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.

Isolation and characterization of a protein with homology to angiotensin converting enzyme from the periacrosomal plasma membrane of equine spermatozoa.

The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68-70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related to 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ejaculated and epididymal spermatozoa. A larger protein (approximately 110 kD) was detected in equine testis. Two antigenically related proteins (64 and 45 kD) were recognized on the plasma membrane of cynomolgus macaque spermatozoa. In vitro sperm-binding assays were performed in the presence of antigen-binding fragments or IgG purified from the polyclonal antiserum to investigate a possible function to the isolated protein in binding of equine spermatozoa to homologous oviductal epithelial cells or zona pellucida. Incubation with antigen-binding fragments or IgG purified from the antiserum did not inhibit binding of equine spermatozoa either to oviductal epithelial cells or the zona pellucida. On ultrastructural examination, the antibody bound exclusively to the cytoplasmic side of the periacrosomal plasma membrane of equine and macaque spermatozoa. Microsequence analysis of 13 residues of sequence showed strong homology with a number of angiotensin converting enzymes: An 84% identity was identified with testis specific and somatic forms of human and mouse angiotensin-converting enzyme. Immunocytochemistry and immunoblot analysis established that the protein is specific for the periacrosomal membrane of ejaculated, epididymal, and testicular stallion spermatozoa.

Antibody directed against plasma membrane components of equine spermatozoa inhibits adhesion of spermatozoa to oviduct epithelial cells in vitro.

Before fertilization, equine spermatozoa adhere to oviduct epithelial cells (OEC) of the mare. The biochemical basis for this adhesion has not been determined. Our objective was to produce an antiserum to block this interaction. Ejaculated spermatozoa were subjected to nitrogen cavitation and spermatozoal plasma membranes enriched by sucrose density gradient centrifugation; membrane enrichment was confirmed by comparative alkaline phosphatase analysis, electron microscopy, and one- and two-dimensional PAGE. Periacrosomal plasma membrane was used as an immunogen for the production of an antiserum, which recognized several components of spermatozoal plasma membrane on Western blots. Antigen-binding fragments (Fab) were isolated by papain digestion from a specific antiserum and from nonimmunized rabbit IgG (control). The periacrosomal regions of epididymal and ejaculated spermatozoa were immunolabeled with antiserum Fab but not control Fab. The immunoneutralizing activity of antiserum Fab was tested in fluorescent cell-binding assays by competitive inhibition of the binding of spermatozoa to OEC monolayers or explants. In both assays, binding of spermatozoa to OEC was reduced as the concentration of specific Fab increased. These results suggest that one or more protein or glycoprotein components of the rostral spermatozoal plasma membrane mediate adhesion between spermatozoa and oviduct epithelium in vitro.

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of bovine oocytes.

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4-24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [35S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation.

Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro.

OBJECTIVE: To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. ANIMALS: 4 reproductively sound stallions, and 1 mare in estrus. PROCEDURES: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fetuin or asialofetuin/ml in modified Tyrode's solution (TALP), or in TALP alone. After 2 hours of coculture, numbers of attached spermatozoa were counted by fluorescence microscopy and analysis of digitized images. In experiment 1b, progressive motility, viability, acrosomal integrity, and capacitation status were determined in spermatozoa incubated for 2 hours in the presence of the respective monosaccharides and glycoproteins or in TALP alone. In experiment 2, proteins isolated from the periacrosomal plasma membrane of equine spermatozoa were subjected to galactose affinity chromatography and subsequent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. RESULTS: Numbers of spermatozoa attached to OEC were reduced (P < 0.05) after all treatments except N-acetyl glucosamine, compared with incubation in TALP alone. The lowest numbers of spermatozoa were bound in cultures incubated in the presence of galactose and asialofetuin. Spermatozoal motility was lower (P < 0.05) after incubation for 2 hours in the presence of fetuin, compared with control, and incubation in the presence of fetuin or asialofetuin caused a significant (P < 0.05) increase in the percentage of capacitated spermatozoa, compared with control. Affinity chromatography of periacrosomal plasma membrane proteins revealed a galactose-binding protein of about 66 kd. CONCLUSION: Recognition of glycoconjugates with exposed galactosyl residues on OEC by galactose-binding proteins on the periacrosomal plasma membrane of equine spermatozoa could mediate the attachment of equine spermatozoa to OEC in vitro.

Characterization of polypeptides synthesized and secreted by oviductal epithelial cell explants obtained from young, fertile and aged, subfertile mares.

OBJECTIVE: To compare the electrophoretic patterns of proteins synthesized and secreted by oviductal epithelial cell (OEC) explants obtained from young, fertile and aged, subfertile mares. ANIMALS: Young, fertile (n = 5; 2 to 7 years old) and aged, subfertile (n = 5; 17 to 24 years old) mares. PROCEDURE: 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computerized densitometry. RESULTS: Variation in the synthesis and secretion of polypeptides from young, fertile mare OEC (YOEC) and aged, subfertile mare OEC (AOEC) was evidenced by differences in the intensity of radiolabeled polypeptides on fluorograms. Fluorograms for 9 acidic (isoelectric point, 5.09 to 6.50) proteins of low to medium molecular mass (18.2 to 85.0 kd) from YOEC had greater intensity than did those from AOEC. Fluorograms for 7 proteins (10.5 to 45.0 kd; isoelectric point, 5.80 to 6.92) from AOEC had greater intensity. CONCLUSION: The differences detected in the fluorographic intensities of secreted proteins from YOEC and AOEC may be related to the disparity in embryo development observed between young, fertile and aged, subfertile mares. CLINICAL RELEVANCE: Failure to maintain pregnancy in aged, subfertile mares may be a result of a suboptimal oviductal environment exerting its effects on the conceptus during early cleavage.

Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells.

Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein secretion by OEC was measured and compared by incorporation of [35S]methionine, and evaluated, using two-dimensional polyacrylamide gel electrophoresis and fluorography. Monolayers of OEC secreted a large number of proteins of molecular mass ranging from 14 to 205 kd. Adhesion of spermatozoa consistently caused reduced synthesis of 2 OEC secretory proteins and new or increased synthesis of 6 proteins. When spermatozoa and OEC were separated by a microporous membrane, some but not all of these changes were duplicated. Synthesis of 3 OEC secretory proteins, unaffected by binding of spermatozoa, was reduced when spermatozoa were prevented from contact with OEC by a microporous membrane. Adhesion of equine spermatozoa to homologous OEC monolayers and presence of equine spermatozoa resulted in qualitative and quantitative changes in synthesis and secretion of proteins by OEC. These changes have implications for storage, longevity, and maturation of spermatozoa.

Initiation of transcription and nucleologenesis in equine embryos.

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 microCi/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counterstained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated 3H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of 3H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos.

Isolation, culture, and characterization of equine oviduct epithelial cells in vitro.

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17 beta or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers.

In vitro interaction between oviduct epithelial and equine sperm.

Coculture of stallion sperm with monolayers of equine oviductal epithelial cells (OEC) was evaluated. Monolayers were obtained from frozen-thawed OEC. Live sperm attached to the OEC in vitro, whereas sperm killed by heat treatment or glutaraldehyde fixation did not. Sperm attached to OEC showed flagellar motion for 4 d in vitro, during which time they gradually became released. Scanning electron-micrographs showed an intimate association between the sperm and OEC. Incubation of sperm for 4 h with either control, heparinized or OEC-conditioned medium (Tyrode's albumin lactate phosphate) resulted in more incapacitated sperm, as determined by chlortetracycline staining patterns. The OEC-conditioned medium caused similar capacitation-like changes to those seen with heparin. Sperm viability as determined by Hoechst 33258 staining was not significantly affected by media type.

De novo protein synthesis by bovine uterine tube (oviduct) epithelial cells changes during co-culture with bull spermatozoa.

Polypeptides secreted by uterine tube epithelial cells (UTEC) may facilitate sperm cell capacitation in vivo. This experiment evaluated the effect of sperm-UTEC co-culture on de novo protein synthesis by epithelial cells of the tubal isthmus. Comparisons of the patterns of proteins secreted into medium were made between four culture groups incubated for 24 h in the presence of 35S-methionine: group 1, sperm cells alone; group 2, control UTEC monolayers; group 3, UTEC co-cultured with sperm cells; and group 4, UTEC partitioned by a diffusible membrane from sperm cells during culture. Two-dimensional PAGE followed by fluorography was used to analyze conditioned medium containing secreted proteins from each group. The experiment was replicated four times. Sperm cells alone secreted no detectable proteins, whereas control UTEC monolayers produced a wide array of polypeptides. Sperm cells attached to UTEC in co-culture within minutes, and the resultant protein profile for these UTEC differed markedly from that of the control UTEC. Several new proteins were seen only from co-cultured cells, whereas other protein groups that were present with UTEC alone were absent in the co-culture medium of group 3. The protein pattern expressed by UTEC partitioned from sperm cells (group 4) was intermediate between that of the group 2 controls and that of co-cultured UTEC (group 3). In summary, the attachment of sperm cells to the UTEC during co-culture changed the types and quantities of proteins secreted into the conditioned medium as compared to those of control UTEC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.

Effect of fibronectin on early embryo development in cows.

Two-cell bovine embryos produced in vitro were cultured in serum-free medium containing the soluble glycoprotein fibronectin (50 micrograms ml-1) to study the function of the extracellular matrix in early development. Some of the embryos (48/164, 29.3%), developed beyond the 16-cell stage compared with none of the 179 controls. Fibronectin at lower (5 micrograms ml-1) or higher (300 micrograms ml-1) concentrations did not promote embryo development (0/89 and 0/82, respectively). Indirect immunofluorescence demonstrated the presence of both fibronectin and its receptor on the surface of eight-cell embryo blastomeres, and biotinylated fibronectin demonstrated that exogenous fibronectin could cross the zona pellucida. These results, demonstrating the successful culture of bovine embryos in serum-free medium, support the hypothesis that the extracellular matrix, specifically fibronectin, plays a role in early development of bovine embryos.

Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle.

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.

On the mechanism by which insulin stimulates protein synthesis in chick embryo fibroblasts.

It has been known that insulin raises the rate of incorporation of [3H]leucine into the total protein of hormone-deficient chick embryo fibroblasts by approximately 1.5-fold. The elevation is not dependent upon the production of new messenger ribonucleic acids (mRNAs). Evidence is now presented in support of the following points: the greater labelling is due to more rapid polypeptide synthesis, not to an increase in the specific activity of leucyl-tRNA; the enhanced synthesis derives largely or entirely from a speeding up of the process of initiation, rather than that of elongation or termination; and the 1.5-fold stimulation is due to the elevated rates of formation of at least many of the fibroblast proteins. The hormone was shown before to stimulate posttranscriptionally and highly preferentially for formation of ribosomal proteins in the resting chick embryo cells. The question has been asked here whether insulin increases the production of total cell ribosomal protein by chemically altering preformed mRNAs. Results obtained by translating messages from deprived and hormone-treated cells in wheat germ and reticulocyte preparations do not support a mechanism involving covalent modification of preformed mRNAs. The observations, coupled with those previously made with inhibitors of translation, lead us to suggest that insulin stimulates protein synthesis in the resting chick embryo cells by activating limiting components of the initiation system. The effects of the hormone are greatest with messages, such as those for the ribosomal proteins, that have low affinities for the limiting initiation components.

Lodish model and regulation of ribosomal protein synthesis by insulin-deficient chick embryo fibroblasts.

The production of ribosomal proteins in chick embryo fibroblasts that have been deprived of insulin is depressed in a much greater degree than that of most or all other cell proteins. Previous observations ruled out explanations for the preferential decrease in ribosomal protein formation that depend upon a selective destruction of ribosomal protein messages or a regulatory role for nascent ribosomal ribonucleic acid. The proposition has now been examined that ribosomal protein messenger ribonucleic acids (mRNAs) in the hormone-deficient chick embryo cells have a lower affinity for a limiting, early acting component of the initiating machinery than do most other cell messages and, in consequence, suffer from a translational disadvantage. The approach that was used depends upon the findings of Lodish and others that all mRNAs are not initiated with equal ease, that inhibitors of elongation favor the initiation of low-affinity mRNAs, and that agents that dampen an early step in initiation discriminate against the low-affinity messages. The idea was tested by comparing the effects of various inhibitors on the rates of synthesis of total cell protein and individual nonribosomal proteins, on the one hand, with those of individual ribosomal proteins, on the other. The results fit the Lodish model and are consistent with the conclusions that ribosomal protein mRNAs are more poorly initiated in the resulting fibroblasts than are most or all other cell messages and that this condition is largely or entirely responsible for the low rate of ribosomal protein formation.

Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period.

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non-ribosomal RNAs: (1) Addition to the "shift-up" medium of either of two inhibitors of RNA formation, camptothecin or 5, 6-dichloro--1-beta-D-ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells through the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced. The possibility is considered that the essential G1 molecules are mRNAs.