Oviduct Epithelial Cell Monolayers Research Articles

Induction of in vivo-like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells†.

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia, and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 × 106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1month after air-liquid interface introduction, >40% and >20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models.

Inducing ciliation in equine oviduct epithelial cell monolayers

Recently, we developed re-differentiated equine oviduct epithelial cell (EOEC) monolayers in Transwell inserts. EOEC monolayers demonstrated various in vivo-like morphological characteristics, but often lacked secondary cilia. The monolayers were classified as low (<1% secondary cilia) or high (>5% secondary cilia) spontaneously re-differentiated, with the latter in the minority (5-10%). Interestingly, only high re-differentiated monolayers responded to Notch inhibition by developing in vivo-like secondary ciliation rates, i.e. over 50% ciliated cells. In this study, we aimed to consistently generate high re-differentiated monolayers. We first assessed whether secondary cilia formation was affected by donor mare, cycle stage and/or experiment. For this, harvested oviduct explants were cultured for 10 days at a concentration of 10 × 106 EOECs per 9.6 cm² well, to yield a confluent de-differentiated monolayer in conventional wells. Subsequently, the monolayers were trypsinized and the EOECs were reseeded onto the microporous membrane of hanging inserts at a concentration of 0.3 × 105 EOECs per 0.33 cm² insert. Three days after seeding, the EOECs were triggered to re-differentiate by air-liquid interface introduction (ALI). After 1 and 2 months in culture, EOEC monolayers were fixed and their phenotype was assessed by combined immunofluorescence staining to detect cilia (mouse anti-acetylated α-tubulin antibody; Alexa 488-conjugated goat anti-mouse secondary antibody),cell nuclei (Hoechst 33342) and actin filaments (Alexa 568-conjugated phalloidin). Neither cell donor nor cycle stage had any effect on secondary ciliation rates, whereas experimental modifications did. In 3/15 experiments, >95% of the monolayers demonstrated high re-differentiated monolayers. To discover the source of experimental variation, the seeding concentration of the de-differentiated EOECs was varied between 0.1 and 10 × 105 EOECs per 0.33 cm² insert; this did not affect secondary ciliation rates. In a third experiment, the effect of varying the seeding concentration of the harvested oviduct explant EOECs used to establish the de-differentiated monolayers, between 1 and 30 × 106 EOECs per 9.6 cm² well, was evaluated. EOECs harvested from de-differentiated monolayers 10 days after oviduct explant seeding at 1 or 5 × 106 EOECs per well supported the formation of high re-differentiated monolayers in >95% of cases. Within 1 month after ALI, >50% and >30% of the re-differentiated EOECs showed secondary cilia after initial seeding of 1 and 5 × 106 oviduct explant EOECs per well, respectively. Notch-inhibition helped to further increase secondary ciliation rates to >70% in the re-differentiated monolayers. We conclude that initial oviduct explant EOEC seeding concentration is critical to subsequently generating monolayers that closely resemble in vivo oviduct epithelia.

77 Notch-inhibition stimulates secondary ciliation in re-differentiated equine oviduct epithelial cell monolayers

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Improving the quality of ovine embryo produced in vitro by culturing zygote in isolated mouse oviduct

Several studies indicate the absence of oviduct or its assistance may compromise the developmental competence and quality of the embryos produced in vitro. This study was aimed to evaluate the quality of embryos produced in vitro by culturing zygotes in isolated mouse oviduct and oviduct epithelial cell monolayer. The in vitro produced ovine zygotes were cultured for 7 days in the following culture systems i) ampullae of isolated C57BL/6 mouse oviduct (IMO), ii) ovine oviduct epithelial cell monolayer (OCM), and iii) chemically semi-defined medium (CSM) as control. In CSM, embryos were cultured in synthetic oviductal fluid medium supplemented with essential and non-essential amino acids and bovine serum albumin (SOFaaBSA). The cryotolerance of resultant blastocysts and their apoptotic cells (TUNEL assay) were then evaluated. The rate of blastocysts in OCM (37.9 ± 16.7) was lower than in IMO (50.6 ± 25) and CSM (49.7 ± 21.9) groups (P < 0.05). In vitrified/warmed blastocysts ICM:TE ratio in IMO (0.33 ± 0.14) was higher than other groups (0.23 ± 0.16 and 0.22 ± 0.10 in CSM and OCM, respectively). Similarly, cryosurvival of vitrified blastocysts and hatching rates tended to be higher in IMO compared to other groups. In vitrified/warmed blastocysts the proportion of tunnel positive cells of ICM in OCM (31.7 ± 11.5) was higher than CSM and IMO groups (23.7 ± 17.9 and 23.0 ± 11.4). In conclusion, culture of ovine zygote in isolated mouse oviduct could increase the quality of resulting blastocysts.

Steroid hormones regulate sperm-oviduct interactions in the bovine.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17β-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

123 GENISTEIN TRANSPORT ACROSS THE BOVINE OVIDUCT EPITHELIUM

The oviduct plays a vital role in regulating the environment surrounding the gametes and early mammalian embryo. However, the permeability of the oviduct to circulating dietary-derived compounds remains relatively unknown. The present study has investigated the barrier properties of the oviduct epithelium in vitro to the movement of genistein, a soya isoflavone and analogue of 17β-estradiol that naturally occurs in the diet and has been reported to exert teratogenic effects. Bovine oviduct epithelial cells (BOECs) were isolated from abattoir-derived reproductive tracts at the mid-luetal phase. The purity of the cell isolate was confirmed using flow cytometry to determine the proportion of cells that expressed CK18 (epithelia), and Vimentin (fibroblasts). Cells were seeded at a density of 1 × 106 mL–1 on polyethylene terephthalate transwell porous supports (Corning) and maintained between two media-filled chambers for 10 to 12 days, until they formed a polarised confluent monolayer, as confirmed by transepithelial resistance (TEER) greater than 700 Ωcm2. To assess the rate of transport, genistein was added to the basal compartment at physiologically relevant levels (100 µM) and the apical compartment was sampled at regular time points for 120 mins. The concentration of genistein in the apical and basal media was measured by HPLC. Furthermore, we compared the rate of genistein transport at physiological (39°C) and room temperature to indicate whether transport was temperature dependent. Rates of transport are expressed as mean apparent permeability coefficient and were compared between groups by a Student’s t-test. Genistein crossed the bovine oviduct epithelium at a linear rate that was higher than spontaneous diffusion across a blank membrane support (12.7 × 10–3 cm–2 µM–1 v. 7.32 × 10–3 cm–2 µM–1, n = 4; P = 0.0075). The rate of genistein transport by epithelial cells was unchanged when cells were assayed at room temperature (12.7 × 10–3 cm–2 µM–1 v. 13.11 × 10–3 cm–2 µM–1, n = 3; P = 0.76), respectively. No significant difference in the directionality of transport was found. Furthermore, TEER was maintained at approximately 700 Ωcm2, indicating that the cells remained confluent for the duration of the experiment. These data suggest that the bovine oviduct epithelial cell monolayer facilitates genistein movement from the basal to the apical compartment in vitro. Furthermore, the observation that the rate of transport is unchanged by temperature suggests a passive, trans-cellular, physicochemical mechanism, rather than an active biological process. Regardless of the mechanism, the oviduct epithelium is permeable to genistein, and may even facilitate its transport into the lumen, suggesting that gametes and early embryos could be exposed to this compound in vivo. This is relevant given the previously reported finding indicating that this naturally occurring dietary isoflavone has detrimental effects on early development (Newbold et al. 2001 Cancer Res. 61, 4325–4328). Furthermore, the results demonstrate the potential use of this epithelial model in characterising the transport or barrier properties of the oviduct epithelium towards a range of circulating xenobiotics.

Effect of PUFA on embryo cryoresistance, gene expression and AMPKα phosphorylation in IVF-derived bovine embryos

The objectives of the present study were to evaluate the effect of conjugated linoleic acid (CLA t10, c12, C18:2), linolenic acid (C18:3) and docosahexaenoic acid (DHA, C22:6) supplementation on in vitro bovine embryo development, embryo survival after cryopreservation, gene expression and AMPKα phosphorylation. Control groups with modified synthetic oviduct fluid (mSOF) ± 100 μM β-mercaptoethanol (β-ME) were performed. The effects of co-culture with bovine oviduct epithelial cell (Boec) monolayers, serum supplementation and embryo development in the ewe oviduct, on gene expression were also examined. Experiments 1 and 2: a lower d 7 embryo survival was found with 100 μM C22:6 and 100 μM C18:2 supplementation compared to 1 μM C22:6 and 100 μM β-ME supplementation ( P < 0.05). C18:3 supplementation had no effect on d 7 embryo survival, but 100 μM C18:3 increased d 8 embryo survival compared to 100 μM β-ME supplementation ( P < 0.05). Experiments 3 and 4: stearoyl-CoA desaturase 1 (SCD1) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNA decreased after 10 μM C22:6 supplementation compared to all other supplementations ( P < 0.05). A lower fatty acid desaturase 2 (FADS2) transcript level was found with 100 μM C18:2, 10 μM C22:6 and 10 μM C18:3 supplementations compared to groups without fatty acid supplementation ( P < 0.05). Acetyl-CoA-carboxylase (ACC), fatty acid synthase (FAS), adipose differentiation-related protein (ADRP), acyl-CoA synthetase long-chain family member 1 (ACSL1), diacylglycerol O-acyltransferase 1 (DGAT1), carnitin palmitoyltransferase-II (CPT-II) mRNAs expression and AMPKα phosphorylation were not modified with PUFA supplementation. Experiment 5: SCD1 and FAS mRNA decrease in Boec group compared to serum supplementation, as SCD1 mRNA in ewe oviduct group ( P < 0.05). In conclusion, this study showed that a PUFA supplementation with C18:2, C18:3 or C22:6 in bovine culture development for 6 days and co-culture with Boec down-regulate mRNA expression of proteins involved in lipid metabolism in d 7–8 embryo (SCD1 and FADS2 desaturases), probably through SREBP1 mRNA regulation after 10 μM C22:6 supplementation, indicating a modification of saturated/unsaturated fatty acid balance in bovine blastocyst.

P.204 Acute HCV infection in haemodialysis patients infected with the same virus strain varies in CD4 T-cell response and viral kinetics

The objectives of the present study were to evaluate the effect of conjugated linoleic acid (CLA t10, c12, C18:2), linolenic acid (C18:3) and docosahexaenoic acid (DHA, C22:6) supplementation on in vitro bovine embryo development, embryo survival after cryopreservation, gene expression and AMPKα phosphorylation. Control groups with modified synthetic oviduct fluid (mSOF) ± 100 μM β-mercaptoethanol (β-ME) were performed. The effects of co-culture with bovine oviduct epithelial cell (Boec) monolayers, serum supplementation and embryo development in the ewe oviduct, on gene expression were also examined. Experiments 1 and 2: a lower d 7 embryo survival was found with 100 μM C22:6 and 100 μM C18:2 supplementation compared to 1 μM C22:6 and 100 μM β-ME supplementation (P < 0.05). C18:3 supplementation had no effect on d 7 embryo survival, but 100 μM C18:3 increased d 8 embryo survival compared to 100 μM β-ME supplementation (P < 0.05). Experiments 3 and 4: stearoyl-CoA desaturase 1 (SCD1) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNA decreased after 10 μM C22:6 supplementation compared to all other supplementations (P < 0.05). A lower fatty acid desaturase 2 (FADS2) transcript level was found with 100 μM C18:2, 10 μM C22:6 and 10 μM C18:3 supplementations compared to groups without fatty acid supplementation (P < 0.05). Acetyl-CoA-carboxylase (ACC), fatty acid synthase (FAS), adipose differentiation-related protein (ADRP), acyl-CoA synthetase long-chain family member 1 (ACSL1), diacylglycerol O-acyltransferase 1 (DGAT1), carnitin palmitoyltransferase-II (CPT-II) mRNAs expression and AMPKα phosphorylation were not modified with PUFA supplementation. Experiment 5: SCD1 and FAS mRNA decrease in Boec group compared to serum supplementation, as SCD1 mRNA in ewe oviduct group (P < 0.05). In conclusion, this study showed that a PUFA supplementation with C18:2, C18:3 or C22:6 in bovine culture development for 6 days and co-culture with Boec down-regulate mRNA expression of proteins involved in lipid metabolism in d 7–8 embryo (SCD1 and FADS2 desaturases), probably through SREBP1 mRNA regulation after 10 μM C22:6 supplementation, indicating a modification of saturated/unsaturated fatty acid balance in bovine blastocyst.

Gametes Alter the Oviductal Secretory Proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.

Successful use of oviduct epithelial cell coculture for in vitro production of viable red deer ( Cervus elaphus) embryos

Techniques for in vitro production (IVP) of viable embryos have been thoroughly developed in several domestic species in view to improve breeding efficiency. When applied to wild life, these techniques may also help the maintenance of biodiversity through amplification of sparse animals offspring and facilitation of genetic material exchange. During the successive steps of IVP, i.e. oocyte in vitro maturation (IVM), fertilization (IVF) and early embryo development (IVD) to the blastocyst stage, gametes and embryos are faced with unusual environment, including oxidative stress, known to be detrimental to their survival. In the present study, starting from methods developed in domestic species, we have adapted IVP to produce viable red deer embryos. In a first experiment, cumulus cells were removed from in vitro matured oocytes either before or after IVF. The presence of cumulus cells during IVF did not affect final cleavage or development rates. In a second experiment, in vitro matured oocytes were fertilized in the presence of cumulus cells and cultured in SOFaaBSA medium alone or in the presence of ovine oviduct epithelial cell (oOEC) monolayer. Whereas, oviduct cells did not improve the cleavage rate, they significantly increased the rate of embryos reaching the blastocyst stage (from 3 to 25% of total oocytes). Ten blastocysts from oOEC coculture were transferred after freezing and thawing to five recipient hinds and gave rise to three pregnancies. The three pregnant hinds gave birth to three live and normal calves.

Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa

The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6 h after culture with or without (Control) oviductal cells (Experiment 1) and 3 h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 ± 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6 h had a higher density than those pre-incubated for 3 h ( P < 0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes ( P < 0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.

In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.

Development of in vitro embryo production systems for red deer ( Cervus elaphus) : Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4–12 h on COEM or for 4 h in TALP medium supplemented with 20 μg of heparin. COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm ( P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24 h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4 h and no motile sperm were observed by 8 h. Overall, the penetration rates of in vitro red deer oocytes were low (5–28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin ( P<0.001). Penetration rates were similar for 4–12 h pre-incubation of sperm on COEM ( P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24 h compared to 12 h ( P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24 h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization

This study was designed to determine the effect of co-culture with porcine oviductal epithelial cell (POEC) monolayers on in vitro fertilization of pig oocytes. The in vitro penetrability of mature (experiment 1) or immature (experiment 2) oocytes was studied in presence or absence of POEC during IVF with fresh semen. In experiment 3, boar and POEC effects were analyzed but in this case with frozen-thawed spermatozoa. In experiment 4, the spermatozoa were pre-incubated before IVF with or without POEC in order to assess their effect on IVF sperm-related parameters. In experiment 5, the effect of POEC was studied by co-culturing them with oocytes before IVF to determine if monospermy was improved. The results showed that high sperm concentration and POEC increase oocyte penetrability ( P<0.01) and decrease monospermy rate ( P<0.01), in both mature and immature oocytes ( P<0.01) with fresh semen and a 18 h culture time. With frozen semen was detected a boar and POEC effect ( P<0.01) on penetration rate. The sperm pre-culture 2 h with POEC also resulted in an increase of sperm penetration in terms of number of sperm per oocyte ( P<0.01) and this treatment did not increase monospermy when contact time between gametes was limited to 6 h although monospermy was higher when POEC were present during IVF. Finally, exposure of oocytes to POEC for 4 h before IVF facilitated monospermic penetration to over 70% ( P<0.01). In conclusion, the use of POEC in porcine IVF systems provides the possibility of working with low sperm concentrations and the effect of POEC on monospermy depends on sperm concentration, boar and contact time between gametes. Moreover, the exposure of oocytes to POEC before IVF improves the rate of monospermy.

Heparin and Ca 2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers

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 Ca 2+-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 × 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 Ca 2+-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 Ca 2+-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 Ca 2+-free medium caused the opposite effect at 0.5 h after addition of treatments. We conclude that both heparin and Ca 2+-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.

Glucose metabolism of in vitro-produced bovine embryos in cell-free and co-culture systems

In vitro-produced (IVP) bovine embryos are used in a wide range of biotechnologies but develop less well than their in vivo counterparts and can give rise to foetal/neonatal anomalies after embryo transfer. The quality of bovine IVP embryos and the systems in which they are produced are traditionally assessed in terms of morphological and developmental criteria; namely, embryo grade and blastocyst formation rate. Lane and Gardner (1996) showed that mouse embryos selected for transfer on the basis of a low glycolytic activity (conversion of glucose to lactate), measured non-invasively, were 4 times more likely to implant than those selected randomly. Comparable data are not available for bovine embryos. The aim of this study was to assess linear glycolytic index of cattle blastocysts in vitro as a marker of viability. We have measured glucose consumption and lactate production by individual bovine IVP embryos grown in cell-free conditions and in a novel co-culture system (Orsi et al., 2000) involving confluent bovine oviduct epithelial cell monolayers on permeable supports. This preparation allows the epithelial cells to be fed by a nutritionally-rich medium via the physiological, basal, route, while the apical medium, containing the embryos, is more dilute, mimicking oviduct fluid.

Sperm binding and penetration of the zona pellucida in vitro but not sperm–egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula)

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.

The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro.

In order to investigate the interaction of fresh and frozen-thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen-thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen-thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen-thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen-thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen-thawed spermatozoa. The findings reported in this study show that fresh and frozen-thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

Induction of thumbtack sperm during coculture with oviduct epithelial cell monolayers in a marsupial, the brushtail possum (Trichosurus vulpecula).

A reorientation of the sperm head so that it is perpendicular to the sperm tail (i.e., T-shape or thumbtack) is considered an indicator of sperm capacitation in the Australian marsupial the brushtail possum (Trichosurus vulpecula). This study describes a method of oviduct epithelial cell monolayer and sperm coculture in the brushtail possum to obtain a high percentage of thumbtack sperm. The oviduct epithelial cell (OEC) monolayers were prepared in vitro from the isthmal and ampullary segments of eCG- and LH-primed brushtail possum oviducts. Coculture experiments demonstrated that cauda epididymidal sperm from the brushtail possum attached equally to the OEC monolayers derived from the isthmal and ampullary segments of the oviduct. After 2 h of coculture, a large number of sperm attached to OEC monolayers (ampulla, 60.1+/-4.7% and isthmus, 63.1+/-5.7%) as well as to controls (tracheal epithelial cell monolayer, 46.2+/-3.7%; Matrigel, 57.4+/-7.7%; plastic, 29.2+/-3.2%). After 6 h, fewer sperm were attached to tracheal epithelial cell monolayers (1.2+/-0.2%; P<0.01) and Matrigel (10.2+/-2.5%; P<0.01), compared to those attached to ampullary and isthmal OEC monolayers (37.9+/-7.2% and 44.6+/-2.2%, respectively), and none were attached to the plastic surface. Fewer sperm were released from the ampullary and isthmal OEC monolayers compared to those from controls (P<0.05). At 6 h of coculture with ampullary and isthmal OEC, the percentage motility of both attached and unattached spermatozoa was maintained at 40-50%, which was higher (P<0.05) than in controls. Progressive motility of unattached sperm was maintained at about 2 (on an arbitrary scale of 1-5) and was not different among treatments until 6 h. More than 60-70% sperm were viable at 6 h of coculture in all the treatments. Coculture of brushtail possum epididymal sperm with OEC monolayers transformed 60% of motile streamlined spermatozoa to thumbtack orientation at 2 h compared to approximately 25% in controls. No acrosomal modifications were induced in spermatozoa in any of the treatments. This study has demonstrated a role of the oviduct in transforming a large number of sperm from a streamlined to thumbtack orientation, which may have relevance in sperm capacitation and fertilization in this species.