Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.
Agricola
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.
Biology of Reproduction 59, 1214-1223 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Characterization of Domains in Mice of Calnexin-t, a Putative Molecular Chaperone Required in Sperm Fertility, with Use of Glutathione S-Transferase-Fusion Proteins

Seiichiroh Ohsako2,a,b, Lynn Janulisa, Yoshihiro Hayashib, , and David Bunick1,a

a Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802 b Department of Veterinary Anatomy, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calnexin was first discovered as one of the proteins composing signal sequence receptor complexes on the endoplasmic reticulum (ER) membrane [1]. Subsequently it was determined that calnexin functions as a molecular chaperone [2, 3]. It associates transiently with many membrane glycoproteins during their maturation in the ER, including major histocompatibility (MHC) class I heavy chain [4], MHC class II [5, 6], T cell receptor [7, 8], integrin ß1 and {alpha}6 chains [9], glucose transporter [10], G protein of vesicular stomatitis virus [11, 12], and human immunodeficiency virus type 1 envelope glycoprotein [13]. Partially trimmed, monoglycosylated, N-linked oligosaccharides have been shown to be recognized by calnexin [14, 15]. Also, in the secretory pathway, calnexin has been shown to retard movement of molecules to the plasma membrane after sequential binding to immunoglobulin heavy chain binding protein (BiP) [11, 16].

As a molecular chaperone, calnexin likely functions as a key ER quality control protein involved in the regulation of folding or assembly of secretory and plasma membrane proteins [17, 18]. Structurally, calnexin has a long internal domain, which resides in the luminal compartment of the ER. This domain is composed of proline-rich repeat sequences bearing a high homology to a similar region in calreticulin [19]. Calreticulin was first thought to be a Ca2+ storage protein in the ER, but it has recently been demonstrated to function as a molecular chaperone in the ER, similar to calnexin [20, 21]. The proline-rich repeat sequence of calreticulin has been identified as a high-affinity and low-capacity Ca2+-binding domain [22]. In contrast to that of calreticulin, the cytoplasmic region of calnexin is relatively short and has several potential casein kinase II phosphorylation sites. Indeed, in vivo, calnexin is isolated as a phosphorylated protein [1], and casein kinase II was identified as the probable kinase for calnexin phosphorylation [23]. Both Ca2+ binding and phosphorylation are likely to be important for the chaperone function of calnexin. Presently, how these changes affect the binding of calnexin to its substrates remains unresolved and is the subject of current investigation [24].

We previously reported the molecular cloning of an abundant male germ cell-specific Ca2+-binding protein, which was initially expressed in middle pachytene spermatocytes and persisted until maturation phase spermatids [25–27]. Since the cDNA and predicted amino acid sequences of this protein showed close similarity to calnexin, we called it calnexin-t as a testis-specific variant. Analogous to the function of somatic calnexin, calnexin-t likely has a role in quality control in the ER for the folding and assembly of glycoproteins that end up in the acrosomal matrix or on the surface of spermatozoa. During spermatogenesis, a number of glycoproteins are synthesized in the ER and transported to the Golgi apparatus and then the acrosome [28]. Such proteins are apt to be important for the process of fertilization. Therefore, an understanding of structural and biochemical differences in calnexin-t during spermatogenesis, and the manner in which they affect its function as an ER chaperone, should provide new insights into the functional morphogenesis of spermatozoa.

Recently a transgenic mouse knockout of calmegin, the gene identical to calnexin-t, was reported by Watanabe et al. [29, 30]. Deletion of the gene resulted in completely infertile male mice in spite of the production of spermatozoa that were completely normal in morphology and motility [31], suggesting that calmegin/calnexin-t is indispensable for sperm function. These results strongly suggest that molecules in the ER that associate with calmegin/calnexin-t, which are postulated to be transported to the sperm membrane during spermiogenesis, must be essential for sperm and egg interaction.

In this study we further investigated the biochemical states of calnexin-t, utilizing recombinant proteins for various domains and antibodies specific to each domain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Restriction endonuclease and DNA-modifying enzymes were obtained from Bethesda Research Laboratories (BRL, Gaithersburg, MD), New England BioLabs (Beverly, MA), and United States Biochemical Corp. (Cleveland, OH). The plasmid pGEX-2T vectors were obtained from Pharmacia LKB Laboratories (Piscataway, NJ). Polyvinylidene difluoride (PVDF) membranes were from Millipore Corp. (Bedford, MA). Potato acid phosphatase was from Boehringer Mannheim Corp. (Indianapolis, IN). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG were from Pierce Chemical Corp. (Rockford, IL). Isopropyl-1-thio-ß-D-galactopyranoside (IPTG), S-linked glutathione-agarose column, and BSA were from Sigma Chemical Co. (St. Louis, MO). Radioisotopes were obtained from Amersham Corp. (Arlington Heights, IL). Staging of spermatogenesis in mouse tubules was carried out with the aid of the specialty software STAGES 2.2, developed by and obtained from Dr. Rex A. Hess (University of Illinois, Urbana, IL).

