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BOR - Papers in Press, published online ahead of print October 1, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020305
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BIOLOGY OF REPRODUCTION 70, 198–203 (2004)
DOI: 10.1095/biolreprod.103.020305
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Evidence for the Regulation of Glycosylation of Golden Hamster (Mesocricetus auratus) Oviductin During the Estrous Cycle1

Deborah S. McBride, Chantale Boisvert, Gilles Bleau, and Frederick W.K. Kan2

Department of Anatomy and Cell Biology,3 Faculty of Health Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada Notre-Dame Hospital Research Center,4 Montreal, Quebec H2L 4M1, Canada Saint-Luc Hospital Research Center,5 Montreal, Quebec H2X 3J4, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oviduct contributes to the reproductive environment by secreting various factors, including a family of glycoproteins termed oviductins. Although many studies have demonstrated that ovarian hormones modulate oviductin gene expression in several mammalian species, there has been controversy surrounding the regulation of golden hamster oviductin. The current study was undertaken to investigate the transcriptional and translational modifications of hamster oviductin during the estrous cycle. First, we verified that hamster oviductin mRNA expression remains constant throughout the estrous cycle by semiquantitative reverse transcription polymerase chain reaction. We then developed a polyclonal antibody against recombinant hamster oviductin (rhaOvm). The anti-rhaOvm antibody was subsequently used in conjunction with quantitative immunocytochemistry to investigate the oviductin levels in the hamster oviduct during the estrous cycle. Quantification of immunolabeling revealed a high, consistent level of glycoprotein throughout the estrous cycle. Therefore, it appears that the production of oviductin is not regulated differentially during the estrous cycle. Size variations in hamster oviductin expression were also investigated by Western blot analysis. The oviduct contains several forms of oviductin at each stage of the estrous cycle, the native glycosylated form(s) of 160–350 kDa, and several precursor forms of 70–100 kDa. Although variations in the intensities of the polydispersed band were not evident during the estrous cycle, additional bands ranging from 90 to 100 kDa were detected in the estrus, metestrus, and diestrus 1 stages. The results from the present investigations suggest that whereas ovarian hormones do not appear to influence the hamster oviductin mRNA and protein expressions, glycosylation of hamster oviductin appears to be differentially regulated during the estrous cycle.

female reproductive tract, gene regulation, oviduct, ovulatory cycle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many important phenomena of mammalian reproduction occur within the female reproductive tract, especially within the oviduct. The mammalian oviduct is a very active secretory organ, providing the optimal milieu for the successful occurrence of sperm capacitation, fertilization, and early embryonic development [1]. The nonciliated epithelial cells of the oviduct actively synthesize and secrete a high-molecular-weight family of glycoproteins termed oviductins [1]. These oviduct-specific proteins have been described in many species, including the hamster [2, 3].

There are several proposed functions for oviductins, which include increased viability and motility of spermatozoa within the female reproductive tract [4] as well as increased sperm capacitation and fertilizing capabilities [5]. The presence of oviductin has been shown to increase sperm-egg binding and penetration rates [6–8] and decrease polyspermy [8, 9]. Oviductin also appears to increase cleavage rates of embryos [9, 10] and the number of embryos reaching the blastocyst stage [8, 10]. Therefore, the presence of oviductins within the oviductal lumen at the time of these events would be beneficial.

Ovarian steroids have been proposed to modulate the expression of oviductins in human [11–13], baboon [12, 14, 15], sheep [16, 17], cow [18, 19], and pig [20, 21]. Previous immunocytochemical studies carried out in our laboratory using a monoclonal antibody specific for a glycosidic epitope of hamster oviductin revealed a variation in protein levels during the estrous cycle [22]. The concentration of oviductin appeared to be highest during the stage of estrus and decreased to reach a minimum at diestrus 2 before beginning to increase again at proestrus. The maximum amount of oviductin observed at estrus coincides with the time of ovulation [22]. However, previous studies have also revealed that hamster oviductin mRNA expression levels do not vary significantly during the estrous cycle [23, 24]. Nevertheless, there appears to be some degree of control over regulation of oviductin mRNA and protein expressions in most mammalian species.

In view of these findings, it is hypothesized that there are estrous cycle-associated modifications in the production of hamster oviductin by the oviductal epithelium. The objective of the current study was to further investigate the regulation of oviductin gene expression during the estrous cycle. Initially, we attempted to verify the regulation of hamster oviductin mRNA expression during the estrous cycle by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Subsequently, we produced a recombinant hamster oviductin (rhaOvm) from a cDNA clone (phage-19) [23] coding for the mucin domain of hamster oviductin and used the recombinant peptide to generate a polyclonal antibody (anti-rhaOvm antibody). The production of the anti-rhaOvm antibody against a peptide portion of hamster oviductin differs from previously used monoclonal antibodies that recognize glycosidic epitopes attached to the protein backbone [2, 25–30]. In the present study, we have applied quantitative immunocytochemistry using the anti-rhaOvm antibody to localize and quantify the corresponding glycoprotein in the secretory granules of the nonciliated cells of the oviductal epithelium. The quantitative immunocytochemical data were complemented by Western blot analysis to examine the size variations in the protein expression of oviductin in the hamster oviduct during the estrous cycle. The present results, taken together with previous findings obtained in our laboratory by use of quantitative immunocytochemistry [22] and lectin cytochemistry [31], indicate that glycosylation of hamster oviductin is temporally and differentially regulated during the estrous cycle.


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

Female golden hamsters (Mesocricetus auratus), 6–8 wk of age (Charles River Inc., St. Constant, QC, Canada), were housed in a temperature-controlled room with a 12L:12D cycle (lights on at 0600). The animals were analyzed for mucous vaginal discharge indicative of the metestrus stage of the estrous cycle and were cycled for 2–3 wk to establish regularity of the cycle. The animals were separated into groups according to the five different stages of the cycle [22]. All animal care practices and experimental procedures complied with the guidelines of the Canadian Council on Animal Care and were approved by the University Animal Use Committee at both Queen's University and the University of Montreal.

Semiquantitative Reverse Transcription Polymerase Chain Reaction

Semiquantitative RT-PCR was performed to examine relative changes in oviductin mRNA expression during the estrous cycle. Oviducts from at least four animals per stage were pooled and homogenized to obtain total messenger RNA representative of each stage of the estrous cycle. Messenger RNA was extracted using Sigma TRI-reagent according to the manufacturer's instructions (Sigma-Aldrich, Oakville, ON, Canada). For reverse transcription, mRNA was subjected to first strand cDNA synthesis using the Omniscript Reverse Transcriptase kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions.

PCR for oviductin semiquantitative mRNA analysis was performed using the sense primer 5'-GACCTGGCTCTGAGAGTCTT-3' and the antisense primer 5'-CACTGTGGCTGTGATCTGTC-3'. The primers were designed to correspond to a region coding for the mucin domain of hamster oviductin, nucleotides 1175–1713 of the published sequence [23]. The length of the amplified region was 538 base pairs (bp). Changes in PCR products of the oviductin gene were evaluated by comparison with those for the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using GAPDH sense primer 5'-ACCACAGTCCATGCCATC-3' and antisense primer 5'-CCTGCTTCACCACCTTCT-3'. The primers were designed within the nucleotide sequence of the golden hamster GAPDH gene [32]. The length of the amplified region was 267 bp. PCR mixtures containing 1x buffer (Qiagen), 2.5 mM MgCl2, 200 µM dNTP mix, 2.5 U Taq DNA polymerase (Qiagen), 0.5 pM selected primers, and sterile H2O were prepared. Amplifications were performed using the following conditions: denaturation at 94°C for 30 sec, annealing at 61°C for 30 sec, and elongation at 72°C for 1 min. A final elongation step was performed by incubation at 72°C for 10 min. Twenty-two cycles were performed for the amplification of oviductin and 32 cycles for GAPDH to ensure that the amplification of each gene was in the linear range and not during the plateau phase. PCR products were separated on a 1.0% agarose gel containing 0.5 µg/ml ethidium bromide. The separated PCR products were photographed under UV light. Semiquantitative analysis was conducted by comparing relative band intensities of oviductin normalized by comparison with GAPDH using SigmaGel software (Jandel Scientific, San Rafael, CA). One-way ANOVA was used for analyzing differences in bands intensities of oviductin products across the five stages of the estrous cycle. Statistical values of P < 0.05 were considered significant.

Cloning of cDNA to Mucin Domain of Hamster Oviductin and Production of rhaOvm

A hamster oviductin cDNA clone (phage-19) [23] was digested with Apa I and EcoR I, producing a fragment designated as haOvm (nt 1140–2276 of the whole cDNA) and coding for the mucin domain of the protein [33]. The nucleotide sequence of the haOvm fragment is unique to hamster oviductin and does not resemble any other glycoproteins. The expression vector pQE-32 (Qiagen) was digested with Sma I. The haOvm fragment and the digested vector were treated with T4 DNA polymerase and ligated using T4 DNA ligase at room temperature for 16 h. The haOvm fragment was cloned in frame with the 6xHis-tag of pQE-32 yielding the plasmid pQE-32-haOvm. The vector-haOvm junction was sequenced by the Sanger method [34] using the Sequenase 2.0 kit (U.S.B., Cleveland, OH). This construct was employed to transform Escherischia coli strain M15 using standard protocols [35]. At a cell density corresponding to A600 = 0.7–0.9 arbitrary units (AU), the expression of recombinant hamster oviductin (rhaOvm) was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG). The transformed cells were incubated for 3 h, harvested by centrifugation, resuspended in lysis buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0), and then incubated overnight at 4°C with shaking.

The lysate was centrifuged, and the supernatant was loaded onto a Ni-NTA column (Qiagen). The column was washed until A280 was less than 0.01 AU, and the bound peptide was eluted and lyophilized according to the manufacturer's instructions. The rhaOvm peptide was resuspended in distilled water.

Production of Antibodies Against rhaOvm

The rhaOvm peptide (5 µg) was analyzed by SDS-PAGE on a 12% polyacrylamide gel followed by staining of the gel with Coomassie Blue. The portion of the gel corresponding to a molecular weight of ~43 kDa was cut and electroeluted (Electro-eluter model 422; Bio-Rad, Mississauga, ON, Canada). For immunization, a male white rabbit (Charles River) received multisite intradermal injections initially with 1 mg of rhaOvm dissolved in PBS and emulsified in complete Freund adjuvant (1:1, v/v). Subsequent booster injections were administered at 4-wk intervals with 1.0, 0.5, and 0.6 mg of the immunogen in PBS emulsified with an equal volume of incomplete adjuvant. Blood samples were collected 2 wk after each booster injection, and serum antibody titers were analyzed by ELISA and Western blot analysis. Controls were performed with the serum of a normal rabbit.

The IgG fractions of the immune and control sera were purified with Affi-Gel protein A agarose beads as described by the manufacturer (Affi-Gel Protein A Kit; Bio-Rad). After protein A purification, the IgG fractions of the immune and control sera were further affinity purified using Affi-Gel 15 resin (Bio-Rad) coupled with rhaOvm according to the manufacturer's instructions. The specificity of the affinity-purified anti-rhaOvm antibody was established by Western blot analysis. Briefly, 5 µg of rhaOvm peptide were separated by SDS-PAGE [36], transferred to a polyvinylidene difluoride (PVDF) membrane [37], and immunodetected with diluted anti-rhaOvm antibody (0.8 mg/ml; 1:1000; 1:5000). Control experiments were performed using 5 µg recombinant dehydrofolate reductase (DHFR; which also contains six histidine residues) to determine whether the histidine residues are immunogenic. The anti-rhaOvm antibody was found not to react with recombinant DHFR. Total protein samples from the ovary, oviduct, and uterus (5 µg total protein) were also examined to test the tissue specificity of the anti-rhaOvm antibody.

Quantitative Immunocytochemistry

Twenty female golden hamsters (Charles River), 6–8 wk of age, were used for this study. The animals were killed by cervical dislocation according to the timing of the different stages of the estrous cycle [38]. Oviducts were excised and processed for immunocytochemistry as described previously [22]. Ultrathin sections of cycled hamster oviduct were immunolabeled as described previously [22] using the anti-rhaOvm antibody (0.8 mg/ml) diluted 1:5 in PBS. Colloidal gold particles of 8 nm diameter [39] were used for preparation of the protein A-gold complex according to the method previously described [40]. Sections were incubated with the protein A-gold complex diluted 1:10 in PBS. The sections were counterstained with uranyl acetate and lead citrate before examination on a Hitachi 7000 Electron Microscope (Hitachi Canada, Ltd., Missisauga, ON, Canada). To assess the specificity of the anti-rhaOvm antibody, control experiments were performed with preimmune serum (diluted 1:5 in PBS). All other steps and conditions remained the same.

The labeling densities of gold particles over the secretory granules were evaluated on electron micrographs, enlarged 31 200 times. For each of the five stages of the estrous cycle, the surface area of each granule and the number of gold particles were determined using a Carl Zeiss MOP-3 modular system (Carl Zeiss, Canada, Ltd., Toronto, ON, Canada). For quantification, 100 granules were evaluated for each of the four animals per stage. For controls, 50 granules were counted for each animal and three animals per stage evaluated. The data were examined using a one-way ANOVA statistical investigation.

Isolation of Total Protein from Oviductal Tissue

The oviducts used for isolation of total protein were collected from 15 mature cycling hamsters divided into groups of three animals each, according to the five stages of the estrous cycle. The oviducts were homogenized, and total protein was prepared as described previously [41]. The total protein contents of the homogenized oviducts were evaluated using the Bio-Rad Protein Assay (Bio-Rad) with bovine serum albumin (BSA) as the protein standard.

SDS-PAGE, Transfer, and Immunodetection of Hamster Oviductin

Oviduct protein samples containing 10 µg total protein from each stage of the estrous cycle were prepared and separated by SDS-PAGE under reducing conditions as described by Laemmli [36]. The proteins were transferred to a PVDF membrane according to the procedure described by Towbin et al. [37]. After transfer of proteins, the membrane was blocked with 10% BSA in 10 mM TBS buffer (pH 8.0) followed by incubation with diluted anti-rhaOvm antibody (0.8 mg/ml; 1:1000 in TBS) for 1 h. The membrane was washed in tris-buffered solution plus Tween 20 (TBST) buffer prior to incubation with the alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10 000) for 1 h. For chemiluminescence detection, the membrane was equilibrated in detection buffer followed by development with CSPD (disodium 3-(4-methoxyspiro{1,2-dioxethane-3,2'-(5'-chloro)tricyclo[3,3.1.13,7]decan}-4-yl) phenyl phosphate) (Roche Diagnostics, Laval, QC, Canada). In order to examine differences in protein expression of the fully glycosylated, mature form of oviductin during the estrous cycle, changes in protein levels of the polydispersed band (160–350 kDa) were normalized by comparison with those of an internal standard, ß-actin. After analysis for hamster oviductin, the blots were washed twice with TBST for 10 min and immunodetected for ß-actin using the monoclonal anti-ß-actin antibody (1:20 000 in TBST; Sigma) according to the procedure recommended by the supplier. The immunoreaction was developed using Western Lightnin Chemiluminescence Reagent Plus (Perkin-Elmer, Woodbridge, ON, Canada). Quantitative analysis was performed on three separate blots and conducted by comparing band intensities of oviductin normalized to ß-actin using SigmaGel Software (Jandel). One-way ANOVA was used for analyzing differences in protein expression during the estrous cycle, and values of P < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Examination of Gene Expression of Hamster Oviductin During the Estrous Cycle

In order to examine the gene expression of oviductin during the estrous cycle, semiquantitative RT-PCR using oviductin-specific primers was performed. Oviductin mRNA was expressed by the hamster oviduct throughout the estrous cycle (Fig. 1a). The relative band intensities of oviductin were compared with the internal control of GAPDH (Fig. 1b). Statistical analysis revealed that the differences in the relative concentration of oviductin/GAPDH between the five stages of the estrous cycle were not statistically significant (P > 0.100).



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  		FIG. 1. Semiquantitative RT-PCR analysis of oviductin gene expression in the hamster oviduct. GAPDH was used as the internal standard. a) Bands representing oviductin and GAPDH are present at all stages of the estrous cycle. Pro, proestrus; Est, estrus; Met, metestrus; Di 1, diestrus 1; Di 2, diestrus 2. b) Histogram demonstrating the relative intensity of oviductin as compared to GAPDH control within the oviduct during the estrous cycle. Ovn, oviductin; A.U., arbitrary units

Development of a Polyclonal Antibody Raised Against Recombinant Hamster Oviductin

In order to further investigate the translational modification of hamster oviductin, we then proceeded to develop a polyclonal antibody against recombinant hamster oviductin. A cDNA clone of hamster oviductin [23] was digested to produce a fragment, designated haOvm. The fragment was cloned into a pQE-32 vector, and the plasmid pQE-32-haOvm was used to transform M15 E. coli cells. Escherischia coli lysate was analyzed for recombinant oviductin expression by SDS-PAGE. Following electrophoresis, the presence of a new peptide, designated rhaOvm, was observed on induction of the M15 cells with IPTG (Fig. 2). This new peptide migrated to a molecular weight of 43 kDa corresponding to the predicted mass based on the size of the cDNA fragment, haOvm (Fig. 2a). The rhaOvm peptide was used to immunize a male white rabbit in order to produce a polyclonal antibody against hamster oviductin. The highest titers were obtained after the third booster injection as measured by ELISA and Western blot (results not shown). The anti-rhaOvm antibody was purified first on a protein A agarose column followed by affinity purification. By Western blot, a positive reaction with rhaOvm was obtained with the protein A-purified IgG fraction at a dilution as low as 1/100 000 and at a dilution of 1/5000 with affinity purified antibody (Fig. 2b). Similar results were obtained by ELISA (not shown). The newly developed antibody reacted with total protein samples from the oviduct but not with total protein samples from the ovary or uterus, indicating the tissue specificity of the anti-rhaOvm antibody (results not shown).



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  		FIG. 2. a) SDS-PAGE analysis of recombinant hamster oviductin (rhaOvm) expression after purification by metal-affinity chromatography and elution with 1 mM phosphate-EDTA buffer. Five micrograms of rhaOvm were analyzed by electrophoresis on a 12% polyacrylamide gel under reducing conditions followed by staining of the gel with Coomassie blue. The recombinant protein, rhaOvm, migrated to a molecular weight of ~43 kDa (arrow). b) Western blot examining the immunoreactivity of affinity-purified anti-rhaOvm antibody. Lanes 1 and 2: 5 µg of rhaOvm detected with affinity-purified anti-rhaOvm antibody diluted 1:1000 and 1:5000, respectively

Immunocytochemical Detection of Oviductin in the Hamster Oviduct During the Estrous Cycle

To examine immunoreactivity in the secretory granules of the nonciliated oviductal cells, a quantitative immunocytochemical study was performed. The application of anti-rhaOvm antibody to thin sections of Lowicryl-embedded hamster oviduct, followed by incubation with protein A-gold complex, resulted in a specific labeling. As all five stages of the estrous cycle presented a similar labeling pattern, only the representative estrus stage is presented here. Within the nonciliated secretory cells of the oviductal epithelium, labeling was observed over the content of the secretory granules located in the apical portion of the cells (Fig. 3a) as well as those positioned adjacent to the Golgi region. The ciliated cells of the epithelium were not labeled with the anti-rhaOvm antibody (not shown). Therefore, the labeling specificity of the anti-rhaOvm antibody for oviductin was established. For the purpose of quantitation, emphasis has been placed on the immunolabeling for oviductin in the secretory granules. Control sections of hamster oviduct incubated with preimmune serum followed by labeling with protein A-gold complex resulted in very few, randomly distributed gold particles over the oviductal sections (Fig. 3b).



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  		FIG. 3. Electron photomicrographs of ultrathin Lowicryl sections of hamster oviduct showing immunolabeling for hamster oviductin by use of the anti-rhaOvm antibody. a) The secretory granules (SG) of the nonciliated secretory cells are intensely labeled (estrus stage; x13 000). b) Negative control section of hamster oviduct incubated with preimmune serum instead of anti-rhaOvm antibody showing the absence of gold particles in the secretory granules (proestrus stage; x15 200). SG, secretory granules; MV, microvilli

Quantitative Evaluation of Immunogold Labeling

In order to evaluate the labeling density of oviductin within the secretory granules of the nonciliated cells, particular consideration was directed toward the number of gold particles located within the granules of oviducts at each of the five stages of the estrous cycle. Only the granules located in the apical portion of the cells were counted, as these are considered to be mature in nature and ready to release their contents into the lumen of the oviduct. The labeling density of oviductin over the secretory granules (Fig. 4) indicated that there were no significant differences (P > 0.05) among the five experimental groups (corresponding to the five stages of the estrous cycle). The labeling densities of gold particles over the secretory granules in the control groups were negligible (results not shown).



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  		FIG. 4. Mean labeling density of hamster oviductin (expressed in the histogram as number of gold particles per µm2 ± standard deviation) in the secretory granules of hamster nonciliated oviductal cells obtained from the five stages of the estrous cycle. Pro, proestrus; Est, estrus; Met, metestrus; Di 1, diestrus 1; Di 2, diestrus 2; SD, standard deviation

Examination of Protein Expression of Hamster Oviductin During the Estrous Cycle

The protein levels of hamster oviductin in the oviduct were examined during the estrous cycle. The protein was detected in the oviductal protein lysates from all stages of the estrous cycle. The anti-rhaOvm antibody reacted with a broad band, ranging from approximately 160 to 350 kDa, corresponding to native form of oviductin (Fig. 5). The antibody was also immunoreactive with several bands ranging between 70 and 90 kDa. In addition to the bands common to all five stages of the estrous cycle, a number of extra bands were repeatedly detected in subsequent experiments at the stages of estrus, metestrus, and diestrus 1 but not found in diestrus 2 and proestrus. Statistical analysis revealed that the relative intensity of the high-molecular-weight polydispersed band of hamster oviductin for each of the five stages of the estrous cycle, after normalization to ß-actin, was not significantly different from each other (P > 0.100) (results not shown).



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  		FIG. 5. Comparative Western blot analysis examining oviductin expression in oviductal tissue from each stage of the estrous cycle after immunolabeling with anti-rhaOvm antibody. Extra bands of ~90–100 kDa are present in the total oviductal homogenates from the estrus, metestrus, and diestrus 1 stages (arrow). Pro, proestrus; Est, estrus; Met, metestrus; Di 1, diestrus 1; Di 2, diestrus 2


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, semiquantitative RT-PCR revealed that there were no significant differences in the relative mRNA concentrations of hamster oviductin between the five stages of the estrous cycle. This technique was utilized in order to verify observations of earlier studies in which less sensitive methods were used. The present findings are consistent with results obtained in previous studies investigating mRNA expression in the hamster by Northern blot analysis [24] and RNase protection assay [23] and provide further evidence that hamster oviductin mRNA expression does not change significantly during the estrous cycle in the hamster. It has been suggested that variation in hamster oviductin concentration during the estrous cycle is controlled at the translational and/or posttranslational level [23].

Hamster oviductin was detected and quantified in the secretory granules of the nonciliated oviductal cells at each stage of the estrous cycle by use of quantitative immunocytochemistry in conjunction with the anti-rhaOvm antibody. The intracellular localization of hamster oviductin within the hamster oviductal epithelium is consistent with findings from previous studies utilizing a monoclonal antibody [2, 22]. Quantification of immunogold particles revealed a high, consistent labeling density over the secretory granules at each of the five stages of the estrous cycle. Statistical analysis, however, indicated that there were no significant differences in labeling density between the five stages, suggesting that the production of oviductin is not differentially regulated during the estrous cycle.

The present results provide new information regarding the biochemical properties of hamster oviductin. In an earlier quantitative immunocytochemical study carried out in our laboratory [22], a maximal labeling for oviductin was observed at the stage of estrus corresponding to the time of ovulation. It was then concluded that the production of hamster oviductin is hormonally regulated [22]. The differences observed between the results obtained in the present study and those from the previous study [22] can be explained as follows: The monoclonal antibody used in our earlier study recognizes the fully glycosylated, native form of hamster oviductin [42]. The newly developed anti-rhaOvm antibody used in the current study recognizes epitopes located on the polypeptide and should therefore be able to detect the nascent, partially glycosylated and mature forms of hamster oviductin. Therefore, based on our data obtained in the present study using semiquantitative RT-PCR, quantitative immunocytochemistry, and Western blot analysis, taken together with our previous findings obtained with a monoclonal antibody [22] and lectin cytochemistry [31], it is reasonable to speculate that ovarian hormones that fluctuate during the estrous cycle regulate glycosylation of hamster oviductin.

From all the information available to date, the mRNA and protein expressions of oviductin in several species appear to be dependent on the stage of the estrous or menstrual cycle. For example in humans, both mRNA [12, 13] and protein [11] expressions of oviductin are high during the periovulatory phase but are markedly reduced or nondetectable at other stages of the menstrual cycle. However, studies of oviductin in several mammalian species including the hamster indicate a difference in the regulation of oviductin expression between species with long durations of the estrous cycle and those with a relatively short cycle (i.e., rodents) [1]. It appears that individual species have developed their own mechanism of control over oviductin expression, whether it occurs at the transcriptional, translational, or posttranslational level.

In the present study, a number of bands were observed at different stages of the estrous cycle: A higher-molecular-weight polydispersed band ranged from ~160 to 350 kDa, and several lower-molecular-weight bands were detected with approximate sizes of 70–100 kDa. The larger band corresponds to the highly glycosylated form of the protein having a molecular size of 160–350 kDa as previously reported [26, 41, 43]. The molecular size of the protein core has been estimated to be 70.89 kDa [44]; therefore, the lower-molecular-weight bands observed in the present study are most likely due to the presence of the nascent protein and partially glycosylated forms of oviductin. In the present study, quantitative analysis of the Western blots failed to demonstrate any significant differences in band intensities between the five stages of the estrous cycle, likely because of the extensive glycosylation of hamster oviductin as revealed by the high-molecular-weight polydispersed band. Western blot analysis does not appear to be as sensitive as quantitative immunocytochemistry, which revealed the maximum labeling of hamster oviductin at estrus when a monoclonal antibody recognizing a carbohydrate epitope was used [22]. Nonetheless, additional bands with molecular weights of approximately 90–100 kDa were observed at the stages of estrus, metestrus, and diestrus 1. These additional bands may indicate stage-specific glycosylation occurring around the time of ovulation. A previous study employing 2D gel electrophoresis has reported the presence of two isoforms in the polydispersed, highly glycosylated band of hamster oviductin [41]. It will be interesting and important to determine in future studies whether glycosylation of one or both of these two isoforms is differentially regulated during the estrous cycle.

In order to fully comprehend the molecular, biochemical, and functional properties of oviductin, further studies must be undertaken. For example, investigation into the glycosyltransferase activity within the hamster oviductal epithelium during the estrous cycle may provide further insight into the regulation of posttranslational glycosylation of hamster oviductin. Although results from a previous study showed a cyclical variation in glycosyltransferase activity within oviductal fluid during the estrous cycle [45], further study is required to determine the source of these enzymes and their relation to posttranslational modifications of hamster oviductin. Perhaps regulation over glycosylation of oviductin is required for the protein to exert its full function during the early processes of the reproductive process. Abnormal glycosylation of oviductin may lead to the inability of oviductin to perform its specific roles, resulting in certain mishaps during the processes of fertilization and/or early embryo development.


   ACKNOWLEDGMENTS
 
The authors would like to express gratitude to Dr. Yat Tse, Ms. Hong Mei Gu, Ms. Verna Norkum, and Mr. Bob Temkin for their technical assistance and reproduction of the original photomicrographs.


   FOOTNOTES
 
1 This work was supported by grants from the Canadian Institutes of Health Research (MOP-44043 to F.W.K.K. and MA 11684 to G.B.). Back

2 Correspondence: Frederick W.K. Kan, Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. FAX: 613 533 2566; kanfwk{at}post.queensu.ca Back

Received: 12 June 2003.

First decision: 13 July 2003.

Accepted: 18 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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D. S. McBride, C. Boisvert, G. Bleau, and F. W.K. Kan
Detection of Nascent and/or Mature Forms of Oviductin in the Female Reproductive Tract and Post-ovulatory Oocytes by Use of a Polyclonal Antibody Against Recombinant Hamster Oviductin
J. Histochem. Cytochem., August 1, 2004; 52(8): 1001 - 1009.
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