Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment.

STUDY QUESTION: Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro? SUMMARY ANSWER: Oocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM. WHAT IS KNOWN ALREADY: Amino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gamete's capacity to undergo fertilization and early cleavage divisions in vitro. STUDY DESIGN, SIZE, DURATION: The study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE: The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and valine depletion or appearance differed in MII versus degenerating oocytes (n = 20) (P < 0.05). Glutamine, arginine, methionine, phenylalanine, total depletion and total turnover all differed in oocytes from patients aged < 35 years versus patients ≥35 years (P < 0.05). MII oocytes obtained following ovarian stimulation with recombinant FSH depleted more isoleucine (P < 0.05) and more alanine and lysine (P < 0.05) appeared than oocytes from hMG-stimulated cycles. MII oocytes from patients with a polycystic ovary (PCO) morphology (n = 33) depleted more serine (P < 0.05) than oocytes from women with normal ovaries (n = 61). LIMITATIONS, REASONS FOR CAUTION: Immature oocytes collected at the time of ICSI were used as the model for human oocyte maturation. These oocytes have therefore failed to respond to the ovulatory hCG trigger in vivo (they are meiotically incompetent), and have limited capacity to support embryo development in vitro. The lack of cumulus cells and stress of the conditions in vitro may have influenced turnover of amino acids, and owing to the small sample sizes further studies are required to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings provide support for the hypothesis that oocyte metabolism reflects oocyte quality. Longitudinal studies are required to link these functional metabolic indices of human oocyte quality with embryo developmental competence. Oocyte amino acid profiling may be a useful tool to quantify the impact of new assisted reproduction technologies (ART) on oocyte quality. STUDY FUNDING/COMPETING INTERESTS: This project was funded by the UK Biology and Biotechnology Research Council (BB/C007395/1) and the Medical Research Council (G 0800250). K.E.H was in receipt of a British Fertility Society/Merck Serono studentship. H.J.L. is a shareholder in Novocellus Ltd, a company which seeks to devise a non-invasive biochemical test of embryo health.

Oocyte developmental failure in response to elevated nonesterified fatty acid concentrations: mechanistic insights.

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid ß-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid ß-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability.

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close-to-physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro-produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited sex-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing "proof of principle" for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births.

Effects of changes in the concentration of systemic progesterone on ions, amino acids and energy substrates in cattle oviduct and uterine fluid and blood.

Early embryo loss is a major factor affecting the conception rate in cattle. Up to 40% of cattle embryos die within 3 weeks of fertilisation while they are nutritionally dependent on oviduct and uterine fluids for their survival. Inadequate systemic progesterone is one of the factors contributing to this loss. We have characterised the effects of changes in systemic progesterone on amino acid, ion and energy substrate composition of oviduct and uterine fluids on Days 3 and 6, respectively, of the oestrus cycle in cattle. Oviduct and uterine fluids were collected in situ following infusion of progesterone. There was no effect of progesterone on oviduct fluid secretion rate; however, uterine fluid secretion rate was lowered. Progesterone increased uterine glucose, decreased oviduct sulfate and, to a lesser degree, oviduct sodium, but had no effect on any of the ions in the uterus. The most marked effect of progesterone was on oviducal amino acid concentrations, with a twofold increase in glycine, whereas in the uterus only valine was increased. These results provide novel information on the maternal environment of the early cattle embryo and provide further evidence of progesterone regulation of oviduct amino acid concentrations in cattle.

Energy metabolism of the equine cumulus oocyte complex during in vitro maturation.

Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate and kinetics of glucose consumption and glycolysis throughout IVM. Approximately 95% of glucose consumed was accounted for by lactate production; however, high concurrent oxygen consumption indicated a comparatively increased role for non-glycolytic oxidative phosphorylation. Up to 38% of equine COC oxygen consumption could be attributed to non-mitochondrial activities and there was a significant loss of spare respiratory capacity over the course of IVM. Notably, our data also revealed that current IVM protocols may be failing to satisfy the metabolic demands of the equine COC. Our findings constitute the first report on mitochondrial efficiency in the equine COC and provide new insight into comparative gamete biology as well as metabolism of the COC during in vitro maturation.

Role of fatty acids in energy provision during oocyte maturation and early embryo development.