Plasmid Construction

Prokaryotic expression vector, pGEX-2T, which encodes a glutathione S-transferase (GST) [32], was digested with EcoRI, and overhanging 5' ends of the DNA were filled in using Klenow fragment of DNA polymerase I (BRL). Calnexin-t cDNA was digested with several restriction enzymes. A 2151-base pair (bp) BamHI/EcoRI fragment (Fig. 1) produces the entire calnexin-t and GST-fusion protein (GST-CNtWhole); a 1455-bp BamHI/PvuII fragment produces the calnexin-t N+P-domain (GST-CNtN+P); a 982-bp BamHI/StyI fragment produces the calnexin-t N-domain (GST-CNtN); a 728-bp StyI/StyI fragment and a 612-bp StyI/PvuII fragment produce the P-domain (GST-CNtP1 and GST-CNtP2, respectively); and a 600-bp PvuII/EcoRI fragment produces the C-domain (GST-CNtC). The DNA fragments were isolated by electroelution from agarose gels, and overhanging ends of the DNA fragments were filled in using Klenow enzyme; 5' ends of all these blunt-ended DNA fragments were in the correct reading frame as GST. The DNAs were then ligated into the prepared pGEX-2T. The ligated plasmids were transformed into Escherichia coli strain DH5{alpha} (BRL). The clones carrying the desired orientation of the fragment were screened by mini-plasmid preparation followed by appropriate restriction enzyme digestion. Recombinant protein was induced by adding 0.1 mM IPTG in each bacterial culture.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1. Domain structure of calnexin-t and construction strategies for recombinant proteins containing each domain. Calnexin-t is a type I transmembrane protein specific to the ER membrane of spermatogenic cells. The protein has a high-affinity calcium-binding domain (P-region) sequestered in the luminal space of the ER, a luminal space region (N-region) that might be involved in binding to newly synthesized glycoproteins, and a short domain (C-region) located in the cytoplasm that has three potential casein kinase II phosphorylation sites. Calnexin-t also has a nuclear localization signal sequence. In order to construct a recombinant protein of each domain, calnexin-t cDNA was digested with several appropriate restriction enzymes and the fragments were cloned into pGEX-2T expression vector. The six expressed GST-fusion proteins were designated GST-CNtWhole, GST-CNtN+P, GST-CNtN, GST-CNtP1, GST-CNtP2, and GST-CNtC.

Purification of the Recombinant Fusion Proteins

GST-fusion proteins were purified by preparative SDS-PAGE followed by electroelution and glutathione-agarose affinity chromatography as described previously [27]. Briefly, the bacterial cell pellets were resuspended in 10-fold lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM PMSF. They were then digested with 0.5 mg/ml chicken lysozyme (Sigma) for 1 h at room temperature (RT) on a magnetic stirrer, after which 0.5 mg/ml deoxycholic acid was added. The viscous bacterial lysate was incubated with 50 mg/ml DNase I for 15 min and centrifuged at 12 000 x g for 30 min. The supernatant was collected (bacterial-soluble fraction) and suspended with 50 volumes of Tris-buffered saline (TBS, pH 7.4) containing 0.1% Triton X-100 (TBS/Triton). Glutathione-agarose was added and the mixture incubated on ice for 20 min, with intermittent swirling. The GST-fusion protein and glutathione-agarose complex were washed with TBS/Triton, and the GST-fusion protein was eluted with 5 mM reduced glutathione, 50 mM Tris-HCl (pH 8.0). Additionally the pellet containing inclusion bodies was resuspended with lysis buffer, and the lysozyme digestion was repeated as described above. Inclusion body GST-fusion protein was purified by preparative SDS-PAGE and electroelution.

Protein Preparation from Testes

Male CD1 mice purchased from Charles River Labs. (Wilmington, MA) were killed under chloroform anesthesia; the testes were excised and homogenized in PBS containing 1% Triton X-100 and 10 mM EDTA. After centrifugation at 20 000 x g for 20 min, the supernatant was collected. CD1 mouse testis microsomes were prepared as described previously [27].

SDS-PAGE and Western Blot

SDS-PAGE and immunoblotting were carried out as described previously [27]. Briefly, the tissues were denatured in SDS sample buffer containing 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 62.5 mM Tris-HCl (pH 6.8) in boiling water for 5 min, and protein samples (5 µg per lane) were subjected to 11% SDS-PAGE and Western blot analysis. Occasionally, the protein samples were concentrated by trichloroacetic acid precipitation. Gel-separated proteins were stained with Coomassie brilliant blue (CBB) and electrophoretically transferred to PVDF membranes. The membranes were blocked with TBS containing 2% BSA. They were then incubated for 1 h with 2 µg/ml primary antibody diluted with 0.2% BSA/TBS. After washing with TBS containing 0.3% Tween 20 (TBS/T), the membranes were incubated for 40 min with 0.4 mg/ml HRP-conjugated secondary antibody. After washing with TBS/T, immunocomplexes were visualized with 0.005% 3,3'-diaminobenzidine tetrahydrochloride, 0.0005% H2O2 in TBS.

45Ca Overlay Assay

A 45Ca overlay assay was performed as described previously [27]. Cells transformed with calnexin-t cDNA domain plasmid constructs were incubated in the presence of IPTG to induce expression. Various amounts of induced crude bacterial lysates were subjected to 11% SDS-PAGE to produce equally intense CBB staining of the various recombinant proteins as assessed by densitometry. The proteins on the gels were transferred to PVDF membrane, and the membrane was incubated with 45Ca. In order to allow detection of any 45Ca binding across all CNt domains on an autoradiograph, roughly equivalent amounts of each expressed recombinant protein domain were Western blotted. This was accomplished by varying the loading amount of crude protein extract from each of the various bacterial-expressed CNt domains run in each lane. Densitometric analysis of the CBB-stained CNt bands was used to confirm that approximately equal amounts of recombinant protein were present in each lane (data not shown). The membrane was washed three times for 30 min each in a solution containing 10 mM Hepes (pH 6.8), 60 mM KCl, and 5 mM MgCl2. The membrane was then incubated for 10 min at RT in the same solution containing 1 mCi/ml (1 mM) 45CaCl2. The membrane was rinsed with 50% ethanol for 5 min, dried, and exposed to x-ray film for 1 day. The presence of a specific signal on the autoradiograph indicated Ca2+ binding.

