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Female Reproductive Tract |
Unidad de Reproducción y Desarrollo,3 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
Millennium Institute for Fundamental and Applied Biology,4
Laboratorio de Inmunología de la Reproducción,5 Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epithelial cells, female reproductive tract, oviduct, rat, sialic acid, sperm, spermatozoa, sperm motility and transport, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is evidence that a small proportion of spermatozoa retained in the isthmic segment bind to epithelial cells [3, 4]. Spermatozoa bind to the oviductal mucosa in several species, including guinea pig [5], rabbit [6], pig [7], ovine [8], bovine [9], hamster [3], human [10], mouse [11], horse [12], and rat [4]. Binding to oviductal epithelial cells maintains spermatozoa in a competent physiological condition to accomplish fertilization when ovulation takes place. Epithelium-bound spermatozoa show extended viability [13] and delayed capacitation [14]. At the time of ovulation, these sperm cells develop hyperactivated motility, they are released and swim to the site of fertilization [15].
Binding of spermatozoa to epithelium is due to interactions between cell-adhesion molecules on the cell surfaces of both the spermatozoon and the epithelial cell [15, 16]. Although these molecules have not been identified, it is known that they are rich in carbohydrate residues that are species-specific [15]. In hamsters, sperm binding to oviductal epithelium is mediated by sialic acid [17], whereas in horses, it is mediated by galactose [14]. The binding of bovine sperm to explants of oviductal epithelium is mediated by fucose [18], and in pigs, the binding involves maltose, lactose, and mannose recognition [19].
Sperm binding to the oviductal epithelium in the rat following intrauterine insemination occurs during proestrus and estrus and is limited to the isthmic segment. Furthermore, attachment of spermatozoa to the epithelium in rats rendered acyclic by treatment with a GnRH antagonist requires priming with estradiol (E2) and progesterone (P) [4]. Thus, adhesion of spermatozoa to the rat oviductal epithelium in vivo is stage and segment specific and requires the combined action of E2 and P.
Here, we assessed the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined the distribution of glycoconjugates in the luminal surface of the oviduct under various hormonal environments and the effect of lectins, monosaccharides, or glycoproteins on rat sperm binding to oviductal epithelium in vivo. The localization of glycoconjugates in the oviductal epithelium and the electrophoretic pattern of oviductal glycoproteins having sialic acid on their oligosaccharide chains were then assessed, and finally the presence of molecules that recognize sialic acid on the plasma membrane of rat spermatozoa was determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Locally bred Sprague-Dawley rats were used. Animals were kept under controlled temperature (21–24°C), and lights were on from 0700 to 2100 h. Water and pelleted rat chow were supplied ad libitum. Males were 4– 5 mo old, weighing 400–450 g, and were of proven fertility. Females weighing 200–220 g and exhibiting 4-day cycles were used. The regularity of the estrous cycle was verified by daily vaginal smears. The day of estrus was considered Day 1 of the cycle. Care and manipulation of the animals was carried out in accordance with the ethical guidelines of our institution.
Treatments
Systemic administration of E2, P4 or Cetrorelix Estradiol (5 µg) or P (5 mg) were injected s.c. as a single dose dissolved in 0.1 ml propylene glycol or olive oil, respectively. The GnRH antagonist Cetrorelix acetate (ASTA Pharma AG, Frankfurt/Main, Germany) was injected s.c. at a daily dose of 30 µg dissolved in 0.1 ml of mannitol 5% for 5 days.
Local administration of lectins Lectins (listed in Table 1) were injected intraoviductally as a single dose at a concentration of 5 µg/µl dissolved in saline solution. Intraoviductal administration was done on the morning of proestrus according to Orihuela et al. [20].
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Preparation of Spermatozoa for Insemination
Each morning, two male rats were killed in order to obtain their epididymal spermatozoa. The four caudae epididymides were removed and cut into several pieces, which were placed in culture dishes containing 1 ml sterile saline, overlaid with mineral oil at 37°C, to let spermatozoa diffuse into the medium. The concentration of spermatozoa in the suspension was determined using a Neubauer Chamber (Cambridge Instruments, Buffalo, NY). Each sperm suspension was used to inseminate four females, each being in a different condition.
Incubation of Spermatozoa with Monosaccharides and Glycoproteins
Sperm suspensions were incubated before insemination at 37°C for 20 min in saline alone or in saline containing mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin (Sigma Chemical Co., St. Louis, MO) at a final concentration of 10 µg/ml. We choose this concentration because, in preliminary experiments, we observed that concentrations above 10 µg/ ml produce hyperactivation of spermatozoa.
Insemination
Females were anaesthetized with ether at 0900 h of proestrus. The uterus was exposed through flank incisions, and 0.1 ml of sperm suspension, containing 10–20 million spermatozoa, was injected into the upper third of each uterine horn. The organs were returned to the peritoneal cavity and muscles and skin were sutured.
Recovery of Spermatozoa
Females were killed and the oviducts were excised and removed free of fat tissue. In separating the oviduct from the uterus, care was taken to leave the intramural segment attached to the oviduct. Spermatozoa were recovered from the oviduct following an adaptation of the technique used by Smith and Yanagimachi [21] in the hamster. Their original technique consists of flushing the oviduct three times, the first two times with 20 µl saline to recover sperm cells lying free in the lumen and loosely attached to the mucosal surface, and the third with 20 µl saline + 0.5% Triton X-100 to dislodge spermatozoa adhering to the epithelium in the mucosal crypts. Triton X-100 is a nonionic detergent that, in the concentration used, dissolves the mucosal surfaces, without affecting sperm morphology. Smith and Yanagimachi determined the origin of the sperm cells in the flushings by direct observation through the oviductal wall. In our previous experiments [4], after the first flushing with 20 or 50 µl saline, no spermatozoa were recovered when the second flushing was performed with saline alone; in the third flushing, performed with saline + 0.5% Triton X-100, sperm cells were recovered. Therefore, the second flushing was deleted and we flushed the oviduct only twice, the first time with 50 µl saline and the second time with 0.5% Triton X-100. Based on the above, we assumed that the first flushing removed spermatozoa lying free in the lumen and that the second one removed sperm cells adhering to the oviductal epithelium. Because we did not examine the oviductal epithelium by histology after the second flushing, we cannot exclude the possibility that some adhering spermatozoa remained attached and were not counted. Counting was done using bright-field microscopy at a magnification of 250x. No attempt was made to assess motility or viability.
Lectin Histochemistry of the Oviduct
Ampullary and isthmic segments were fixed in Bouin solution for 6 h, dehydrated in ethanol 70–100%, embedded in paraffin, sliced into 10-µm-thick sections with the aid of a microtome (Leica RM 2135; Lab-Line Instruments Inc., Melrose Park, IL) and mounted on gelatin-coated slides. Sections were deparaffinized and hydrated and were incubated with 10 µg/ml of one of the fluorescein isothiocyanate (FITC)-lectins PNA, DBA, ECA, HPA, UEAI, or WFA (Sigma Chemical Co.), rinsed with PBS, and mounted with Fluoromount G (Electronic Microscopy Science, Washington, PA). All sections were visualized using an Optiphot Epifluoresence Microscope (Olympus, Middlebush, NJ). Negative controls consisting of oviductal samples without labeled lectins were also included.
Lectin Immunohistochemistry of the Oviduct
Six oviducts obtained from three rats in proestrus were separated into ampulla and isthmus. To remove excess mucus, each segment was flushed with 50 µl saline. Tissues were processed as above except they were sliced into 4-µm-thick sections. Sections were blocked with BSA 1% diluted in PBS for 1 h, followed by incubation with 5 µg/ml of the sialic acid-binding lectin Tritrichomonas mobilensis (TML; Calbiochem, La Jolla, CA) overnight at 4°C. Slides were rinsed with PBS and incubated with mouse anti-TML (Calbiochem) diluted 1:300. After washing with PBS, the preparations were incubated for 2 h with rabbit anti-mouse IgG FITC conjugate (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1: 1000. Sections were washed and counterstained with 1 µg/ml of propidium iodide (Sigma Chemical), washed again, and then mounted in Fluoromount G. All sections were examined using an Axiovert 100 M Confocal Microscope (Zeiss, Göttingen, Germany). Negative controls consisting of oviductal samples without TML or mouse anti-TML were also included.
Extraction of Epithelial Glycoproteins
Oviducts in groups of eight (obtained from four rats) were separated into ampulla and isthmus. Both segments were flushed with 50 µl saline to remove excess mucus, and then the epithelial cells were collected by stripping the segments into a glass-plate dish. Epithelial cells from ampullary and isthmic segments were separately homogenized with a polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) in 1 ml of phosphate-buffered saline (PBS, pH 7.4) containing 0.1 M phenylmethylsulfonyl fluoride, and centrifuged at 1000 x g for 5 min at 4°C. The pellet was resuspended with 1 ml extraction buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 5% ß-mercaptoethanol) and boiled for 5 min. Samples were centrifuged at 15 000 x g for 5 min at 4°C. The supernatant containing the glycoproteins was precipitated with cold acetone 80% (v/v) for 1 h at –20°C and then was centrifuged at 2300 x g for 5 min at 4°C. The pellet was dissolved in 100 µl of 100 mM Tris-HCl, pH 6.8, + 0.05% Tween 20 [22] and stored at –20°C until electrophoretic analysis. The concentration of proteins was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
Protein Electrophoresis and Lectin Immunoblotting
Aliquots of the dissolved pellets containing the same amount of protein (25 µg) were dissociated for 3 min at 95°C with a buffer containing 10% SDS, 20% glycerol, 5% ß-mercaptoethanol, and 0.012% bromophenol blue. Samples were run on SDS 12% polyacrylamide slab gels according to the method of Laemmli [23], utilizing a mini PROTEAN electrophoretic chamber (Bio-Rad). Proteins resolved in the gels were electroblotted onto nitrocellulose membranes (Bio-Rad). Nitrocellulose blots were blocked using 3% BSA in TTBS (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% v/v Tween 20) for 2 h. Membranes were incubated with TML 1 µg/ml in TTBS for 2 h and then rinsed three times in TTBS, 5 min each, and incubated for 2 h with a mouse anti-TML antibody (Calbiochem) diluted 1:300. Blots were rinsed three times for 5 min each in TTBS and were incubated for 2 h in TTBS containing 1:5000 dilution of rabbit anti-mouse IgG alkaline phosphatase conjugate (Calbiochem). The alkaline phosphatase activity was detected by color development during incubation of blots in 100 mM Tris/HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2, containing BCIP/NBT tablets (1 tablet in 10 ml; Sigma). Negative controls consisting of blots incubated without TML or mouse anti-TML were also included.
Labeling of Spermatozoa
Sperm suspensions were obtained from rat epididymides as described earlier. Spermatozoa were fixed with formaldehyde 2% in microfuge tubes. Ten microliters of fixed cells were dried on a gelatin-coated slide, washed with PBS, and incubated for 1 h with 150 µg/ml of the sialic acid 
-NeuAc-PAA-FITC conjugate (Glycotech, Rockville, MD). The slides were then counterstained with propidium iodide and mounted with Fluoromount G. Spermatozoa were visualized using a confocal microscope. A negative control, consisting of sperm samples without labeled sialic acid, was also included.
Extraction of Sperm-Membrane Proteins
Epididymal spermatozoa were recovered as described earlier and washed two times with protein-free HEPES-balanced salt solution (HBS) supplemented with a cocktail of protease inhibitors (1 tablet in 50 ml; Roche Applied Science, Mannheim, Germany). HBS consisted of 25 mM HEPES, 130 mM NaCl, 5 mM KCl, 0.36 mM NaH2PO4, 0.49 mM MgCl2, and 2.4 mM CaCl2. Washed sperm were resuspended in HBS at 2 x 107 sperm cells/ml. Sperm membrane proteins were extracted with 0.5% Triton X-100 in HBS by gentle mixing on a rotary shaker for 1 h at 4°C. Following extraction, sperm were removed by centrifugation at 2300 x g for 5 min and discarded. Pellet was dissolved in 150 µl of HBS + 0.5% Triton X-100, assayed for protein content (DC protein assay; Bio-Rad), and stored at –20°C.
