Biomedical subjects

R G Wales

Publications and source records attributed to R G Wales.

At least 37 records · Page 2Linked to original sources

PubMed1989

Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism.

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.

Animals↗

PubMed1989

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes.

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30-90 min after insemination (10(5) pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.

Acrosome↗

PubMed1989

Development of sheep preimplantation embryos in media supplemented with glucose and acetate.

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF)+sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.

Journal Article↗

PubMed1989

Morphology and chemical analysis of the sheep conceptus from the 13th to the 19th day of pregnancy.

Between the 13th and 19th day of pregnancy the sheep conceptus developed into a structure showing considerable differentiation and all the extraembryonic membranes were established. Both length and dried weight of the embryo increased exponentially during this period. A highly significant regression of dried weight on length of embryos was found but measurement of the additional variable, width, did not improve the accuracy of estimating weight from the embryo's dimensions. The mass of the extraembryonic membranes also increased greatly. The dried weight of the trophoblast increased 90-fold over this period; that of the yolk sac increased 17-fold from day 15 to day 19. The protein content of each of the structures making up the sheep conceptus approached 50% of dried weight, which is similar to the proportion in adult soft tissues. The contribution of glycogen to dried weight was low in the sheep embryo and embryonic membranes when compared with estimates in the mouse blastocyst. However, at about the time of implantation the level of this polymer in the embryo was high compared with that in adult soft tissues and approached the level found in adult muscle. Concentrations of DNA and RNA in the sheep conceptus are much higher than the levels in most adult soft tissues and probably reflect higher synthetic rates and a smaller cytoplasmic volume in the embryonic cells.

Allantois↗

PubMed1989

Effects of macromolecules recovered from uterine luminal fluid on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

Day-4 mouse embryos grew well in culture media supplemented with macromolecular components of uterine fluids recovered on day 3, 4 or 5 of pregnancy and pseudopregnancy. Addition of these components to media during a 2-h pulse culture had no significant effect on the incorporation of glucose carbon by morulae/early blastocysts. However, various fractions of uterine luminal macro-molecules significantly increased the turnover of glucose carbon incorporated into acid-soluble and acid-insoluble glycogen, into nucleic acids and into proteins during a 24-h chase culture. These effects were due mainly to components with a molecular weight between 1000 and 10,000 Da and the activity was most marked in fluids collected on day 5 of pregnancy or pseudopregnancy. Oxidation of glucose during a 4-h incubation was inhibited in the presence of certain uterine macromolecules but most consistently by the large molecular weight component (greater than 300,000 Da). Some differences were noted in the inhibitory activity of macromolecules obtained from pregnant and pseudopregnant sources. There was little evidence of an effect of uterine-fluid components on lactate production from glucose.

Animals↗

PubMed1989

Effects of oxygen concentration on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The utilization of the acid-soluble glycogen pool in pulse-labelled embryos was significantly enhanced during 24- and 48-h chase culture under low oxygen concentrations of 5, 2.5 and 1%. The lower the oxygen tension the greater was the turnover in the pool. The morphological development of embryos was equally as good at very low oxygen concentrations as when embryos were cultured in 5-20% oxygen. Reduction in oxygen concentration enhanced the oxidative utilization of substrate, as measured by rate of carbon dioxide production. The present study could provide an explanation for the discrepancy in glycogen content between mouse blastocysts developing in utero and in vitro and for the reported beneficial effects of low oxygen concentration during development of embryos in culture.

Animals↗

PubMed1989

Incorporation of glucose by the sheep conceptus between days 13 and 19 of pregnancy.

Incorporation of glucose into the internal biochemical pools of the sheep embryo and samples of extraembryonic membranes was measured during a 2.5 h incubation in the presence of radiolabelled glucose. Very little glucose was incorporated into the glycogen pools by either the embryo or its membranes and never represented more than 5% of total incorporation. Approximately 65% of label was isolated in the non-glycogen acid-soluble fraction of samples and the remainder was incorporated into non-glycogen macromolecules. The embryonic disc of the day-13 conceptus had the highest rate of incorporation per mg dried weight of any structure studied. Synthesis of non-glycogen macromolecules by the day-13 disc was five to six times that of either day-15 or day-17 embryos. On day 19 very low rates of incorporation into the isolated embryo were found during culture. Evidence suggests that this was a result of limitations on the diffusion of substrate into the embryo because incubation of fragmented embryos produced rates similar to those found on days 15 and 17. Incorporation of glucose into the intracellular pools of extraembryonic membranes per mg dried weight remained relatively low and stable over the period studied and there were only minor differences in the rate of incorporation between membranes. Incorporation of glucose by embryos and extraembryonic membranes was equally as good in phosphate-buffered media as in bicarbonate-buffered solutions and was unaffected by changes in the concentration of lactate and pyruvate in the culture medium.

