Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro.

The relationship between the oviduct and embryo development in the mouse was investigated and the period at which the influence of oviduct can be concerned in the development of mouse embryos in vitro was identified. In addition, the relative molecular weight of oviductal factors that promote embryo development was demonstrated. Mouse zygotes developed to the blastocyst stage when co-cultured with ampulla. The period of embryo co-culture significantly affected the further development of the embryos. Fewer one-cell embryos co-cultured with dissected ampullae for less than 24 h developed to blastocysts than those co-cultured for more than 28 h (P < 0.001). A high percentage of embryos co-cultured with ampullae after 24 h of culture in vitro developed to the blastocyst stage, which suggests that the influences of ampulla on the development of mouse embryos are restricted to a specific period at the two-cell stage (about 55-56 h after hCG injection) in vitro. Mouse ova that were cultured in media conditioned by ampullae could also develop to the blastocyst stage. The fractionated medium that contained low molecular weight fractions was more effective (P < 0.001) on the development of embryos to the blastocyst stage than that containing high molecular weight fractions. These results suggest that the low molecular weight oviductal factors play an important role in the development of mouse embryos at a certain critical age in vitro.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

In vitro development of day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles

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 exmplants 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.     

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P = 0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P /= 0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P = 0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P < 0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P = 0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P >/= 0.535).     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Nonspecies-specific effects of mouse oviducts on the development of bovine IVM/IVF embryos by a serum free co-culture

In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.     

Oviductal and uterine influence on the development of Day-2 equine embryos in vivo and in vitro

The objective of this experiment was to contrast the influence of the oviductal and uterine environments on development of Day-2 embryos. Embryos were transferred to oviducts or uteri of synchronous recipient mares, or were incubated in oviductal co-culture, in uterine co-culture or in defined culture medium. Significantly more (P < 0.02) embryos transferred to the oviduct versus the uterus survived until Day 11 after ovulation (5 7 vs 0 7 , respectively). Significantly more (P < 0.001) embryos developed to expanded and hatched blastocysts in uterine co-culture than in culture medium (6 7 vs 0 7 , respectively). The rate of embryo development to expanded blastocysts was not significantly different (P > 0.1) in oviductal co-culture versus uterine co-culture (3 7 vs 6 7 , respectively), or in oviductal co-culture versus culture in medium (3 7 vs 0 7 , respectively). Three of 7 and 6 of 7 embryos developed to hatched blastocysts greater than 2000 mum in diameter during oviductal and uterine co-culture, respectively, while 0 of 7 embryos cultured in medium expanded to greater than 500 mum in diameter. Proportions of embryos that developed for at least 9 days.     

Aberrant fetal growth and development after in vitro culture of sheep zygotes.

The effects of in vitro culture systems for sheep zygotes on subsequent fetal growth and development to day 61 and day 125 of gestation were studied. Zygotes recovered from superovulated Scottish Blackface ewes approximately 36 h after intrauterine insemination using semen from a single Suffolk sire were cultured for 5 days in (a) a granulosa cell co-culture system (co-culture); (b) synthetic oviductal fluid medium without serum (SOF-); and (c) synthetic oviductal fluid medium supplemented with human serum (SOF+). Control embryos were recovered from superovulated donor ewes at day 6 after oestrus. Embryos were transferred at day 6 to synchronous Scottish Blackface recipient ewes. In total, 146 gravid uteri were recovered, comprising 97 at day 61 (20 co-culture, 27 SOF-, 25 SOF+ and 25 control) and 49 at day 125 (13 co-culture, 8 SOF-, 6 SOF+ and 22 control) of gestation. Fetuses derived from co-cultured embryos were 14% heavier (P < 0.01) by day 61 of gestation than those derived from control embryos. Growth coefficients derived from the linear allometric equation logey = logea + b logex (where y = organ mass; x = fetal mass) were significantly greater (P < 0.05) for liver, heart, kidneys and plantaris muscle in fetuses derived from co-cultured embryos, and for liver in fetuses derived from SOF+ embryos than those for control fetuses. Fetuses derived from co-cultured embryos were 34% heavier (P < 0.001) and fetuses derived from SOF+ embryos were 18% heavier (P < 0.01) by day 125 of gestation than those derived from control embryos. Growth coefficients for liver and heart for fetuses derived from co-culture and SOF+ embryos were also significantly greater (P < 0.05) at this stage of gestation than those for control group fetuses. In contrast, allometric coefficients for these organs in fetuses derived from embryos cultured in SOF without serum supplementation were not different from those for controls. Excessive volumes of amniotic fluid (polyhydramnios) were observed in 23% of conceptuses derived from co-cultured embryos. In vitro embryo culture can significantly influence fetal growth and this study provides quantitative evidence of major shifts in the patterns of organ and tissue development.     

In vitro development of DNA-injected embryos co-cultured with goat oviduct epithelial cells in Korean native goats (Capra hircus aegagrus)

In vitro development of Korean native goat embryos was investigated in 2 different culture systems with and without goat oviduct epithelial cells (GOEC). Estrus was synchronized by inserting intravaginal progestagen-impnegnated sponge (Veramix) containing 60 mg medroxyprogesterone acetate (MAP) for 14 d. Superovulation was induced with follicle stimulating hormone (FSH). Goat ova were surgically obtained by retrograde flushing the oviducts of does at 66 to 68 h after MAP removal. Mean number of recovered ova per doe was 7.28 +/- 3.91, and the proportion of fertilized embryos in recovered ova was 66.5% (121/182 ). Fertilized embryos were cultured for 9 d in CR1aa medium supplemented with 10% estrous goat serum (EGS) at 38.5 degrees C, 5% CO(2) in air. There was no difference in development of the embryos to the morula stage between the 2 culture systems (84.4 and 84.0%, respectively). However, developmental rate to blastocysts (65.6%) of the embryos co-cultured with GOEC was significantly higher than of those (12.0%) cultured without GOEC (P < 0.001). Goat zygotes were injected with bovine beta-casein/human lactoferrin cDNA fusion gene (pBL1). When the DNA-injected embryos were co-cultured with GOEC, developmental rates of the embryos to the morula and blastocyst stages were 82.9 and 36.6%, respectively. The results obtained in this study indicate that "blocking" of in vitro development of Korean native goat embryos appears to occur at the morula stage, but can be overcome to some extent by co-culture with GOEC. In the co-culture system, DNA-injected goat embryos could successfully develop to normal hatching blastocysts.     

Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.     

共培养体系在牛核移植胚体外发育培养中的应用

采用电融合法构建牛体细胞核移植重构胚,分析共培养细胞类型、传代次数、细胞冻-融以及蛋白质添加物(BFF和FBS)对牛体细胞核移植胚体外发育的影响,探讨胚胎体外共培养的条件,以建立优化的共培养体系。结果表明与非共培养组相比,共培养组重构胚的囊胚发育率以及胚胎细胞数显著增加(P<0.05),而输卵管上皮细胞共培养组同颗粒细胞共培养组相比胚胎细胞数显著增加(P<0.05),更适合做共培养细胞;随着共培养细胞传代次数的增加重构胚囊胚发育率及胚胎细胞数显著下降(P<0.05),共培养细胞在冷冻处理后重构胚的囊胚率和胚胎细胞数都显著下降(P<0.05);BFF较FBS更能促进牛核移植胚的囊胚发育率(P<0.05)。表明应用新鲜原代输卵管上皮细胞进行牛核移植胚胎的共培养,并在SOFaa添加10?F能够有效促进核移植胚胎的体外发育。     

Comparison of conditioned medium and direct co-culture of human granulosa cells on mouse embryo development

Although numerous investigations have demonstrated the beneficial effects of co-culture system of different somatic cells on in vitro development of embryos, the effects of conditioned-media of co-culture cells have not been well documented. The objective of this study was to compare the effects of human granulosa cells co-culture system and its conditioned medium on the developmental rate of mouse embryos in vitro. Two sets of experiments were undertaken: in the first one 317 mouse one-cell embryos were cultured in human granulosa cell co-culture system (GC). Ham's F10 medium conditioned with granulosa cells (CM) and non-conditioned Ham's F10 for 120 h. In the second experiment. 391 late two-cell embryos were cultured in the 3 fore-mentioned culture treatments for 72 h. Embryos were obtained from NMRI mice. Granulosa cells were collected from patients undergoing an IVF program during oocyte pickup. In the first set of experiments, 23.6, 14.5 and 11.1% of one-cell embryos passed two-cell block and continued growing to 4-cell in GC, CM and HF, respectively. This index in GC was significantly different from two other treatments. Also significantly more embryos reached blastocyst stage in GC compared with two other treatments. The blastocyst rate was not significantly different between CM and HF. In the second set of experiments the proportion of blastocyst stage was significantly higher in CM than that in HF and lower than that in GC. In conclusion, although human granulosa cell-conditioned medium has beneficial effects on mouse embryo development, it was not as effective as co-culture of these cells.     

Improved development of DNA-injected bovine embryos co-cultured with mouse embryonic fibroblast cells

The in vitro development of DNA-injected bovine zygotes, produced in vitro, was compared when cultured with or without mouse embryonic fibroblasts (MEF). The in vivo viability of the embryos produced in these in vitro culture systems was assessed by single or double transfer to recipients taken to term. For these experiments, in vitro fertilized oocytes were not injected (Experiment 1) or were injected with pBL1 gene (Experiment 2) and then cultured for 2 days in CR1aa medium supplemented with 3 mg/ml BSA at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air. Embryos that developed to the 4- to 8-cell stage at the end of this period were randomly assigned to the two cultured systems and cultured for a further 5 days in groups of 10 to 15 embryos in 0.75 ml medium. These two culture systems were CR1aa medium alone or co-culture with MEF in CR1aa medium supplemented with 10% fetal bovine serum (FBS). Every 48 h, 0.5 ml of the medium was replaced with fresh CR1aa medium and at Day 5 of culture, both media were supplemented by the addition of 5.56 mM glucose and 1x GMS-X supplement solutions. Results were assessed as morphological development of the embryos and data were analyzed by Chi-square test or Student's t-test.The development rate of in vitro fertilization (IVF)-derived embryos co-cultured with MEF (24.4%, 49/201) was significantly higher than those cultured alone (14.4%, 28/194; P<0.05) in Experiment 1. There was a similar difference between the treatments in the proportions of embryos which reached the hatching stage or hatched (10.9%, 22/201 vs. 4.1%, 8/194, respectively; P<0.05). DNA-injected embryos co-cultured with MEF (13.7%, 28/205) showed a higher developmental rate than that of the embryos cultured without MEF (6.7%, 13/193; P<0.05) in Experiment 2. Following the transfer to recipients of one or two DNA-injected blastocysts, the pregnancy rates for two culture systems were similar (MEF co-culture 27.4%, 23/84; CR1aa culture 24. 2%, 16/66). However, the numbers of calves born alive from these pregnancies were higher on the MEF co-culture group (82.6%, 19/23) than the CR1aa culture group (56.2%, 9/16). It was concluded that in vitro embryo development to the blastocyst stage and subsequent in vivo development to term of DNA-injected bovine embryos was improved in comparison to culture in CR1aa alone when the last 5 days of in vitro culture were in a MEF co-culture system.     

Culture of in vitro fertilized bovine embryos with bovine oviductal epithelial cells, Buffalo rat liver (BRL) cells, or BRL-cell-conditioned medium

Co-culture with various cell types can enhance development of bovine embryos, especially through the transition from maternal to embryonic mRNA utilization, a stage of growth refractory to most in vitro methods. Bovine oviductal epithelial (BOE) cells have been particularly successful for culturing embryos through the refractory stage; however, Buffalo rat liver (BRL) cells are a readily available, long-lived, easy-to-care-for alternative. This study compared the embryotrophic activity of BOE to BRL cells with particular emphasis on the transition stage of growth. A total of 7158 immature bovine oocytes, matured and fertilized in vitro, were divided into 4 different culture treatments: Treatment 1: BRL conditioned medium for 72 h then BRL co-culture; Treatment 2: BRL co-culture; Treatment 3: BOE co-culture for 72 h in 5% oxygen then BRL co-culture; and Treatment 4: BOE co-culture for 72 h in 5% oxygen followed by BOE co-culture in air. Those same treatments were used to evaluate embryotrophic differences of early (4 to 5) versus late (14 to 15) passage BRL cells maintained in M-199 medium with 10% serum. Two bulls were also evaluated to determine if there exists a bull-by-culture system interaction. Treatment 3 resulted in the best development after 9 d; 9.1% of selected immature oocytes developed to expanded blastocyst. Early passage BRL cells were significantly more embryotrophic than later passage cells; this was most pronounced for Treatment 2. There was a treatment-by-bull interaction, which should be considered when comparing results among similar studies.     

In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products.

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.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1 x 10(5) or 1 x 10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1 x 10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1 x 10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1 x 10(5) and 1 x 10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

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)     

Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)