Safety issues in assisted reproduction technology: Should men undergoing ICSI be screened for chromosome abnormalities in their sperm?

The incidence of aneuploidy in gametes of men undergoing ICSI has raised the prospect of there being risks associated with ICSI and the question of whether or not to screen men for sperm aneuploidy before treatment. We report results of a questionnaire undertaken to address how IVF staff perceive this problem, whether ICSI men are already being screened for sperm aneuploidy and the extent to which IVF specialists feel that there is merit in such a test. The results suggest that this is seen as a problem but most feel the risks outweigh the benefits. Most claimed their clinics do not screen sperm for aneuploidy but feel that there is merit in doing so. There are considerable benefits to screening i.e. couples would get additional information about the genetic repercussions of ICSI and could make informed decisions before treatment; screening would also facilitate the design of a large research study to give clearer answers on the safety of ICSI. However, we acknowledge counter arguments i.e. families would not necessarily benefit as most would have the ICSI procedure regardless of screen results; sex chromosome trisomies clinically are not severe enough to worry about in this context and there are other potential risks of ICSI that screening would not address.

Effects of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine on sperm function in vitro.

This in-vitro study was designed to investigate the effects of commonly prescribed antibiotics on sperm movement characteristics, viability and the ability of spermatozoa to undergo the acrosome reaction. Spermatozoa were obtained by swim-up from normozoospermic semen and cultured for 24 h with increasing concentrations of co-trimoxazole, erythromycin, amoxycillin, tetracycline and chloroquine. Tetracycline at concentrations as low as 2.5 microg/ml led to a significant dose-dependent inhibition in percent rapid-moving spermatozoa, mean path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL), but at 50 microg/ml tetracycline all spermatozoa were static. Erythromycin had significant effects on rapid movement, VAP, VSL and VCL only at concentrations >100 microg/ml. In contrast, percent rapid-moving spermatozoa was significantly enhanced at low concentrations of chloroquine (5 microg/ml), but significantly inhibited by higher concentrations. Co-trimoxazole did not adversely affect percent rapid-moving spermatozoa below 500 microg/ml, at which concentration movement was decreased by 34%. The mean lateral head displacement (ALH) was significantly enhanced by 5 microg/ml co-trimoxazole and reduced at 1 mg/ml erythromycin. The effects of these drugs were mostly irreversible. Amoxycillin had no effect on sperm movement characteristics over the dose range used, though it inhibited viability at high doses. Viability was significantly reduced at concentrations of all drugs which affect rapid sperm movement; these concentrations of drugs did not appear to affect the ability of spermatozoa to undergo the acrosome reaction. The results from this study, when combined with known effects on spermatogenesis, should facilitate the choice of drugs for the treatment of both genitourinary and unrelated infections in men who are attempting to conceive.

A bridge to intracytoplasmic sperm injection--high insemination concentrations benefit patients who have a reduced chance of fertilization with standard in-vitro fertilization.

This study was carried out to determine whether high insemination concentrations (HIC) could improve fertilization and pregnancy rates in patients who had either previously demonstrated poor fertilization rates in vitro using standard protocols (Group 1) or in whom a reduced chance of fertilization was indicated at semen assessment prior to in-vitro fertilization (IVF) (Groups 2 and 3). Forty nine patients were recruited for the study. Standard IVF was carried out in 1 ml volumes using 10(5) spermatozoa/ml. HIC treatment involved co-culture of spermatozoa and oocytes in microdroplets with insemination concentrations increased 10-50 fold higher than standard IVF. Fertilization and pregnancy rates were compared between standard IVF and HIC in individual patients either in consecutive cycles (Group 1) or using sibling oocytes in the same cycle (Group 2). Group 3 patients were treated with HIC for their first treatment cycle. HIC significantly improved the fertilization rate compared with standard IVF for Groups 1 (59.7 +/- 10.7 versus 19.6 +/- 5.4% respectively) and 2 (54.9 +/- 8.5 versus 34.0 +/- 8.5% respectively). HIC increased the pregnancy rate from 0% with standard IVF to 20% per embryo transfer in Group 1 patients. A single pregnancy derived from the transfer of HIC and IVF embryos occurred in Group 2. The fertilization rate (47.2 +/- 7.6%) and pregnancy rate (31.3% per embryo transfer) for Group 3 patients was higher than predicted. There was no increase in the rate of polyploidy with HIC. Provided there are sufficient numbers of motile spermatozoa, HIC may be offered as an initial form of treatment, thus permitting referral of only the poorest responders for intracytoplasmic sperm injection (ICSI).

