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Human sperm function was compared in co-culture with monolayers of oviduct epithelial cells (OEC) from three species, human, macaque and bovine. For all species, freeze-thawed and passaged OEC from females in the periovulatory phase were used. OEC cultured on an extra-cellular matrix (Matrigel) formed a monolayer which supported human sperm attachment to OEC from all three species. Spermatozoa in co-culture with OEC from all three species showed prolonged survival and improved motility characteristics over those cultured in medium alone. This paper describes an efficient, repeatable co-culture system for human spermatozoa which supports sperm attachment to OEC and subsequently improves sperm function over that seen in control medium cultures. Because the improved sperm function in co-culture did not differ significantly between human and bovine OEC for those attributes studied, it is proposed that bovine OEC could be used as an alternative to human OEC in certain human sperm co-culture studies. Follicular phase bovine OEC from reproductively normal donors are far more accessible than their human counterparts, thus making this co-culture system more widely available for the study of human spermatozoa-female tract interactions. [ABSTRACT FROM PUBLISHER]

The ability to rapidly and reliably screen for bacterial vaginosis (BV) during pregnancy is of great significance for maternal health and pregnancy outcomes. In this proof-of-concept study, we demonstrated the potential of carbon nanotube field-effect transistors (NTFET) in the rapid diagnostics of BV with the sensing of BV-related factors such as pH and biogenic amines. The fabricated sensors showed good linearity to pH changes with a linear correlation coefficient of 0.99. The pH sensing performance was stable after more than one month of sensor storage. In addition, the sensor was able to classify BV-related biogenic amine-negative/positive samples with machine learning, utilizing different test strategies and algorithms, including linear discriminant analysis (LDA), support vector machine (SVM), and principal component analysis (PCA). The biogenic amine sample status could be well classified using a soft-margin SVM model with a validation accuracy of 87.5%. The accuracy could be further improved using a gold gate electrode for measurement, with accuracy higher than 90% in both LDA and SVM models. We also explored the sensing mechanisms and found that the change in NTFET off current was crucial for classification. The fabricated sensors successfully detect BV-related factors, demonstrating the competitive advantage of NTFET for point-of-care diagnostics of BV.

The success of assisted reproductive techniques, such as IVF, could be enhanced by being able to select the most competent spermatozoa in a sample. Attachment and subsequent release of spermatozoa from oviductal epithelial cells (OEC) could provide populations of functionally superior spermatozoa for use in these protocols. The objective of the present study was to investigate the ability of heparin and Ca2+-free medium to induce spermatozoa release from bovine OEC. Epithelial cells were grown to confluence in 24-well plates and pooled frozen bull semen was added to a final concentration of 1 x 10(6) spermatozoa/well. Spermatozoa were allowed to bind to OEC for 2 h. Medium with unbound spermatozoa was removed and replaced by Sperm-TALP, only (control), with heparin (5, 10, or 15 IU/mL), or Ca2+-free with 2 mM EGTA. Treatments were left on sperm-OEC co-cultures for 0.5, 1, 2, 3, or 5 h. At each time, the media were recovered and spermatozoa from each treatment were counted and evaluated for acrosome integrity and motility. The total number of spermatozoa attached to OEC after 2 h of co-culture was considered 100%. Spermatozoa release is expressed as percentage of the total number of sperm cells bound to OEC after 2 h of co-culture. Data were analyzed by ANOVA and results are expressed as mean +/- SEM from three independent replicates. Beginning at 0.5 h, more sperm cells (P < 0.05) were released from OEC in the heparin groups (10 and 15 IU/mL, 77.3 +/- 6.2% and 84.0 +/- 6.2%, respectively) as compared to the control (46.4 +/- 6.2%). The Ca2+-free medium also induced spermatozoa release when compared with the control, but the effect was not significant until 3 h (38.2 +/- 1.9% vs 59.5 +/- 6.9%; P < 0.05). The percentage of acrosome reacted spermatozoa was not affected by heparin treatment. Heparin at 10 IU/mL increased (P < 0.05) the percentage of motile spermatozoa, whereas Ca2+-free medium caused the opposite effect at 0.5 h after addition of treatments. We conclude that both heparin and Ca2+-free medium are able to promote spermatozoa displacement from OEC attachment. Based on motility and acrosome status data, we predict that released sperm cells may be used for IVF and other assisted reproductive techniques.

