993; 48:674-682. 21. Kashiwabara S, Arai, Y, Kodaira K, Baba T. Acrosin biosynthesis in meiotic and postmeiotic spermatogenic cells. Biochem Biophys Res Commun 1990; 173:240245. 22. Escalier D, Gallo J-M, Albert M, Meduri G, Bermudez D, David G, Schrevel J. Human acrosome biogenesis: immunodetection of proacrosin in primary sper-

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24.

25.

26.

27.

28.

29. 30.

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37. 38.

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matocytes and of its partitioning pattern during meiosis. Development 1991; 113:779-788. Anakwe 0OO, Gerton GL. Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis. Biol Reprod 1990; 42:317-328. Ludwig T, Riither U, Metzger R, Copeland NG, Jenkins NA, Lobel P, Hoflack B. Gene and pseudogene of the mouse cation-dependent mannose 6-phosphate receptor. J Biol Chem 1992; 267:12211-12219. Li M, Jourdian GW. Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose 6-phosphate receptor from bovine testis. J Biol Chem 1991; 266:17621-17630. Dahms NM, Lobel P, Breitmeyer J, Chirgwin JM, Kornfeld S. 46 kd mannose 6phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor. Cell 1987; 50:181-192. Meijer D, Hermans A, von Lindern M, van Agthoven T, de Klein A, Mackenbach P, Grootegoed A, Talarico D, Valle GD, Grosveld G. Molecular characterization of the testis specific cabl mRNA in mouse. EMBO J 1987; 6:4041-4048. Oppi C, Shore SK, Reddy EP. Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation. Proc Natl Acad Sci USA 1987; 84:8200-8204. Ponzetto C, Wolgemuth DJ. Haploid expression of a unique c-abl transcript in the mouse male germ line. Mol Cell Biol 1985; 5:1791-1794. Shaper NL, Wright WW, Shaper JH. Murine 1,4-galactosyltransferase: both the amounts and structure of the mRNA are regulated during spermatogenesis. Proc Natl Acad Sci USA 1990; 87:791-795. Hake LE, Alcivar AA, Hecht NB. Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse. Development 1990; 110:249-257. Fujimoto H, Erickson RP, Tone S. Changes in polyadenylation of lactate dehydrogenase-X mRNA during spermatogenesis in mice. Mol Reprod Dev 1988; 1:2734. Alcivar AA, Trasler JM, Hake LE, Salehi-Ashtiani K, Goldberg E, Hecht NB. DNA methylation and expression of the genes coding for lactate dehydrogenases A and C during rodent spermatogenesis. Biol Reprod 1991; 44:527-535. Vassalli J-D, Huarte J, Belin D, Gubler P, Vassalli A, O'Connell ML, Parton LA, Rickles RJ,Strickland S. Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev 1989; 3:2163-2171. Richter JD. Translational control during early development. Bioessays 1991; 13:179183. Salles FJ, Darrow AL, O'Connell ML, Strickland S. Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation. Genes Dev 1992; 6:12021212. Tanaka S, Fujimoto H. A postmeiotically expressed clone encodes lactate dehydrogenase isozyme X. Biochem Biophys Res Commun 1986; 136:760-766. Sakai I, Sharief FS, Li S S-L. Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse. Biochem J 1987; 242:619-622.