Serum is more effective than albumin in promoting human embryo development and implantation.

OBJECTIVE: To compare the effects of serum with those of Albuminar-5 (Armour Pharmaceutical Co., Eastbourne, Sussex, United Kingdom) as medium supplements to Earl's balanced salt solution (EBSS) for IVF and subsequent embryo development. DESIGN: A retrospective study. Gametes and embryos from 318 patients were cultured in the presence of serum (group 1). Gametes and embryos from 130 patients were cultured in the presence of Albuminar-5 (group 2). Embryos obtained from IVF were replaced into the uterus within 48 hours after insemination. Surplus bipronucleate embryos were cultured up to 14 days with either serum or Albuminar-5. SETTING: Two tertiary referral fertility clinics; university teaching hospital. PATIENTS: Four hundred forty-eight patients with a wide spectrum of causes of subfertility, ranging in age from 24 to 43 years. MAIN OUTCOME MEASURES: Fertilization rate, pregnancy rate (PR), implantation rate, and surplus embryo development in vitro. RESULTS: The PR for group 1 patients was higher than that of group 2 (27.0% versus 15.4%, respectively). Although fertilization rates were identical in the two groups, cumulative embryo scores and implantation rates were significantly higher in group 1. There was no difference between the groups in age distribution, types of ovarian stimulation, numbers of patients with day 1 or day 2 transfers, or luteal phase support. Of 31 embryos cultured with serum, 54.8% reached the fully expanded blastocyst stage and 25.8% hatched. Of 19 embryos cultured with Albuminar-5, only 5.3% reached the fully expanded blastocyst stage and none hatched. CONCLUSIONS: The results suggest that, under certain conditions, serum supplementation yields better results than protein supplementation alone. The latter may be suitable only in conjunction with additional components.

Calcium and meiotic maturation of the mammalian oocyte.

The role of calcium in the regulation of both the meiotic and mitotic cell cycles has been the subject of considerable investigation in the nonmammalian field. In contrast, the mechanisms for signalling meiotic maturation in the mammalian oocyte are not as well documented nor as clearly defined. In the mammalian oocyte, calcium is associated with both spontaneous and hormone-induced meiotic maturation. A transient release of endogenously stored calcium precedes germinal vesicle breakdown and can override cyclic AMP maintained meiotic arrest; it thus may signal the resumption of meiosis. Additionally, extracellular calcium is apparently required for meiotic progression past metaphase I. The time sequence for meiotic resumption and progression is very varied between species. The timing of cell cycle protein synthesis during meiosis suggests that cyclins may be expressed in oocytes of some species much earlier in their development than in others. A generic model is proposed for the mechanism for triggering meiotic resumption in the mammalian oocyte. In this model, the critical components of meiotic resumption involve the temporal relationship of cyclin synthesis and the subsequent activation of the MPF complex by the calcium signal generated, which accounts for differences among species.

A cytosolic sperm factor triggers calcium oscillations and membrane hyperpolarizations in human oocytes.

Intracellular free Ca2+ concentrations ([Ca2+]i) and membrane potentials were measured in mature human oocytes. Injection of cytosolic extracts made from human or hamster spermatozoa triggered oscillations in [Ca2+]i in human oocytes similar to those described previously in mouse and hamster oocytes. In contrast, injection of carrier buffer caused no [Ca2+]i increase and injection of Ca(2+)-containing solutions caused only a single [Ca2+]i transient. Injection of human sperm extracts also triggered [Ca2+]i oscillations in mature mouse oocytes. The [Ca2+]i oscillations in human oocytes were accompanied by hyperpolarizations in membrane potential. Perfusing oocytes with the sulphydryl reagent thimerosal also caused oscillations in the free [Ca2+]i concentration simultaneously with membrane potential hyperpolarizations. These data suggest that human oocytes possess a similar mechanism for generating [Ca2+]i oscillations to those described in other mammalian oocytes and a membrane potential response similar to that seen previously specifically in hamster oocytes. The data also support the view that human oocytes are activated at fertilization by diffusion of a protein from the spermatozoa into the ooplasm after gamete membrane fusion.

Lack of evidence for the production of interferon-alpha-like species by the cultured human pre-embryo.