Antibodies

Polyclonal antibody against GST-calnexin-t 130-kDa fusion protein was generated by injecting rabbits with GST-CNtWhole, which had been prepared earlier by preparative SDS-PAGE and electroelution [27]. The IgG fraction from serum was purified by using HiTrap Protein-A prepacked column (Amersham). Calnexin-t cytoplasmic domain-specific antibody (Anti-C) was generated by immunoabsorption against excess amounts of bacterial lysates containing GST-CNtN+P, GST/CNtN, GST/CNtP1, and GST/CNtP2 followed by protein-A column purification. Completely immunoabsorbed antibody (Abso) was generated by the same procedure using all recombinant proteins. Monoclonal antibody 1C9 [25, 26] was obtained from ascites fluid of a BALB/c mouse. The immunoglobulin fraction was separated by ammonium sulfate (33%) precipitation.

Immunocytochemistry

CD1 mouse testes were fixed with Bouin's solution and embedded in paraffin. Deparaffinized sections were blocked with PBS (pH 7.4) containing 20% normal goat serum and 1% BSA for 1 h at RT to minimize nonspecific staining. After rinsing with PBS, the sections were incubated for 1 h at RT with 40 µg/ml primary antibody suspended in PBS containing 0.1% BSA (BSA/PBS). They were then washed three times with PBS and incubated for 1 h at RT with 8 µg/ml HRP-conjugated goat anti-mouse or -rabbit IgG suspended in BSA/PBS. After washing with PBS, 0.05% diaminobenzidine tetrahydrochloride and 0.01% H2O2 in PBS were applied to the sections for 3 min to develop the peroxidase reaction.

Protein Dephosphorylation and Re-Phosphorylation

Protein samples, including purified recombinant proteins and mouse testicular microsomes, were treated with 2 mU potato acid phosphatase in 150 mM citric acid solution (pH 5.5) at 25°C. In each incubation, 1 µg of mouse testicular microsomes, or 2 ml of bacterial-soluble fraction, or 40 ng of purified GST-fusion proteins was treated with 10 µU phosphatase. Incubation times for each reaction are indicated in the figure legends. The treated samples were precipitated with 5% trichloroacetic acid, denatured in sample buffer, subjected to SDS-PAGE, Western blot-analyzed using monoclonal antibody 1C9, and immunostained with polyclonal anti-GST-CNtWhole antibody. For re-phosphorylation, an aliquot of dephosphorylated testicular microsomes was resuspended in 40 mM Hepes buffer (pH 7.9), 200 mM NaCl, 160 mM KCl, and 32 mM MgCl2. Then 30 µg/ml HeLa S100 fraction, prepared as described previously [33], was mixed with the sample and incubated at 37°C for 15 min. The mixture was denatured by addition of sample buffer and analyzed by Western blot with polyclonal anti-GST-CNtWhole antibody. Densitometric analysis was performed on the two forms of the protein, and the amount of the phosphorylated form of calnexin-t in each lane was calculated as a percentage of total antibody-detected protein.

Labeling of E. coli-Synthesized Proteins With [32P]Phosphate

Bacteria transformed with expression plasmids were incubated with [32P]phosphate according to the protocol described by Lindberg and Pasquale [34]. Briefly, bacteria were grown to an early log phase at 37°C in 5 ml of M9 medium containing 0.5% casamino acids, 1 mM MgSO4, 0.1 mM CaCl2, 0.02% glucose, 10 µg/ml thiamin, 0.5 µg/ml NaCl, 1 µg/ml NH4Cl, 50 µg/ml ampicillin, 6 µg/ml NaH2PO4-7H2O, and 3 µg/ml KH2PO4. The culture was spun down and the pellet resuspended in 200 µl of phosphate-free M9 medium containing 1 mM IPTG. Isotope, 25 ;gml of 10 mCi/ml of [32P]orthophosphate, was added, followed by incubation for 12 h at 37°C. The cell pellet was then directly denatured with 100 µl SDS-sample buffer, and 10 µl was subjected to SDS-PAGE and autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of the Recombinant Proteins in E. coli

The various recombinant domain-specific proteins (Fig. 1) were differentially soluble as well as differentially expressed in the bacteria. In the preparation of recombinant proteins, it was very difficult to dissolve GST-CNtWhole, GST-CNtN, and GST-CNtP2 from the bacterial inclusion bodies for isolation on glutathione-agarose columns. Only small amounts of GST-CNtN+P and GST-CNtP1 could be purified on glutathione-agarose columns, whereas GST/CNtC could be easily solubilized and column purified. Purification of small amounts of the recombinant proteins required the use of both glutathione-agarose column chromatography and SDS-PAGE separation followed by electroelution. Thus, whole bacterial lysates were used for SDS-PAGE analysis unless otherwise noted. The amount of the crude bacterial cell extract loaded per lane, on gels containing multiple domain-specific proteins, was adjusted so that the recombinant protein band in each extract gave approximately equal CBB staining as assessed by densitometry (data not shown). As seen in Figure 2A, all six recombinant proteins were detected as the major product in each lane at approximately equal levels, whereas bacterial proteins varied in abundance. Table 1 represents the calculated molecular weights and the estimate of molecular weights based on actual migration positions of the recombinant GST-fusion proteins. Due to the relatively acidic polypeptide domains and casein kinase II phosphorylation sites, the C-region recombinant protein GST/CNtC migrated much more slowly on the gel than expected from the predicted molecular weight.



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 2. SDS-PAGE and 45Ca overlay assay of the recombinant proteins. A) CBB staining of the gel. Lane 1, cell lysate containing GST-CNtWhole (130 kDa); lane 2, GST-CNtN+P (95 kDa); lane 3, GST-CNtN (59.3 kDa); lane 4, GST-CNtP1 (59.3 kDa); lane 5, GST-CNtP2 (55.8 kDa); lane 6, GST-CNtC (53.7 kDa); lane 7, GST (28.6 kDa). B) Autoradiography of the membrane. GST-CNtWhole, GST-CNtN+P, GST-CNtP1, and GST-CNtP2 had strong 45Ca binding. GST-CNtN showed relativity weak binding. GST-CNtC and GST did not show any binding. Asterisk indicates an unidentified endogenous calcium-binding protein in E. coli.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of the calculated and estimated molecular weights of the recombinant GST-fusion proteins.

