Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro.

The composition of fluids within the bovine oviduct and uterine lumen, important in fertilisation and early embryonic development, is ultimately determined by the transport properties of the epithelial cells which line the lumen. A preparation has therefore been devised to study the role of these cells in oviduct and uterine fluid formation. Pure preparations of epithelial cells, as judged immunocytochemically, were isolated by enzyme digestion, and grown on collagen filters in primary culture. The cells re-establish intercellular junctions to form a confluent epithelial layer. Serial samples from the apical and basal media were analysed for K+, Na+, Ca2+, glucose and lactate. Bovine oviduct epithelial cells maintained gradients of K+ and Ca2+ (apical > basal) for up to 14 days after confluence, while bovine uterine epithelial cells maintained apical > basal gradients of K+. Both types of epithelium exhibited a small transepithelial electrical potential difference and a higher uptake of glucose and production of lactate in the basal, as opposed to apical medium. There were no consistent differences in any of these parameters with the stage of the oestrous cycle at which the cells were removed. The data indicate that bovine oviduct and uterine epithelia may be isolated and grown as polarised layers in primary culture. The preparations will now enable the mechanisms underlying the secretion of ions and non-electrolytes to be determined.

Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos.

The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0-6 of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l-1, 10 mmol D,L-lactate l-1 and 1.5 mmol glucose l-1 and also 3 mg BSA ml-1 and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0-4 of culture (0.24-0.27 nl per embryo h-1), but increased with the initiation of compaction (0.39 nl per embryo h-1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h-1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake at day 0 of culture (putative zygotes), increasing to 70% by day 2 (eight-cell stage) and 100% of glucose uptake from day 4 of culture onwards. ATP production followed a similar pattern to that of oxygen consumption (60-85 pmol per embryo h-1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h-1) and blastulation (221 pmol per embryo h-1). For precompaction stages, 93-96% of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.

Intracellular calcium in cultured rabbit oviduct epithelial cells.

Oviduct fluid is the medium in which fertilization and early embryonic development occur but little is known about the ionic basis of fluid secretion or its control. Since calcium ions (Ca2+) are involved in the mechanism of secretion in other epithelia, the intracellular calcium concentration ([Ca2+]i) was measured in single, rabbit oviduct epithelial cells in primary culture using the fluorescent dye Fura-2. The resting [Ca2+]i was constant (115 nM) in cells cultured for 2-7 days. Ion substitution experiments demonstrated the presence of a Na+/Ca(2+)-exchange system in the plasma membrane, whereas influx through channels was found to have only a minor role maintaining the resting [Ca2+]i. The addition of dibutyryl cAMP (db cAMP) induced two types of response: the first was an increase in [Ca2+]i, dependent on the presence of extracellular Ca2+, and the second was a zero response. Extracellular ATP induced a transient increase in [Ca2+]i owing to the release of Ca2+ from intracellular stores and Ca2+ entering the cell across the plasma membrane. It is proposed that these effects may be due to the presence of two types of cell in culture-the ciliated and non-ciliated (secretory type) oviduct epithelial cells.

Effect of purinergic stimulation on intracellular calcium concentration and transepithelial potential difference in cultured bovine oviduct cells.

Epithelial cells were removed from bovine oviducts by enzyme digestion and either cultured on laminin-coated coverslips (for determination of [Ca2+]i) or on collagen filters (for determination of transepithelial potential difference [pd]). Cells on coverslips were loaded with Fura-2 to monitor [Ca2+]i. Application of extracellular ATP induced a transient increase in [Ca2+]i in a dose-dependent manner. This response was abolished by thapsigargin, indicating that the rise in [Ca2+]i was derived from intracellular stores. The order of potency of the nucleotide-induced rise in [Ca2+]i was uridine triphosphate (UTP)>ATP>ADP. Epithelial cells were grown on collagen filters, and when mounted in a modified Ussing chamber exhibited an electrical pd of 1.00 +/- 0.36 mV with the apical side negative with respect to the basal. Application of UTP, ATP, and ADP to the basal side induced transient increases in pd of 1.15 +/- 0.21, 0.77 +/- 0.16, and 0.26 +/- 0.06 mV, respectively. The order of potency of the nucleotides in eliciting transient increases in [Ca2+]i and pd suggests the presence of a P2u purinergic receptor in the bovine oviduct epithelium that could play a role in transepithelial ion movements and hence the control of oviductal fluid formation.