help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0630
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O’Bryan, M. K.
Right arrow Articles by Hedger, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O’Bryan, M. K.
Right arrow Articles by Hedger, M. P.
Endocrinology Vol. 145, No. 11 5231-5242
Copyright © 2004 by The Endocrine Society

Identification of a Novel Apolipoprotein, ApoN, in Ovarian Follicular Fluid

Moira K. O’Bryan, Lynda M. Foulds, James F. Cannon, Wendy R. Winnall, Julie A. Muir, Kim Sebire, A. Ian Smith, Hooi-Hong Keah, Milton T. W. Hearn, David M. de Kretser and Mark P. Hedger

Monash Institute of Reproduction and Development (M.K.O., L.M.F., J.F.C., W.R.W., J.A.M., K.S., D.M.d.K., M.P.H.), The Australian Research Council (ARC) Centre of Excellence in Biotechnology and Development (M.K.O., D.M.d.K.), and The ARC Special Research Centre for Green Chemistry (H.-H.K., M.T.W.H.), Monash University, Clayton 3168; and The Baker Medical Research Institute (A.I.S.), Prahran 3181, Melbourne, Victoria, Australia

Address all correspondence and requests for reprints to: Moira O’Bryan, Ph.D., The Centre for Molecular Reproduction and Development, Monash Institute of Reproduction and Development, 27–31 Wright Street, Clayton 3168, Melbourne, Victoria, Australia. E-mail: moira.obryan{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel apolipoprotein, designated ApoN, has been identified in bovine ovarian follicular fluid using chromatographic purification methods, amino acid sequence analysis, molecular biology, and bioinformatics. The apolipoprotein is a hydrophobic 12-kDa protein processed from the C terminus of a 29-kDa precursor expressed in a number of tissues, including the ovary, testis, the anterior chamber of the eye, skeletal muscle, uterus, and liver. Bovine, porcine, and murine ApoN display significant homology at the amino acid level across the entire precursor sequence. Surprisingly, there appears to be no orthologous protein in the human, although an APON-like pseudogene is found on chromosome 12. The N-terminal fragment of the ApoN precursor shows significant homology with the N-terminal sequence of the precursor of the cholesterol transport regulatory protein ApoF, but the corresponding C-terminal sequences of ApoN and ApoF possess no homology. ApoN is present in the high-density lipoprotein fraction of bovine serum and both the high-density lipoprotein and low-density lipoprotein fractions of bovine follicular fluid and is found in several tissues that are associated with local immunological privilege. These data suggest that ApoN may play a role in steroidogenesis and/or immunoregulation in the gonads of nonhuman species, as well as similar roles in other tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DEVELOPMENT OF THE germ cells occurs within immunologically sequestered compartments of the testis and ovary. In the case of the male, most of this development takes place after maturation of the immune response. As a consequence of this temporal and physical separation from the immune system, male and female germ cells are profoundly autoantigenic (1, 2, 3), and protection of these cells from immunological attack is essential for maintaining fertility (4, 5).

It is well established that testicular secretions inhibit T-lymphocyte activation and proliferation (6, 7), and similar activity has been reported in the fluid surrounding the egg in the ovarian follicles (8, 9). This follicular fluid is rich in both ovarian-derived and serum-borne proteins (10, 11). During attempts to purify factors responsible for the inhibition of T-lymphocyte function, an apolipoprotein-rich fraction was isolated from bovine follicular fluid (bFF). This fraction was composed of several high-density lipoprotein (HDL)-associated proteins, including a previously uncharacterized protein, which we have identified as a novel apolipoprotein related to the cholesterol transport regulatory protein, ApoF (12, 13). Whereas certain lipoproteins and their associated apolipoproteins have been shown to possess immunoregulatory properties (14), HDLs specifically play a major role in the regulation and support of ovarian steroidogenesis (15, 16). Consequently, the discovery of a unique apolipoprotein in the follicular fluid is likely to be highly significant for understanding a number of aspects of ovarian function. Herein, we describe the isolation, sequencing, and partial characterization of this novel apolipoprotein, which we have designated ApoN. ApoN is only the second member to be identified in a protein family that currently is represented by ApoF alone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of tissues
Bovine tissues were collected from young adult and mature cows at an abattoir. bFF was collected from ovarian antral follicles by aspiration on ice and stored at –20 C before extraction. The remaining tissues were immersion-fixed in Bouin’s fluid, or snap-frozen in isopentane/dry ice. Mature bull testis tissue was obtained after castration, and fragments were immersion-fixed in paraformaldehyde-lysine-periodate (17). Fragments of porcine ovarian tissue, obtained from an adult pig postmortem, were snap-frozen and the remainder fixed in Bouin’s fluid. All fixed tissues were processed into paraffin for sections, as described previously (18). Rodent tissues were obtained from adult Sprague Dawley rats and inbred Dark Agouti rats (70–100 d of age; Monash University Central Animal Services, Clayton, Victoria, Australia) and adult BALB/c mice (36–100 d of age). Human blood for genomic DNA (gDNA) extraction and lipoprotein fractionation was obtained from healthy adult volunteers, as approved by the Monash Medical Centre Human Ethics Committee. Human testis tissue, obtained with consent from a healthy human donor with unexplained testicular pain requiring orchidectomy, was snap-frozen immediately after resection. A portion of the testis was fixed with Bouin’s solution, processed into paraffin and stained as described previously (19). Testicular histology was examined by an experienced andrologist and found to be quantitatively and qualitatively normal. gDNA was extracted from the blood or liver of several species, using standard protocols (20). All animal studies were performed in accordance with the National Health and Medical Research Council Guidelines on Ethics in Animal Experimentation and were approved by the Monash Medical Centre Animal Experimentation Ethics Committee.

Methanol extraction and reversed phase HPLC (RP-HPLC) fractionation of bFF
Before processing, multiple aliquots of bFF were thawed and pooled and aliquots of 25 ml were re-frozen in the absence of protease inhibitors. Each aliquot was processed as follows: the bFF was thawed and 2 x 12.5 ml was transferred to 250-ml Sorvall polypropylene centrifuge bottles, and each had 187-ml ice-cold HPLC grade methanol/50 mM NH4HCO3 (pH 7.5) added slowly with agitation at 4 C. This mixture was left to stand for 10 min at 4 C with occasional agitation, then centrifuged at 10,000 x g for 30 min at 4 C. Water and trifluoroacetic acid (TFA; Pierce, Rockford, IL) were added to a final concentration of 30% methanol/0.1% TFA. This solution was clarified through several 0.5-µm filters and degassed before loading at 1.0 ml/min through a Waters Pump 6000A (Waters Corp., Milford, MA) onto a C3 semipreparative RP-HPLC column (Ultrapore C3, Beckman Coulter, Fullerton, CA), after a 50-ml wash with 30% methanol/0.1% TFA. The loaded column was transferred to an HPLC system (LKB Produckter AB, Bromma, Sweden) and washed with a further 50 ml of 30% methanol/0.1% TFA before elution. The column was then subjected to RP-HPLC using a 0.1% TFA/30%-100% methanol staggered gradient over 45 min at a flow rate of 3.0 ml/min. Collected fractions (3 ml) were assessed for biological activity in a lymphocyte proliferation bioassay, as described below.