The aim of this study was to determine if the human pre-embryo produces a substance similar to the trophoblast interferon secreted by ruminant trophoblasts. Human embryos surplus to in-vitro fertilization (IVF) treatment were cultured up to 14 days following IVF. Viable cultures were determined by microscopic examination and by assay of the culture medium for human chorionic gonadotrophin (HCG). Four stages of development were visually identified: pre-blastocyst, unhatched, part-hatched and fully hatched blastocyst. HCG was detected in medium which had contained the more developmentally advanced embryos. A total of 62 samples were assayed for human interferon-alpha (IFN-alpha), including all cultures presumed viable. None contained detectable IFN-alpha immunoreactivity. Out of 14 candidate samples subjected to cytopathic effect reduction assay, none contained antiviral activity. We suggest that a trophoblast-derived interferon, unlike HCG, does not play a significant role in the maternal recognition of pregnancy in humans.

The role of calcium in mammalian oocyte maturation and egg activation.

The maturation of the immature oocyte and the fertilization of a mature egg are two absolute prerequisites for mammalian embryo development. There is increasing evidence in mammals that both oocyte maturation and egg activation at fertilization are controlled by changes in intracellular free Ca2+ levels. The role of Ca2+ changes at fertilization is clear in that they are both required and sufficient for egg activation. However, it is not established how the sperm causes Ca2+ changes in eggs at fertilization, nor how different patterns of Ca2+ change affect embryo development. The role of Ca2+ in triggering oocyte maturation is less clear, although preventing intracellular Ca2+ changes can inhibit meiotic maturation at specific stages. Studies on how Ca2+ regulates meiosis and fertilization in mammals may provide new insights into the causes of failed fertilization in human IVF procedures.

Defining a role for calcium in the resumption and progression of meiosis in the pig oocyte.

The role of calcium (Ca++) during spontaneous meiotic maturation of pig oocytes was examined. The hypothesis that elevations of endogenously derived intracellular Ca++ are prerequisite for germinal vesicle breakdown (GVBD) and progression through meiosis was tested. In addition, investigations were carried out to determine whether GVBD and meiotic progression were dependent upon extracellular Ca++ influx. Elevation of endogenously derived Ca++ was inhibited directly by loading cells with BAPTA, a specific Ca++-chelator, or indirectly with neomycin. Extracellular Ca++ influx was prevented by culturing oocytes in Ca(++)-deficient medium, with EGTA, or in the presence of the Ca++ channel blocker verapamil. Pretreatment with BAPTA/AM and subsequent culture in the absence of added exogenous Ca++ resulted in a similar inhibition of GVBD (1 microM BAPTA/AM vs. untreated, P < 0.01). After 4 h following follicular release, oocytes were no longer sensitive to BAPTA/AM treatment. Neomycin also significantly inhibited GVBD (0.5 mM neomycin vs. untreated, P < 0.05) as well as meiotic progression past metaphase I (0.25 mM neomycin vs. untreated, P < 0.05). The incidence of GVBD was not significantly affected when oocytes were simply cultured in Ca++ deficient medium or when cultured in the presence of EGTA or verapamil. However, progression of meiosis past GVBD to metaphase II was suppressed by reducing levels of Ca++ in the culture medium (0.68 mM Ca++ vs. 1.7 mM Ca++, P < 0.05) and by treatment with verapamil (0.25 mM verapamil vs. untreated, P < 0.05). Meiotic progression was completely blocked at metaphase I in the presence of 0.85 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomal analysis of meiotic stages of human oocytes matured in vitro: benefits of protease treatment before fixation.

OBJECTIVE: To modify Tarkowski's air-dry technique for mouse oocytes to develop a rapid, consistent procedure for human oocytes that enables accurate scoring of meiotic stage. DESIGN, SETTING, AND PATIENTS: Meiotically immature human oocytes, obtained after oophorectomy, were cultured for various periods and then subjected to Tarkowski's air-dry procedure (n = 104) or to our modified procedure (n = 175) that used a brief exposure to protease (20 to 40 seconds) before fixation. MAIN OUTCOME MEASURES: Air-dried oocytes were assessed for readability and for whether they contained overspread or overlapping chromosomes. In addition, discrete meiotic stages in human oocytes were identified. RESULTS: Our protease procedure significantly increased readability of air-dried oocytes (96% versus 79% readable for protease versus Tarkowski, respectively; P less than 0.001) by significantly reducing the number of preparations with either overscattered (0.7% versus 3.4% for protease versus Tarkowski, respectively, P less than 0.05) or overlapping (1.3% versus 18% for protease versus Tarkowski, respectively, P less than 0.001) chromosomes. CONCLUSIONS: Protease exposure of oocytes, combined with a modification of Tarkowski's procedure, resulted in high quality air-dries of human oocytes. This rapid and reliable procedure should have clinical application in in vitro fertilization programs for meiotic assessment of oocytes failing to fertilize.