Calcium Binding of Recombinant CNt Domains

Using the recombinant proteins (Fig. 2A), a 45Ca overlay assay was performed. After 1 day of autoradiography, recombinant protein-specific binding could be seen (Fig. 2B). The control GST tag-expressed protein did not show any binding to calcium. As expected, GST/CNtP1 and GST/CNtP2, containing only the internal proline-rich repeat sequence domain of calnexin-t, showed high calcium binding. GST/CNtN+P, which also contains the proline-rich repeat sequence, showed strong calcium binding as well. GST/CNtN showed only faint binding, especially as compared to GST/CNtP1 and GST/CNtP2, which are of similar size and thus were present in the gel at similar levels, whereas GST/CNtC did not show any binding.

Specificity of Anti-C and Determination of the Domain Recognized by 1C9

Polyclonal antibody (anti-GST-CNtWhole) raised against electroelution-purified GST-CNtWhole protein could react to all recombinant proteins (Fig. 3A). C-region-specific antibody (Anti-C), which was prepared by immunoabsorption by N- and P-region proteins, reacted only to GST-CNtWhole and GST-CNtC (Fig. 3B), showing that this antibody specifically recognizes only calnexin-t cytoplasmic domain. No reaction was observed (Fig. 3C) when Abso was used. Using mouse tissue extracts including testis, liver, kidney, brain, spleen, and heart, the antibody Anti-C did not show any reactivity to tissues other than testis (data not shown). The monoclonal antibody 1C9, which was used as a probe for calnexin-t cDNA cloning [27], reacted to GST-CNtWhole, GST-CNtN+P, GST-CNtP1, and GST-CNtP2 on the Western blot (Fig. 3D). Therefore it is likely that the epitope recognized by 1C9 is in the P-region.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 3. Western blot of the recombinant proteins. The same samples of bacterial cell lysates seen in Figure 2 were diluted 30-fold, subjected to 11% SDS-PAGE, and transferred to PVDF membranes. The membranes were immunostained with rabbit anti-GST-CNtWhole polyclonal antibody (A), anti-cytoplasmic domain antibody (B, Anti-C), completely absorbed IgGs (C, Abso), and the monoclonal antibody 1C9 (D). Lane 1, cell lysate containing GST-CNtWhole; lane 2, GST-CNtN+P; lane 3, GST-CNtN; lane 4, GST-CNtP1; lane 5, GST-CNtP2; lane 6, GST-CNtC; lane 7, GST. The Anti-C IgGs reacted only with recombinant proteins containing the C-region, i.e., lanes 1 and 6. Monoclonal antibody 1C9 recognized only the calnexin-t P-region. Lower molecular weight bands in positive lanes seen below the recombinant proteins are degraded or incomplete translation products.

Developmental Stage-Specific Changes of the Reaction to 1C9 and Anti-C

The monoclonal antibody 1C9 and polyclonal antibody Anti-C were used on a Western blot of extracts from the testes of mice that were 10 days of age (spermatogonia rich), 18 days (pachytene stage spermatocyte rich), 26 days (round spermatid rich), and 60 days (containing all developmental stages of spermatogenic cells). Both antibodies recognized the 101-kDa protein as the major band in the testes after 18 days of development (Fig. 4), indicating that the 101-kDa protein form of calnexin-t is the only form found in any of the developmental stages of spermatogenic cells expressing calnexin-t.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 4. Western blot of mouse testis extracts using Anti-C and 1C9. Extract from testes of 10-day-old mice (Day 10) represents a spermatogonia-rich sample; testes from 18-day-old mice (Day 18) represent samples that also have pachytene spermatocytes; testes from 26-day-old mice (Day 26) represent samples that also have spermatids; and testes from 60-day-old mice (Day 60) have the complete adult complement of germ cells. Tissues were extracted with 1% Triton X-100, and the protein samples (5 µg in each lane) were subjected to 11% SDS-PAGE and Western blot analysis. The membranes were immunostained with 1C9, Anti-C, and Abso. Both 1C9 and Anti-C detected the 101-kDa calnexin-t in Day 18, 26, and 60 testes.

Differential Reactivity of 1C9 and Anti-C to Spermatogenic Cells in Adult Testis Sections

Mouse seminiferous epithelium staging was carried out using the STAGES 2.2 software. In paraffin sections of the adult testis immunostained with anti-GST-CNtWhole, 1C9, and Anti-C, the initial staining appeared in middle pachytene spermatocytes and persisted until maturation phase spermatids (step 15)&; same results as previously reported in the hamster testis [26]. No staining difference was observed between anti-GST-CNtWhole and Anti-C; however, staining intensity of 1C9 to positive spermatogenic cells was quite different from that of anti-GST-CNtWhole and Anti-C. Although the Anti-C staining intensity in the positive spermatogenic cells was constant, 1C9 stained middle pachytene stage spermatocytes less intensely than it stained spermatids residing just above them in the same region of seminiferous epithelium. As seen in Figure 5A, seminiferous tubules at stages VII and VIII stained with 1C9 showed spermatids that stained intensely but spermatocytes that stained only slightly. On the other hand, as illustrated in Figure 5B, stages VII and VIII stained with Anti-C showed spermatocytes staining as intensely as spermatids. Comparison of 1C9 and Anti-C staining at stage III showed that there was no visible 1C9 staining of spermatocytes but slight staining with Anti-C (Fig. 5, A and B). At stage IV, 1C9 staining of pachytene spermatocytes was much weaker than that for step 4 spermatids (Fig. 5F), but Anti-C staining of spermatocytes was as intense as that for step 4 spermatids (Fig. 5G). At stage VII, the 1C9 staining of pachytene spermatocytes was still weaker than that for step 7 spermatids (Fig. 5H), but Anti-C staining of spermatocytes and step 4 spermatids showed the same strong intensity (Fig. 5I). Figure 5D and 5E show stage VIII–IX seminiferous tubules. Step 8–9 spermatids were stained intensely with both 1C9 and Anti-C; however, pachytene spermatocytes just below the spermatids were stained moderately with 1C9 but strongly with Anti-C. At the later stages, such as stage XI, the difference in staining intensity between diplotene spermatocytes and step 13 elongating spermatids was the same, but 1C9 staining of both spermatocytes and spermatids was less intense than Anti-C staining (Fig. 5, A and B).