In vitro lymphocyte proliferation inhibition assay
Mechanically isolated thymic cells from young adult (60–80 d old) male inbred Dark Agouti rats were cultured (37 C, 96 h) in 96-well culture plates (0.8x 106 cells/well), in DMEM with 1% heat-inactivated fetal calf serum and 25 µM ß-mercaptoethanol, in the presence of a suboptimal dose of mitogen (2–8 µg phytohemagglutinin/well), as previously described (7). The isolated thymic cells were preplated for 30 min at 37 C to reduce adherent cells before transfer to culture. Test samples were added at the start of the culture, and cell growth was determined by the incorporation of [3H]thymidine added during the final 16–20 h of culture.

SDS-PAGE
Fractions obtained from the RP-HPLC fractionation of bFF were run on 18% PAGE gels in Tris-tricine buffer by a modification of the method of Schägger and von Jagow (21) using a single electrode buffer under nonreducing and reducing conditions. Protein bands were identified by silver staining (22).

Amino acid sequencing
Two bioactive fractions from each of two RP-HPLC profiles were lyophilized, redissolved in Tris-tricine sample buffer, and heated at 95 C for 5 min. Pooled samples were run in duplicate lanes, together with known molecular weight markers on two sides of an 18% Tris-tricine PAGE reducing gel. At the conclusion of the electrophoresis, the separated proteins on one side of the gel were transferred to a polyvinylidene fluoride membrane, then stained with Coomassie blue R-250 for 30 sec followed by destaining in 16.5% methanol/5% acetic acid /78.5% water. The protein bands of interest were excised from the membrane and applied to an on-line Applied Biosystems (Foster City, CA) 470A gas phase sequencer equipped with an on-line Applied Biosystems 120A HPLC for analysis of the phenylthiohydantoin-derivatized amino acids.

The second half of the gel was stained for 15 min with Coomassie blue R-250 and the bands of interest excised and subjected to digestion with trypsin alone or trypsin/V8 in ammonium bicarbonate at 37 C for 40 h, as described previously (23), followed by reduction and alkylation with ß-mercaptoethanol at 37 C for 1 h and 4-vinylpyridine at 37 C for 1 h. The tryptic digest was fractionated on a microbore Poly LC C8 column at 50 C using a 0.1% TFA/60% acetonitrile gradient over 1 h and the collected digest peaks were sequenced as described above.

After amino acid sequencing and database interrogations, one of the protein bands isolated from bFF corresponding to a molecular mass of 12 kDa on SDS-PAGE under reducing conditions was found to be a previously unidentified protein. This protein was designated apolipoprotein N (ApoN), following in rank order from the most recently described apolipoprotein, ApoM (24).

Cloning of porcine ApoN
Full sequence details of all primers used in this study are listed in Table 1Go. The bovine peptide sequences obtained from Edman degradation sequencing were used to perform a basic local alignment search tool (BLAST) search of the GenBank expressed sequence tag (EST) database. At the time of protein sequencing, a single EST (GenBank accession no. AW359350) derived from pooled Sus scofa endocrine tissues was identified with significant homology to the bovine ApoN peptides FF#3 and FF#4 (see Fig. 1Go). This fragment was subsequently amplified using RT-PCR from porcine ovarian follicle tissue. For RT, no more than 2 µg of total RNA were reverse transcribed using the primer CDSP using standard methods. ApoN cDNA was subsequently amplified in the presence of 1 U Taq polymerase, 10x PCR buffer [0.15 mM MgCl2 (pH 8.3)], 2 mM deoxynucleotide triphosphates and 1 µM of primers P3 and P5. Reactions were thermocycled under the following conditions: an initial denaturation of 94 C for 3 min followed by 35 cycles of 94 C for 30 sec, 58 C for 30 sec and 72 C for 30 sec. Cycling was followed by a final extension of 72 C for 5 min. The partial AW359350 sequence was TA cloned into the PCRII plasmid (Invitrogen Life Technologies, Carlsbad, CA) as described by the manufacturer. Additional 3'-sequence was obtained by 3'-rapid amplification of cDNA ends (RACE) as described previously (25) using the cDNA-specific primers P6 and AP3. To ensure the veracity of this sequence, and all others in this study, inserts from at least two clones were sequenced in both directions using standard protocols in The Wellcome Trust sequencing facility, Monash Medical Centre.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Full sequence details of primers used

 


View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1. Isolation and partial sequencing of bovine ApoN. A, RP-HPLC purification profile of methanol extracted bFF (absorbance measured at 214 nm) and inhibition of [3H]thymidine uptake in a lymphocyte proliferation assay. The dashed line indicates the [3H]thymidine uptake induced by the mitogen, phytohemagglutinin, in the absence of inhibitory activity. B, SDS-PAGE analysis of fractions 22–28 from the purification profile in panel A, under nonreducing and reducing conditions. Numbers on the left and right indicate the relative molecular mass of the standards. The location of ApoN is indicated by the solid arrows (12 kDa), and the ApoAII monomer is indicated by the open arrows (7 kDa). C, N-terminal and internal (tryptic digest) amino acid sequences of the 12-kDa band comprising bovine ApoN. PHA, Phytohemagglutinin.

 
Additional 5'-sequence was obtained from porcine ovary mRNA using an Invitrogen Life Technologies 5'-RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit, as recommended by the manufacturer. ApoN cDNA was reverse transcribed using Superscript II reverse transcriptase and the porcine ApoN-specific oligonucleotide P3. The 5' end of ApoN was amplified using the reverse ApoN-specific primers P2 and P4 for the first and second round amplification respectively. A band of approximately 400 bp was purified using gel extraction, TA-cloned into PCRII and sequenced.

The 5'-RACE strategy resulted in an additional 207 bp to the EST sequence originally obtained by database searching. This assembled partial porcine ApoN cDNA sequence was then used for a further BLAST search of the GenBank EST database. This analysis revealed the existence of an additional EST obtained from pooled porcine endocrine tissues with identity over a region of the 5' end of the assembled sequences (GenBank accession no. BF078743). The four porcine cDNA fragments obtained from database searching and RACE were assembled into the full-length porcine ApoN sequence, and this sequence was subsequently used for database and sequence analyses. The full-length porcine ApoN sequence was subsequently amplified by RT-PCR and sequenced from porcine ovary and testis.