Changes in linoleic acid during follicular development and inhibition of spontaneous breakdown of germinal vesicles in cumulus-free bovine oocytes.

The fatty-acid composition of follicular fluid from small and large developing follicles was analysed and the effects of saturated and unsaturated fatty acids on spontaneous breakdown of germinal vesicles were investigated. Fatty acids were bound to bovine serum albumin and cultured with oocytes at 100 mumol/l. Linoleic acid (18:2) was the only fatty acid tested that significantly inhibited breakdown of germinal vesicles (P less than 0.01). The effect was dose-dependent and was greatest at 50 mumol fatty acid/l (% breakdown of control, 81.1 +/- 6.8 vs. 50 mumol linoleic acid/l, 35.4 +/- 7.3; P less than 0.02). Linoleic acid was the major fatty acid, constituting about a third of the total fatty acid in the follicular fluid; followed by 18.9 +/- 1.0% and 16.9 +/- 1.3% oleic acid (18:1) in small and large follicles, respectively. Saturated fatty acids accounted for less than 30% of the total fatty acid composition. There was a marked absence of tetraenoic acids in small and large follicles. Proportions of linoleic acid were significantly lower in follicular fluid from large follicles (31.1 +/- 1.2% of total fatty acid) than from small follicles (34.8 +/- 0.7% of total fatty acid) (P less than 0.05) and there was a significant inverse correlation between follicle diameter and percentage of linoleic acid in the follicular fluid (r = -0.6966; P less than 0.05). There was no significant alteration in any other fatty acid during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: effects of a cyclic AMP phosphodiesterase inhibitor.

The turnover of [32P]orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in [32P]phosphatidic acid (PA) and an increase in [32P]-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in [32P]PC and [32P]phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in [32P]PC and [32P]PE which accompanied GVBD. The increase in [32P]phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid. The third is associated with GVBD, and is cAMP-sensitive, and may represent stimulation of de novo synthesis of phospholipid, thereby permitting disruption of the nuclear membrane.

Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation.

The possibility that the intracellular signals generated upon phosphoinositide hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTA/AM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1) Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1 nM PMA has a stimulatory effect while 1 microM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Verapamil inhibits phosphatidic acid formation and modifies phosphoinositide metabolism in stimulated platelets.

Rabbit platelet-rich plasma was incubated with [32P]orthophosphate, after which the platelets were washed, further incubated in the absence or presence of verapamil and subsequently stimulated with PAF-acether or thrombin. In the absence of verapamil, a rapid increase in radioactivity in phosphatidic acid was observed in platelets stimulated with PAF-acether or thrombin. This was inhibited by verapamil over the concentration range 10(-7) to 10(-4) M, at which concentration the rise in phosphatidic acid was completely abolished. In unstimulated platelets, 10(-4) M verapamil induced an increase in radioactivity in polyphosphoinositides but not significantly in phosphatidylinositol. When these verapamil-treated platelets were stimulated with PAF-acether or thrombin, there was a rapid, sustained loss of the additional radioactivity induced in the polyphosphoinositides by verapamil. Polyphosphoinositide radioactivity remained unchanged in platelets stimulated in the absence of verapamil. Verapamil may stimulate formation of a separate pool of polyphosphoinositide which is susceptible to agonist-induced phospholipase C, and failure to re-synthesize this polyphosphoinositide could result from inhibition of phosphatidic acid synthesis.

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium.

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated subpopulations of mass-harvested pig oocytes and their characterization by size, incidence of atresia and competence to mature in culture.

Pig oocytes were hand collected after follicular puncture or were mass harvested with stainless-steel screens and purified on sucrose density gradients. Most oocytes obtained by mass harvesting with a 75 micron screen and selecting on a 0 and 30% discontinuous sucrose gradient were meiotically incompetent and atretic after culture. Two enriched populations of oocytes were isolated from the 0-15% and 15-30% interfaces with a 106 micron screen and a 0%, 15% and 30% 2-step discontinuous sucrose gradient. These oocyte populations differed significantly in size (P less than 0.001, t test) and in meiotic competence. More than 50% of the larger oocytes matured compared with 14% of the smaller oocytes (P less than 0.002). Of the larger oocytes, 21% progressed beyond meiotic metaphase I, whereas all of the maturing small oocytes were arrested at first metaphase after culture (P less than 0.002). Yields were 45 times greater for oocytes mass harvested with a 106 microns screen and a 15% sucrose density gradient than for oocytes hand collected in the same period of time. Layering these mass-harvested oocytes over a second 15% sucrose gradient or hand sorting the mass-harvested oocytes after culture increased the proportion of maturing oocytes compared with oocytes which were mass harvested before culture and produced higher proportions of oocytes at telophase I or beyond (P less than 0.002). The results demonstrate that mass harvesting of pig oocytes is more efficient than hand selection, and that oocytes collected and selected in this way are at least as capable of meiotic maturation as are oocytes hand selected before culture.

