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a Departments of Medicine and of Biochemistry, University of Montreal, Guy-Bernier Research Centre, Montreal, Quebec, Canada H1T 2M4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The BSP proteins exhibit multiple binding and functional properties. They bind to choline phospholipids of cauda epididymal spermatozoa at ejaculation [11, 12]. Also, these BSP proteins bind to sperm capacitation factors, namely, heparin [13] and high-density lipoproteins (HDL) [14]. Recently it has been shown that BSP proteins potentiate the capacitation of bovine epididymal sperm induced by heparin [15] and HDL [16]. The mechanism underlying the heparin-induced capacitation is unknown, but the binding of BSP proteins to the sperm membrane appears to increase the number of heparin-binding sites on the sperm surface [15–17]. Recent results suggest that the mechanism by which HDL and BSP proteins stimulate sperm capacitation involves cholesterol efflux [17].
Phosphorylcholine-binding proteins from the seminal fluids of various mammalian species (including humans, hamsters, mice, rats, and pigs) share antigenic determinants with bovine BSP-A1/A2, BSP-A3, and BSP-30-kDa [18]. More recently, proteins that display heparin- and phosphorylcholine-binding properties have been characterized from stallion (HSP-1 and HSP-2; [19]) and boar seminal plasma (pB1; [20, 21]). These proteins are structurally related and belong to the same protein family as the bovine BSP proteins and therefore seem to be important in reproduction [22, 23]. However, only the cDNA coding for BSP-A1/A2 has been sequenced [24, 25]. Also, little is known about their tissue specificity of expression, since testis, epididymis, and other accessory sex glands as well as other organs have never been investigated for the production of these proteins. In the present study, we report the cloning of the cDNAs coding for the BSP-A3 and BSP-30-kDa proteins, as well as the expression of all BSP proteins mRNAs in several bovine tissues. We also investigated the presence of BSP protein homologous genes in human, rat, hamster, and rabbit genomic DNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seminal vesicles and tissues from two bulls were collected immediately after slaughter from Abattoir Les cèdres (St-Lazare, PQ, Canada). Rabbit kidney and rat spleen were removed and processed as indicated later. Human prostate was obtained from Québec Transplant (Montréal, PQ, Canada). All the tissues were rapidly frozen in liquid nitrogen and stored at -80°C.
RNA Isolation and cDNA Synthesis
Total RNA from seminal vesicles and tissues was isolated following the acid guanidinium-phenol-chloroform method [26]. Ten micrograms of total RNA from seminal vesicles was reverse transcribed using a poly(T) primer (RACE-dT [5'-GCTAAGCTAGCGCTAAGAGCGGCCG-CAAGC(T)15-3']) and the Superscript II enzyme (GibcoBRL, Burlington, ON, Canada). Reverse-transcribed samples were purified using the Geneclean Kit II (BIO 101, La Jolla, CA) and kept at -20°C before the amplification reactions. One tenth of each reaction was used in the polymerase chain reaction (PCR) amplifications.
Amplification of Partial cDNA Sequences Encoding BSP Proteins
Degenerate PCR primers (Table 1) for BSP-A3 and BSP-30-kDa were designed on the basis of the primary sequence of the protein [4, 6], in regions where the amino acids showed less degeneracy in their genetic codons. For BSP-A3, primers A3deg.for. and A3deg.rev. were based on amino acid regions 12–19 and 106–114, respectively [4]; the predicted length of the amplified fragment was 308 nucleotides (nt). For BSP-30-kDa, primers 30Kdeg.for and 30Kdeg.rev were based on amino acid regions 2–7 and 152–158, respectively [6]; the predicted length of the amplified fragment was 471 nt. PCR primers for BSP-A1/A2 were designed on the basis of the published cDNA sequence [24]. The sequence of the forward primer (A1/A2for.) corresponds to the beginning of the 5' untranslated region, and the reverse primer (A1/A2rev.) is located 328 nt downstream of the 5' untranslated region primer. The predicted length of the amplified fragment was 371 nt.
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Using these primers, 35 PCR cycles were performed on reverse-transcribed RNA from bovine seminal vesicles. Cycling conditions consisted of 1-min denaturation at 94°C, 1-min annealing (40°C for BSP-A3, 51°C for BSP-30-kDa, and 50°C for BSP-A1/A2), and 1-min primer extension at 72°C. A 3-min denaturation step at 94°C preceded cycling; and at the end, a final 20-min primer extension at 72°C was performed, followed by a 4°C soak. The "hot start" procedure was accomplished by adding 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Laval, PQ, Canada) to each reaction tube only after the block temperature had risen above 80°C during the initial denaturation step.