Cloning of bovine ApoN
The fragment of the orthologous bovine ApoN sequence was initially cloned from bovine gDNA using degenerate primers, P6 and B19, based on the peptides derived from protein sequencing, using standard PCR conditions. This approach was eventually chosen because of several failed attempts to amplify ApoN from ovary and testis cDNA using degenerate primers. To increase the signal before cloning, a second round of amplification was undertaken using the degenerate primer B20 and the AP3 primer, the sequence of which had been incorporated into the 5' end of B19. The 3' end of bovine ApoN was cloned from liver and skeletal muscle RNA using 3'-RACE, as described above, using the ApoN-specific primers B21 and B22 for the first and second round amplifications, respectively. The putative 5' end of bovine ApoN was also cloned from gDNA, using a forward primer (B23) based on the porcine 5'-untranslated region (UTR) and a reverse primer derived from the known bovine ApoN sequence (B24). PCR-amplified fragments were cloned into PCRII and sequenced in both directions. 5'-RACE failed to generate a specific bovine ApoN sequence (data not shown).

Cloning of murine ApoN
The mouse ortholog of ApoN was virtually cloned based on homology between the bovine and porcine ApoN sequences and mouse ESTs. Homology was determined using the BLAST and tBLASTx algorithms (26) and the partial expression and chromosome localization revealed by searching the various National Center for Biotechnology Information (NCBI) databases (http://www.ncbi.nlm.nih.gov/).

Cloning of human ApoN-like sequence
The full-length porcine and bovine ApoN sequences were used to perform a BLAST search of the publicly accessible high-throughput genomics sequence database available through NCBI. This analysis revealed highly significant homology with a sequence within the working draft of human chromosome 12 (GenBank accession no. AC025574). Oligonucleotide primers were designed to regions of high homology between porcine/bovine and human sequences (H9 and H10) and were used to amplify a fragment of human APON-like sequence from gDNA-free human testis and ovarian RNA as already described above. The 3' end of the human APON-like sequence was subsequently cloned from ovary and testis RNA by 3'RACE using the forward primers H7 and H8. An analysis of the ApoN-like sequence indicated an insertion of 20 bp approximately 140 bp 3'-of the predicted ATG, compared with the bovine sequence. This resulted in a frame shift and ultimately the introduction of a premature stop codon. To explore the possibility that this cDNA was from a pseudogene and a functional APON sequence remained to be determined, touch-down PCR was undertaken on human ovary and testis cDNA using various combinations of the primers H11, H12, H13, and H14 in a previously described protocol (25). PCR products were cloned into PCRII and multiple APON-positive colonies from each amplification were chosen for sequence analysis.

Localization of the human APON-like gene
The existence of a human APON-like gene was initially indicated from a BLAST search of the NCBI human genome sequence, which revealed two regions of significant homology between the bovine ApoN sequence and an unordered bacterial artificial chromosome (BAC) sequence derived from chromosome 12 (GenBank accession no. AC025574). In parallel, the precise localization of the human APON-like gene was determined using a radiation hybrid protocol as outlined by the manufacturer (Research Genetics Inc., Huntsville, AL). To achieve this, three sets of primers were designed based on the region of highest homology between the porcine/bovine cDNA sequence and the human chromosome 12 BAC sequences. Primer sets included H1 and H2, H3 and H4, and H5 and H6. Before analysis on radiation hybrid plates, primer sets were tested on several samples of human gDNA and Chinese hamster ovary cell gDNA and were found to reliably detect PCR products of the predicted size from human gDNA, but resulted in no amplification products from hamster gDNA.

Translation of the human APON-like cDNA sequence indicated that the sequence obtained resulted in a truncated protein with very little homology to bovine ApoN. To determine whether an alternative APON gene was present within the human genome a Southern blot was carried out using gDNA derived from bovine, porcine, mouse, rat, and human gDNA. gDNA was extracted, HindIII digested, and size separated, as described previously (20).

Protein sequence analysis
Protein characteristics were predicted using the analysis tools available through the ExPASy molecular biology server (http://expasy. proteome.org.au).

Peptide synthesis and antibody production
Peptide sequences for antibody production were derived from the bovine ApoN peptide sequencing results. Peptide BL1 corresponded to amino acids SVTLPEACRQED of the N-terminal sequence of the purified bovine 12-kDa ApoN (peptide FF#1). Peptide BL3 corresponded to amino acids AFTMPMQDQLYF of the bovine ApoN peptide (FF#3) obtained after tryptic digestion. Peptide BL3 was synthesized with a terminal cysteine residue for the purposes of conjugating to a carrier protein, keyhole limpet hemocyanin (KLH). Peptide BL1 contained an internal cysteine that was used for conjugation. Solid-phase peptide synthesis, conjugation to KLH, and antisera generation were carried out as described previously (19). Briefly, antisera were raised by injecting (im) 300 µg of the peptide-KLH complex emulsified in Freund’s complete adjuvant into virgin New Zealand White female rabbits, followed by two booster injections in Freund’s incomplete adjuvant over a period of up to 12 wk. For clarity, antisera generated from these peptides were given the same nomenclature as the immunizing peptides.

Isolation of lipoproteins
Bovine follicular fluid, bovine serum and human plasma (collected in EDTA-coated tubes) were separated into chylomicron/very low-density lipoprotein (VLDL; <1.006 g/ml), low-density lipoprotein (LDL; 1.006–1.063 g/ml), HDL (1.063–1.21 g/ml), and lipoprotein-deficient (>1.21 g/ml) fractions by sequential potassium bromide density ultracentrifugation, using standard methods (27). Fractions were dialyzed against PBS to remove the potassium bromide, and total protein content was measured using the DC Protein assay (Bio-Rad, Hercules, CA).

Western blot analysis
To detect the presence of ApoN in ovarian and testicular extracts, and in bovine and human lipoprotein fractions, samples were prepared for Western blot analysis as described previously (19). In all cases, 20 µg of protein was size fractionated on 15% Tris-glycine SDS-PAGE gels and probed with the anti-BL1 sera at a 1:1000 dilution using a standard chemiluminescence method. The molecular mass values of the detected proteins were estimated by comparison to prestained protein markers.

ApoN expression in bovine tissues
The expression of ApoN in various bovine tissues was determined using RT-PCR. mRNA was reverse transcribed, as described above. ApoN cDNA was amplified using a nested PCR with the primers B25 and B26 in the first reaction and B27 and B28 in the second reaction under the conditions described above. To ensure that the DNA being amplified was derived from cDNA rather than gDNA, each reaction contained a no-RT control, and a sample amplified with primers complementary to bovine LH introns (LHF and LHR). The presence of mRNA in the original sample was ensured by the amplification of bovine gapdh cDNA using the primers GAPF and GAPR.

