Glycobiology Program, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037, USA
Received on January 29, 2002; revised on April 3, 2002; accepted on April 11, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: congenital disorder of glycosylation/KDN/Mpi/phosphomannose isomerase/testis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Immunohistochemistry
To further determine the PMI expression pattern, we immunohistochemically localized the PMI antigen using the purified anti-mouse PMI antibody. Most cell types showed diffuse cytosolic staining. The staining was specific, because sections treated with purified pre-immune IgG, yeast PMI preadsorbed antibody, or secondary antibody alone did not give positive staining. As expected, the strongest signal was observed in testis and the weakest in liver. Round spermatids and residual bodies showed very strong immunoreactivity. Pachytene spermatocytes showed moderate reactivity, whereas spermatogonia, primary spermatocytes, spermatozoa, and Sertoli cells, were faint even at high concentration of antibody (Figure 5).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interestingly, we found that mouse Mpi mRNA is alternatively spliced in the 3'-UTR region. The 3'-UTR of mRNAs play important roles in regulating their translation, allowing them to be used at different times and in specific subcellular locations (Conne et al., 2000). Such uncoupling between transcription and translation is required, for example, in gametogenesis, embryogenesis, and for the targeting of specific mRNA into neuronal dendrites (Wu et al., 1997
; Conne et al., 2000
). Furthermore, mouse Mpi mRNA contains putative H and Y elements which were first identified in protamine genes (Wu et al., 1997
). These elements bind to TB-RBP, resulting in mRNA transport to microtubules and arrested translation (Hecht, 2000
). The level of mRNAs in pachytene spermatocytes and round spermatids is comparable. However, the level of protein in pachytene spermatocytes is much lower than it is in round spermatids, suggesting that PMI translation is delayed during spermatogenesis. PMI has a stage-specific expression pattern and may have a stage-specific role during spermatogenesis. We speculate that Mpi expression is regulated not only at the transcription level by the TASS-1 element but also at the translational level by H and Y elements and the 3'-UTR.
Differential expression of PMI activity, especially in the testis, suggests that it may be needed either to supply precursors for glycosylation or for catabolism. It seems unlikely that increased PMI activity converting Man-6-P to Fru-6-P would make a significant contribution to glycolysis. The very sharp rise in PMI antigen in round spermatids, however, suggests a particularly important function at that stage. One possibility for this increase is that more Man-6-P is needed for synthesis and accumulation of KDN, which has been found in large amounts in rainbow trout testis (Angata et al., 1999a) and in small amounts in mouse melanoma B16 and African green monkey kidney COS-7 cell lines (Angata et al., 1999b
). In the latter, KDN synthesis is stimulated when mannose is provided in the culture medium (Angata et al., 1999b
). We found that bound KDN in testis is nearly sixfold higher than in liver when normalized to protein. However, we do not know if increased KDN synthesis occurs in the round spermatids. Another possibility is that Man-6-P is needed for N-linked oligosaccharide synthesis by round spermatids engaged in acrosome formation and packaging of degradative enzymes into this specialized lysosome (Tulsiani et al., 1998
). Lysosomal enzymes typically have glycan chains of the high-mannose type, rather than complex type. Recent results (Chayko and Orgebin-Christ, 2000
) suggest that the targeting of several acrosomal proteins does not require either of the Man-6-P receptors.