View larger version (151K):
[in this window]
[in a new window]
  		FIG. 5. Immunocytochemical observation of adult mouse testis using Anti-C and 1C9. Mouse testis sections were stained with the monoclonal antibody 1C9 (A, D, F, H), the anti-cytoplasmic domain antibody Anti-C (B, E, G, I), or completely absorbed IgGs, Abso (C), as described in Materials and Methods. Roman numerals in the panels indicate the stages of the spermatogenic cycle of seminiferous tubules. In D, F, and H, the staining intensity of middle pachytene stage spermatocytes (Sc) is weaker than that of the spermatids (St) located above them. In E, G, and I, however, staining intensity of middle pachytene stage spermatocytes (Sc) is of the same level as that of the spermatids (St) located above them. A–C) x80; D and E) x200; F–I) x240.

Dephosphorylation of Native and Recombinant Calnexin-t

To examine whether calnexin-t is phosphorylated in vivo, a mouse testicular microsome fraction was incubated with potato acid phosphatase. Figure 6 shows the results of a time course incubation analyzed by Western blot. A 93-kDa major band was detected after phosphatase digestion, suggesting that the nonphosphorylated form of calnexin-t is 93 kDa. Similar results were obtained with rat and hamster samples (data not shown). Surprisingly, the 130-kDa recombinant GST-fusion protein (GST-CNtWhole) was reduced to a 120-kDa form by phosphatase digestion (Fig. 7A). However, GST-CNtN+P did not show any effect of the phosphatase (Fig. 7B). In a time course digestion, column-purified 53.7-kDa GST-CNtC was converted to a 48-kDa form, and the band intensity became weaker (Fig. 7C). To check for protease activity we treated glutathione-agarose-bound GST-CNtC with phosphatase and eluted the dephosphorylated proteins; however, the same amount of 48-kDa form protein was visualized on a CBB-stained gel (data not shown). Therefore the reduction of band intensity is likely due to a decrease in the antibody affinity to the 48-kDa form of the protein. Purified GST-CNtP1 did not show any reduction in antibody affinity after phosphatase digestion (Fig. 7D).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 6. Dephosphorylation of testis protein extracts using potato acid phosphatase. The numbers on the top are incubation times in each reaction.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 7. Dephosphorylation of purified recombinant proteins using potato acid phosphatase. Bacterial-soluble fractions or GST-fusion proteins purified with glutathione-agarose were incubated with potato acid phosphatase at 25°C and subjected to SDS-PAGE and Western blot analysis using anti-GST-CNtWhole antibody. A) Bacterial-soluble fractions (2 µl) containing GST-CNtWhole (130 kDa) were treated with 10 mU phosphatase. After phosphatase incubation, the molecular mass of GST-CNtWhole was reduced to 120 kDa. Lower molecular weight bands seen below the recombinant proteins are degradation products. B) GST-CNtN+P (95 kDa) was incubated with phosphatase as in A. No change in mobility was seen after phosphatase incubation. C) Purified GST-CNtC (40 ng) was incubated with 1 mU of phosphatase. The numbers on the top are incubation times of each reaction. The 53.7-kDa form was gradually reduced to 48 kDa. D) Purified GST-CNtP1 (40 ng) was incubated as in C. No effect was observed.

Phosphorylation of Calnexin-t

To confirm that the apparent molecular weight shift of calnexin-t resulting from potato acid phosphatase treatment was due to actual dephosphorylation, phosphatase-digested mouse microsome samples were treated with HeLa cell S100 fraction, which has strong kinase activities including casein kinase II. After dephosphorylation, the 101-kDa form composed only 14% of the total calnexin-t as determined by densitometry (Fig. 8, lane 1). Treatment with HeLa S100 fraction resulted in an increase to 49% in the relative proportion of calnexin-t present as the 101-kDa form (Fig. 8, lane 2). In addition, GST-CNtC was induced in the bacterial expression system in the presence of [32P]orthophosphate. A 53.7-kDa GST-CNtC expression product was labeled with radioactive phosphate (Fig. 9), indicating that GST/CNtC was phosphorylated even in the bacterial culture.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 8. Re-phosphorylation of the dephosphorylated testis microsome sample with HeLa cell S100 fraction. Dephosphorylated samples of mouse testicular microsomes were incubated with HeLa S100 fraction for 15 min. Lane 1, dephosphorylated mouse testicular microsome (1 µg); lane 2, dephosphorylated microsomes (1 µg) incubated with HeLa S100 fraction (4 µg); lane 3, HeLa S100 fraction alone (4 µg). After incubation with HeLa S100 fraction, an increase in the relative level of the 101-kDa form of calnexin-t is clearly seen, lane 2. Densitometry indicated that the 101-kDa form calnexin-t composes only 14% of the dephosphorylated calnexin-t but 49% of the re-phosphorylated calnexin-t (lanes 1 and 2, respectively).



View larger version (106K):
[in this window]
[in a new window]
  		FIG. 9. Labeling with 32P of recombinant protein in bacterial culture. GST-CNtC recombinant proteins were induced by IPTG in the presence of 1 mCi/ml [32P]orthophosphate. Bacterial lysates were subjected to SDS-PAGE, and the dried gel was exposed to x-ray film. CBB, staining of total proteins on the gel; Autora, autoradiograph of the gel. GST-CNtC (53.7 kDa), which was induced in this system, incorporated 32P. Molecular weight markers are indicated on the left in kDa.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A series of recombinant proteins for each domain of calnexin-t were generated to investigate the biochemical state of calnexin-t. As expected, the highest binding capacity of Ca2+ was observed in the proline-rich repeat sequence (P-region) having close similarity to the calreticulin internal domain, documented as a high-affinity and low-capacity Ca2+-binding site [22, 35]. The N-region showed weaker Ca2+ binding, whereas the C-region did not show any binding. These properties are different from those of calreticulin, in which the C-region is a high-capacity Ca2+-binding domain likely involved in Ca2+ storage [22]. Therefore calnexin-t appears to mainly bind calcium ions in the ER luminal domain.

The epitope recognized by monoclonal antibody 1C9 was shown to be located in the P-region. Our previous report of the immunogold electron microscopic localization of calnexin-t using antibody 1C9 indicated that the immunogold localized mostly to the luminal side of the ER [26]. These results are in agreement with that predicted by Wada et al. [1] for somatic calnexin.

The 101-kDa form of testis calnexin-t was converted into a smaller 93-kDa form after acid phosphatase treatment. HeLa cell S100 extract used as a source of kinase, including casein kinase II, could restore the 93-kDa dephosphorylated form to the 101-kDa size. These results suggest that the 101-kDa protein is a phosphorylated form. In our previous study, the calculated molecular weight of calnexin-t (Mr 69 454) predicted from cDNA sequence data was much smaller than the 101-kDa migration size on SDS-PAGE [27]. This large difference could be due to the high level of phosphorylation. Also, somatic calnexin was initially purified as a phosphoprotein [1, 23]. Even recombinant proteins, which included the calnexin-t C-region, were already phosphorylated when isolated from the bacterial expression system. Several protein kinases have been identified in E. coli [36]. However, three identified phosphorylation sites in the calnexin-t cytoplasmic domain are targets for casein kinase II [27], which is a eukaryotic protein kinase [37].

In the Western blots of testis extracts from various developmental times, the monoclonal antibody 1C9 and polyclonal antibody Anti-C stained only a 101-kDa band in the testes after 18 days of age (Fig. 4). This indicates that the 101-kDa phosphorylated form is always the predominant form in calnexin-t-positive spermatogenic cells. In addition, Anti-C stained middle pachytene spermatocytes and spermatids as shown in Figure 5. Anti-GST/CNtWhole, which recognizes all domains but cross-reacts with the tag protein GST (Fig. 3A), also starts to show staining of the middle pachytene spermatocyte, the same result as with Anti-C, indicating that calnexin-t begins to be synthesized at the middle pachytene stage and that calnexin-t molecules are abundant in spermatocytes and spermatids. Taken together, the Western blot results and the immunocytochemical data indicate that phosphorylation appears to occur immediately after calnexin-t is synthesized in the pachytene spermatocyte. Germ cell staining by Anti-C, anti-GST/CNtWhole, and 1C9 indicates that immunoreactive calnexin-t persists until step 16 spermatids. Therefore phosphorylation seems to be constant and stable throughout calnexin-t expression during spermatogenesis. Probably this phosphorylation is essential for the proper functioning of calnexin-t. For somatic calnexin, there is still no report on the function of the phosphorylation of the cytoplasmic domain [17].

In contrast to observations with Anti-C, the staining intensity with monoclonal antibody 1C9 in middle pachytene stage spermatocytes was much weaker than that against spermatids residing just above them in the same seminiferous epithelium (Fig. 5). The difference in staining intensity between spermatocytes and spermatids was seen in our previous studies in the hamster testis by immunofluorescence on cryosections [25] and with avidin-biotin-peroxidase complex methods on paraffin sections [26]. Since calnexin-t molecules are as rich in pachytene spermatocytes as in spermatids, this faint staining is likely due to reduced 1C9 binding to the P-region. We hypothesize that the structures around the 1C9 epitope in the middle pachytene spermatocyte are somewhat different than in the spermatid.

Somatic calnexin and calreticulin have been reported to bind newly synthesized glycoproteins [3, 11, 16, 38, 39]. These proteins interact specifically with monoglucosylated N-linked oligosaccharides [14, 21]. Using deletion mutants, the glycoprotein-binding site in these proteins was localized to the proline-rich P-region. Furthermore, the binding of Ca2+ to this region has been found to be essential for the glycoprotein-binding function of these proteins [40]. This may reflect the need for these proteins to be in a certain conformation before they are competent chaperones, a conformation that could be induced by Ca2+ binding. Indeed Ca2+ has been shown to alter the conformation of calnexin [24]. Thus, calcium ions might control the chaperone function of calnexin in a manner similar to that for other chaperone proteins [41]. Therefore we speculate that the reduced 1C9 binding in spermatocytes is likely a result of conformational differences due to changes in Ca2+ binding or the binding of germ cell-specific glycoprotein precursors. The strong staining of 1C9 to spermatids might mean a change in calnexin-t interaction with germ cell-specific glycoprotein precursors after meiotic division.