Immunohistochemistry
The distribution of ApoN protein in normal bovine tissues was determined using an avidin-biotin amplified technique, as described previously (18). Tissues tested were bovine ovary, testis, spleen, lymph node, thymus, whole eye, uterus, pancreas, heart, liver, and kidney. The specificity of the antisera (anti-BL1 and anti-BL3) in immunohistochemical protocols was determined by preabsorbing antisera with the synthetic peptides to which they were raised. An approximate 10-fold excess (wt/wt) of the immunizing peptide was added to the antiserum and incubated at 37 C for 1 h. Subsequently, the whole solution was used as the primary antibody during the immunohistochemical procedure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ApoN purification
After extraction of bFF with methanol and separation by RP-HPLC on a C3 column, fractions were collected and aliquots assessed in the in vitro T-lymphocyte proliferation bioassay and separated on SDS-PAGE Tris-tricine gels. A peak of inhibition of T-lymphocyte proliferation, as indicated by a reduction in [3H]thymidine incorporation, was found for fractions 25–27 from the C3 column (Fig. 1AGo). Examination of these fractions identified a ubiquitous band at approximately 7 kDa, a weak band at 14 kDa, and a distinct 10- and 12-kDa doublet, which became a single band of approximately 12 kDa on reduction (Fig. 1BGo). Multimers of the 7-kDa band were also present at approximately 15, 21, and 28 kDa and were particularly prominent in the nonreduced gels. All bands were subjected to N-terminal and/or internal sequencing. The major band (7 kDa) appeared to be N terminally blocked, but internal sequencing after digestion with trypsin/V8 protease identified the peptide sequence ... SNLQSLVSQYRQTVADYGK..., which possessed 55.5% identity with the murine apolipoprotein AII (ApoAII) precursor and was 82.3% identical over 19 amino acids to human ApoAII precursor, indicating that this protein band represented the bovine ortholog of ApoAII. A partial peptide sequence of the 14-kDa band (... GSVGAGEP...) identified this protein as bovine transthyretin, a member of the lipocalin protein family of extracellular proteins that binds and transports small hydrophobic molecules (28). The second most abundant band (12 kDa) provided an N-terminal sequence and several internal sequences, indicating that this was a unique, previously unidentified protein (Fig. 1CGo), which we eventually designated ApoN. After extraction from the gel and analysis in the lymphocyte proliferation assay, the purified 12-kDa protein was not bioactive in the lymphocyte proliferation assay (data not shown). Moreover, alternate purification of the bFF extract using extended gradient conditions on a C4 RP-HPLC column separated both the 12- and 14-kDa bands from the peak of biological activity (data not shown).

Cloning and identification of ApoN
A search of the NCBI EST database using peptide sequences obtained by Edman degradation (Fig. 1CGo) revealed significant homology between the bovine peptide sequences BL3 and BL4 and a 297-bp porcine EST (AW359350) obtained from tissues of endocrine origin (GenBank accession no. AW359350). Virtual translation of this EST in the 5'->3' direction in the third reading frame resulted in a predicted peptide of ...DFTMPMDDQIYEVKRSVAILARVILE.... This sequence shared 50% identity and 69% homology with bovine peptides BL3 and BL4 obtained by tryptic digestion (Figs. 1CGo and 2Go). As such, it was interpreted that AW359350 represented a partial clone of the porcine ApoN homolog. The sequence corresponding to AW359350 was subsequently amplified and sequenced. The cloned insert corresponded to nucleotides 626–917 of the full-length porcine ApoN shown in Fig. 2Go. The 3' end of porcine ApoN was obtained by 3'RACE, as described.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2. The cDNA and predicted amino acid sequences of porcine ApoN. The numbers on the left indicate the relative position of the nucleotides. The numbers on the right indicate the relative position of the predicted amino acids. The predicted signal peptide sequence is shown in bold. The arrow indicates the predicted proteolytic cleavage site, which would generate a 12-kDa protein homologous to the bovine ApoN sequence.

 
An additional 209 bp of the porcine ApoN sequence was obtained by 5'-RACE. This plasmid contained nucleotides 413–621 of the full-length porcine sequence (Fig. 2Go). Comparison of the predicted partial porcine sequence, with the amino terminal sequence of the 12-kDa ApoN (FF#1) derived from bFF showed 90% identity and 95% homology (Figs. 1CGo and 2Go). This result also strongly suggested that the 12-kDa protein found within bFF was the product of cleavage from a higher molecular mass precursor protein and that the cDNA identified in porcine ovary tissue was an ortholog of bovine ApoN.

A further BLAST search using the sequence obtained by 5'-RACE revealed partial identity with an additional porcine 5'-EST (GenBank accession no. BF078743) obtained from tissues of endocrine origin. Tiling of the four porcine ApoN fragments obtained by database searching and RACE resulted in the full-length porcine sequence of 917 nucleotides, which was deposited in the NCBI database as GenBank accession no. AY583018. This sequence contained 74 bp of 5'-UTR, 762 coding nucleotides, and 81 bp of 3'-UTR. The first methionine is predicted to be the initiation start site as it proceeded by a modified Kozak sequence and an in-frame stop codon.

Cloning of the bovine ApoN cDNA
Using a combination of standard PCR on gDNA and 3'-RACE from both liver and skeletal muscle RNA the putative sequence of bovine ApoN cDNA was determined (Fig. 3AGo). In our hands, it was not possible to amplify the full-length bovine ApoN from liver or muscle RNA. This was most likely due to RNA degradation before its collection at the abattoir. It was, however, possible to reliably amplify the 5' end of ApoN (data not shown). The putative bovine ApoN sequence has been deposited within the NCBI database as GenBank accession no. AY583019.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 3. A, The cDNA and predicted amino acid sequences of bovine ApoN. The numbers on the left indicate the relative position of the nucleotides. The numbers on the right indicate the relative position of predicted amino acids. The predicted signal peptide sequence is shown in bold. The boxed areas indicate the position of peptides derived from sequencing the amino terminus of the mature peptide or tryptic fragments (see Fig. 1CGo). Note the discrepancy between the N-terminal amino acids assigned for FF#3 and FF#4, and the predicted amino acids from the nucleotide sequence of the cloned protein. B, Hydrophobicity plot of bovine ApoN. The arrow indicates the position of the R-S cleavage site, which would generate the 12-kDa protein purified from bovine follicular fluid.