While much is known about the metabolism of exogenous nutrients such as glucose, lactate, pyruvate, amino acids by oocytes and pre-implantation mammalian embryos, the role of endogenous stores, particularly lipid, has been largely overlooked. The presence of lipid within oocytes and early embryos has been long known, and comparisons between species indicate that the amounts and types of lipid present vary considerably. Large amounts of intracellular lipid can compromise the success of cryopreservation and the removal of such lipid has been the subject of considerable effort. In this review, we present evidence that strongly suggests a metabolic role for lipid, specifically with regard to energy provision, in the late-stage oocyte and the pre-implantation embryo. We focus initially on oxygen consumption as a global indicator of metabolic activity, before reviewing different approaches that either have been designed to investigate directly, or have revealed indirectly the role of endogenous lipid in energy generation. These fall under five headings: (i) fatty acid oxidation; (ii) inhibition of triglyceride oxidation; (iii) culture in the absence of exogenous substrates; (iv) cytoplasmic organization; and (v) delipidation. On the basis of the data derived from these studies, we conclude that there is strong evidence for the utilization of endogenous lipid as an energy substrate by oocytes and early embryos.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Energy substrates in bovine oviduct and uterine fluid and blood plasma during the oestrous cycle.

Up to 40 percent of cattle embryos die within 3 weeks of fertilization but there is little or no published information on the composition of the oviduct and uterine fluids essential for their survival during this time. We have measured the concentrations of the energy substrates, glucose, lactate, and pyruvate in cattle oviduct fluid on Days 0, 2, 4, and 6 and uterine fluid on Days 6, 8, and 14 of the oestrous cycle and corresponding blood samples. Oviduct and uterine fluids were collected in situ. Glucose concentrations in oviduct and uterine fluids were similar on all days and lower than in plasma (P < 0.05). Oviduct lactate concentration was up to eightfold higher than uterine or plasma concentration (P < 0.01). Oviduct pyruvate concentrations were similar on all days and lower than plasma concentrations on Days 0 and 2 (P < 0.005). Pyruvate concentrations were similar in the uterus and in plasma except on Day 14 when the concentration in plasma was higher (P < 0.05). There were no associations between systemic progesterone or oestradiol and glucose, lactate or pyruvate. There was a linear positive relationship (P < 0.001) between oviduct fluid secretion rate and oviduct glucose concentration and a linear negative relationship (P < 0.001) between oviduct fluid secretion rate and oviduct lactate, but no association between uterine fluid secretion rate and energy substrates. The different concentrations and associations between the energy substrates in oviduct and uterine fluids and blood plasma indicate a differential regulation of the secretion of these energy substrates by the oviduct and uterine epithelium.

Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.

Amino acid metabolism of the porcine blastocyst.

The pattern of depletion and appearance of a mixture of amino acids by single porcine blastocysts incubated in two different media has been determined non-invasively using high performance liquid chromatography. Zygotes were produced by the in vitro fertilisation of in vitro-matured, abattoir-derived immature oocytes and cultured in medium NCSU 23 with or without amino acids. Embryos grown in the absence of amino acids up to the blastocyst stage were transferred to amino acid-containing culture medium for measurement of turnover (Experiment 1). Blastocysts grown in NCSU 23+amino acids were transferred into fresh droplets of the same medium (Experiment 2). Although the specific pattern of amino acid production and depletion varied between experiments, a general pattern emerged, with arginine being significantly depleted (p<0.001) and alanine consistently appearing in the media, in quantities that varied depending with culture conditions. The data suggest that arginine is important during porcine blastocyst development, most likely contributing to the formation of nitric oxide and polyamines and that alanine is produced as a means of disposing of excess amino groups. A model for the interactions of amino acids during porcine early embryo development is proposed. The profile of amino acid metabolism by porcine blastocysts is qualitatively and quantitatively similar to that given by human embryos during the morula:blastocyst transition suggesting that the porcine blastocyst is a good model for the human.

Culture of human preimplantation embryos to the blastocyst stage: a comparison of 3 media.

Following culture for 2 days in Earle's balanced salt solution (EBSS), human embryos which remained after transfer were cultured in one of 3 media for 4 days, from the 2- to 4-cell stage to the blastocyst stage. Sibling embryos were divided equally between treatments. Throughout the 4 day culture period, embryos were assessed for morphology and development, as well as uptake and production of energy substrates. Cell numbers in the inner cell mass and trophectoderm were determined for embryos which reached the blastocyst stage. No significant effect was observed in the extent or rate of development to the 8-cell, morula or blastocyst stage between treatments. Uptake of pyruvate was related to concentration in the medium and no differences in glucose uptake were observed between media. Endogenous energy metabolism, as measured by lactate production, was significantly higher in Ham's F12 than in EBSS from day 3.5 onwards. Blastocyst cell numbers were also increased; 79.6+/-7.7 in Ham's F12 (n=17) and 57.8+/-5.2 in EBSS (n=19), p<0.05. Of the embryos which reached the blastocyst stage by day 5, 36% (14/25) had degenerated by day 6 in EBSS compared to only 19% (5/27) in Ham's F12 (p=0.06). Slightly higher rates of embryo survival between day 5 and 6 in Ham's F12 may account for the observed increase in blastocyst cell number. The results do not suggest that improved embryo development can be obtained using human tubal fluid or Ham's F12, in preference to EBSS during early cleavage stages, but the use of Ham's F12 may improve embryo survival at later stages of development.

The effects of phenformin on the transport and metabolism of sugars by the rat small intestine.

The effects of 0.25-10 mM phenformin on sugar transport and metabolism have been studied in a preparation for the combined perfusion of the vascular bed and the lumen. At all concentrations the effects of vascular phenformin were more pronounced than those of luminal phenformin. Phenformin inhibited galactose transport across the intestine, the pattern of inhibition depending on whether the phenformin was added to the luminal or vascular compartments. The active accumulation of galactose in the mucosal epithelial cells was also abolished. There was a linear relationship between the percentage reduction in mucosal ATP levels and vascular phenformin concentration. Phenformin reduced the rate of glucose uptake from the lumen, and the proportion of this glucose which reached the vascular effluent. Most of the glucose which did not reach the vascular side could be accounted for by the formation of lactic acid. Vascular phenformin increased glucose uptake from the vascular medium by ca 88%, 97% of which could be accounted for by lactate formation. Phenformin was sequestered by the mucosa when added to the vascular, but not the luminal, perfusates. There was very little translocation of intact phenformin across the gut in either the mucosal or serosal directions. It is suggested that the effects of phenformin on the gut mainly derive from an inhibition of mitochondrial oxidative phosphorylation, with a small contribution from a direct effect on the brush border, more pronounced at high phenformin concentrations. The results are consistent with the idea that phenformin delays sugar absorption in man, and that the intestine may be a significant source of lactate production in lactic acidosis.

Application of a marker of ciliated epithelial cells to gynaecological pathology.

BACKGROUND: The assessment of neoplastic disease in gynaecological histopathology can be complicated by the high incidence of metaplasia seen in tissues of the female genital tract. There is a need to identify specific tissue markers which can be applied in routine histopathological practice. AIM: To examine the clinical potential of a monoclonal antibody, LhS28, which reacts with basal bodies of ciliated epithelial cells. METHODS: A panel of normal and pathological gynaecological tissues was processed and labelled with LhS28. RESULTS: LhS28 immunoreactivity was found in the normal Fallopian tube where it was confined to ciliated rather than secretory epithelial cells. In the remaining specimens, LhS28 was associated exclusively with ciliated cells in tubal metaplasias of the cervix and endometrium and in benign serous lined inclusion cysts. CONCLUSIONS: LhS28 may be a valuable marker for identifying metaplasia of tubal type and may find application in distinguishing tubal metaplasia from low grade cervical glandular intraepithelial neoplasia.

Glucose-free medium in human in vitro fertilization and embryo transfer: a large-scale, prospective, randomized clinical trial.

OBJECTIVE: To determine whether excluding glucose from the culture medium used in a clinical IVF program improves human embryo quality and pregnancy rates. DESIGN: Randomized controlled trial. SETTING: Clinical assisted conception laboratory in a large teaching hospital. PATIENT(S): Seven hundred forty-one patients undergoing IVF-ET. INTERVENTION(S): Embryos were cultured from the pronucleate stage to ET in medium with glucose for patients in the control group and without glucose for patients in the trial group. MAIN OUTCOME MEASURE(S): Comparison of embryo quality and pregnancy rates between the two groups. RESULT(S): Embryo quality was enhanced with the use of glucose-free medium but pregnancy rates were similar. CONCLUSION(S): Although pregnancy rates remained similar in the two groups, a reduction in the glucose concentration of the medium used for embryo culture from the pronucleate stage to ET on day 2 or 3 is prudent.

Sugar absorption by the mouse small intestine following infection with Schistosoma mansoni.