Increased PMI activity in round spermatids may also be needed to ensure sufficient Man-6-P production from Fru-6-P generated through gluconeogenesis (Figure 1). This may be necessary because the glycolytic enzymes, specifically hexokinase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase, known to be rate-limiting enzymes in glycolysis, are very low in round spermatids (Nakamura et al., 1982). In fact, glucose is toxic in round spermatids because it depletes ATP supplies (Nakamura et al., 1986
). Lactate or pyruvate are the preferred sources for energy (Jutte et al., 1981
; Mita and Hall, 1982
; Grootegoed et al., 1984
) and can be catabolized through the tricarboxylic acid cycle. Alternatively, they can yield Fru-6-P through the gluconeogenesis pathway. Labeling experiments using isolated guinea pig spermatocytes and spermatids failed to show [3H] mannose incorporation into glycoconjugates, whereas other monosaccharides showed substantial incorporation (Joshi et al., 1990
). The reason radiolabeled mannose was not incorporated is unknown, but high PMI activity would favor diverting [2-3H] Man-6-P into catabolism through the hexose monophosphate pathway (HMP pathway), leaving less for incorporation into glycoproteins.
The HMP pathway begins with glucose-6-P dehydrogenase (G6PDH) which generates NADPH that is used for reduction of highly reactive and potentially damaging lipid hydroperoxides (Sikka, 1996, 2001). There is evidence that human sperm may be limiting in G6PDH (Storey et al., 1998
). Postmeiotic male germ cells contain NADPH-requiring lanosterol 14
-demethylase (CYP51), which is required for sterol synthesis and thought to be involved in signaling (Stromstedt et al., 1998
; Rozman and Waterman, 1998
). On the other hand, reactive oxygen species are required for fertilization (Aitken et al., 1995
). Therefore, it may be important to balance the amount of highly reactive species at different stages of spermatogenesis. High expression of PMI, G6PDH (Peter et al., 1997
), and CYP51 (Rozman and Waterman, 1998
) in the same population of cells may balance the needs of the HMP shunt and protein glycosylation. Additional studies including isolation of round spermatids will be needed to clarify the role of PMI in testis. These studies may be important for determining whether male CDG-Ib patients may have impaired fertility.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of mouse Mpi cDNA
To isolate mouse Mpi homolog, we searched EST database with human PMI protein sequence (accession: CAA53657). With mouse Mpi genespecific primers designed according to EST sequences, the forward primers 5'-ACGTTCCTGAAGCCGGGT (accession AU080858), the reverse primer, 5'-CTGTATCCTTCAACACAAGTG (accession AK003289), RT-PCR was performed to amplify mouse Mpi cDNA. Two independent PCR products were cloned and sequenced. Using mouse testis Marathon-Ready cDNA as a template, RACEPCR was used to confirm the sequences of 5' and 3' flanking regions of the open reading frame (ORF).
Expression and alternative splicing assay of mouse Mpi with RT-PCR
Mouse (five male Sprague-Dawley mice C57BL/6) mRNAs from various tissues were isolated with RNeasy Mini Kit following the instructions of manufacturer. Three primers were designed according to mouse Mpi cDNA sequence: one forward 5'-TACGTTCCTGAAGCCGGG, and two reverse primers, 5'-CTACAGCAGACAGCAGGC for the coding region; 5'-TACGTTCCT GAAGCCGGG for the full-length cDNA. A set of primers against mouse ß-actin cDNA was used as RT-PCR control (the forward primer 5'-TTTGAGACCTTCAACACCCC, the reverse primer 5'-TGATCCACATCTGCTGGAAG according to cDNA; accession X03765). RT-PCR was performed with Superscript one-step RT-PCR kit and the conditions were 50°C for 30 min, 94°C for 1 min, 30 (ß-actin) or 35 (Mpi) cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 2 min. The RT-PCR products were separated with agarose gel and visualized with ethidium bromide. The products from testis were eluted from agarose gels and confirmed by sequencing.