 
Virtual translation of the bovine ApoN cDNA revealed an open reading frame of 259 amino acids with a predicted molecular mass of 29.1 kDa and an isoelectric point (pI) of 5.49. Sequence analysis predicted a signal peptide region between amino acids 1–19, which when cleaved would result in a protein of 27.2 kDa with a pI of 5.36. All four of the peptide sequences obtained from automated sequencing were visible within the predicted sequence, although there was a minor discrepancy between the N-terminal amino acids assigned to FF#3 and FF#4, and the predicted amino acids from the nucleotide sequence of the cloned protein (Fig. 3AGo). These differences were attributable to ambiguities in the original peptide sequencing. Interestingly, the sequence corresponding to FF#1, which had been obtained by N-terminal sequencing, occurred at amino acids R151–S152 of the cloned ApoN sequence, indicating that the ApoN purified from bFF was a C-terminal cleavage product of a larger precursor protein. The same monobasic arginine cleavage site was also seen in the predicted porcine ApoN sequence. The cleavage sites in both sequences occur in a relatively hydrophilic region of the molecule (Fig. 3BGo), consistent with solvent accessibility and potential for cleavage under in vivo conditions. The molecular mass of the predicted C-terminal ApoN fragment was 12.7 kDa, and a pI of 4.97 and was consistent with the ApoN protein purified by HPLC and its relative hydrophobicity (Fig. 1Go, A and B). The predicted molecular mass of the N-terminal fragment (minus the signal peptide) was 14.5 kDa and a pI of 5.79. The molecule contains no glycosylation sites but does contain four cysteine residues outside of the signal peptide region, thus raising the possibility for intramolecular disulfide bonding. In addition bovine ApoN contains potential protein kinase C phosphorylation motifs at residues 114–116, 164–166, and 192–194, potential casein kinase II phosphorylation motifs at residues 33–36, 55–58, 136–139, and 148–151, a tyrosine kinase phosphorylation site at 194–202, a tyrosine sulfation motif at 195–209 and a potential myristylation motif at residues 102–107. Furthermore, sequence analysis revealed several regions likely to form {alpha} helices within the mature protein (Fig. 4Go).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 4. A, Comparison of the full protein sequence of ApoN from bovine (B), porcine (P), murine (M), and human (H) sources determined in the present study, and the published sequences of human and murine ApoF. The annotations on the left indicate the species from which the sequence was derived. Numbers on the right indicate the amino acid position relative to bovine ApoN. The open arrow indicates the potential signal peptide cleavage site for ApoN, and the solid arrows indicate the cleavage points for the mature ApoN/ApoF from the pro-peptide. Shading indicates regions of amino acid identity with the bovine ApoN sequence, after spacing to improve alignment (——-). Bold overlining indicates areas of predicted ApoN {alpha}-helix secondary structure. The boxed region of the human ApoN amino acid sequence represents the region affected by a putative frame shift mutation that leads to loss of homology, and a premature truncation of the predicted protein product (*).

 
Consistent with the cloning and sequence analysis results, an approximately 29-kDa anti-ApoN immunoreactive band was seen within bovine ovarian extracts, as well as extracts of whole rat and mouse ovary and testis (Fig. 5Go). A 12-kDa band was never detected within any whole tissue extract, and was only observed in extracts of bFF (Fig. 6Go), suggesting that the ApoN precursor is cleaved into N-terminal and 12-kDa C-terminal components upon secretion into the follicular fluid.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 5. A Western blot analysis of ApoN in cow, rat, and mouse ovarian and testicular extracts using the anti-BL1 serum. The relative molecular mass of standards is indicated on the left. Arrows on the right indicate the presence of the likely ApoN-precursor protein at approximately 29 kDa and of a larger immunoreactive band in the rat tissues at approximately 60 kDa.

 


View larger version (40K):
[in this window]
[in a new window]
  		FIG. 6. Western blot analysis for ApoN in bovine plasma (A) and follicular fluid (B) lipoprotein fractions, and in a partially purified fraction of bFF from the C3 RP-HPLC column (WP1) using the anti-BL1 serum. All lanes contain equal amounts of total protein (20 µg). A, Bovine plasma: a faint band corresponding to the 12-kDa ApoN protein extracted from bFF is visible in the HDL fraction, but not in LDL, VLDL or lipoprotein-deficient fractions of bovine plasma. B, Bovine follicular fluid: a band corresponding to the 12-kDa ApoN protein was visible in both HDL and LDL fractions, but not in VLDL or lipoprotein-deficient fractions of bFF. The WP1 sample was included as a positive control.

 
Further analysis of the bovine sequence revealed significant homology between the N-terminal fragment of bovine ApoN and apolipoprotein F (ApoF) (GenBank accession no. NM_001638) i.e. 42% homology and 31% identity over the first 195 amino acids. No significant homology was seen between the C-terminal fragment of ApoN and any proteins within any publicly accessible protein database. It was, however, possible to see ESTs corresponding to the likely mouse ortholog of ApoN (see below). Collectively, these data suggested that ApoN is the second distinct member of the ApoF-like subfamily of apolipoproteins.

Identification of murine ApoN
Searching the NCBI database with either the bovine ApoN protein or cDNA sequences revealed significant homology with several ESTs (for example, accession nos. BI246520, BI147630, and NM_133996). These sequences corresponded to the Mus musculus UniGene MGI (Mm30121). Translation of these sequences revealed significantly higher homology with bovine ApoN than with mouse ApoF (GenBank accession no. AAL06341), i.e. 52% identity/59% homology and 33% identity/52% homology respectively (Fig. 4Go). Further database analysis revealed that mouse ApoN-like sequence is located on chromosome 10 in a region syntenic to human chromosome 12, and it is expressed in at least the liver, aorta and vein, gall bladder, muscle, and in hepatic tumors. These data strongly suggested that the product of UniGene Mm30121 is mouse ApoN.

This conclusion was supported by the identification of an anti-ApoN (using the anti-BL1) immunoreactive band in mouse ovary and testis extracts at 29 kDa, consistent with the size of the ApoN pre-pro-protein (Fig. 5Go). A similar 29-kDa immunoreactive band was seen within rat ovary and testis extracts (Fig. 5Go). The identity of the approximately 60-kDa bands in the rat ovary and testis extracts was not determined, but these may represent multimers of the rat protein due to hydrophobic interactions, comparable with the multimers observed for ApoAII in the bFF extracts (see Fig. 1BGo).