Rapid Amplification of cDNA Ends (RACE) Experiments
To perform 3' RACE, we used cDNA synthesized from total seminal vesicle RNA with the RACE-dT primer. Subsequently, for BSP-A3, a touchdown PCR [27] was performed using the RACE-2A primer and the gene-specific primer A3-pos34. The beginning annealing temperature was 55°C (minus 1°C per cycle for 8 cycles); there were then 30 cycles at 47°C. A DNA fragment at about 450 base pairs (bp) was isolated from a 1% agarose gel and melted at 95°C in 300 µl of water. Then, 2 µl of this dilution was used to perform a nested PCR with the primer pair RACE-2A + A3-pos57 with the same cycling conditions. For BSP-30-kDa, PCR was performed using the RACE-2A primer and the gene-specific primer 30k-pos109 with the cycling conditions described for the BSP-A3 3' RACE experiment. A small piece of agarose gel containing a DNA fragment at about 400 bp was isolated and melted at 95°C in 300 µl of water. Then, 2 µl of this dilution was used to perform a nested PCR with the primer pair RACE-2A and 30k-pos143 with the same cycling conditions.
For 5' RACE experiments, the 5' RACE System (GibcoBRL) was used. Ten micrograms of total bovine seminal vesicle RNA was reverse transcribed using the specific primer A3-Apos115 or 30k-Apos146 (for BSP-A3 and BSP-30-kDa, respectively). The RNA was degraded with RNAse H, and the cDNA was purified using the GlassMax DNA Isolation Spin Cartridge (GibcoBRL). Subsequently, a poly(C)+ tail was added at the 3' end of the cDNA using terminal transferase and dCTP. For BSP-A3, the dC-tailed cDNA was amplified using the Abridged Anchor Primer and primer A3-Apos102. The annealing temperature began at 60°C (minus 1°C per cycle for 8 cycles); there were then 27 cycles at 52°C. A small piece of agarose gel containing a DNA fragment at about 450 bp was isolated and melted at 95°C in 500 µl of water. Then 2 µl of this dilution was used to perform a nested PCR with the primer pair A3-Apos30 and the Abridged Universal Amplification Primer. The annealing temperature began at 60°C (minus 1°C per cycle for 9 cycles), followed by 26 cycles at 51°C.
For BSP-30-kDa, the dC-tailed cDNA was amplified using the Abridged Anchor Primer and primer 30k-Apos146. The annealing temperature began at 60°C (minus 1°C per cycle for 9 cycles), followed by 28 cycles at 51°C. As no product was visible on an ethidium bromide-stained gel, 1 µl of the first PCR was diluted 100-fold with water and 5 µl of this dilution was reamplified using Abridged Universal Amplification Primer and the nested primer 30k-Apos77. The annealing temperature began at 60°C (minus 1°C per cycle for 8 cycles); 30 cycles at 52°C followed. In both RACE 3' and RACE 5' experiments, control reactions without reverse transcriptase or without RNA were performed. For 5' RACE, additional control reactions without terminal transferase were performed.
Cloning and Sequencing
All PCR products were cloned into the SmaI site of the pGEM3zf vector (Promega, Madison, WI) via the T-A overhang method [28]. The sequencing was performed on both strands with the Fidelity DNA Sequencing kit (Oncor, Gaithersburg, MD) and [
-35S]dATP (Amersham, Oakville, ON, Canada) using either SP6 or T7 primers (Table 1).
RNase Protection Assays
The pGEM3zf vectors containing the partial cDNA encoding for each BSP protein as described earlier were linearized separately and then transcribed using the MAXIscript SP6/T7 kit (Ambion, Austin, TX) in the presence of [-32P]UTP (Amersham) to obtain uniformly labeled antisense strands. The BSP-A3 and the BSP-A1/A2 clones were linearized by the EcoRI enzyme, giving an antisense RNA transcript of 375 bp for BSP-A3 and of 438 bp for BSP-A1/A2 with the SP6 RNA polymerase. The BSP-30-kDa clone was linearized by the SmlI enzyme, generating a BSP-30-kDa antisense RNA transcript of 392 bp with the T7 RNA polymerase. The sequence of each BSP protein cDNA transcribed for the RNase protection assays is shown in Figure 1. Full-length RNA probes were recovered from 6% 8 M urea acrylamide gels by submerging gel slices in elution buffer containing 500 mM ammonium acetate, 1 mM EDTA, and 0.2% SDS overnight at room temperature. The diluted RNA probes were precipitated by adding 10 µg of yeast RNA and 2.5 volumes of cold absolute ethanol. RNase protection assays were performed with the RPA II RNase protection kit (Ambion). Briefly, 10 µg of total RNA from adult bovine tissues (the amount and quality of the RNA used for the protection assays had been previously checked on formaldehyde agarose gel to ensure equal loading of RNA in all samples) was hybridized for 20 h at 45°C with one of the radiolabeled RNA probes using 1.8 x 105 cpm of the BSP-A3 RNA probe, or 1.5 x 105 cpm of the BSP-30-kDa RNA probe, or 3.4 x 105 cpm of the BSP-A1/A2 RNA probe. The specific activity of the probes was greater than 7.5 x 108 cpm/µg. RNase digestion of the unhybridized RNA was performed at 37°C for 2 h with 0.1 U of RNase A and 20 U of RNase T1. The protected RNA fragments were separated on 6% polyacrylamide 8 M urea gels. The gels were exposed to Biomax MS x-ray film (Eastman Kodak, Rochester, NY) with the Biomax MS intensifying screen at -80°C overnight and for 1 wk. For each experiment, two control tubes containing the same amount of labeled probe and 10 µg of yeast RNA were incubated with or without RNases. RNA markers were 32P-labeled transcripts from the century marker templates from Ambion.
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Southern Analysis
High molecular weight genomic DNA from various species was prepared as described previously [28]. For rabbits, rats, humans, hamsters, and cattle, the DNA was extracted, respectively, from kidney, spleen, prostate, CHO cells (Chinese hamster ovary cells), and lung. Twenty-five micrograms of genomic DNA from each species was digested with the EcoRI restriction enzyme (New England Biolabs, Mississauga, ON, Canada). The fragments were separated on a 1% agarose gel, transferred to Duralon-UV nylon membranes (Stratagene, La Jolla, CA), and hybridized as described below. The BSP-A1/A2 and BSP-A3 DNA probes were, respectively, the 371-bp and the 308-bp PCR products obtained as described above. The BSP-30-kda DNA probe was a 435-bp PCR product amplified using primers 30Kdeg.for. and 30k-Apos146. The sequence of each probe is shown in Figure 1. Each of these fragments was labeled using [-32P]dCTP (Amersham) and the Ready-to-Go DNA labeling kit (Pharmacia, Baie d'Urfé, PQ, Canada). Membranes were prehybridized in 4-strength saline-sodium phosphate-EDTA buffer, 0.1% sodium pyrophosphate, 5% SDS, and 100 µg/ml heparin for 5 h at 68°C. Hybridization was carried out overnight at 68°C; the membrane was washed three times at room temperature for 5 min in double-strength sodium chloride/sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) with 0.1% SDS, followed by a 60-min wash at 65°C in 0.1-strength SSC/0.1% SDS (high-stringency washing) and three final washes at room temperature in 0.1-strength SSC. The membranes were exposed to Biomax MS x-ray film (Kodak) at -80°C with the Biomax MS intensifying screen overnight or up to 10 days. Size markers were 
-DNA digested with HindIII. The percentages of nucleotide homologies between the probes and the various BSP cDNAs are given in Table 2. The BSP-30-kDa probe contains the region coding for the unique amino-terminal sequence, and this DNA region has no homology with the other BSP DNA probes (Fig. 1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR fragments covering the complete BSP-A3 and BSP-30-kDa cDNAs were cloned and sequenced. First, a specific fragment of each cDNA was amplified by PCR using degenerate primers designed on the basis of the primary sequence of the proteins. The nature of the fragments obtained was verified by comparing their deduced amino acid sequences with the published protein sequences. Then, specific primers were designed on the basis of these sequences. These primers were used to amplify the 3' and 5' untranslated cDNA regions by the 3' and 5' RACE techniques, and the fragments were cloned and sequenced (Figs. 2 and 3). For both cDNAs, the initiation methionine occurred within the sequence CTACCATGG, highly homologous to the Kozak consensus sequence CCA(G)CCATGG that controls translational efficiency of mammalian mRNAs [29–31]. Furthermore, both possessed the consensus polyadenylation signal AATAAA, 13 or 12 nt upstream of the poly(A) tail, respectively, for the BSP-30-kDa cDNA and the BSP-A3 cDNA.
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The complete cDNA of BSP-A3, excluding the poly(A) tail, was 633 nt long (Fig. 2). The cDNA coding for BSP-A3 contained an open reading frame of 420 nt starting at position 19. The deduced amino acid sequence, excluding the signal peptide, was identical to the primary sequence previously reported [4] except at position 2, where a glutamate (E) was found instead of a glutamine (Q), and at position 90, which was an isoleucine (I) rather than a lysine (K). The cleavage site for the precursor peptide occurred between amino acid -1 and +1 as determined by the primary sequence previously reported [4] (Fig. 2). The theoretical molecular weight of the 140-amino acid residue precursor polypeptide of BSP-A3 is 16 584.
The complete cDNA of BSP-30-kDa, excluding the poly(A) tail, was 782 nt long (Fig. 3). The cDNA coding for BSP-30-kDa had an open reading frame of 549 bp starting at position 24. The deduced amino acid sequence, excluding the signal peptide, was the same as the primary sequence obtained earlier [6], but we found at position 13 a proline (P) rather than a serine (S). The six unidentified residues in the reported peptide sequence were found to be threonine, as suggested by the previous authors. The cleavage site for the precursor peptide occurred between amino acid -1 and +1 as determined by the primary sequence previously reported [6] (Fig. 3). The theoretical molecular weight of the 183-amino acid residue precursor polypeptide of BSP-30-kDa is 21 721. The aligned sequences of the cDNA encoding for the various BSP proteins are presented in Figure 1, and the degrees of residue homologies between these cDNAs are presented in Table 2. The sequence region between the three BSP cDNAs that had the most identical nucleotides was the one encoding the putative signal peptide (88% homology), and the sequence region that had the highest nucleotide variations and gap frequencies was the one encoding the N-terminal extension.
The first 25 amino acid residues of the BSP-A3 and BSP-30-kDa precursor proteins had a hydrophobic character; they displayed, respectively, 92% and 76% similarity with those residues of the BSP-A1/A2 precursor polypeptide (Fig. 4). They also displayed a significant similarity (80% for BSP-A1/A2, 80% for BSP-A3, and 68% for BSP-30-kDa) with the first 25 amino acid residues of the precursor polypeptide of boar pB1 recently sequenced by Plucienniczak and coworkers (GenBank #AF047026).
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Tissue-Specific Expression of BSP Proteins
The results of the RNase protection assay for BSP-A3 mRNA are depicted in Figure 5. In seminal vesicle tissue and ampullae, strong expression of BSP-A3 mRNA was observed, since the expected 308-bp RNA fragment was detected in these tissues (Fig. 5A, lanes 2–7; Fig. 5B, lanes 4–7). Also a second major RNA fragment at approximately 270 bp was protected. No BSP-A3 mRNA expression was revealed in artery, small intestine, adipose tissue, brain, kidney, heart, lung, large intestine, liver, epididymis, testis, or prostate (Fig. 5C). The results of the RNase protection assays for BSP-30-kDa and BSP-A1/A2 mRNAs are depicted, respectively, in Figures 6 and 7. These BSP protein mRNAs showed strong expression only in seminal vesicle tissue and in the ampullae (Figs. 6A and 7A, lanes 2–7; Figs. 6B and 7B, lanes 4–6), since the expected major RNA fragments (344 bp for BSP-30-kDa mRNA or 371 bp for BSP-A1/A2 mRNA) were detected only in these two tissues (Figs. 6C and 7C).
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Southern Analysis of Genomic DNA in Various Species
A comparative genomic analysis employing a BSP-30-kDa-specific DNA probe and high-stringency washing conditions yielded a band at 3.7 kilobases (kb) in human, rabbit, rat, and hamster DNA (Fig. 8A). In the same conditions, bands at 3.7 and 9.8 kb were seen in male and female bovine DNA. The use of a BSP-A1/A2- or a BSP-A3-specific DNA probe with low-stringency washing conditions yielded no signal with human, rabbit, rat, and hamster DNA (data not shown). Using high-stringency conditions with male and female bovine DNA, bands at 4.2 and 3.9 were detected with the BSP-A3 probe (Fig. 8B), while bands at 10.5, 1.6, and 1.0 kb were detected with the BSP-A1/A2 probe (Fig. 8C). The bands at 4.2 and 3.9 kb (detected with the BSP-A3 probe) were also detected faintly in male and female DNA in medium-stringency conditions by the BSP-A1/A2 probe (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The deduced amino acid sequences indicate one (for BSP-30-kDa) or two (for BSP-A3) amino acid differences from the primary sequences previously reported [4, 6]. These differences have been found consistently in several clones from different PCR reactions, excluding the possibility that these differences arise from amplification or sequencing errors. Also, the presence in RNase protection assays of a fragment at the expected size for the full-length BSP-A3 mRNA protected probe (308 bp) confirms that the base change (position 362 on the cDNA sequence) detected in the sequencing is present in the mRNA (Fig. 5). Polypeptide sequencing errors are unlikely for BSP-A3, since among known members of the BSP protein family in cattle and other species, at the position of the first difference, the amino acid seems to be a conserved glutamine (Q), and at the second different position a conserved basic amino acid, lysine (K) or arginine (R), except for a threonine (T) for HSP-1 (Fig. 9). No such comparison can be made concerning the proline (P) at position 13 in BSP-30-kDa, since this region does not show any homology to known protein sequences [6, 21].
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These differences could be due to polymorphism in the genes coding for the BSP proteins. Several arguments converge in this direction. The presence of a smaller RNA fragment (at approximately 270 bp) with the same intensity as the full-length fragment in the BSP-A3 RNase protection assay (Fig. 5) suggests the possibility that this bull is heterozygous for the gene coding for BSP-A3. The total RNA used in these experiments was extracted from tissues of a bull different from the one used for the cloning of the BSP cDNAs. This smaller RNA fragment is not the result of cross-binding between the BSP-A3 probe and the BSP-A1/A2 or BSP-30-kDa RNA, since there are many nucleotide differences between them (Fig. 1). Also, some amino acid differences are seen between the published primary sequence of boar pB1 [21] and the deduced amino acid sequence of its cDNA recently cloned by Plucienniczak and coworkers (Genbank #AF047026). Further studies are required in order to determine whether these differences are due to polymorphism in the BSP genes or in homologous genes in other species.
The amino acid difference in BSP-30-kDa and the first one in BSP-A3 occur in the amino-terminal region. The effect of this substitution is unknown since the role of the amino-terminal region in each BSP protein is unknown. The second substitution in BSP-A3 occurs in the second type II domain of the protein (domain b). These type II domains are important for the biological function of the BSP proteins [9]. In fact, in BSP-A1/A2, they constitute binding units for sperm membrane choline phospholipids and heparin, and the presence of both type II domains is essential for the expression of functional properties, namely lipid efflux and sperm capacitation [9]. So, if an amino acid substitution in domain b affects its binding properties, the stimulation of sperm cholesterol efflux and sperm capacitation by the BSP protein will be affected. These amino acid substitutions could modify the affinity of the BSP proteins for sperm membrane choline phospholipids and thus affect sperm capacitation induced by heparin or HDL. Conversely, they could influence their affinity toward heparin or HDL and thus change the sperm capacitation rate.
As seen in the RNase protection assays, all the BSP proteins mRNAs are synthesized in the seminal vesicles and the ampullae (Figs. 5A, 6A, and 7A, lanes 2–6; Fig. 5B, lanes 4–7; Figs. 6B and 7B, lanes 4–6). The expression is very strong in these tissues, since a very intense radioactive signal appears on the autoradiogram after only one night of exposure. Each BSP protein mRNA is detected in only one nanogram of total seminal vesicle RNA (Figs. 5B, 6B, and 7B, lane 6; Fig. 5C, lane 1; Fig. 6C, lane 8; and Fig. 7C, lane 15). No signal was detected in 10 µg RNA from testis, epididymis, and other male accessory sex glands or other tissues (Figs. 5C, 6C, and 7C). This suggests that the BSP protein mRNA expression in these tissues is at least 10 000 times less than in the seminal vesicles. However, even a very low expression of BSP proteins in other tissues could have a physiological significance, since the large mass of other tissues, even with a very low level of expression, could produce large enough quantities of BSP proteins in the bloodstream to influence some physiological processes, such as cholesterol efflux, for example [33]. Only more sensitive techniques, such as RNase protection on isolated mRNA or nested reverse transcription-PCR, could determine whether expression of BSP genes in other tissues is totally abolished. The presence of BSP proteins in female bovine tissues is unknown.
Comparison of the signal obtained with RNA from ampullae and seminal vesicles suggests that BSP-A1/A2 and BSP-30-kDa mRNAs are expressed at about the same level in the ampullae and seminal vesicles. This is not the case for BSP-A3 mRNA, which is about 50 times less expressed in the ampullae than in seminal vesicles. This difference in the level of expression in the ampullae was confirmed by Northern blot analysis (data not shown). Using probes of approximately the same specific radioactivity, comparison of the signal strength suggests that the amount of BSP-A1/A2 mRNA is higher than that of the BSP-30-kDa mRNA, and the amount of BSP-30-kDa mRNA is slightly higher than that of BSP-A3 mRNA in the seminal vesicles (Figs. 5B, 6B, and 7B). This correlates with the proportion of each protein in the seminal fluid (BSP-A1/A2, ~65%; BSP-A3, ~15%; BSP-30-kDa, ~20% as determined by respective RIAs [5]). These results suggest that BSP protein expression is controlled at the transcriptional level.
Using a BSP-30-kDa-specific cDNA probe and high-stringency Southern hybridization, a homologous sequence at 3.7 kb was detected in genomic DNA from the human, rat, hamster, and rabbit (Fig. 8A), suggesting that the genomic DNA of these species possess a gene with a significant homology to the bovine BSP-30-kDa gene. This is in agreement with the fact that in human, hamster, and rat seminal plasma, phosphorylcholine-binding proteins immunologically related to the bovine BSP-30-kDa protein have been detected [18]. Southern analysis with a BSP-A1/A2- or a BSP-A3-specific cDNA probe at high-stringency conditions of hybridization yielded no signal with the human and other mammalian species (Fig. 8, B and C). Even using low-stringency conditions of hybridization, these two probes yielded no signal with the human or other mammalian species (data not shown). The BSP-30-kDa probe contains the sequence coding for the amino-terminal region without homology to known protein sequences (Fig. 1). The detection of homologous genes in different species using this probe suggests that this unique region has been well conserved in mammals, while genes homologous to BSP-A1/A2 and BSP-A3, if present, seem to have diverged rapidly during evolution. It is unlikely that they are absent in other species, since in human, hamster, and rat seminal plasma, phosphorylcholine-binding proteins immunologically related to the bovine BSP-A1/A2 and BSP-A3 proteins have been detected [18]. Furthermore, analogous proteins have been purified from stallion [19] and boar seminal plasma [20, 21]. Analysis of genes homologous to the BSP-A1/A2 and BSP-A3 genes in other species will be necessary to confirm this supposed high mutation rate; such a high rate could suggest that these proteins have an important role in reproduction. On the other hand, as mentioned above, one or two amino acid changes could influence bull fertility; then, several differences between species could, for example, contribute to a cross-species barrier in reproduction.
In contrast, the fact that the BSP-30-kDa unique region is more conserved among species could suggest a common role for this domain in the reproduction processes in several species. Among the bovine BSP proteins, BSP-30-kDa is the best mediator of sperm capacitation induced by heparin [15] or HDL [16] on epididymal spermatozoa, and its presence on the sperm membranes corresponds to increased fertility of bulls [34–36]. The common role of this protein in different species could be to accelerate capacitation by increasing lipid modifications in the sperm membrane.
In summary, we have determined the complete sequences of the two cDNAs coding for BSP-A3 and BSP-30-kDa proteins. Two amino acid changes for BSP-A3 and one for BSP-30-kDa were found in the deduced amino acid sequences as compared with the previously reported primary sequences. We propose that the amino acid substitutions in BSP-A3 may be due to genetic polymorphism. All BSP proteins are not only synthesized by the seminal vesicles but also produced by the ampullae. We suggest that these proteins are important mostly in reproduction, since their mRNAs are expressed at a high level only by these two accessory sex glands. Finally, we propose that the BSP-analogous proteins in the human, rat, hamster, and rabbit may have numerous amino acid differences, since no homologous genes were detected with the BSP-A1/A2 and BSP-A3 cDNA probes using either high- or low-stringency Southern hybridization conditions. These differences may be important in blocking cross-species fertilization.
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FOOTNOTES |
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2 Correspondence: P. Manjunath, Guy-Bernier Research Centre, 5415 Boul. de l'Assomption, Montreal, PQ, Canada H1T 2M4. FAX: 514 252 3430; manjunap{at}ere.umontreal.ca
Accepted: February 26, 1999.
Received: December 7, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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