Organization of mouse Mpi gene
The mouse Mpi genomic clones were isolated from the 129/SvJ phage library, using mouse Mpi cDNA as a probe. A 12-kb genomic DNA fragment that covers exon 3 to exon 8 was isolated and subcloned into pBluescript KS vector at NotI site. Restriction endonuclease digestion and Southern blot analysis using 32P-labeled oligonucleotides corresponding to mouse Mpi cDNA determined exonintron boundaries. Exonintron boundaries of exon 1 to intron 3 were defined by sequencing of PCR products of genomic DNA, using 129/SvJ mouse genomic DNA library as template. Six primers were used: primer 1, 5'-TTTCAGGCAACTGGGATGGC-3'; primer 2, 5'-GCCACTTCACTTTTGGAGC-3'; primer 3, 5'-CTTTCCTGTGTGGTGCA-3'; primer 4, 5'-GTTAGGGTGTGCCTGGATAG-3'; primer 5, 5'-GCTCAAAGGTCAAAAACAC C-3'; and primer 6, 5'-ATCAGCAACTGGAACTC-3'.
Construction of prokaryotic expression plasmids
The full length cDNA encoding the entire ORF of mouse Mpi was expressed as a glutathione S-transferase (GST) fusion protein with a Factor Xa recognition site using the pGEX-5X-2 system at EcoRI site (Pharmacia Biotech, Inc., Uppsala, Sweden). This generated the sequence encoding an N-terminal GST tag followed by a Factor Xa recognition site, followed by the Mpi gene under the control of the inducible lac promoter.
Preparation and purification of recombinant enzyme
The pGEX-5X-2 plasmid encoding PMI-GST was transformed into E. coli strain BL21 (DE3) as described (Sambrook et al., 1989). A single colony of transformed cells was picked and cultured at 37°C overnight in Luria Bertani (LB) medium supplemented with ampicillin. The colony was propagated by incubating 100 µl of the transformed cells in 5 ml LB medium. The expression was induced by adding isopropyl-1-thio-ß-D-galactopyranoside to a final concentration of 0.1 mM. After 3 h at 37°C, the cells were harvested and frozen at 20°C or processed immediately.
For the purification of the recombinant enzyme, the bacterial cells were suspended in cold phosphate buffered saline (PBS), pH 7.2, containing 1 µg/ml each of aprotinin, leupeptin, and pepstatin A and 1% Triton X-100; sonicated; and spun at 15,000 x g for 20 min at 4°C. The supernatant was applied to glutathione Sepharose 4B column (bed volume 8 ml) equilibrated with PBS. After washing with PBS/1% Triton X-100 and PBS, the fusion protein was eluted with 5 mM glutathione followed by second elution with 3 M NaCl, 6 M urea in PBS. The A280nm peak was pooled; dialyzed against 10 mM CaCl2, 100 mM NaCl in 50 mM TrisHCl, pH 8.0; and digested with 40 U of Factor Xa for 60 h at room temperature. The digest was reapplied over glutathione Sepharose 4B to remove the GST tag, and the run-through was pooled as enzyme source. Purity of the enzyme was confirmed by SDSPAGE and enzyme assays confirmed that the expressed protein had PMI activity.
Preparation of PMI antibodies
The purified recombinant mouse PMI was used as antigen in New Zealand rabbits. The animals were boosted repeatedly with 50 µg antigen. The rabbit antisera were partially purified by the caprylic acid method, followed by Protein GSepharose column (Harlow and Lane, 1988). The GST-Affigel-10 column eluate was affinity purified on a yeast PMI-Affigel-10 column; washed with 20 volumes of 0.1 M TrisHCl, pH 8.0; eluted with 0.1 M glycine-HCl, pH 3.0; and neutralized with 1 M TrisHCl, pH 8.0. The A280nm peak was pooled, concentrated to 750 µg/ml, and stored frozen at 20°C and used for the experiments.
PMI assay
PMI activity was measured in 100,000 x g supernatant of freshly homogenized tissues using a coupled assay (Westphal et al., 2001). In brief, the assay was carried out in 50 mM HEPES, pH 7.1, containing 50 µg protein, 5 mM MgCl2, 0.25 mM NADP, and 1 U/ml each of PGI and G6PDH, and the NADPH produced was measured at A 340nm.