Cloning of the human ApoN-like cDNA and gene localization
A BLAST analysis of the publicly accessible human genome sequence with the full-length bovine or pig ApoN clone revealed two regions with significant homology within the working draft of human chromosome 12 (GenBank accession no. AC25574). This result was confirmed by radiation hybrid analysis, which indicated the human APON-like gene was located at 12q13.2 (data not shown). Primers were designed to likely exonic sequence in the most homologous region and used to amplify the portion of the predicted protein-coding region of human APON from human ovarian and testicular RNA. The 515-bp product was sequenced. From this, it was possible to determine the sequence of a human APON-like cDNA. An additional approximately 250 bp of 3'-UTR sequence was obtained by 3'-RACE from testis RNA, showing that a APON-like mRNA was transcribed. Despite multiple attempts at 5'-RACE using several combinations of primers, it was not possible to amplify the 5' end of a human APON-like sequence using this technique (data not shown). It was however, possible to align the obtained cDNA perfectly with the sequence of BAC AC25574. From this alignment and based on homology with the bovine and porcine ApoN cDNA sequence and putative translational products, it was possible to predict the position of the initiating methionine and at least a proportion of the 5'UTR of a human APON-like protein. Based on this alignment, the human APON-like gene appeared to be a 2 exon gene with the entire protein coding region contained within the second exon. From these data, a forward primer was designed to lie just 3' of the transcription initiation site (B25). A more proximal primer was not chosen because of the additional region of significant homology with the bovine ApoN on chromosome 12 and the associated possibility of obtaining multiple amplification products. This primer was then used in a nested RT-PCR on both human testis and ovary RNA. This RT-PCR successfully generated an additional approximately 155 bp of 5' human APON-like cDNA sequence. The human APON-like sequence has been deposited within the NCBI database as GenBank accession no. AY583020. Significantly, the human APON-like sequence lies adjacent to the APOF gene on chromosome 12. Furthermore, the APON-like gene sequence, in common with the APOF gene, possesses two exons, the second of which contains the entire protein coding region.

Although the human APON-like cDNA sequence was found to have very high sequence identity with the bovine and porcine cDNAs, upon translation of the human cDNA very little homology was predicted. A 20-bp insertion resulted in a frame shift and the introduction of a stop codon, with the result that the human APON-like mRNA was predicted to produce a protein of only 72 amino acids with only partial homology to bovine ApoN. The insertion and the subsequent premature truncation were also seen in the publicly accessible gDNA sequence, thus confirming the veracity of the human cDNA sequence.

Attempts to amplify mRNAs/cDNAs transcribed from the region of human chromosome 12 with lesser homology (at both an mRNA and predicted protein level) to bovine and porcine ApoN failed to amplify an APON-like sequence from several tissues, including testis and ovary, despite the use of several sets of nested PCRs capable of discriminating between the two potential APON-like sequences from cDNA (data not shown). This suggested that the APON-like sequence was the only such sequence in the human transcriptome. The absence of an alternative APON gene within the human genome was confirmed using Southern blotting on human gDNA, which revealed two bands only, with sizes consistent with those generated from the two APON-like genes described above (data not shown). At the time of writing this manuscript, no human ESTs (other than APOF) with high levels of homology to the bovine or porcine ApoN sequence had been deposited in any publicly accessible database.

ApoN in lipoprotein fractions
To establish whether ApoN was an integral lipoprotein component, bovine plasma and follicular fluid were separated into their various lipoprotein fractions. The yields of protein for the various lipoprotein fractions from plasma were: HDL, 70 mg/ml; LDL, 0.14 mg/ml; VLDL, 0.12 mg/ml. The protein yields from bFF were: HDL, 3.06 mg/ml; LDL, 0.01 mg/ml; VLDL, 0.031 mg/ml. Equal amounts, on a protein basis, of each lipoprotein fraction, lipoprotein-depleted plasma, and bFF were subjected to Western blot analysis. Western blotting of bovine plasma lipoproteins with the anti-ApoN (anti-BL1) serum revealed a weakly immunoreactive band of approximately 12 kDa in the HDL fraction. Bands were not observed in either the LDL or VLDL fractions, nor in lipoprotein-depleted plasma (Fig. 6AGo). In bFF fractions, ApoN was detected in association with both HDL and LDL, but not in association with VLDL, nor was it detectable in the lipoprotein-depleted bFF (Fig. 6BGo). As predicted by the database searching and cloning data, no specific bands were detected in any lipoprotein fractions of human plasma (data not shown).

ApoN tissue expression
ApoN mRNA was detected by RT-PCR in bovine ovary, liver, skeletal muscle, and uterus (Fig. 7Go). ApoN was not detected in heart, placenta, kidney, spleen, or thymus tissue by this method. It is of note that the RT-PCR on cDNA from skeletal muscle resulted in two bands, the shorter of which corresponded to ApoN. The sequence of the longer band bore no identity to ApoN and was thus likely a product of nonspecific priming.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 7. The expression of ApoN in bovine tissues as determined by RT-PCR. Tissues are indicated along the top. Reaction set 1: LH intronic primers to detect the presence of contaminating gDNA. Reaction set 2: Gapdh cDNA primers to confirm the successful production of cDNA. Reaction set 3: Bovine ApoN primers B25 (forward) and B26 (reverse). Reaction set 4: Bovine ApoN primers B27 (forward) and B28 (reverse). O, Ovary; L, liver; H, heart; M, skeletal muscle; P, pancreas; U, uterus; K, kidney; T, thymus; S, spleen; No RT, no RT (negative control); No Temp, no template (negative control); gDNA, positive control.

 
Immunohistochemistry
The cellular localization of ApoN was assessed in a range of bovine tissues using antisera from several rabbits immunized with either the BL1 and BL3 peptides. The pattern of staining was very similar between the two sera; however, the intensity of staining and therefore resolution was greater with the anti-BL3 sera (Fig. 8Go). In the case of both the anti-BL1 and anti-BL3 sera, omission of the primary antisera or preabsorption of the immunizing peptide before immunohistochemistry resulted in no staining, confirming staining specificity (Fig. 8BGo). As anticipated from the RP-HPLC purification, specific staining was seen within the follicular fluid of antral and Graafian follicles (Fig. 8AGo). Staining was also seen within the cytoplasm of granulosa cells of secondary through to Graafian follicles and in the cumulus oophorus of antral-Graafian follicles. The oocyte was not stained for ApoN (not visible in this section). The majority of cells within the theca and stroma were negative, but the endothelial cells of ovarian blood vessels were positively stained. Primary follicles were not stained, and only a very few cells, possibly macrophages, in the corpora lutea and corpora nigricans were positive.



View larger version (140K):
[in this window]
[in a new window]
  		FIG. 8. Immunolocalization of ApoN in bovine tissues. Panel A, An antral follicle showing positive staining for ApoN in granulosa cells (G), in follicular fluid within the atrium (A) and in isolated theca (T) cells surrounding the follicle. Panel B, Preabsorption of the sera with a 10-fold (wt/wt) excess of immunizing peptide confirmed the specificity of staining. Panel C, Within the testis ApoN was localized to Sertoli cell cytoplasm (SC) and round spermatids (RS) within the seminiferous epithelium and to Leydig cells (LC) within the intertubular space. Panel D, Within the spleen, as in several other tissues, positive ApoN staining was seen within neutrophils (white arrows), in this instance within a germinal center (GC) surrounded by white pulp (W). Panel E, Within the uterus ApoN was localized to the epithelium (E) of the uterine crypts (UC). Stromal cells (S) were not stained. Panel F, ApoN staining was seen within nonpigmented epithelial (NP) cells of the eye, but was not observed within pigmented epithelial cells (asterisk).