Lipid analysis of immature pig oocytes.

The detailed analysis of the lipid composition of immature pig oocytes represents the first such study carried out on mammalian eggs. In order to undertake a large scale lipid analysis using conventional extraction and chromatographic techniques a procedure for mass harvesting relatively large numbers of pig oocytes (200-300 oocytes/ovary) was developed. The study revealed that triacylglycerol was the major lipid component (100.71 nmol/mg protein) followed by cholesterol (32.71 nmol/mg protein). Phosphatidylcholine constituted the major phospholipid component (27.83 nmol/mg protein). Pig oocytes contained relatively low proportions of phosphatidylethanolamine (16.41% total phospholipid) and relatively high proportions of lysophosphatidylcholine (4.68% total phospholipid). The free fatty acid pattern was strikingly similar to the fatty acid composition of phosphatidylcholine. This observation, in conjunction with the observed high levels of lysophosphatidylcholine and the low ratio of phosphatidylethanolamine to phosphatidylcholine, suggests a fast rate of phospholipid turnover in the immature pig oocyte. Analysis of fatty acids esterified to the individual phospholipids and neutral lipids has shown that in all the classes examined, particularly in the neutral lipid fractions, there are high levels of the saturated fatty acid palmitic acid (16:0) and the monounsaturated fatty acid oleic acid (18:1). Triacylglycerol, free fatty acids and most of the phospholipids, particularly phosphatidylethanolamine, are considerably enriched in n-6 polyunsaturated fatty acids, specifically linoleic (18:2), arachidonic (20:4) and adrenic (22:4) acids. This may indicate an ability of oocytes to synthesize prostaglandins and leukotrienes. The results show that the lipid environment of the immature pig oocyte may be adapted to the highly specialized requirements of the cell, promoting growth and development with a potential role in the regulation of maturation.

Unsaturated fatty acids stimulate the formation of lipoxygenase and cyclooxygenase products in rat spleen lymphocytes.

Rat spleen lymphocytes prelabelled with [14C] arachidonate were suspended in fresh medium in the presence or absence of exogenous non-radioactive fatty acids added in ethanol. It was found that fatty acids stimulate thromboxane B2, PGE2, HHT and HETE formation. The effect is specific for unsaturated fatty acids. It occurs at 50 micron concentrations and is apparent after 20 minutes incubation. Unsaturated fatty acids may serve an important role in the regulation of prostaglandin and hydroxy fatty acid metabolism in vivo. This may indicate a mechanism for the action of fatty acids on the immune response.

Formation of diacylglycerol and degradation of phosphatidylinositol induced in rat lymphocytes by non-esterified oleic or linoleic acid.

Rat spleen lymphocytes were incubated for 3 h with [14C]arachidonic acid in foetal calf serum. It was found that arachidonic acid distributed into phospholipids in the order phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol. After labelling with arachidonic acid the lymphocytes were washed, and incubated for up to 2 h with non-radioactive palmitic, oleic or linoleic acid dissolved in ethanol. The presence of ethanol or palmitic acid during a 2 h post-incubation had little effect on the amount of radioactivity found in different lipid fractions. Both oleic acid and linoleic acid, however, brought about an accumulation (up to 8-fold) of radioactivity in the diacylglycerol fraction. These fatty acids also brought about a change of radioactivity in several phospholipids, notably in phosphatidylinositol, which lost more than 50% of its counts during the 2 h incubation. Although maximum effects were seen at 2 h, diacylglycerol radioactivity was increased by 100% within 5 min after adding the fatty acids. The minimum concentration of fatty acids used (50 microM) gave an almost maximum response. The results indicate that unsaturated fatty acids may activate phosphatidylinositol phosphodiesterase in lymphocytes, as they do in brain. The possibility that a phospholipase A is activated is discussed. Possible implications for any experiments in which cells are incubated with fatty acids are pointed out.