The effect of acute schistosomiasis on the structure and function of the mouse small intestine has been examined. Whole loops of small intestine from normal mice, and mice seven weeks post-infection were incubated in an in vitro perfusion apparatus. The transport of glucose, 3-0-methylglucose, sorbitol and fluid were all markedly impaired in loops from the infected animals. Kinetic analysis of the data for glucose transport indicated that schistosome infection brought about a reduction in the total number, but not the affinity, of the absorptive sites for glucose. Under the scanning electron microscope, villi from normal mice were tall and erect, with intact surfaces devoid of mucus, whereas the villi from the infected animals were partially covered with strands of mucus and appeared swollen and eroded, with lesions around their apices.

Role of developmental factors in the switch from pyruvate to glucose as the major exogenous energy substrate in the preimplantation mouse embryo.

Preimplantation mouse embryos, cultured in vitro and those freshly flushed from the reproductive tract, exhibit a switch in energy substrate preference, from pyruvate during the early preimplantation stages, to glucose at the blastocyst stage. Although the biochemical basis of this phenomenon is quite well characterized, its timing and possible association with developmental factors have not been considered. We have therefore examined the role of five developmental factors in determining the timing of the switch, namely: (1) embryo age (in hours post hCG); (2) developmental stage; (3) cytokinesis; (4) cell number; and (5) activation of the embryonic genome. One-cell embryos, which develop more slowly than 2-cell embryos in vitro, were used to investigate the role of embryo age and developmental stage. Cytochalasin D, which inhibits cytokinesis and delays the timing of compaction and cavitation, was used to investigate the role of cell division and developmental stage. Finally, transcription of the embryonic genome was examined with the inhibitor, alpha-amanitin. Pyruvate and glucose consumption by single embryos were measured using a noninvasive ultramicrofluorometric technique. The results showed that the timing of the switch in energy substrate preference is precisely regulated in the mouse preimplantation embryo. Activation of the embryonic genome is a prerequisite for the switch and its timing is closely associated with developmental stage, specifically compaction and/or cavitation. Cell number, cytokinesis and embryo age appeared to be unrelated to the timing of the switch. These conclusions may well be extrapolated to other species, since an increase in net glucose uptake, if not always at the expense of pyruvate, is a feature of preimplantation embryo metabolism in all mammals studied.

Consumption of amino acids by bovine preimplantation embryos.

Bovine embryos produced in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus, were cultured in a physiological mixture of amino acids. Depletion of amino acids from the medium and, in a few cases, their appearance, was measured by high performance liquid chromatography. Amino acids were depleted at widely differing rates. The depletion of amino acids was higher when embryos at later developmental stages were cultured, implying an increase in amino acid requirement with development. Threonine was the only amino acid to be depleted at all stages of development; depletion increased from 0.18 +/- 0.07 pmol embryo-1 h-1 at the putative zygote stage to 1.96 +/- 0.49 pmol embryo-1 h-1 at the blastocyst stage. Glutamine was depleted at the putative zygote stage and the 4-cell stage (0.76 +/- 0.05 and 0.94 +/- 0.10 pmol embryo-1 h-1 respectively), but was not significantly depleted at the later stages. Alanine was the only amino acid that appeared consistently in the medium and its production increased progressively throughout development. Aspartate, glutamate, threonine and lysine were depleted significantly by blastocysts derived both in vitro and in vivo; the embryos in vivo also depleted arginine, phenylalanine, isoleucine and tyrosine. These results indicate that individual amino acids are depleted at different rates by bovine preimplantation embryos and suggest that amino acid requirements change during development.

Effects of insulin-like growth factors I and II on tumour-necrosis-factor-alpha-induced apoptosis in early murine embryos.

The proposition that members of the insulin-like growth factor superfamily act as rescue factors from apoptosis in murine preimplantation embryos was tested. The cytokine tumour necrosis factor alpha (TNFalpha) was used to induce apoptosis. Zygotes were cultured for 5 days to the blastocyst stage in the presence or absence of TNFalpha and in the presence or absence of the insulin-like growth factors, IGF-I or IGF-II. Tumour necrosis factor alpha significantly increased the percentage of apoptotic cells and reduced the total cell count in Day 5 blastocysts. When IGF-I or IGF-II were added to the culture medium in the presence of TNFalpha, the cell number and apoptotic dead cell index (DCI) were restored to control values. Insulin-like growth factor-I alone had a greater effect on total cell number than IGF-II alone, but did not significantly decrease the apoptotic DCI. In contrast, IGF-II significantly reduced the number of apoptotic cells. This study shows that IGFs may play a role as apoptotic survival factors in the early mouse embryo.

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.

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.