 
Within bovine testes, positive staining was seen within Sertoli cell cytoplasm, round and elongating spermatids in the region of the developing acrosome and within the interstitial space in cells with morphology consistent with Leydig cells (Fig. 8CGo). Staining was also seen within neutrophils and some macrophages within several tissues, including the kidney, lymph nodes, and in the spleen (Fig. 8DGo). ApoN staining was also observed in epithelial cells lining the uterine crypts (Fig. 8EGo) and in the ciliary processes of the retina in the nonpigmented epithelial cells (Fig. 8FGo). Consistent with its distribution in lipoproteins, serum and lymph showed some positive staining for ApoN within the majority of tissues (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During protein fractionation of bovine ovarian follicular fluid, we identified and sequenced a novel protein, called ApoN. Its restriction to the HDL and LDL particles in follicular fluid and HDL in serum, copurification with other lipoprotein components (i.e. ApoAII and transthyretin), and its structural relationship to the ApoF gene, strongly support the identification of this new protein as an apolipoprotein. ApoN was subsequently identified in the cow, pig, mouse, and rat as a 29-kDa protein, which could be cleaved by an enzyme with monobasic arginine specificity (i.e. kallikrein-like specificity) (29), and the C-terminal fragment appeared within follicular fluid and plasma as a 12-kDa protein associated with the lipoprotein fractions. This 12-kDa fragment has no homology with any known protein in the publicly accessible databases. Although it appeared that the LDL fraction of follicular fluid contained a larger proportion of ApoN, it should be noted that ovarian follicular fluid contains principally HDL and relatively little LDL (11, 30, 31), i.e. the LDL preparation was concentrated to a much greater extent (more than 300-fold) during preparation compared with the adjacent HDL fraction. Consequently, the bulk of ApoN in both follicular fluid and in plasma is associated with HDL, which is entirely consistent with its copurification with both ApoAII and transthyretin (32).

Production of apolipoproteins by the ovary is not unexpected. Most notably, ApoE is synthesized in both the ovary (33, 34) and testis (35, 36), where it has been shown to play a number of roles in gonadal physiology. Based on the available data, however, the biological activity of ApoN cannot be inferred from its partial homology with ApoF. The active fragment of ApoF and the lipoprotein-associated portion of ApoN, are both C-terminal fragments of a specific cleavage event, with the products having almost no sequence homology (12, 13). The homology between the N-terminal fragments of ApoF and ApoN may indicate a common protein processing or anchoring mechanism. Nonetheless, the association between ApoN and HDL/LDL in follicular fluid and serum may be indicative of biological activity associated with cholesterol and/or fatty acid transport, and ultimately in gonadal steroidogenesis. Finally, several lipoproteins and apolipoproteins have been shown to have immunosuppressive activities, particularly the ApoE-rich LDLs (14, 37). Although HDLs generally have not been associated with immunoregulatory activity, transthyretin itself has been reported to inhibit production of IL-1 by monocytes and endothelial cells (38). Consequently, the distribution of ApoN in the epithelia of several immunoprivileged tissues (i.e. the testis, ovary, placenta, uterus, and the anterior chamber of the eye) and in macrophages and neutrophils, may indicate some role for ApoN in immunoregulation. Proof of this hypothesis, however, requires further experimentation.

Despite extensive searching using a number of techniques, there does not appear to be a functional human ortholog of ApoN. A gene with a high level of sequence homology to both bovine and porcine ApoN cDNA, and the predicted mouse cDNA, is clearly present on human chromosome 12 in a region syntenic to the position of the putative mouse ApoN ortholog (UniGene Mm30121). The human APON-like gene is transcribed to mRNA in at least the human testis and ovary. The presence of an insertion within the sequence, however, means that the predicted translation product would share little homology with the bovine ApoN protein sequence and would be prematurely truncated by the introduction of a frame shift derived stop codon. Significantly, the human APON-like sequence lies adjacent to the APOF gene on chromosome 12. Furthermore, the APON-like gene sequence, in common with the APOF gene, possesses two exons, the second of which contains the entire protein coding region. These data, and the moderate level of homology between the N-terminal fragment of both APOF and APON, suggest that the APON gene arose as an APOF gene duplication event (or vice versa), but the C-terminal fragments in particular progressively diverged during evolution. At some time subsequent to the divergence of a common ruminant and primate ancestor, however, the human APON-like gene acquired frame shift mutations, which effectively resulted in the ablation of human APON.

In summary, the above data clearly identify the second member of the ApoF-related protein family in a number of species and its association with HDL, and with LDL in ovarian follicular fluid. Although the precise functions of ApoN remain to be determined, a role in either the regulation of steriodogenesis or immunosuppression is suggested by its molecular characteristics and distribution.


   Acknowledgments
 
Thanks are due to Kim Edgar, Mary Matthew, and Tracy Warner for expert technical assistance. This work was supported by a Program Grant (14786) from the National Health and Medical Research Council (NHMRC) of Australia, and an NHMRC Institute Block Grant (182813). M.K.O., A.I.S., and M.P.H. are supported by NHMRC Fellowships 143781, 182814, and 143788, respectively.


   Footnotes
 
Abbreviations: ApoF, Cholesterol transport regulatory protein; ApoAII, apolipoprotein AII; ApoN, novel apolipoprotein; BAC, bacterial artificial chromosome; bFF, bovine follicular fluid; BLAST, basic local alignment search tool; EST, expressed sequence tag; gDNA, genomic DNA; HDL, high-density lipoprotein; KLH, keyhole limpet hemocyanin; LDL, low-density lipoprotein; pI, isoelectric point; RACE, rapid amplification of cDNA ends; RP-HPLC, reversed phase HPLC; TFA, trifluoroacetic acid; UTR, untranslated region; VLDL, very low-density lipoprotein.

Received May 17, 2004.