Okabe and coworkers [29, 31] recently made a calmegin (calnexin-t) knockout mouse. The knockout of calmegin resulted in completely infertile male mice [31]. Interestingly, the spermatozoa from the homozygous mice not only showed normal morphology but also perfect motility. Moreover, some acrosomal antigens could be detected by monoclonal antibodies, and the membrane proteins from homozygous spermatozoa showed no difference from the wild type on SDS-PAGE. These results suggest that although calmegin/calnexin-t is temporarily expressed during spermatogenesis, it is indispensable for sperm function. Probably calmegin/calnexin-t substrate molecules in the ER, which are postulated to be transported to sperm membrane during spermiogenesis, are essential for sperm and egg interaction. Indeed, several molecules on the sperm membrane that play an important role in sperm and egg interaction have been reported, for example, galactosyltransferase [42, 43], PH-20 [44], and SP-10 [45]. Antibodies against these molecules can inhibit sperm-zona adhesion, suggesting that these sperm antigens are the receptors for zona pellucida. Precise conformation of these molecules should be necessary for proper adhesion. Our data indicate that in spermatocytes the calnexin-t ER-internal domain is antigenically different than in spermatids. We hypothesize that this could mean that substrate molecules are likely to interact differently with this chaperone during the diploid and the haploid stages of spermatogenesis. The data presented here suggest that the stage of germ cell development needs to be considered in efforts to detect the substrates of calmegin/calnexin-t in spermatogenic cells.

In summary, the internal proline-rich repeat sequence of calnexin-t has been shown to bind Ca2+ with high affinity. The proline-rich repeat in the ER luminal domain of calnexin-t shows some antigenic structural difference between middle pachytene spermatocytes and spermatids. Also, calnexin-t was highly phosphorylated in the testis. Phosphorylation appears to occur just after protein synthesis and is not restricted to specific developmental stages of spermatogenic cells. To date, the substrates for calnexin-t are unknown, but detection of the substrate proteins should prove to be very important for a more complete understanding of male fertility.


   FOOTNOTES
 
1 Correspondence: David Bunick, Department of Veterinary Biosciences, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802. FAX: 217 244 1652; d-bunick{at}uiuc.edu Back

2 Current address: National Institute of Environmental Studies, 6–2 Onogawa, Tsukuba 305–0031, Japan. Back

Accepted: July 8, 1998.

Received: February 27, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJII, Louvard D, Bell AW, Dignard D, Thomas DY, Bergeron JJM. SSRa and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 1991; 266:19599–19610.[Abstract/Free Full Text]
  2. Ou WJ, Cameron PH, Thomas DY, Bergeron JJM. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 1993; 364:771–776.[CrossRef][Medline]
  3. Jackson MR, Cohen-Doyle MF, Peterson PA, Williams DB. Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90). Science 1994; 263:384–387.[Abstract/Free Full Text]
  4. Tector M, Zhang Q, Salter RD. Beta 2-microglobulin and calnexin can independently promote folding and disulfide bond formation in class I histocompatibility proteins. Mol Immunol 1997; 34:401–408.[CrossRef][Medline]
  5. Anderson KS, Cresswell P. A role for calnexin (IP90) in the assembly of class II MHC molecules. EMBO J 1994; 13:675–682.[Medline]
  6. Arunachalam B, Cresswell P. Molecular requirements for the interaction of class II major histocompatibility complex molecules and invariant chain with calnexin. J Biol Chem 1995; 270:2784–2790.[Abstract/Free Full Text]
  7. van Leeuwen JEM, Kearse KP. Calnexin associates exclusively with individual and T cell antigen receptor (TCR) proteins containing incompletely trimmed glycans that are not assembled into multisubunit TCR complexes. J Biol Chem 1996; 271:9660–9665.[Abstract/Free Full Text]
  8. Huppa JB, Ploegh HL. In vitro translation and assembly of a complete T cell receptor-CD3 complex. J Exp Med 1997; 186:393–403.[Abstract/Free Full Text]
  9. Lenter M, Vestweber D. The integrin chains beta 1 and alpha 6 associate with the chaperone calnexin prior to integrin assembly. J Biol Chem 1994; 269:12263–12268.[Abstract/Free Full Text]
  10. Oliver JD, Hresko RC, Mueckler M, High H. The glut 1 glucose transporter interacts with calnexin and calreticulin. J Biol Chem 1996; 271:13691–13696.[Abstract/Free Full Text]
  11. Hammond C, Helenius A. Folding of VSV G protein: sequential interaction with BiP and calnexin. Science 1994; 266:456–458.[Abstract/Free Full Text]
  12. Cannon KS, Hebert DN, Helenius A. Glycan-dependent and -independent association of vesicular stomatitis virus G protein with calnexin. J Biol Chem 1996; 271:14280–14284.[Abstract/Free Full Text]
  13. Otteken A, Moss B. Calreticulin interacts with newly synthesized human immunodeficiency virus type 1 envelope glycoprotein, suggesting a chaperone function similar to that of calnexin. J Biol Chem 1996; 271:97–103.[Abstract/Free Full Text]
  14. Hammond C, Braakman I, Helenius A. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 1994; 91:913–917.[Abstract/Free Full Text]
  15. Zapun A, Petrescu SM, Rudd PM, Dwek RA, Thomas DY, Bergeron JJ. Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. Cell 1997; 88:29–38.[CrossRef][Medline]
  16. Rajagopalan S, Xu Y, Brenner MB. Retention of unassembled components of integral membrane proteins by calnexin. Science 1994; 263:387–390.[Abstract/Free Full Text]
  17. Bergeron JJ, Brenner MB, Thomas DY, Williams DB. Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci 1994; 19:124–128.[CrossRef][Medline]
  18. Zhang JX, Braakman I, Matlack KE, Helenius A. Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell 1997; 8:1943–1954.[Abstract/Free Full Text]
  19. Michalak M, Milner RE, Burns K, Opas M. Calreticulin. Biochem J 1992; 285:681–692.
  20. McDonnell JM, Jones GE, White TK, Tanzer ML. Calreticulin binding affinity for glycosylated laminin. J Biol Chem 1996; 271:7891–7894.[Abstract/Free Full Text]
  21. Wada I, Kai M, Imai S, Sakane F, Kanoh H. Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin. EMBO J 1997; 16:5420–5432.[CrossRef][Medline]
  22. Baksh S, Michalak M. Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains. J Biol Chem 1991; 266:21458–21465.[Abstract/Free Full Text]
  23. Cala SE, Ulbright C, Kelley JS, Jones LR. Purification of a 90-kDa protein (band VII) from cardiac sarcoplasmic reticulum. Identification as calnexin and localization of casein kinase II phosphorylation sites. J Biol Chem 1993; 268:2969–2975.[Abstract/Free Full Text]
  24. Ou WJ, Bergeron JJM, Li Y, Kang CY, Thomas DY. Conformational changes induced in the endoplasmic reticulum luminal domain of calnexin by Mg-ATP and Ca. J Biol Chem 1995; 270:18051–18059.[Abstract/Free Full Text]
  25. Ohsako S, Kurohmaru M, Nishida T, Hayashi Y. Analysis of various antigens in golden hamster testis by monoclonal antibodies. J Vet Med Sci 1991; 53:969–974.[Medline]
  26. Ohsako S, Bunick D, Hess RA, Nishida T, Kurohmaru M, Hayashi Y. Characterization of a testis specific protein localized to endoplasmic reticulum of spermatogenic cells. Anat Rec 1994; 238:335–348.[CrossRef][Medline]
  27. Ohsako S, Hayashi Y, Bunick D. Molecular cloning and sequencing of calnexin-t: an abundant male germ cell-specific calcium-binding protein of the endoplasmic reticulum. J Biol Chem 1994; 269:14140–14148.[Abstract/Free Full Text]
  28. Clermont Y, Tang XM. Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat Rec 1985; 213:33–43.[CrossRef][Medline]
  29. Watanabe D, Yamada K, Nishida Y, Tajima Y, Koshimizu U, Nagata A, Nishimune Y. Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development. J Biol Chem 1994; 269:7744–7749.[Abstract/Free Full Text]
  30. Watanabe D, Okabe M, Hamajima N, Morita T, Nishina Y, Nishimune Y. Characterization of the testis-specific gene "calmegin" promoter sequence and its activity defined by transgenic mouse experiments. FEBS Lett 1995; 368:509–512.[CrossRef][Medline]
  31. Ikawa M, Wada I, Kominami K, Watanabe D, Toshimori K, Nishimune Y, Okabe M. The putative chaperone calmegin is required for sperm fertility. Nature 1997; 387:607–611.[CrossRef][Medline]
  32. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 1988; 67:31–40.[CrossRef][Medline]
  33. Bunick D, Johnson PA, Johnson TR, Hecht NB. Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system. Proc Natl Acad Sci USA 1990; 87:891–895.[Abstract/Free Full Text]
  34. Lindberg RA, Pasquale EB. Isolation of cDNA clones that encode active protein-tyrosine kinases using antibodies against phosphotyrosine. Methods Enzymol 1991; 200:557–564.[Medline]
  35. Fliegel L, Burns K, MacLennan DH, Reithmeier RAF, Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 1989; 264:21522–21528.[Abstract/Free Full Text]
  36. Stock JB, Ninfa AJ, Stock AM. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 1989; 53:450–490.[Abstract/Free Full Text]
  37. Litchfield DW, Luscher B. Casein kinase II in signal transduction and cell cycle regulation. Mol Cell Biochem 1993; 127–128:187–199.
  38. Nauseef WM, McCormick SJ, Clark RA. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. J Biol Chem 1995; 270:4741–4747.[Abstract/Free Full Text]
  39. Peterson J, Ora A, Van PN, Helenius A. Calreticulin is a lectin-like molecular chaperone for glycoproteins in the endoplasmic reticulum. Mol Biol Cell 1995; 6:1173–1184.[Abstract]
  40. Krause KH, Michalak M. Calreticulin. Cell 1997; 88:439–443.[CrossRef][Medline]
  41. Suzuki CK, Bonifacino JS, Lin AY, Davis MM, Klausner RD. Regulating the retention of T-cell receptor alpha chain variants within the endoplasmic reticulum: Ca(2+)-dependent association with BiP. J Cell Biol 1991; 114:189–205.[Abstract/Free Full Text]
  42. Gong X, Dubois DH, Miller DJ, Shur BD. Activation of a G protein complex by aggregation of beta-1,4-galactosyltransferase on the surface of sperm. Science 1995; 269:1718–1721.[Abstract/Free Full Text]
  43. Miller DJ, Macek MB, Shur BD. Complementarily between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature 1992; 357:589–593.[CrossRef][Medline]
  44. Hunnicutt GR, Primakoff P, Myles DG. Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding. Biol Reprod 1996; 55:80–86.[Abstract]
  45. Reddi PP, Naaby-Hansen S, Aguolnik I, Tsai JY, Silver LM, Flickinger CJ, Herr JC. Complementary deoxyribonucleic acid cloning and characterization of mSP-10: the mouse homologue of human acrosomal protein SP-10. Biol Reprod 1995; 53:873–881.[Abstract]


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
J. Oyhenart, S. Benichou, and N. Raich
Putative Homeodomain Transcription Factor 1 Interacts with the Feminization Factor Homolog Fem1b in Male Germ Cells
Biol Reprod, April 1, 2005; 72(4): 780 - 787.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
N. H. Ing, A. M. Laughlin, D. D. Varner, T. H. Welsh Jr., D. W. Forrest, T. L. Blanchard, and L. Johnson
Gene Expression in the Spermatogenically Inactive "Dark" and Maturing "Light" Testicular Tissues of the Prepubertal Colt
J Androl, July 1, 2004; 25(4): 535 - 544.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. Oyhenart, R. Le Goffic, M. Samson, B. Jegou, and N. Raich
Phtf1 Is an Integral Membrane Protein Localized in an Endoplasmic Reticulum Domain in Maturing Male Germ Cells
Biol Reprod, March 1, 2003; 68(3): 1044 - 1053.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.
Agricola
Right arrow Articles by Ohsako, S.
Right arrow Articles by Bunick, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS