Binding patterns of bovine seminal plasma proteins A1/A2, 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid

Previous studies from our laboratory have reported empirical associations between bovine seminal plasma protein(s) (BSP) A1/A2 and 30 kDa and osteopontin (OPN) in accessory sex gland fluid and bull fertility. These BSP and OPN are believed to bind to sperm at ejaculation and to remain bound until sperm reach the oviduct. The objective of the present study was to evaluate the topographical distribution of BSP A1/A2, 30 kDa and OPN binding on: (1) bovine ejaculated sperm; (2) ejaculated sperm incubated with isthmic oviductal fluid (ODF); (3) ejaculated sperm+isthmic ODF incubated in ampullary ODF. From each of these media, aliquots of sperm for BSP and OPN were processed for immunocytochemistry and analysis by laser scanning confocal microscopy. Isthmic and ampullary ODF was collected from indwelling catheters and used as pools from three cows in the non-luteal phase of the estrous cycle. Anti-BSP A1/A2 was detected bound to the midpiece, post-equatorial and equatorial segments and acrosome of sperm after ejaculation and after incubation with isthmic and ampullary ODF. The BSP A1/A2 fluorescence was more concentrated on the midpiece and increased as acrosome-intact sperm came in contact with ODF. As compared with acrosome-intact sperm, non-intact acrosome intact sperm had 39 and 68% reductions of acrosome fluorescence and 36% and 90% increases of post-equatorial fluorescence after contact with isthmic and ampullary ODF (P<0.05). Anti-BSP 30 kDa was more intense on the midpiece than on post-equatorial, equatorial and acrosome regions of sperm after ejaculation and contact with ODF. However, equatorial fluorescence was 141% and 89% more intense and acrosome stainning was 80% and 76% less (P<0.05) in non-intact acrosome sperm than in acrosome intact cells, during all ODF incubations. Anti-OPN was identified on the acrosome of ejaculated sperm, but with less fluorescence (P<0.05) on the post-equatorial segment and midpiece. Incubation of sperm with isthmic ODF increased fluorescence on post-equatorial segment (P<0.05). There were 72% and 78% reductions (P<0.05) of acrosome fluorescence and intensification (P<0.05) in equatorial fluorescence in non-intact acrosome sperm as compared with acrosome intact cells incubated with isthmic and ampullary ODF. In summary, interactions of BSP A1/A2 and 30 kDa and osteopontin with the sperm membrane undergo modifications dictated by the oviductal fluid. The BSP are thought to modulate cholesterol and phospholipid movement from the sperm membrane and help sperm binding to the oviductal epithelium. Furthermore, our model suggests that OPN participates in sperm-oocyte interaction, affecting fertilization and early embryonic development.     

Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct

Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 microg, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 microg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.     

Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Identification and characterization of bovine oviductal glycoproteins synthesized at estrus

Published reports indicate that in several mammalian species the oviduct synthesizes and secretes specific glycoproteins which are components of the luminal fluids at the time of ovulation and fertilization. The present study characterized the secretory glycoproteins synthesized by the bovine oviduct at estrus. Oviducts obtained from four crossbred cows in estrus were flushed with saline, and segments of the ampullary and isthmic regions were fixed for immunocytochemical analyses. The remainder of the tissue was subjected to explant culture for 24 h in medium containing either 3H-leucine or 3H-glucosamine. Analysis of culture media by one- and two-dimensional SDS-PAGE followed by fluorography indicated that both the ampullary and isthmic regions synthesized a major class of Mr 97,000 glycoproteins with isoelectric points ranging from 5.5 to 8.1. A polyclonal antibody was generated to the glycoproteins after their isolation by gel filtration followed by electrophoretic separation. Western blot analysis of oviductal culture media indicated that the antisera cross-reacted with a doublet at Mr 97,000 and to a lesser extent with two additional bands at Mr greater than 200,000. Immunoreactive antigens were not identified in serum or in culture media of ovary, uterus, and nonreproductive tract tissues. The Mr 97,000 glycoproteins were present in oviductal flushings obtained from cows in estrus. They were also detected to a lesser degree in oviductal flushings obtained from cows at Days 5, 10, 15, and 18 of the estrous cycle, with the least amount of immunoreactivity being observed in Day 10 samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm migration into and through the oviduct following artificial insemination at different stages of the estrous cycle in the rat

In order to examine whether sperm migration into and through the oviduct follows an invariable pattern or is subject to regulation, rats in proestrus, estrus, metestrus, or diestrus were inseminated in the upper third of each uterine horn with 10-20 million epididymal spermatozoa. Three or eight hours later, the numbers of spermatozoa free and adhering to the epithelium in the ampullary and isthmic segments were determined. A significantly higher number of spermatozoa were recovered in estrus than in other stages, at 3 h than at 8 h, and at all stages from the isthmus than from the ampulla. Spermatozoa adhering to the epithelium were observed only in proestrus and estrus and in the isthmus. The effect of exogenous estradiol-17beta (E2) and progesterone (P4) on sperm migration was investigated in rats in which the estrous cycle was inhibited pharmacologically. E2 facilitated sperm migration into the oviduct and P4 antagonized this effect, whereas P4 alone had no effect. Concomitant treatment with E2+P4 induced adhesion of spermatozoa to the oviductal epithelium. In conclusion, the pattern of sperm migration into and through the rat oviduct varies with the stage of the cycle, being dependent on E2 and P4. The adhesion of spermatozoa to the rat oviductal epithelium is stage- and segment-specific and requires the combined action of both hormones.     

