Microfluidic devices for the study of sperm migration.

Microfluidics technology offers us an opportunity to model the biophysical and biochemical environments encountered by sperm moving through the female reproductive tract and, at the same time, to study sperm swimming dynamics at a quantitative level. In humans, coitus results in the deposition of sperm in the vagina at the entrance to the cervix. Consequently, sperm must swim or be drawn through the cervix, uterus, uterotubal junction and oviductal isthmus to reach the oocyte in the oviductal ampulla. Only a very small percentage of inseminated sperm reach the ampulla in the periovulatory period, indicating that strong selection pressures act on sperm during migration. A better understanding of how sperm interact with the female tract would inspire improvements in diagnosis of fertility problems and development of novel-assisted reproductive technologies that minimize damage to sperm and mimic natural selection pressures on sperm.

Mathematical modeling of calcium signaling during sperm hyperactivation.

Mammalian sperm must hyperactivate in order to fertilize oocytes. Hyperactivation is characterized by highly asymmetrical flagellar bending. It serves to move sperm out of the oviductal reservoir and to penetrate viscoelastic fluids, such as the cumulus matrix. It is absolutely required for sperm penetration of the oocyte zona pellucida. In order for sperm to hyperactivate, cytoplasmic Ca(2+) levels in the flagellum must increase. The major mechanism for providing Ca(2+) to the flagellum, at least in mice, are CatSper channels in the plasma membrane of the principal piece of the flagellum, because sperm from CatSper null males are unable to hyperactivate. There is some evidence for the existence of other types of Ca(2+) channels in sperm, but their roles in hyperactivation have not been clearly established. Another Ca(2+) source for hyperactivation is the store in the redundant nuclear envelope of sperm. To stabilize levels of cytoplasmic Ca(2+), sperm contain Ca(2+) ATPase and exchangers. The interactions between channels, Ca(2+) ATPases, and exchangers are poorly understood; however, mathematical modeling can help to elucidate how they work together to produce the patterns of changes in Ca(2+) levels that have been observed in sperm. Mathematical models can reveal interesting and unexpected relationships, suggesting experiments to be performed in the laboratory. Mathematical analysis of Ca(2+) dynamics has been used to develop a model for Ca(2+) clearance and for CatSper-mediated Ca(2+) dynamics. Models may also be used to understand how Ca(2+) patterns produce flagellar bending patterns of sperm in fluids of low and high viscosity and elasticity.

Regulation of sperm storage and movement in the ruminant oviduct.

Three regions of the ruminant oviduct play different roles in the progress of sperm: the uterotubal junction, isthmus, and ampulla. The uterotubal junction acts as a point of selection of sperm, requiring that sperm are progressively motile and express specific proteins in order to enter the oviduct. The isthmus stores sperm, preserving motility and viability until ovulation. Sperm are stored in the isthmus by binding to its mucosal epithelium. In bovine sperm, binding to the oviductal epithelium is promoted by proteins that are secreted by the seminal vesicles and coat the heads of sperm by associating with plasma membrane phospholipids. Putative oviductal receptors for the seminal vesicle proteins are members of the annexin protein family. Release of sperm from the storage site in the isthmus is gradual, which serves to ensure that sperm in the proper physiological state reach the oocytes at the appropriate moment and also to reduce incidence of polyspermic fertilization. The ampulla supports fertilization and may participate in guiding sperm toward the eggs. Further studies are needed to improve our understanding of the interactions between sperm and the female reproductive tract, in order to develop means to improve fertility in ruminants.

Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes.

Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The objective of this study was to characterize stallion sperm hyperactivation and to test whether hyperactivation of capacitated sperm supported equine IVF. Treatment of sperm with procaine, an anesthetic shown to induce hyperactivation in other mammalian species, resulted in the decrease of three motility variables indicative of hyperactivation: straight line velocity (P = 0.029), straightness (P = 0.001), and linearity (P = 0.002). We demonstrated that procaine-induced hyperactivation was not regulated by changes in protein tyrosine phosphorylation and that it did not induce acrosomal exocytosis in capacitated sperm compared with calcium ionophore (P > 0.05), similar to findings in the bovine. Most notably, by coupling our capacitating conditions with the induction of hyperactivation using procaine, we have achieved the novel result of substantial and reproducible percentages of fertilized mare oocytes (60.7%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with procaine did not fertilize (0%). These results support the hypothesis that capacitation and hyperactivation are required for successful IVF in the equine.

Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction.

Artificial insemination with sexed semen, in vitro fertilisation and intracytoplasmic sperm injection have been used to reproduce animals, but often not as successfully as natural mating. Learning more about how spermatozoa normally interact with the female tract can provide inspiration for developing improvements in assisted reproduction. The present review focuses on Bos taurus, because more is known about this species than others. At coitus, bull spermatozoa are deposited into the anterior vagina, where they rapidly enter the cervix. Cervical mucus quickly filters out seminal plasma from spermatozoa, unlike most assisted reproduction protocols. Spermatozoa that reach the uterus may require certain cell surface proteins to swim through the uterotubal junction. Shortly after passing through the junction, most spermatozoa are trapped in a storage reservoir by binding to oviducal epithelium, in the case of cattle via bovine seminal plasma (BSP) proteins coating the sperm head. As ovulation approaches, spermatozoa capacitate and shed BSP proteins. This reduces sperm binding to the epithelium and releases them from storage. Motility hyperactivation assists spermatozoa in leaving the storage reservoir, swimming through oviducal mucus and the cumulus oophorus, and penetrating the oocyte zona pellucida. Chemotactically regulated switching between asymmetrical (i.e. hyperactivated) and symmetrical flagellar beating may also guide spermatozoa to the oocyte.

Sperm transport in the female reproductive tract.

At coitus, human sperm are deposited into the anterior vagina, where, to avoid vaginal acid and immune responses, they quickly contact cervical mucus and enter the cervix. Cervical mucus filters out sperm with poor morphology and motility and as such only a minority of ejaculated sperm actually enter the cervix. In the uterus, muscular contractions may enhance passage of sperm through the uterine cavity. A few thousand sperm swim through the uterotubal junctions to reach the Fallopian tubes (uterine tubes, oviducts) where sperm are stored in a reservoir, or at least maintained in a fertile state, by interacting with endosalpingeal (oviductal) epithelium. As the time of ovulation approaches, sperm become capacitated and hyperactivated, which enables them to proceed towards the tubal ampulla. Sperm may be guided to the oocyte by a combination of thermotaxis and chemotaxis. Motility hyperactivation assists sperm in penetrating mucus in the tubes and the cumulus oophorus and zona pellucida of the oocyte, so that they may finally fuse with the oocyte plasma membrane. Knowledge of the biology of sperm transport can inspire improvements in artificial insemination, IVF, the diagnosis of infertility and the development of contraceptives.

Color vision pigment frequencies in wild tamarins (Saguinus spp.).

The adaptive importance of polymorphic color vision found in many New World and some prosimian primates has been discussed for many years. Polymorphism is probably maintained in part through a heterozygote advantage for trichromatic females, as such individuals are observed to have greater foraging success when selecting ripe fruits against a background of forest leaves. However, recent work also suggests there are some situations in which dichromatic individuals may have an advantage, and that variation in color vision among individuals possessing different alleles may also be significant. Alleles that confer a selective advantage to individuals are expected to occur at a higher frequency in populations than those that do not. Therefore, analyzing the frequencies of color vision alleles in wild populations can add to our understanding of the selective advantages of some color vision phenotypes over others. With this aim, we used molecular techniques to determine the frequencies of color vision alleles in 12 wild tamarin groups representing three species of the genus Saguinus. Our results show that allele frequencies are not equal, possibly reflecting different selective regimes operating on different color vision phenotypes.

Hyperactivation of mammalian sperm.

Mammalian sperm commonly show hyperactivated motility just before fertilization. The movement of hyperactivated sperm appears different in fluids of different viscosity and elasticity and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Hyperactivation may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none have yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, divergent pathways regulate the two events.

Hyperactivated motility in sperm.

Hyperactivation is a movement pattern seen in sperm at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated sperm appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, the two events are regulated by somewhat different pathways.

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.

Hyperactivation of mammalian spermatozoa: function and regulation.

Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry. Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified. There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct. Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. The process may also involve increases in intracellular cAMP, which at least is required to support motility in general. Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways.

Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir.

Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.

Carbohydrate-mediated formation of the oviductal sperm reservoir in mammals.

Mammalian sperm are trapped in a reservoir in the oviduct until ovulation is imminent. Then, they are gradually released, such that a few meet the oocytes as they enter the ampulla of the oviduct. In the three eutherian species studied to date, sperm are trapped in the reservoir by carbohydrate-mediated binding to the oviductal mucosa. Evidence indicates that a molecule on the surface of the plasma membrane overlying the acrosome binds to a carbohydrate moiety on the surface of the oviduct. While sperm remain bound, they appear to be protected from degradation. When sperm become capacitated, they lose binding affinity for the oviductal mucosa. The mechanism initiating capacitation in the reservoir is unknown; however, it must be tied to the hormonal signalling of ovulation. Hyperactivated motility may assist sperm in pulling off from the mucosal surface as binding affinity declines. The function of the reservoir appears to be to prevent polyspermy and ensure fertilization by providing a small number of sperm in the proper physiological condition for fertilization at the time the oocytes enter the oviduct.

Physiological state of bull sperm affects fucose- and mannose-binding properties.

In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean +/- SD:167 +/- 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by capacitating washed sperm in medium containing 10 microg/ml heparin (50 +/- 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 +/- 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.

Three-dimensional structure of the Golgi apparatus in mouse spermatids: a scanning electron microscopic study.

In this study, the three-dimensional organization of the Golgi apparatus in mouse spermatids was elucidated by preparing testicular tissue with the osmium-DMSO-osmium method and examining it by stereo-scanning electron microscopy. The cis-most saccule was found to be a regular network of anastomotic membranous tubules covered by a single cisterna of ER. The trans-Golgi network was seen to be composed of irregular saccules perforated by pores at the edge. It appears that the anastomosing trans-Golgi network breaks down into strings of connected vesicles which arise from the edge of the saccules during the cap phase of spermiogenesis. Many apparently individual vesicles seen in thin sections through the trans-Golgi network are actually joined in continuous strings. This was the first time that these structures could be visualized directly without three-dimensional image reconstruction. By correlating the morphology of the Golgi apparatus with the stage of acrosome formation, the Golgi cisternae were found to change dynamically in a cis-trans direction from fenestrated saccules to continuous strings of vesicles, which finally dissipated as transport vesicles at the trans aspect. This suggests that the hypothetical model of cisternal maturation, which dictates that cargo moves through the Golgi apparatus without leaving the cisternal lumen and the secretion occurs by progressive maturation of the Golgi cisternae as they move in the cis-trans direction, may be applicable to acrosome formation.

Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes Lewis-a trisaccharide.

Sperm binding to oviductal epithelium produces a reservoir in vivo that may serve to maintain sperm fertility and provide sperm for fertilization when ovulation occurs. Previously, it was determined that bull sperm binding could be blocked by fucoidan and its component fucose; furthermore, treatment of epithelium with fucosidase prevented binding. The present study was conducted to further characterize binding. Because fucose would probably be present on the epithelium as part of oligosaccharide moieties of glycoproteins and/or glycolipids, competitive binding inhibition activity was tested for fucose in five linkages commonly found in oligosaccharides. Binding inhibition was assayed by determining the concentration of motile, frozen/thawed sperm bound to fresh epithelial explants in the presence of test inhibitors. Initially, 5 monosaccharides were tested at 30 mM (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose), and only fucose significantly reduced sperm binding compared to vehicle control (p = 0.03). Of the oligosaccharides tested (lacto-N-fucopentaose I, 3-fucosyllactose, Lewis-X, Lewis-a, and GlcNAcbeta1-4[Fucalpha1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.

Bovine sperm binding to oviductal epithelium involves fucose recognition.

Sperm binding to oviductal epithelium probably serves to form the isthmic sperm reservoir. This interaction of sperm and oviductal epithelium may involve species-specific carbohydrate recognition. We tested a series of carbohydrates and glycoproteins for inhibition of bovine sperm binding to oviductal epithelium in vitro. Explants of isthmic and ampullar epithelium were obtained from oviducts that had been surgically removed from preovulatory heifers. The explants were incubated (39 degrees C, 5% CO2) with fetuin, asialofetuin, ovalbumin, fucoidan, fucose, N-acetyl glucosamine, or N-acetyl glucosamine sulfate dissolved in a modified Tyrode's balanced salt solution, termed sperm-TALP (pH 7.4, 295 mOsm) for 10 min before frozen-thawed motile sperm obtained by swim-up were added. After 15 min, the explants were rinsed, and sperm binding density was evaluated. Oviductal explants treated with fucoidan (3 mg/ml; p < 0.001, n = 5) or fucose (31 mM, p < 0.01, n = 6) had reduced densities of bound sperm compared to the controls. Incubation of explants in increasing concentrations of fucose (4-62 mM) resulted in increased inhibition of sperm binding. Pretreating explants with fucosidase also reduced sperm binding (p < 0.001, n = 3) compared to that in controls containing the fucosidase inhibitor deoxyfuconojirimycin. The presence of fucosylated molecules on the surface of the oviductal epithelium was confirmed by labeling with fucose lectins from Ulex europeus and Lotus tetragonolobus. We conclude that fucose is involved in a specific interaction between bovine sperm and oviductal epithelium.

Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro.

Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca2+]i in spermatozoa still attached to OEC. In addition, [Ca2+]i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca2+]i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca2+]i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca2+]i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca2+]i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca2+]i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization.

Distribution of mucus and sperm in bovine oviducts after artificial insemination: the physical environment of the oviductal sperm reservoir.

There is a sperm reservoir in the caudal oviduct of cattle and other mammals. We had observed trapping of sperm by mucus produced by explants of bovine oviductal epithelium in vitro; therefore, we used techniques designed for preserving mucus and luminal dimensions to determine whether mucus is associated with the reservoir in vivo. Heifers were synchronized by prostaglandin injections and inseminated during estrus. Oviducts on the side of ovulation were surgically removed either 8-10 h after insemination (preovulatory) or 50-55 h after insemination (postovulatory). Segments (1 cm) taken from the uterotubal junction (UTJ), caudal 3 cm of isthmus, and mid ampulla were snap frozen. Frozen sections were coated with collodion and postfixed in phosphate-buffered formaldehyde containing cetyl-pyridinium chloride. Sections were alternately stained with periodic acid-Schiff stain (PAS) or alcian blue/PAS. Most of the oviductal lumen was highly branched with passages that measured only a few microns across and were filled with mucus. In limited areas, the lumen opened to 100 microm across and was only lightly stained for mucus. Overall, the lumen was much narrower than in sections prepared by standard fixation and paraffin embedding. Sperm were found scattered throughout the lumen of the UTJ and isthmus, in both the narrow, deeply stained luminal areas and the wider, lightly stained areas. The numbers tapered off cranially, especially prior to ovulation. In conclusion, the combination of narrow passages and mucus would appear to impede sperm progress, contributing to the creation of a reservoir.