Western blot
Western blots were carried out as described (Towbin et al., 1979). In short, 10 µg of 100,000 x g supernatant of mouse tissue proteins were resolved on 8% SDSPAGE and transferred to the nitrocellulose, incubated with purified anti-mouse PMI (0.2 µg/ml) and anti-rabbit IgG-alkaline phosphatase and detected with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. The intensity of the immunoreactivity was integrated using the NIH Image program.
Native gel electrophoresis zymograms
The zymograms were performed as described with slight modifications (Hendriksen et al., 1997). Briefly, the frozen tissues were homogenized in PBS, pH 7.2, containing protease inhibitors (Boehringer Mannheim, Germany, cat. no. 1697498) and 0.2 % (v/v) Triton X-100 and ultracentrifuged for 60 min at 100,000 x g. The supernatants were used as an enzyme source. The native PAGE was run at 5% stacking gel and 7.5% separating gel. Aliquots (20 µg) of protein were applied and electrophoresed at constant current (30 mA) at 10°C. The gels were stained for PMI activity with a mixture containing 50 mM TrisHCl, pH 7.2, containing 10 mM Man-6-P, 0.25 mM NADP, 0.057 mM phenazine methosulfate, and 0.21 mM nitroblue tetrazolium for 2 h at 37°C in the dark.
Immunohistochemistry
Organs from 8-week-old male Sprague-Dawley mice (C57Bl/6) were removed (Davis and Freeze, 2001), fixed in 4% paraformaldehyde overnight at 4°C and transferred to PBS. Organs were embedded in paraffin blocks and 5-µm-thick sections were made. The sections were dewaxed and treated with 0.25% trypsin for 30 min at 37°C to unmask the cryptic epitope, washed in 0.2% Tween 20/PBS (PBST), blocked with 5% goat serum, and treated with purified anti-mouse PMI at 0.5 µg/ml in 0.1% bovine serum albumin/PBS overnight at 4°C in humid chamber. After three washes with PBST, the sections were treated with 0.3% H2O2 for 15 min at room temperature to block endogenous peroxidase activity. For amplification, the slides were treated with anti-rabbit IgG-horseradish peroxidase (HRP) (1:500), washed with PBST, and treated with peroxidase substrate diaminobenzidine (DAB). The slides were sealed in mounting medium and examined in light microscopy. As controls, the sections were treated with preadsorbed anti-mouse PMI, pre-immune IgG, or without primary IgG and analyzed the same way as the test sample.
Northern blot analysis
Total RNA from pachytene spermatocytes, round spermatids, and Sertoli cells were isolated as described (Shaper et al., 1990). Northern blot analysis was performed using the probe (344 nt) labeled with 32P generated by PCR with specific primers, forward 5'-CTTTCCTGTGTGG TGCA, reverse 5'-AGATGCAGCTTCTCTGC (Sambrook et al., 1989
). Each lane was loaded with 10 µg RNA and stained with ethidium bromide to confirm equal loading of each lane (data not shown).
Quantitation of protein-bound KDN in mouse testis and liver
Protein-bound KDN in mouse testis and liver were quantitated as described with some modifications (Angata et al., 1999a). All procedures were carried out at 4°C unless otherwise stated. Testis and liver (about 0.5 g each) from 35-week-old mice were homogenized in 1.5 ml of 50 mM TrisHCl buffer, pH 8.0, containing 0.1 M NaCl using a Polytron homogenizer (Kinematica, Switzerland). The homogenate was spun at 100,000 x g, and 200 µl supernatant was treated with 0.1 N HCl at 80°C for 1 h and ultrafiltered through Microcon 10 (Amicon), and the run-through was passed over a Dowex-1 column and eluted with 1 M formic acid, and then derivatized with 1,2-diamino-4,5 methylenedioxybenzene dihydrochloride (DMB). Ten microliters of the reaction mixture was applied to a C18 reversed-phase (4.6 x 250 mm) high-performance liquid chromatography (HPLC) column, eluted (1 ml/min) with acetonitrile:methanol:water (9:7:84, v/v/v) and detected by fluorescence (excitation, 373 nm; emission, 448 nm). As an internal marker, a known amount of [9-3H] sialic acid was added during the procedure and typically 90% radioactivity was recovered. Quantitation was adjusted based on recovery. DMB derivatives of KDN and Neu5Ac were separated and the areas of the peaks were quantitated and normalized to protein.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mouse Mpi cDNA sequence was submitted to GenBank under accession number AF244360. The mouse Man-6-P isomerase gene is termed as Mpi, instead of the old name Mpi1, because isoforms of this gene have not been reported yet. The terms Man-6-P isomerase and PMI are interchangeable.
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 To whom correspondence should be addressed; E-mail: hudson{at}burnham.org
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F.A. (1999a) Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis. J. Biol. Chem., 274, 2294922956.
Angata, T., Nakata, D., Matsuda, T., and Kitajima, K. (1999b) Elevated expression of free deaminoneuraminic acid in mammalian cells cultured in mannose-rich media. Biochem. Biophys. Res. Commun., 261, 326331.[CrossRef][ISI][Medline]
Babovic-Vuksanovic, D., Patterson, M.C., Schwenk, W.F., OBrien, J.F., Vockley, J., Freeze, H.H., Mehta, D.P., and Michels, V.V. (1999) Severe hypoglycemia as a presenting symptom of carbohydrate-deficient glycoprotein syndrome. J. Pediat., 135, 775781.[ISI][Medline]
Charron, M., Shaper, N.L., Rajput, B., and Shaper, J.H. (1999) A novel 14-base pair regulatory element is essential for in vivo expression of murine ß1, 4-galactosyltransferase in late pachytene spermatocytes and round spermatids. Mol. Cell. Biol., 19, 58235832.
Chayko, C.A. and Orgebin-Christ, M.C. (2000) Targeted disruption of the cation-dependent or cation-independent mannose 6-phosphate receptor does not decrease the content of acid glycosidases in the acrosome. J. Androl., 21, 944953.
Conne, B., Stutz, A., and Vassalli, J-D. (2000) The 3' untranslated region of messenger RNA: a molecular "hotspot" for pathology? Nat. Med., 6, 637641.[CrossRef][ISI][Medline]
Davis, J.A. and Freeze, H.H. (2001) Studies of mannose metabolism and effects of long-term mannose ingestion in the mice. Biochim. Biophys. Acta, 1528, 116126.[ISI][Medline]
Grootegoed, J.A., Jansen, R., and van der Molen, H.J. (1984) The role of glucose, pyruvate and lactate in ATP production by rat spermatocytes and spermatids. Biochim. Biophys. Acta, 767, 248256.[ISI][Medline]
Harlow, E. and Lane, D. (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Hecht, N. (2000) Intracellular and intercellular transport of many germ cells mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP). Mol. Reprod. Dev., 56, 252253.[CrossRef][ISI]
Hendriksen, P.J.M., Hoogerbrugge, J.W., Baarends, W.M., de Boer, P., Vreeburg, J.T.M., Vos, E.A., van der Lende, T., and Grootegoed, J.A. (1997) Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse. Genomics, 41, 350359.[CrossRef][ISI][Medline]
Joshi, M.S., Anakwe, O.O., and Gerton, G.L. (1990) Preparation and short-term culture of enriched populations of guinea pig spermatocytes and spermatids. J. Androl., 11, 120130.
Jutte, N.H., Grootegoed, J.A., Rommerts, F.F., and van der Molen, H.J. (1981) Exogenous lactate is essential for metabolic activities in isolated rat spermatocytes and spermatids. J. Reprod. Fertil., 62, 399405.[Abstract]
Lonlay, P.D., Cuer, M., Vuillaumier-Barrot, S., Beaune, G., Castelnau, P., Kretz, M., Durand, G., Saudubray, J.M., and Seta, N. (1999) Hyperinsulinemic hypoglycemia as a presenting sign in phosphomannose isomerase deficiency: a new manifestation of carbohydrate-deficient glycoprotein syndrome treatable by mannose. J. Pediatr., 135, 379383.[ISI][Medline]
Mita, M. and Hall, P.F. (1982) Metabolism of round spermatids from rats: lactate as the preferred substrate. Biol. Reprod., 26, 445455.[Abstract]
Nakamura, M., Fujiwara, A., Yasumasu, I., Okinaga, S., and Arai, K. (1982) Regulation of glucose metabolism by adenine nucleotides in round spermatids from rat testes. J. Biol. Chem., 257, 1394513950.
Nakamura, M., Okinaga, S., and Arai, K. (1986) Studies of metabolism of round spermatids: glucose as unfavorable substrate. Biol. Reprod., 35, 927935.[Abstract]
Niehues, R., Haslik, M., Alton, G., Korner, C., Schiebe-Sukumar, M., Koch, H.G., Zimmer, K.P., Wu, R., Harms, E., Reiter, K., and others. (1998) Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy. J. Clin. Invest., 101, 14141420.
Payton, M., Rheinnecker, M., Klig, L.S., DeTiani, M., and Bowden, E. (1991) A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase. J. Bacteriol., 173, 20062010.[ISI][Medline]
Peter, J.M., Hendriksen, P.J.M., Hoogerbrugge, J.W., Barrends, W.M., De Boer, P., Vreeburg, J.T.M., Vos, E.A., van der Lende, T., and Grootegoed, J.A. (1997) Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse. Genomics, 41, 350359.[CrossRef][ISI][Medline]
Proudfoot, A.E., Turcatti, G., Wells, T.N.C., Payton, M.A., and Smith, D.J. (1994) Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase. Eur. J. Biochem., 219, 415423.[Abstract]
Rozman, D. and Waterman, M.R. (1998) Lanosterol 14 -demethylase (CYP51) and spermatogenesis. Drug Metab. Dispos., 26, 11991201.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular cloning: a laboratory manual, 2d ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 1.741.84, 7.377.52.
Shaper, N.L., Wright, W.W., and Shaper, J.H. (1990) Murine ß 1, 4-galactosyltransferase: Both the amounts and structure of the mRNA are regulated during spermatogenesis. Proc. Natl Acad. Sci. USA, 87, 791795.
Sikka, S.C. (1996) Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front. Biosci., 1, 7886.
Sikka, S.C. (2001) Relative impact of oxidative stress on male reproductive function. Curr. Med. Chem., 8, 851862.[ISI][Medline]
Storey, B.T., Alvarez, J.G., and Thompson, K.A. (1998) Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH. Mol. Reprod. Dev., 49, 400407.[CrossRef][ISI][Medline]
Stromstedt, M., Waterman, M.R., Haugen, T.B., Tasken, K., Parvinen, M., and Rozman, D. (1998) Elevated expression of lanosterol 14-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats. Endocrinology, 139, 23142321.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 43504354.[Abstract]
Tulsiani, D.R.P., Abou-Haila, A., Loeser, C.R., and Pereira, B.M.J. (1998) The biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization. Exp. Cell Res., 240, 151164.[CrossRef][ISI][Medline]
Westphal, V., Kjaergaard, S., Davis, J.A., Peterson, S.M., Skovby, F., and Freeze, H.H. (2001) Genetic and metabolic analysis of the first adult with congenital disorder of glycosylation type 1b: long-term outcome and effects of mannose supplementation. Mol. Genet. Metab., 73, 7785.[CrossRef][ISI][Medline]
Wu, X.Q., Gu, W., Meng, X., and Hecht, N.B. (1997) The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations. Proc. Natl Acad. Sci. USA, 94, 56405645.