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct

In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P=0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5+/-10.49 mg/L in ampullar ODF, compared to 43.2+/-10.74 mg/L in isthmic ODF); least square mean (L.S.M.)+/-standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5+/-10.54 mg/L on the ovulation side, compared to 43.1+/-10.71 mg/L in the oviduct on the contralateral side (L.S.M.+/-S.E.M., P=0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.     

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin–horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.     

Unexpected flagellar movement patterns and epithelial binding behavior of mouse sperm in the oviduct

In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.     

Antioxidant defenses are modulated in the cow oviduct during the estrous cycle

The balanced presence of reactive oxygen species and antioxidants has a positive impact on sperm functions, oocyte maturation, fertilization, and embryo development in vitro. The mammalian oviduct is likely to provide an optimal environment for final gamete maturation, sperm-egg fusion, and early embryonic development. However, the expression and distribution of antioxidant enzymes in the bovine oviduct are poorly characterized. We analyzed the mRNA expression and enzymatic activities of major antioxidants glutathione peroxidase (GPx), superoxide dismutase (Cu,ZnSOD), and catalase in the bovine oviduct throughout the estrous cycle. The high levels of expression for GPx-3 in the isthmus were in contrast to expression of GPx-1 and GPx-2, which occurred mostly in the ampulla and infundibulum of the oviduct. The highest levels of mRNA expression for GPx-1 were observed toward the end of the estrous cycle before ovulation, whereas GPx-2 was mostly expressed at midcycle. Catalase and Cu,ZnSOD mRNA analyses revealed a homogenous expression along the oviduct. The highest levels of glutathione and enzymatic activities for GPx and catalase occurred at the middle (10-12 days) and end (18-20 days) of the estrous cycle, whereas total SOD activity remained constant throughout the estrous cycle in the oviductal fluids. These findings underscore the importance of hydrogen peroxide and hydroperoxide removal by GPx in the oviduct. The heterogeneous expression of antioxidants such as GPx along the oviduct is a possible indication of their physiological role in the events leading to successful fertilization and implantation in vivo.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.     

Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.     

Spermadhesin AQN1 is a candidate receptor molecule involved in the formation of the oviductal sperm reservoir in the pig

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.     

Histological changes and transglutaminase 2 expression in the oviduct of advanced pregnant cows

The oviduct is a dynamic organ that has not been assigned specific functions during advanced pregnancy. However, since changes in the oviductal epithelium during the estrous cycle are attributed mainly to variations in estradiol (E2) levels, and E2 levels increase along pregnancy, we hypothesized that advanced pregnant cows should present changes in the oviductal epithelium. In advanced pregnant cows, the oviducts showed higher leaf-like folds and lower mucosa width and epithelium height than those of cycling animals. Also, PAS-positive apical protrusions and TUNEL-positive extruded cytoplasmic material were observed in advanced pregnant cows. Oviductal fluid from advanced pregnant cows showed lower protein concentration than that from cycling cows. Transglutaminase 2 (TG2) was detected exclusively in oviductal fluid of pregnant cows but not in cells from any stage, whereas its mRNA was detected in different amounts in cells from all stages. This protein was identified by LC/MS-MS and its identity was corroborated by Western blot. The observations in histology of the epithelium and the presence of TG2 in oviductal fluid correlate with high levels of E2 in serum. In conclusion, important histological changes in the oviductal epithelium and secretion of TG2 to the oviductal fluid appear to be triggered by the high E2 levels exclusive of advanced pregnancy.     

Sperm binding to oviductal epithelial cells in the rat: role of sialic acid residues on the epithelial surface and sialic acid-binding sites on the sperm surface

The aim of this study was to assess the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined, by lectin labeling, the distribution of glycoconjugates in rat oviducts obtained under various hormonal environments. Several classes of glycoconjugates were abundant in the epithelium, and the expression of some of these molecules varied differentially in ampulla and isthmus, along the estrous cycle and with estradiol and progesterone treatment. Proestrous rats were intraoviductally injected with lectins Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Arachis hypogea, Ulex europaeus I, Triticum vulgaris, or Tritrichomonas mobilensis and were inseminated with 10-20 million epididymal spermatozoa in each uterine horn. Three hours later, the total number of spermatozoa present in the oviduct and the proportion adhering to the epithelium were determined. Intraoviductal administration of lectins did not affect the total number of spermatozoa recovered from the oviduct and only the sialic acid-binding lectin TML decreased the percentage of sperm cells adhering to the epithelium. The involvement of sialic acid in sperm-oviduct adhesion was further explored, inseminating spermatozoa preincubated with mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin. Sialic acid and fetuin inhibited sperm-oviduct binding while other carbohydrates had no effect. Using TML lectin immunohistochemistry, we found that sialic acid-rich glycoconjugates are equally localized in the epithelium of ampulla and isthmus of proestrous rats. The electrophoretic pattern of sialic acid-rich glycoproteins of the epithelium showed 15 major protein bands, for which molecular mass ranged from 200 to 50 kDa with no difference between ampulla and isthmus or between estrous cycle stages. Binding sites for sialic acid-fluorescein isothiocyanate were demonstrated on the surface of rat spermatozoa, and biotinylated sialic acid recognized 11 plasma membrane proteins of sperm cells. These groups of sialic acid-rich glycoproteins in the oviductal epithelium and of sialic acid-binding proteins in the plasma membrane of sperm cells are good candidates for further studies to characterize the molecules responsible for sperm binding. We conclude that there are segment-specific changes of sugar residues present in the oviductal epithelium associated with different endocrine environments. Sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the epithelial cells with sialic acid-binding proteins on the sperm surface. This replicates the situation previously found in hamsters, disclosing for the first time that species-specificity in the sugar involved in sperm binding is not absolute.     

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO₂. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.