Formation of a prefertilization sperm reservoir in mammals is thought to occur via sperm cell attachment to fallopian tube or oviduct epithelial cells (OEC). Recent data suggests that such an interaction also occurs for human sperm in the fallopian tube. We have previously validated an in vitro sperm-OEC coculture system utilizing bovine OEC monolayers to study postejaculatory human sperm physiology. This study was done to evaluate aspects of human sperm attachment to OEC in coculture and to determine if such attachment and subsequent sperm survival differ between fresh and cryopreserved human sperm. In experiment 1, aliquots of fresh (n = 4) or cryopreserved sperm (n = 3) from normospermic donors were placed into coculture with OEC monolayers at dilutions ranging from 2 x 10(5) to 15 x 10(6) sperm per well. Numbers of each type of sperm attaching to OEC at each concentration were determined. In experiment 2, fresh and cryopreserved sperm from the same donors (n = 4) were put into OEC coculture to observe numbers attaching and subsequent survival time for each sperm type. Sperm attachment to OEC occurred in a linear, dose-dependent manner for fresh and cryopreserved sperm in experiment 1, both as a function of total sperm numbers and as a function of numbers of motile sperm applied (R2 > or = 0.79). However, cryopreserved sperm attached to the OEC at a slower rate than fresh (as a function of the average increase in the number of sperm attaching per unit increase in the number of sperm applied; P < 0.05), with an overall lower percentage of the total and motile sperm applied attaching to OEC (P < 0.01) for cryopreserved versus fresh sperm. Fewer cryopreserved sperm also attached to the OEC, as compared with fresh sperm, in experiment 2 (P < 0.05), even after correcting for motility differences between the sperm types. Sperm survival time in coculture was also decreased for cryopreserved sperm as compared with fresh sperm (P = 0.005). Understanding the kinetics of sperm and OEC interactions may be useful for developing improved cryopreservation protocols or bioassays of sperm function.

Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitro handling of stallion sperm may be isolated from OEC secretory products. Experiment 1 compared first service conception (FSC) rates resulting from the use of cryopreserved sperm of seven stallions, with sperm function in co-culture such as attachment to OEC and subsequent survival time. Stallions were grouped by cumulative FSC rates observed over three seasons as having average (44 +/- 3%) or high (65 +/- 2%) fertility over a total of 217 first services (31 +/- 9 per stallion). Samples from stallions in the high fertility group had more (P = 0.04) sperm attached to OEC and longer subsequent sperm survival in co-culture (P = 0.05) as compared with those from the average fertility group. FSC rates correlated with numbers of sperm attaching to OEC and their survival time in co-culture (r > or = 0.71). In Experiment 2, the function of cryopreserved stallion sperm was evaluated in culture with OEC secretory products from three different sources. After 5 h of culture, sperm incubated with medium conditioned by bovine OEC which had been 'bioactivated' (e.g. previously exposed to sperm in culture) were found to be more (P < or = 0.05) motile and capacitated as compared to sperm in basal TALP medium alone. Sperm in this conditioned medium also survived longer (P = 0.05; 27 +/- 5 h vs. 17 +/- 4 h) than did those in control medium.

Objective: To determine if sperm attachment to oviduct epithelial cells (OEC) in vitro is selective for higher quality sperm and if the system requires homologous species OEC., Design: Controlled prospective study with outcomes assayed by a technician blind to sperm treatment groups., Setting: An academic research laboratory., Patient(s): Experiment 1: normospermic donors with children (4 donors, 7 ejaculates). Experiment 2: cryopreserved donor samples (4 donors)., Intervention(s): Semen collection by masturbation after 48 hours of abstinence., Main Outcome Measure(s): Experiment 1: sperm assays of motility, morphology, membrane integrity, and capacitation status. Experiment 2: sperm chromatin (DNA) integrity and condensation., Result(s): Experiment 1: sperm not attaching to OEC had lower motility, more membrane disruptions, and more acrosome reactions than did control sperm. This selectivity was equivalent for sperm in coculture with all OEC types. Experiment 2: sperm attached to OEC had fewer abnormalities in chromatin structure compared with sperm that were not attached., Conclusion(s): Selective attachment of functionally superior sperm to OEC is likely important during sperm reservoir formation in vivo and may be exploitable in vitro as a method to isolate high-quality sperm for clinical procedures. Such a system does not require human origin OEC.

Objective: To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA., Sample Population: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase., Procedure: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozoal survival and motility characteristics were observed over time for all culture methods. Number of spermatozoa attaching to OEC were compared for cocultures., Results: Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture. A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ. Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures. More spermatozoa were able to attach to OEC in TM without BSA., Conclusions: Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC., Clinical Relevance: Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa. Removal of BSA from such media improves spermatozoal quality and survival.

The hypothesis was tested that frozen bovine oviduct epithelial cells (BOEC) could provide a physiologic and standardized system for studying motility and acrosomal changes of fresh and frozen-thawed sperm. The BOEC were collected by lavage from cows and monolayers prepared from both fresh and frozen-thawed cells. Fresh and frozen semen processed from five bulls was extended in a modified Tyrode's medium and coincubated with both types of monolayers at 39 degrees C in 5% CO2 and 95% humidified air. After 5, 10 and 24 h of incubation, percentages of motile, progressively motile and acrosome-reacted unattached sperm were measured. The percentage of motile fresh sperm exceeded the motility of frozen sperm, and more fresh sperm attached to BOEC. More frozen sperm than fresh sperm were acrosome reacted. Fresh and frozen epithelial cells produced similar effects, except sperm motility was higher after 5 h of incubation with fresh BOEC. Thus, the convenient frozen-thawed coincubation protocol provides a practical in vitro system for studying capacitation and the acrosome reaction.

Objective: To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells., Design: Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course., Setting: An academic research laboratory., Patient(s): Normospermic donors with children., Intervention(s): Semen collection through masturbation after 48 hours of abstinence., Main Outcome Measure(s): The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ., Result(s): The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter., Conclusion(s): Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

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.

It has been suggested that superovulation in cattle is impaired if FSH injections are initiated in the presence of a dominant follicle, but the results of experiments to test this hypothesis have been contradictory. However, previous experiments were conducted during mid-cycle, when the absence or presence of a dominant follicle is difficult to assess. We took a different approach by comparing the effects of initiating superovulatory injections of FSH (11 equal doses of FSH-P, every 12 h) on Day 1 of the bovine estrous cycle, when a dominant follicle clearly is not present, vs initiation on Day 6, when a dominant follicle clearly is present and actively growing (n = 17 heifers in a "crossover" design). In 8 17 heifers initiation of FSH injections in the presence of a dominant follicle (Day 6 group) caused ovulation of the dominant follicle within 1 to 2 days and formation of a smaller than normal CL. These animals had higher than normal concentrations of plasma progesterone around the time of expected estrus (P < 0.05) and failed to exhibit estrus. Although the mean number and diameter of the follicles recruited in response to FSH injections in heifers that ovulated the dominant follicle prematurely were not different from the other heifers in the Day 6 group, no ovulations were observed, and no embryos or ova were recovered 6 d after insemination. Conversely, when FSH injections were initiated on Day 1 in these 8 heifers, they exhibited estrus, and their plasma progesterone around the time of estrus, mean ovulation rate, and number of total and transferable embryos recovered did not differ from the responses observed in the remaining 9 heifers treated either on Day 1 or on Day 6. Taken together, these results indicate that a dominant follicle does not affect the ability of smaller follicles to be recruited in response to exogenous FSH, but may impair their ovulation. These findings provide an explanation for previous reports of decreased superovulatory responses during times of the cycle when a dominant follicle would be expected to be present.

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.

Oocytes were harvested from mare ovaries obtained at slaughter and were divided into 3 groups based on the age of the donor. The age groups consisted of young (2 to 7 yr), middle-aged (8 to 14 yr) and aged (>or=15 yr) mares. There were no differences between age groups in the proportions of follicles available for examination or the proportions of normal, abnormal or total oocytes collected. After 24 h of culture, the overall maturation rate to the second metaphase (MII) was 52.7%. Maturation rates for oocytes obtained from young and middle-aged mares were similar, but oocytes from aged mares were only approximately 25% as likely to reach MII and they were 3 times more likely to remain at metaphase I. Twelve oocytes had chromosome spreads suitable for counting; 6 were haploid, 2 were hyperhaploid and 4 were hypohaploid. Insufficient numbers of readable spreads precluded comparisons of chromosome complements between age groups.

This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 x 2 crossover design. Young, fertile mares (n = 19; 2-7 years of age) and aged, subfertile, mares (n = 16; 17-24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare oviductal epithelial cells and eight pairs were co-cultured with aged mare oviductal epithelial cells. Five additional embryos obtained from young mares were co-cultured with oviductal epithelial cells from either young mares or aged mares but were not paired. Embryos were co-cultured for 7 days at 38.5 degrees C in 5% CO2 or until morphological degeneration was detected. The proportions of paired embryos that reached the blastocyst stage were similar for embryos obtained from young mares and embryos obtained from aged mares after co-culture with oviductal epithelial cells from young mares (6 of 8 versus 5 of 8) or from aged mares (6 of 8 versus 5 of 8), respectively. Although the overall rate of development of embryos to blastocyst from both young mares and aged mares was similar, blastocysts developing from embryos obtained from aged mares were inferior to blastocysts obtained from young mares in terms of number of cell nuclei, quality score, and diameter at day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliable capacitation of equine spermatozoa has been a major obstacle in the development of equine in vitro fertilization. Experiments were done to compare in vitro capacitation of equine spermatozoa by use of heparin/caffeine, calcium ionophore, uterine tube epithelial cell (UTEC)-conditioned medium, and direct culturing of spermatozoa with UTEC (coculturing). Capacitation-like changes, as determined by chlortetracycline membrane staining patterns, developed with UTEC-conditioned medium and coculturing, equivalent to that with calcium ionophore. Both of these treatments induced more (P < 0.05) capacitation-like changes than did the control, a modified Tyrode's medium. More (P < 0.05) spermatozoa were viable after 24 hours of UTEC coculturing than in the control incubation.

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.

Development of 1- to 2-cell in vivo fertilized equine embryos cultured with or without uterine tubal epithelial cells (UTEC) was studied. One- to 2-cell embryos (n = 26) were collected surgically from the uterine tubes of pony mares 1 day after ovulation. Four- to 8-cell embryos (n = 9) were collected 2 days after ovulation. Presumptive zygotes and 2-cell embryos were cultured with (n = 17) or without (n = 9) UTEC, and all 4- to 8-cell embryos were cocultured with UTEC as positive controls. Uterine tubal epithelial cells were used as cell suspensions within 2 weeks after initiation of cultures. Embryos were cultured to blastocysts or until the embryo had morphologic degeneration. Six presumptive zygotes failed to cleave in vitro. Development to blastocysts of 1-cell (4 of 11) and 2-cell (2 of 6) embryos cocultured with UTEC was similar. Coculture of 1- to 2-cell embryos with UTEC significantly (P = 0.05) improved development to blastocysts, compared with culture in medium alone (35 vs 0%, respectively); however, development to blastocysts of 1- to 2-cell embryos cocultured with UTEC was less (P < 0.025) than that of 4- to 8-cell embryos cocultured with UTEC (35 vs 89%, respectively).