Ligand Blot
Sperm membrane proteins (30 µg) were dissolved in a buffer containing 1.25% SDS, 16.25% glycerol, and 0.012% bromophenol blue. Samples were run on SDS 12% polyacrylamide slab gels according to the method of Laemmli [23], utilizing a mini-PROTEAN electrophoretic chamber (Bio-Rad). Proteins resolved in the gels were electroblotted onto nitrocellulose membranes (Bio-Rad). Sialic acid-binding proteins were detected by probing blots with a biotinylated sialic acid. Briefly, blots were incubated overnight at 4°C in binding buffer (2 mM CaCl2, 1 mM NiCl2, 154 mM NaCl, and 0.05% Tween-20) to block any nonspecific sites and to renature blotted proteins [24]. Membranes were probed at room temperature with 500 ng/ml of the sialic acid -NeuAc-PAA-biotin (Glycotech) in binding buffer for 2 h. Blots were rinsed three times for 5 min each in binding buffer and were incubated for 1 h in binding buffer containing 1: 5000 dilution of avidin-alkaline phosphatase conjugate (Sigma). The alkaline phosphatase activity was detected by color development during incubation of blots in 100 mM Tris/HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2, containing BCIP/NBT tablets (1 tablet in 10 ml; Sigma). A negative control consisting of blots incubated without labeled sugar was also included.
Densitometry of the Immunoblots
Immunoblots were scanned using an Epson model Expression 636 scanner. This scanner digitizes an image into 64 000 pixels and automatically assigns a gray value (range, 0–255) to each pixel. Only major bands that were present consistently in all the replicates and that were neatly separated were subjected to densitometric analysis. This method has the limitation of not measuring all the bands, but it permits precise measurement of selected bands. The immunoblots were quantitatively analyzed with the NIH Image 1.61 Software and an iBook computer (Apple Computer Inc., Cupertino, CA). The selected bands were plotted and the area of the peaks was measured to calculate the intensity of the bands, expressed as pixel2. More details are found in the web site http://rsb.info.nih.gov/nih-image/manual/contents.html.
Statistical Analysis
The data are presented as mean ± SEM. Overall analysis was performed by the Kruskal-Wallis test followed by the Mann-Whitney test for pairwise comparisons when overall significance was detected.
Experimental Design
Distribution of glycoconjugates in the rat oviduct Using lectin-labeling, we assessed the distribution of glycoconjugates in rat oviducts under conditions in which sperm binding is expected to occur and in oviducts in which binding is absent [4]. Thirty-six oviducts were obtained from 18 rats at different stages of the estrous cycle, from rats rendered acyclic by treatment with Cetrorelix or acyclic rats primed with E2 and P, then separated into ampulla and isthmus and processed by lectin histochemistry as described earlier. This experiment consisted of three replicas for each condition.
Effect of lectins on sperm binding to oviductal epithelium This experiment determined whether intraoviductal administration of lectins prior to insemination could affect sperm binding to oviductal epithelium in the rat. Forty-eight female rats were locally injected with PNA, ECA, HPA, UEA-I, or TML in the morning of proestrus and then were artificially inseminated. Three hours after insemination, the oviducts were flushed to determine the number of spermatozoa lying free in the lumen or adhering to the epithelium. Replicas of this experiment are stated in Figure 2.
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Effect of monosaccharides and glycoproteins on sperm binding to oviductal epithelium This experiment determined whether exposure of spermatozoa to carbohydrates prior to insemination affects sperm binding to oviductal epithelium. Thirty-four female rats were inseminated in the morning of proestrus with spermatozoa previously coincubated with mannose, fucose, galactose, sialic acid, or saline. Three hours after insemination, the oviducts were flushed to determine the number of spermatozoa lying free in the lumen or adhering to the epithelium. Another 16 female rats were inseminated in the morning of proestrus with spermatozoa previously coincubated with fetuin, asialofetuin, or saline. Fetuin is a sialic acid-rich glycoprotein, while asialofetuin is fetuin with its terminal sialic acid removed [25]. Replicas of this experiment are stated in Figure 3.
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Cellular localization of sialic acid-containing glycoconjugates in oviductal tissue This experiment examined the presence and distribution of glycoconjugates containing sialic acid on their oligosaccharide chains, in oviductal tissue layers. A total of six oviducts from three proestrous rats were separated in ampulla and isthmus and then processed for lectin immunohistochemistry. This experiment consisted of three replicas.
Electrophoretic pattern of glycoproteins of oviductal epithelial cells This experiment attempted to detect fluctuations in the level of sialic acid-containing glycoproteins in the ampullary and isthmic epithelium throughout the estrous cycle. Groups of 16 rats were used at each stage of the cycle. Epithelial cells from ampullary and isthmic segments were separately obtained and the sialic acid-containing glycoproteins were subjected to electrophoresis and lectin-immunoblotting. This experiment consisted of three replicas for each stage of the cycle.
Localization and electrophoretic pattern of sialic acid-binding molecules on the surface of rat spermatozoa The presence of sialic acid-binding molecules on the surface of rat sperm was investigated. A total of 10 caudae epididymides were excised from five male rats and their epididymal spermatozoa were obtained, labeled with sialic acid-FITC, and examined by confocal microscopy. This experiment consisted of five replicas. Other epididymal spermatozoa from two rats were extracted with Triton X-100 and their plasma membrane proteins were subjected to SDS-PAGE followed by ligand blot with sialic acid-biotin. This experiment consisted of two replicas
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of lectins on sperm binding to oviductal epithelium As shown in Figure 2, intraoviductal administration of lectins did not change significantly the total number of spermatozoa recovered from the oviduct. However, TML decreased the percentage of spermatozoa adhering to the epithelium (Fig. 2B), while other lectins had no effect.
Effect of monosaccharides and glycoproteins on sperm binding to oviductal epithelium Figure 3 shows that incubation of spermatozoa with carbohydrates did not decrease the total number of spermatozoa recovered from the oviduct. However, incubation with sialic acid decreased the percentage of spermatozoa adhering to the epithelium while other sugars had no effect. As shown in Figure 4, treatment with fetuin or asialofetuin had no effect on the total number of spermatozoa recovered from the oviduct. Coincubation of spermatozoa with fetuin decreased the percentage of spermatozoa adhering to the epithelium while asialofetuin had no effect. The results of these experiments single out free and protein-bound sialic acid as a candidate monosaccharide playing a role in the binding of spermatozoa to epithelial cells.
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Cellular localization of sialic acid-containing glycoconjugates in oviductal tissue As shown in Figure 5, strong staining of sialic acid-containing glycoconjugates was only observed in the apical membrane of epithelial cells. No differences were found between ampullary and isthmic segments with regard to immunostaining of these glyconjugates.
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Electrophoretic pattern of sialic acid rich-glycoproteins of oviductal epithelial cells TML lectin immunoblots showed 15 major protein bands for which molecular mass ranged from 200 to 50 kDa. Only six of these protein bands were quantitatively analyzed. No difference was found between ampulla and isthmus or between estrous cycle stages (Figs. 6 and 7).
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Localization and electrophoretic pattern of sialic acid-binding molecules on the surface of rat spermatozoa Confocal microscopy showed that FITC-labeled sialic acid stained the apical portion of the head and along the tail (Fig. 8), while ligand blot showed that sialic acid-biotin reacted with 11 major proteins of plasma membrane for which molecular mass ranged from 250 to 25 kDa (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of various types of sugars on the epithelial surface suggests that some of these molecules might participate in sperm binding to these cells. We explored this possibility by injecting intraoviductally several classes of lectins prior to insemination. Lectins were expected to bind selectively to its corresponding sugar residues, and this complex would prevent sperm binding. We found that only TML blocked sperm binding without preventing migration of spermatozoa to the oviduct. TML binds specifically to sialic acid-rich glycoconjugates, thus, sialic acid might mediate adhesion of spermatozoa to the oviductal epithelium in the rat. This possibility was further explored inseminating spermatozoa preincubated with monosaccharides or glycoproteins. Inhibition of sperm-epithelium binding by sialic acid and fetuin but not by other carbohydrates tested confirmed that this phenomenon involves a specific interaction with a sialic acid-like moiety. Furthermore, asialofetuin lacking the terminal sialic acid residues was ineffective. None of the molecules tested inhibited sperm migration from the uterus into oviduct because the total number of spermatozoa recovered was not different from the saline-treated control. It is unlikely that monosaccharides and glycoproteins tested in our experiments affected sperm capacitation because it has been shown that carbohydrates do not affect motility or acrosome reaction at these concentrations during short-term incubation [29–31].
DeMott et al. [17] showed in hamster that sperm binding to oviductal epithelium in vitro is mediated by sialic acid-containing glycoproteins present in the oviduct. Although previous studies revealed species specificity in the residues of carbohydrates involved in sperm-oviduct adhesion [15], our results show, for the first time, this is not absolute. The number of species studied in this regard is too small to formulate a phylogenetic-based algorithm describing species specificity in the sugars involved in sperm binding. At this point, we can say that the only two rodents studied, the hamster and rat, bear in common sialic acid as the major sugar involved in sperm-epithelium binding.
Using a lectin battery, Menghi et al. [32] showed that the predominant sugar moieties present in the rat oviduct are sialic acid and fucose. Our studies, using TML lectin, also suggest the presence of glycoproteins with sialic acid termini on the surface of epithelial cells lining the rat oviduct. These sialoglyconjugates were found in a patchy distribution in the isthmus, supporting the notion that they are involved in sperm binding because it has been shown that attachment of spermatozoa to the oviduct in vivo occurs only in discrete regions of the isthmic segment [11, 21]. However, the localization of sialoglycoconjugates in the ampulla and the similar banding pattern of sialoglycoproteins between ampulla and isthmus suggest that sperm-binding sites may be also present in the ampulla. In fact, pig, equine, and bovine sperm binding in vitro does not present regional differences in relation to the oviductal segments [12, 33, 34]. Although we previously demonstrated that sperm binding to the rat oviductal epithelium is limited to the isthmic segment in vivo, it is possible that sperm binding appears limited to this segment because it is the first region encountered by spermatozoa when they enter the oviduct [25].
Previously, we reported that sperm binding to oviductal epithelium is limited to proestrus and estrus and requires the combined action of E2 and P [4]. However, contrary to our expectation, we found here that the banding pattern of the six more abundant sialoglycoproteins did not change along the estrous cycle. One likely explanation is that the amount of the sialoprotein involved in sperm binding is too small, in relationship to other sialoglycoproteins (compare Figs. 1 and 5), to be assessed by the detection technique utilized. Besides the possibility that sex steroid hormones act directly on sperm cells [35–37], influencing their ability to bind to oviductal epithelium, they may regulate the expression of other molecules that can mask the sperm-binding sites in the epithelium.
The localization of sialic acid-binding molecules over the apical surface of the sperm head is consistent with a role of these molecules in sperm binding because, in other species, spermatozoa attach to oviductal epithelial cells via the plasma membrane overlaying the acrosome [15, 38, 39]. Ligand blots labeled with sialic acid detected several sperm membrane proteins that are good candidates for further studies to characterize the sialic acid-binding molecule responsible for sperm adhesion to the epithelium.
In summary, this study shows that sugar residues present in the rat oviductal epithelium vary among different regions of the oviduct and along the estrous cycle or by E2 and P treatment. Furthermore, local injection of the specific-sialic acid lectin TML and coincubation of epididymal rat spermatozoa with sialic acid or fetuin prevents adhesion of spermatozoa to oviductal epithelium in vivo. Moreover, oviductal epithelial cells express sialic acid-rich glycoproteins and, in the sperm head surface, there are sialic acid-binding proteins. We concluded that sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the oviductal epithelial cells with sialic acid-binding proteins on the sperm surface. In addition, we demonstrated for the first time that species specificity in the sugar involved in sperm binding is not absolute.
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FOOTNOTES |
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2 Correspondence: P.A. Orihuela, Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. FAX: 562 222 5515; porihuel{at}genes.bio.puc.cl
Received: 15 January 2004.
First decision: 6 February 2004.
Accepted: 1 June 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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b, P < 0.05