Accepted for publication July 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tung KS, Teuscher C, Meng AL 1981 Autoimmunity to spermatozoa and the testis. Immunol Rev 55:217–255[CrossRef][Medline]
  2. Paterson M, Jennings ZA, van Duin M, Aitken RJ 2000 Immunocontraception with zona pellucida proteins. Cells Tissues Organs 166:228–232[CrossRef][Medline]
  3. Herr JC 1996 Update on the Center for Recombinant Gamete Contraceptive Vaccinogens. Am J Reprod Immunol 35:184–189
  4. Tung K 1987 Immunologic basis of male infertility. Lab Invest 57:1–4[Medline]
  5. Dunbar BS, Prasad S, Carino C, Skinner SM 2001 The ovary as an immune target. J Soc Gynecol Investig 8(1 Suppl Proc):S43–S48
  6. Pöllänen P, Söder O, Uksila J 1988 Testicular immunosuppressive protein. J Reprod Immunol 14:125–138[CrossRef][Medline]
  7. Hedger MP, Nikolic-Paterson DJ, Hutchinson P, Atkins RC, de Kretser DM 1998 Immunoregulatory activity in adult rat testicular interstitial fluid: roles of interleukin-1 and transforming growth factor ß. Biol Reprod 58:927–934[Abstract/Free Full Text]
  8. Watson ED, Zanecosky HG 1990 Immunosuppressive properties of follicular fluid from preovulatory horse follicles. J Reprod Fertil 89:627–632
  9. Fahmi HA, Hunter AG, Markham RJ, Seguin BE 1985 Immunosuppressive activity of bovine follicular fluid on bovine T lymphocytes in vitro. J Dairy Sci 68:3312–3317
  10. Edwards RG 1974 Follicular fluid. J Reprod Fertil 37:189–219
  11. Shalgi R, Kraicer P, Rimon A, Pinto M, Soferman N 1973 Proteins of human follicular fluid: the blood-follicle barrier. Fertil Steril 24:429–434[Medline]
  12. Day JR, Albers JJ, Gilbert TL, Whitmore TE, McConathy WJ, Wolfbauer G 1994 Purification and molecular cloning of human apolipoprotein F. Biochem Biophys Res Commun 203:1146–1151[CrossRef][Medline]
  13. Wang X, Driscoll DM, Morton RE 1999 Molecular cloning and expression of lipid transfer inhibitor protein reveals its identity with apolipoprotein F. J Biol Chem 274:1814–1820[Abstract/Free Full Text]
  14. Traill KN, Huber LA, Wick G, Jurgens G 1990 Lipoprotein interactions with T cells: an update. Immunol Today 11:411–417[CrossRef][Medline]
  15. Gwynne JT, Mahaffee DD 1986 Lipoproteins and steroid hormone-producing tissues. Methods Enzymol 129:679–690[Medline]
  16. Andersen JM, Dietschy JM 1978 Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J Biol Chem 253:9024–9032[Free Full Text]
  17. Hancock WW, Becker GJ, Atkins RC 1982 A comparison of fixatives and immunohistochemical technics for use with monoclonal antibodies to cell surface antigens. Am J Clin Pathol 78:825–831[Medline]
  18. O’Bryan MK, Schlatt S, Gerdprasert O, Phillips DJ, de Kretser DM, Hedger MP 2000 Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility. Biol Reprod 63:1285–1293[Abstract/Free Full Text]
  19. O’Bryan MK, Sebire K, Meinhardt A, Edgar K, Keah HH, Hearn MT, de Kretser DM 2001 Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa. Mol Reprod Dev 58:116–1125[CrossRef][Medline]
  20. Sambrook J, Russell DW 2001 Molecular cloning. A laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  21. Schägger H, von Jagow G 1987 Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379[CrossRef][Medline]
  22. Wray W, Boulikas T, Wray VP, Hancock R 1981 Silver staining of proteins in polyacrylamide gels. Anal Biochem 118:197–203[CrossRef][Medline]
  23. Tetaz T, Bozas E, Kanellos J, Walker I, Smith AI 1993 In situ tryptic digestion of proteins separated by SDS-PAGE. Improved procedures for extraction of peptides prior to microsequencing. In: Techniques in protein chemistry. San Diego: Academic Press; 389–397
  24. Xu N, Dahlbäck B 1999 A novel human apolipoprotein (apoM). J Biol Chem 274:31286–31290[Abstract/Free Full Text]
  25. O’Bryan MK, Sebire KL, Gerdprasert O, Hedger MP, Hearn MT, de Kretser DM 2000 Cloning and regulation of the rat activin ßE subunit. J Mol Endocrinol 24:409–418[Abstract]
  26. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ 1990 Basic local alignment search tool. J Mol Biol 215:403–410[CrossRef][Medline]
  27. Havel RJ, Eder HA, Bragdon JH 1955 The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345–1353
  28. Ganfornina MD, Gutierrez G, Bastiani M, Sanchez D 2000 A phylogenetic analysis of the lipocalin protein family. Mol Biol Evol 17:114–126[Abstract/Free Full Text]
  29. Del Nery E, Chagas JR, Juliano MA, Juliano L, Prado ES 1999 Comparison of human and porcine tissue kallikrein substrate specificities. Immunopharmacology 45:151–157[CrossRef][Medline]
  30. Le Goff D 1994 Follicular fluid lipoproteins in the mare: evaluation of HDL transfer from plasma to follicular fluid. Biochim Biophys Acta 1210:226–232[Medline]
  31. Perret BP, Parinaud J, Ribbes H, Moatti JP, Pontonnier G, Chap H, Douste-Blazy L 1985 Lipoprotein and phospholipid distribution in human follicular fluids. Fertil Steril 43:405–409[Medline]
  32. Sousa MM, Berglund L, Saraiva MJ 2000 Transthyretin in high density lipoproteins: association with apolipoprotein A-I. J Lipid Res 41:58–65[Abstract/Free Full Text]
  33. Driscoll DM, Getz GS 1984 Extrahepatic synthesis of apolipoprotein E. J Lipid Res 25:1368–1379[Abstract]
  34. Polacek D, Beckmann MW, Schreiber JR 1992 Rat ovarian apolipoprotein E: localization and gonadotropic control of messenger RNA. Biol Reprod 46:65–72[Abstract]
  35. Law GL, McGuinness MP, Linder CC, Griswold MD 1997 Expression of apolipoprotein E mRNA in the epithelium and interstitium of the testis and the epididymis. J Androl 18:32–42[Abstract/Free Full Text]
  36. Schleicher RL, Zheng M, Zhang M 1993 Immunocytochemical localization and endogenous synthesis of apolipoprotein E in testicular Leydig cells. Biol Reprod 48:313–324[Abstract]
  37. Kelly ME, Clay MA, Mistry MJ, Hsieh-Li HM, Harmony JA 1994 Apolipoprotein E inhibition of proliferation of mitogen-activated T lymphocytes: production of interleukin 2 with reduced biological activity. Cell Immunol 159:124–139[CrossRef][Medline]
  38. Borish L, King MS, Mascali JJ, Johnson S, Coll B, Rosenwasser LJ 1992 Transthyretin is an inhibitor of monocyte and endothelial cell interleukin-1 production. Inflammation 16:471–484[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Lamant, F. Smih, R. Harmancey, P. Philip-Couderc, A. Pathak, J. Roncalli, M. Galinier, X. Collet, P. Massabuau, J.-M. Senard, et al.
ApoO, a Novel Apolipoprotein, Is an Original Glycoprotein Up-regulated by Diabetes in Human Heart
J. Biol. Chem., November 24, 2006; 281(47): 36289 - 36302.
[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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O’Bryan, M. K.
Right arrow Articles by Hedger, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O’Bryan, M. K.
Right arrow Articles by Hedger, M. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals