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Female Reproductive Tract |
a Institute for Research in Reproduction, Indian Council of Medical Research, Parel, Mumbai 400012, India
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100–110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.
estradiol, fallopian tubes, female reproductive tract, oviduct, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epithelial cells in most mammalian oviducts secrete a major estrogen-induced glycoprotein known as oviduct-specific glycoprotein (OGP; also called MUC9).The protein is elaborated at the time of ovulation, fertilization, and early embryonic development [11–14]. The most significant finding among all the species studied so far is the association of OGP with the zona pellucida and its detection within the perivitelline space of ovulated oocytes and embryos. Studies with sheep OGP have also revealed its presence on the surface of blastomeres [15]. Binding sites on the acrosome in the sperm of hamsters [16] and bovine [17] have also been reported.
In hamsters, in vitro, the presence of antibodies raised against the well-conserved region of OGP led to a significant decline in the number of sperm that attached to the zona pellucida [18]. Observation of enhanced sperm penetration in the presence of human OGP and its inhibition by baboon OGP despite a high degree of homology (96%) suggests the possibility of two different binding sites on the gametes [19]. On the other hand, studies with porcine OGP have shown a lower incidence of polyspermy in oocytes [20] with no effect on sperm penetration. Taken together, these data indicate a critical role for OGP in fertilization.
Complementary DNA cloning of most mammalian OGPs has been achieved [21], and information gleaned from the nucleotide sequence analysis indicated several interesting features of this class of molecules. From mouse to humans, the N-terminal region of the molecule (1–600 base pairs) shows a higher degree of homology than the C-terminal region. The C-terminal region possesses several insertions or deletions in the nucleotide sequence, and it is worth noting that all the glycosylation sites are also located in this region. Recently, genomic organization of the 5' flanking region of the mouse OGP has been enumerated to reveal the presence of 10 palindromic estrogen-responsive elements [22].
Despite these advances in our knowledge of OGP, several fundamental questions remain unanswered. Issues associated with the protein's physiological function and its role in hormonal regulation need to be addressed. Reports exist to show that LH could induce OGP expression [23], and recently in rabbits it was determined that the endocervix is another site of its synthesis [24]. Targeted disruption of the OGP gene will aid in resolving several key points in OGP function and its role during fertilization and preimplantation events. Use of a suitable animal model, availability of monospecific antibodies, and a molecular probe would greatly assist in understanding the hormonal modulation of OGP. However, the mode of OGP association with gametes needs to be examined. In particular, answers to the following questions are needed: 1) Is the mode of OGP association with gametes through a protein backbone or is it through carbohydrate residues? 2) Is there a separate and distinct binding domain on OGP that interacts with sperm and zona/eggs? and 3) What are the putative partners involved in binding OGP with sperm?
These issues can be resolved when bulk preparation of OGP, and in particular their glycosylated and nonglycosylated forms, are available either by isolating and purifying them from oviductal fluid or through the recombinant approach. In this endeavor, we have successfully cloned a partial cDNA that encodes bonnet monkey (Macaca radiata) OGP [25]. The nucleotide sequence (AF132 215) shows 96% homology to human, baboon, and rhesus monkey OGP and 84%–88% homology to other mammalian oviductal protein. In this paper we report overexpression of monkey OGP, characterization of the recombinant protein, and antibodies to it generated in rabbits.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bacterial Strains and Growth Condition
Escherichia coli strain DH5
(Bethesda Research Laboratories, Bethesda, MD) was used as the host strain for propagating the plasmid for cloning and sequencing the OGP gene. BL21 was used for bacterial expression of pET20b and pET20b containing the OGP gene. E. coli strains with plasmids were grown in Luria-Bertani (LB) medium containing 50µg/ml of ampicillin as described by Sambrook et al. [26]. Oligo nucleotide primers were designed on the basis of reported nucleotide sequence of human OGP [27] and were synthesized locally (Bangalore Genei). The sequence of the forward primer was 5'-GGGCTGGTTCATATGCTGAAACACCAC, corresponding to the 27–58 nucleotide sequence with a slight modification made to include a restriction site for NdeI. The reverse primer contained a XhoI site, and the sequence corresponding to 403–426 was GAAAAGCTCGAGACCATCAAAGTCATG. The restriction enzyme sites were included to aid directional cloning into the pET20b vector for protein expression. The plasmid pBluescript OGP was used as a template to amplify the OGP DNA. The conditions for polymerase chain reaction (PCR) were as follows: denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension for 2 min at 72°C, and an additional elongation step at the end of the 30th cycle for 10 min.
PCR product was resolved on 1% agarose gel. After ascertaining the presence of correct-size PCR product (400 base pairs [bp]), further aliquots of PCR product were separated on 1% low-melting-point agarose and gel-purified. The PCR product and pET20b vector were digested with NdeI and XhoI. The enzyme-digested PCR product was ligated to pET20b with T4 DNA ligase. The ligation mixture was incubated at 16°C for 16 h, and following this, the plasmid was transformed into DH5. Recombinant clones were selected by plating them on LB agar plates containing ampicillin (50 µg/ml). The plasmids harboring OGP were ascertained for size as well as by release of the insert following double digestion with NdeI and XhoI.
Expression of OGP in E. coli BL21
The recombinant plasmids were isolated and transformed in BL21. The E. coli cells harboring the pET20b-OGP plasmid were grown in 10 ml of LB medium containing 50 µg/ml of ampicillin. Overnight cultures of bacteria (2 ml) were diluted with fresh medium and grown at 37°C until the cell density reached an OD600 of 0.6, after which IPTG at different concentrations (0–1 mM) was added to the bacterial culture. The cells were harvested 4 h later and the cell pellet was resuspended in sonication buffer (50 mM sodium phosphate pH 8.0 containing 300 mM NaCl). The cell suspension was sonicated until it became optically clear and was then centrifuged at 10 000 x g for 30 min. The supernatant was used for SDS-PAGE [28]. For large-scale expression, an overnight culture of 25 ml was added to 500 ml of LB medium containing ampicillin (50 µg/ml), grown to an OD600 of 0.6–1.0 at 37°C, IPTG was then added to a final concentration of 0.4 mM, and then incubation continued for 4 h at 37°C.
Isolation and Purification of Recombinant OGP
The cell lysate was loaded onto a Sephadex G75 column (1 x 50 cm) that had been previously equilibrated with 10 mM Tris-HCl pH 7.4 containing 0.5 M NaCl. One-milliliter fractions were collected, and OD at 280 nm was monitored. Three 280-nm absorbing peaks were obtained and the fractions corresponding to these were pooled, dialyzed, lyophilized, and analyzed by SDS-PAGE followed by Western blotting for the presence of OGP.
Immunological Identification of Mouse OGP
In order to demonstrate the ability of antibodies generated against the monkey recombinant OGP (rOGP) to recognize mouse OGP, immunoblot analysis was performed using a mouse OGP clone that expresses rOGP (generated in our laboratory; unpublished results). The following procedure was used for cloning. In brief, based on the reported nucleotide sequence of mouse OGP [29], two specific oligo nucleotide primers were designed to amplify the DNA nucleotide sequence 76–2165 region corresponding to the mature protein. Mouse oviductal RNA was isolated and reverse transcribed to prepare cDNA. This cDNA was used as a template for PCR amplification. The PCR product was then cloned into expression vector pTriEx 1.1, and the protein was expressed and analyzed according to the procedure described for monkey OGP.
Production of Antibodies to Recombinant OGP
Three rabbits were used in the production of antibodies. Use of rabbits for immunization was approved by the Animal Ethics Committee of the Institute and Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India. A primary dose of 50 µg of purified rOGP emulsified in Freunds complete adjuvant was administered s.c. to rabbits at multiple sites followed by two doses of the protein (25 µg) emulsified in Freunds incomplete adjuvant at 3–4 weekly intervals. Ten days after the second booster, rabbits were bled and the serum was separated.
Enzyme-Linked Immunosorbent Assay
A 100-µl solution of rOGP at 10 µg/ml in 0.05 M carbonate buffer pH 9.6 was coated onto the wells of an ELISA plate and allowed to rest overnight at 4°C. The solution was discarded, the plate was washed with PBS-Tween 20 (50 mM phosphate buffer pH 7.4 containing 0.15 M NaCl and 0.5% Tween 20), and the plate was incubated with 2% BSA in PBS in order to block nonspecific sites. Then various dilutions of polyclonal antibody to rOGP were added to the wells and incubated for 45 min at 37°C. This was followed by washing with PBS-Tween 20. Following this, anti-rabbit IgG conjugated to HRP was added and incubation was continued for 1 h. The bound peroxidase activity was detected using O-nitrophenylenediamine (OPD). The reaction was terminated with 4 N H2SO4 and absorbance at 492 nm was recorded using an ELISA reader. All measurements were made in duplicate. The dilution at which the rOGP antibody gave an OD of 0.7–1.0 was determined. Following two boosters, an OD of 0.7 was seen at an antibody dilution of 1:20 000.
SDS-PAGE and Immunoblotting
Protein samples for Western blotting either from E. coli lysates or purified recombinant proteins were resolved on 15% SDS-PAGE and transferred onto a nitrocellulose membrane according to a procedure detailed elsewhere [30]. Briefly, following electrophoresis, the gel was equilibrated for 1 h at room temperature in 20 mM phosphate buffer pH 6.8 containing 20% methanol and 0.05% SDS, and transfer occurred in the same buffer at 10 V overnight at 4°C. After transfer, the nitrocellulose was stained with Ponceau S to visualize the protein bands and to ascertain proper transfer of the protein. Following destaining with distilled water, the blots were incubated with 2% fat-free milk proteins for 1 h followed by incubation with polyclonal antibodies to baboon OGP, human OGP, and antipeptide antibodies preadsorbed with E. coli lysate. These antibodies were generously provided by Prof. H.G. Verhage at the University of Chicago (Chicago, IL). After washing the membrane extensively with PBS-Tween 20, the blots were incubated with anti-rabbit IgG conjugated to HRP. Immunoreactive bands were visualized using the chromogen 3,3-diaminobenzidine (DAB) substrate in PBS pH 7.4 containing 0.03% H2O2.
Dot Blot Analysis
Proteins (2.5 µl; 1–2.5 µg) were loaded onto nitrocellulose strips and allowed to air-dry. The strips were incubated with 2% fat-free milk proteins for 1 h followed by incubation with polyclonal antibodies to recombinant monkey OGP for 1 h. The remainder of the procedure for detecting immunoreactive protein was performed as described above.
Molecular Weight of rOGP
The molecular weight of rOGP was determined by SDS-PAGE and by matrix-assisted laser adsorption time-of-flight (MALDI-TOF). MALDI mass spectrometry was performed using a Kratos pc-kompact Maldi Mass Spectrometer (Shimadzu, Tokyo, Japan) fitted with a standard nitrogen laser (337 nm). An aliquot of 0.5 µl of rOGP (10 pmol) was mixed on a probe slide with 0.5 µl of saturated sinapinic acid in 0.1% trifluroacetic acid/acetonitrile (2% v/v). Sinapinic acid was used as matrix. The sample and the matrix mixture were air-dried and the spectra were taken [31].
N-Terminal Amino Acid Sequencer
The N-terminal amino acid sequence of rOGP was obtained by subjecting the purified protein to SDS-PAGE [15%] and electroblotting to a PVDF membrane in 10 mM CAPS buffer pH 11 [32 ]. Following the transfer, the PVDF blot was removed, rinsed with distilled water, stained with 0.1% Coomassie brilliant blue in 50% methanol and 10% acetic acid for 3 min, and destained in 50% methanol in 10% acetic acid. The band corresponding to rOGP was excised and sequenced on a Shimadzu PSQI gas phase sequencer (Tokyo, Japan) at the Indian Institute of Science in Bangalore, India.
Enrichment of Human OGP
Hydrosalpinx fluid was obtained by aspirating hydrosalphinges in women undergoing follicular aspirations as part of IVF cycles using transvaginal ultrasound. Each patient provided informed consent. The fluid was centrifuged to remove particulate material and then dialyzed overnight against 10 mM Tris-HCl pH 7.4. Dialyzed and clarified fluid was fractionated on a DEAE cellulose column (20 ml bed volume) pre-equilibrated with the same buffer. Proteins were fractionated by increasing the ionic strength of the buffer (0.1–0.5 M NaCl). The protein fractions eluted with 0.1 M NaCl that were immunopositive with anti-rOGP on dot blots were pooled, dialyzed, and lyophilized. This protein fraction was henceforth referred to as enriched human OGP preparation.
Immunocytochemistry
Deparaffinized and rehydrated fallopian tube and human endometrial sections were treated with 0.5% of 30% H2O2 in methanol for 10 min to remove endogenous peroxidase activity followed by washing with PBS. The sections were briefly incubated with trypsin (0.1%) for 2 min. Following this, the slides were washed and incubated with BSA (1% w/v) for 30 min. BSA was used as an enzyme blocking solution. This procedure was used for antigen retrieval [33]. The sections were then incubated with rabbit anti-rOGP (IgG, 10 mg/ml) for 1 h at room temperature, and after washing in PBS (three times for 5 min each) they were incubated with HRP-conjugated goat anti-rabbit IgG at a dilution of 1:100. Following extensive washing with PBS the sections were incubated with chromogen 0.05% DAB in 10 ml of PBS and 0.03% hydrogen peroxide for 10 min at room temperature. The reaction was stopped by washing with PBS. The sections were counterstained with hematoxylin and mounted in Canada balsam for examination with a microscope. In control experiments, sections were incubated with normal rabbit serum (IgG, 10 mg/ml).
Immunocytochemical Localization of OGP Binding> with Sperm
The method we used was essentially the same one reported for bovine OGP [17]. Semen samples collected from normal bonnet monkeys were allowed to liquefy at 37°C for 30 min. Sperm in 1 ml of semen were washed twice with 9 ml of Ham F-10 medium and centrifuged at 1000 x g for 20 min. The pellet was resuspended in the same medium and divided into two aliquots. One aliquot (1 x 109/ml sperm) was incubated with 50 µg of human-enriched OGP at 37°C for 4 h, and washed twice with medium after incubation. The other aliquot did not receive the enriched OGP preparation. Smears were made on glass slides with 10 µl of sperm suspension from both aliquots, fixed in methanol for 20 min, and frozen until required.
Methanol-fixed slides were washed twice at room temperature with 0.01 M PBS pH 7.2. Sperm membranes were partially permeabilized by incubation at 4°C for 30 min in 0.01 M PBS containing 0.002% sodium deoxycholate pH 7.2. Following this, slides were washed twice with PBS containing 0.5% Tween 20 pH 7.2. To visualize the site of OGP binding, immunocytochemistry was performed; rabbit anti-rOGP was used as primary antibody (10 mg/ml), goat anti-rabbit HRP conjugate served as the secondary antibody, and DAB served as the chromogen. At least 100 sperm per slide were evaluated with light microscopy. Slides incubated with normal rabbit serum also served as negative controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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14-kDa protein following IPTG addition could be seen; however, when the IPTG concentration rose beyond 0.5 mM, protein synthesis was inhibited. The protein was expressed in a soluble form. From the densitometric scan of the gel, it appeared that an IPTG-induced product would be a major protein, one that was well separated from other E. coli proteins. Therefore, a simple gel filtration on Sephadex G75 was attempted (Fig. 3A). The silver-stained protein profile on SDS-PAGE electropherotogram (Fig. 3B) shows an apparent majority of E. coli protein eluted in the void volume and shows that the second peak contained the overexpressed protein. The third peak did not reveal the presence of any macromolecules.
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Characterization of rOGP
The identity of the purified recombinant protein was verified to be a part of monkey OGP by its ability to interact with antibodies to baboon OGP, human OGP, and antipeptide antibodies. The amino acid sequence of the synthetic peptide is well conserved across all forms of mammalian OGP (amino acid sequence 52–68 of hamster OGP, GenBank accession number U15048; shown in Fig. 1b). A single immunoreactive peak with the purified protein confirmed the identity of the overexpressed purified protein (Fig. 4). Purified protein (2.5 mg) could be made from 50 ml of induced culture. The molecular weight of the expressed protein was 14 439 as shown by MALDI-TOF (Fig. 5, mass/charge one). The second peak corresponding to 7272.9 is due to mass/charge two. The N-terminal amino acid analysis revealed an amino acid sequence of VHML.
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Characterization of Antibodies to Recombinant Protein
Polyclonal antibodies to rOGP were raised in three rabbits, and each rabbit generated antibodies that could interact with OGP in ELISA. Rabbit antibodies were thoroughly characterized in one of the three ways described below.
Antibodies that show high specificity
Figure 6a depicts the protein profile of hydrosalpinx fluid fractionated on DEAE cellulose with SDS-PAGE. Antibodies to rOGP were able to immunostain enriched preparations of human OGP with an expected molecular weight of 105 kDa (Fig. 6b). Antibodies were highly specific and did not stain high concentrations of hydrosalpinx fractions (peaks 2 and 3) that were devoid of OGP, thus establishing the monospecificity of the antibody.
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Antibodies could also recognize recombinant mouse OGP in crude E. coli lysate (Fig. 7) with a molecular weight of 66 kDa. The molecular size corresponds to the expected size from the deduced amino acid sequence.
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Immunolocalization of OGP secreting cells Immunohistochemical studies were performed on paraffin-embedded (5 µm) sections of fallopian tubes to determine the cellular localization of human OGP that cross-reacted with antibodies to rOGP on Western blots. Fallopian tube specimens obtained at the follicular phase of the cycle exhibited intense immunoperoxidase staining in the luminal epithelia of oviductal mucosa. Immunostaining to a lower extent was also noticed in tissues taken from women in menopause. No immunostaining was observed when preimmune sera were substituted for the primary antibody (Fig. 8). Immunostaining was specific to fallopian tube sections and was absent in endometrium.
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Localization of OGP binding sites on monkey sperm Ability of anti-rOGP to localize OGP bound to monkey sperm was examined. Figure 9 depicts the presence of binding sites for OGP at the head and tail of sperm. No staining was seen when the OGP enriched preparation was omitted or when the primary antibody was substituted with preimmune sera. The specific localization is similar to that observed with bovine sperm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overexpression of the recombinant protein in response to IPTG as a major protein was apparent and the molecular weight was similar to the expected size (Fig. 2). From the SDS-PAGE profile, the protein was well separated from E. coli proteins, therefore, purification was attempted using simple gel permeation chromatography on Sephadex G75 (Fig. 3A). Most of the overexpressed protein appeared in the second peak as a homogeneous fraction (Fig. 3B), and the molecular weight of the protein as determined by MALDI-TOF was 14 439 daltons (Fig 5). The N-terminal determination of VHML also matched the sequence deduced from the nucleic acid sequence (Fig. 1b). The identity of the protein was confirmed by Western blot analysis using antibodies generated against baboon and human OGP and antipeptide antibody (Fig. 4). The characterization of OGP antibodies to baboon [34], human [35], and antipeptide antibodies [18] have been described. The peptide sequence against the antibodies that were generated is conserved across mammalian species, including that of monkey OGP (Fig. 1b, sequence shown in gold). All three antibodies immunostained a single protein with a molecular size of 14 kDa in purified preparations (lane 1) and a major protein of the same size in E. coli lysate, indicating antigenic identity among OGP from monkeys, baboons, and humans. The ability of antipeptide antibodies to immunostain rOGP confirms the presence of this sequence in monkey OGP. Taken together, these results confirm the homology data derived from nucleotide and deduced amino acid sequence depicted in Figure 1b. Following this, rabbit antibodies were generated against rOGP, and the monospecificity of the antibodies was ascertained by examining the ability of antibodies to immunostain human OGP enriched preparation on Western blots. A complete absence of immunostaining of fractions devoid of human OGP (peaks 2 and 3 on DEAE cellulose) indicated an ability of the antibodies to rOGP to immunoreact exclusively with OGP (Fig. 6b), indicating that it was possible to generate an antibody that could be used across the species. Antisera could immunostain mouse OGP expressed in a bacterial expression system in response to IPTG. Antisera immunostained a major protein band corresponding to the expected molecular weight from the several E. coli lysate proteins (Fig. 7). Fallopian tube sections obtained from menopausal as well as follicular phases of the cycle indicated the presence of epithelial cells secreting OGP (Fig. 8). Regulation of hamster OGP expression as ascertained by in situ hybridization revealed that OGP gene expression did not disappear completely during the estrous cycle or in aged animals (18 mo) with low levels of estrogen [36]. We earlier demonstrated by Western blot analysis the presence of estrogen receptor in fallopian tubes taken from menopausal tissue [30]. It is possible that a low amount of estrogen from nonovarian tissue was sufficient to maintain OGP synthesis. There is also evidence to suggest that in the bovine oviduct, LH stimulates the expression of OGP [23]. However, regulation of OGP via LH in the human fallopian tube needs to be examined.
Finally, anti-rOGP was employed to demonstrate the binding site for OGP on sperm. It has been accepted particularly in farm animals that after ejaculated sperm travel through the reproductive tract they are stored in the apical region of epithelial cells of the isthmus. This has been observed both in vivo and in vitro in many species, including humans [37]. This interaction occurs through the exposed carbohydrate branches on the surface of epithelial cells with lectin-like molecules on the sperm membrane. Several functions have been attributed to this association: first, to maintain fertilizing capacity; second, to prevent polyspermic fertilization by controlling sperm transport to the ampulla, which has been demonstrated to be important in farm animals; and third, to modulate the capacitation process by synchronizing with the time of ovulation, which in turn is controlled by Ca2+ ions. It has been suggested that sperm detachment and acquisition of hyperactivation, which is necessary for fertilization, occurs via its interaction with an oviductal protein. OGP binding at the tail and head regions of sperm reported here (Fig. 9) together with earlier studies using bovine [17] and hamsters [16] raises the important question of whether OGP binding on sperm could contribute to the process of hyperactivation and prevention of polyspermy. Studies with porcine OGP have already shown that OGP prevents polyspermy [20]. Preliminary results do show that antibodies to rOGP drastically reduce sperm attachment to zona free murine eggs, but clearly, more rigorous analysis is required to validate this premise. In summary, we have succeeded in expressing and purifying monkey OGP. The usefulness of rabbit antibodies to rOGP has also been demonstrated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Usha Natraj, Deputy Director, Institute for Research in Reproduction (I.C.M.R.), Jehangir Merwanji Street, Parel, Mumbai 400012, India. FAX: 91 022 4139412; dirirr{at}vsnl.com or ushan3{at}rediffmail.com
Received: 10 October 2001.
First decision: 2 November 2001.
Accepted: 3 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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