The objective of this study was to determine whether coculture of stallion spermatozoa and mare oviductal (uterine tubal) epithelial cells induced sperm cell capacitation in vitro. Capacitation as determined by zona binding and chlortetracycline staining of the sperm cells was compared for stallion spermatozoa: (1) incubated with medium alone (negative control), (2) treated with calcium ionophore A23187 (positive control) or (3) cultured with mare oviductal epithelial cells (OEC) for 4 h. Chlortetracycline staining patterns of sperm cells bound to the zonae were used to group spermatozoa as uncapacitated, capacitated or acrosome reacted. The zonae and attached spermatozoa were stained for evaluation after initial binding (pulse) and after 1 h of co-incubation (chase). More sperm cells in the ionophore and OEC treatments bound to the zonae at both the pulse and chase than in control medium (P < 0.001). More bound sperm cells were capacitated at the pulse, and acrosome reacted at the chase, for the ionophore and co-culture groups than for the controls (P < 0.001). Staining patterns for sperm cells not bound to the zona pellucida in each of the treatments differed (P < 0.05) from the population of sperm cells that bound to the zona pellucida. There was a higher percentage of capacitated spermatozoa and a lower percentage of acrosome-reacted spermatozoa bound to the zonae at the pulse than were represented in the treatment suspensions of sperm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Our knowledge of the control of preimplantation embryo development and growth is deficient in many aspects as is evidenced by the great difficulty there is in growing embryos of many species in vitro while maintaining viability. This review discusses recent findings on the roles of nutrients, peptide growth factors and co-culture cells in embryo growth and development in vitro.

The bovine oviduct provides the environment for sperm transport and capacitation, oocyte transport and maturation, fertilization and early embryonic cleavage. Gamete interactions in the tube occur in contact with both the tubal epithelium and the oviduct fluid secreted by these cells. Current research continues to reveal the active role of the oviduct and its products play in normal fertilization and embryo development. This paper reviews the anatomy and physiology of the oviduct of the cow, including the specific events of reproduction which occur in this organ.

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.

This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Ham's F12 and Dulbecco's Modified Eagle's Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluorescence to determine the number of nuclei present. Six of seven embryos co-cultured with oviductal examples developed to the morula/blastocyst stage, while four of seven embryos co-cultured with trophoblastic vesicles developed to the morula stage. More (P = 0.1) embryos co-cultured with oviductal explants reached the blastocyst stage than embryos co-cultured with trophoblastic vesicles (3/7 vs 0/7, respectively). The number of cells was higher (P = 0.1) for embryos co-cultured with oviductal explants than for embryos co-cultured with trophoblastic vesicles (162.6 +/- 32 vs 87.3 +/- 28, respectively). The number of cells for embryos co-cultured with either oviductal explants or trophoblastic vesicles appeared to be lower than for embryos matured in vivo that were recovered from the uterus at Day 6 (378, 399, >1000). The co-culture of early equine embryos in a completely defined medium with either trophoblastic vesicles or oviductal explants can support development to at least the morula stage. The co-culture of embryos with oviductal explants resulted in superior development of four-to eight-cell embryos, as indicated by the proportion that reached the blastocyst stage and by the number of cells.

Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture.

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)

A superovulatory and surgical protocol was developed for recovery of bovine zygotes. Holstein cows and heifers were given follicle-stimulating hormone and cloprostenol to induce superovulation. Surgical cannulation and lavage of the uterine tube was performed 40 to 48 hours after the start of standing estrus. In general, cows had more corpora hemorrhagica than did heifers, but a higher percentage (P less than 0.05) of ova recovered from cows were infertile. Several heifers were subjected to the procedure twice, and embryo recovery rates were equivalent both times.

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.