In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells.

A reliable procedure was developed to harvest purified populations of bovine uterine epithelial cells from the luminal endometrium that allowed cells to be maintained in vitro for multiple subpassages. This method allowed adequate numbers of cells for in vitro culture studies to evaluate uterine secretion rates, uterine-embryo interactions and a new embryo co-culture system. In a second study, cells were isolated from the uterus and oviducts collected from cattle on day of estrus, on d 4 to 6, d 8 to 10 and d 14 to 16 of the cycle to monitor growth and development following incubation in vitro. The highest percent cell viability and attachment during primary culture was noted for cells isolated on the day of estrus or between d 4 to 6 for both uterine and oviductal cell populations. The effects of different culture media and incubation temperatures on growth and proliferation of uterine and oviduct epithelial cells in vitro were evaluated. Cells incubated in CMRL and MB2 had the highest growth rates, however, there were no differences in proliferation rates among media evaluated. There were no differences in growth and proliferation rates between different incubation temperatures (37°C vs 39°C). Morphological observations of cell integrity and quality were made using image analysis during primary culture and following the first and third subpassages. Optical density readings were influenced by cell subpassage and quality. Cell quality following in vitro culture could be effectively monitored using image analysis. Bovine oocytes were matured and fertilized in vitro in different culture media. Culture medium did not influence fertilization or cleavage rates. Early embryo development was enhanced following incubation in CMRL and TCM media. Embryo viability percent improved when morulae were maintained on oviductal cells compared with those cultured in medium alone. Bovine embryos derived from in W/ro-fertilization procedures were co-cultured with oviductal cells isolated between cycle d 4 to 6 or 14 to 16 and proteins secreted by oviductal cells monitored. Cycle stage oviductal cells were isolated did not influence developmental rates or proteins secreted. However, co-culture with oviduct epithelial cells were superior to culture in medium alone. IN T R O D U C T IO N The use of embryo transfer methodologies in research has evolved from simple embryo collection and transfer to recipient females in early years to the more technical and complex embryo biotechnologies. These new embryo biotechnology procedures encompass a wide range of laboratory techniques such as cryopreservation, micro­ manipulation, in vitro fertilization (TVF), gene insertion and embryonic cloning. The development of IVF procedures for hum animals in the late 1980’s (48,94) has provided increased numbers of laboratory-derived embryos available for research purposes. The access to large numbers of early-stage embryos has increased research efforts in the areas of embryo bisection methodology (63), DNA insertion (69) and nuclear transfer techniques (120,130,163) for farm animals. For many of these new embryo biotechnologies to become successful, embryos will have to be maintained and develop in vitro prior to recipient transfer. In addition, the chances of successful pregnancies increase in farm animals if embryos develop to the morulae or blastocyst-stages prior to their transfer. In most instances this requires maintenance of early-stage embryos in vitro for up to 7 days. However, the specific requirements for in vitro development of early-stage embryos of domestic animals are still not fully understood (see review by 167). In 1965, Cole and Paul (32) first reported that a high percentage of mouse embryos developed and hatched from their zona pellucida when cultured in vitro with a feeder layer of irradiated HeLa cells. This initial study provided a basis for embryo co-culture methodology currently in use today. The development of embryo co-culture systems using various types of "helper cells" has provided more efficient means of maintaining embryos in vitro for extended periods with minimal reductions in viability (see review by 124). In addition, embryo 2 co-culture systems may offer the unique advantage of improving pregnancy rates of marginal-quality embryos prior to recipient transfer. The development of embryo co-culture systems has resulted in the emergence of three different types of co-culture systems (trophoblastic vesicles, cellular monolayers and chick embryo co-culture). In virtually all instances, co-culture systems have improved embryo development over that of incubating similar embryos in culture medium alone. The present hypotheses explaining the beneficial effects of embryos co-culture systems are that monolayers remove embryotoxic substances from the medium surrounding the developing embryo, secrete embryotropic factor(s) to enhance embryo development and/or a combination of both (see review by 8,124). Although the beneficial effects of co-culture systems have been demonstrated by many different laboratories in recent years, there are still many biochemical components and interactions of cell monolayers that are not fully understood. Developmental capacity of individual embryos in vitro can be enhanced with co­ culture, however, most scientists agree that optimum success rates have likely not been achieved. The variability of success rates using similar co-culture systems across studies and among different laboratories can be attributed to the cells of the monolayer or the environmental conditions that the cells are maintained during incubation. It would seem logical that conditions altering monolayer cell growth or characteristics while maintained in vitro may ultimately affect embryotropic factor(s) produced during co-culture. This review will discuss current in vitro co-culture systems used for mammalian embryos and in vitro culture studies conducted on bovine epithelial monolayers of uterine and oviductal origin. L IT E R A T U R E R E V IE W In Vitro Co-Culture of Mammalian Embryos Trophoblastic Vesicle Co-Culture. In the mid-1980’s French scientists (27,72) developed a unique embryo co-culture system using trophoblastic tissue segments of d 12 to d 14 elongating bovine concentuses. The trophectoderm of elongating cattle and sheep blastocyst were sectioned and incubated in vitro. These trophoblastic segments subsequently formed spherical vesicles (trophoblastic vesicles), and were suggested to secrete luteotropic and embryo development promoting factors (72). Camous et al. (27) investigated the ability of oneto eight-cell bovine embryos to overcome the eightto 16-cell block stage in vitro by co-culturing with d 13 or d 14 bovine trophoblastic vesicles. In this study, 46% of the oneto eight-cell embryos developed to the morula stage when cultured with trophoblastic vesicles for 3 to 4 days in Menezo’s B2 medium (MB2). In contrast, <20% of similar stage embryos reached the morula-stage when embryos were cultured in the control MB2 medium alone. In a later study, Heyman et al. (73) co-cultured oneto eight-cell bovine embryos with d 14 bovine trophoblastic vesicles and 46% reached the morula stage compared with only 18% reaching the morula stage in control medium. In a second experiment, 55 one-cell embryos were co-cultured with trophoblastic vesicles and 44 % cleaved beyond the eight-cell stage compared with only 13% of the one-cell embryos cultured in medium alone. In addition, when one-cell ovine embryos were co-cultured with d 12 sheep trophoblastic vesicles 75% of the embryos reached the morula stage compared with 35% of the embryos cultured in control medium alone. Camous et al. (27) and Heyman and Menezo (71) have suggested that tropho­ blastic vesicles may provide important metabolic component(s) (e.g. lipids), that are