Animals↗

PubMed1989

Volume of fluid and concentration of cations and energy substrates in the uteri of mice during early pseudopregnancy.

Estimates of the volume of fluid and the concentration of cations in the uterus were made by flushing the uterine cavity of mice mated to vasectomized males and measuring the concentrations of sodium and potassium cations in the recovered fluid. On day 1 following mating, large volumes of fluid (greater than 90 microL) were found in the uterus but, from days 2 to 5, only 2-5 microL of fluid were present. The ratio of sodium to potassium fell from 4.5:1 on day 1 to 1.8:1 on days 2 to 5 of pseudopregnancy, indicating that uterine fluid in the mouse has a high K+ content ranging from 35 mEq L-1 on day 1 to 75 mEq L-1 on day 5. Glucose, lactate and pyruvate in uterine flushings were also assayed and their concentration in uterine fluid calculated using the volumes found above. The level of all substrates was low on day 1 after mating. From day 2 onwards approximately 1 mM glucose was present in the fluids. The concentration of lactate was more variable, and peaked at 4 mM on day 2 of pseudopregnancy. In general, the concentration of pyruvate was 10% of the lactate value.

Animals↗

PubMed1987

Effects of coculture with uterine epithelial cells on the metabolism of glucose by mouse morulae and early blastocysts.

Coculture of mouse morulae/early blastocysts with isolated endometrial epithelial cells reduced incorporation of glucose carbon into embryonic glycogen but had no significant effect on incorporation into other internal carbon pools during a 5-h culture in serum-supplemented Dulbecco's modification of Eagle's minimum essential medium. Turnover of glycogen pools during 24-h chase culture of pulse-labelled embryos was unaffected by the presence of uterine epithelial cells recovered from day-4 pregnant or non-pregnant mice. However, significantly more label was retained in non-glycogen macromolecules during chase in the presence of endometrium recovered from non-pregnant than from pregnant uteri.

Animals↗

PubMed1987

Effects of oestradiol and progesterone on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The addition of progesterone (10(-7) to 10(-5) M) and/or oestradiol (10(-10) M) during 24-h chase culture of pulse-labelled morulae-early blastocysts did not affect the degradation of radiolabelled glycogen or other biochemical fractions. The presence of a high concentration of progesterone (10(-5) M) during 5-h pulse culture significantly inhibited incorporation of substrate carbon from [U-14C]glucose into both the acid-soluble and acid-insoluble glycogen fractions, but had no effect on non-glycogen fractions. Catabolic utilization of glucose as estimated by the rate of carbon dioxide and lactate production was not affected by the presence of progesterone (10(-7) to 10(-5) M), oestradiol (10(-10) to 10(-8) M) or a combination of both. The results indicate that ovarian steroids at expected physiological concentrations do not directly influence embryonic energy metabolism.

Animals↗

PubMed1987

Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro.

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.

Animals↗

PubMed1987

Metabolism of glucose by human embryos.

Glucose turnover, as measured by CO2 production, lactate accumulation and carbon incorporation from [U-14C]glucose as sole energy substrate, was low on the 2nd day of culture of human embryos resulting from in-vitro fertilization but above that of unfertilized oocytes. In general, all parameters of metabolism increased substantially during the following 2 days of development but the rate of increase in lactate production was greater than that of CO2, especially between Days 3 and 4. Within developing embryos, no correlation was evident between the metabolic turnover of glucose and the method of patient stimulation, the morphological quality of embryos or the apparent rate of cleavage in culture. The results indicate that, before Day 3 of development, glucose is not effective as an energy source for the human embryo because of a blockade to glycolysis similar to that in mouse embryos.

Blastocyst↗

PubMed1986

Measurement of metabolic turnover in single mouse embryos.

The method for estimation of metabolic turnover by a single embryo at each stage of preimplantation development is based on the incubation of an embryo in the presence of labelled substrate at high specific activity in a miniaturized incubation chamber and the subsequent radioassay of metabolic products. Preliminary tests indicated that the treatment did not affect embryos adversely. Estimates of glycolysis, substrate oxidation and incorporation by mouse embryos throughout the whole of the preimplantation period of development were made. This technique could prove particularly useful for investigating substrate requirements and metabolic turnover in species for which few embryos are available for study.

Animals↗

PubMed1985

Influence of environmental factors on the metabolism of glucose by preimplantation mouse embryos in vitro.

The metabolism of glucose by late preimplantation mouse embryos was studied in a variety of media whose composition had been changed to reflect the environmental conditions in the uterus more closely than do standard culture media. The effects of combinations of energy substrates, the presence or absence of amino acids and the level of potassium in the medium were investigated. The use of energy substrates for in vitro culture at levels present in the uterine environment resulted in rates of synthesis and degradation of glycogen pools similar to those obtained using standard in vitro culture conditions but elevated incorporation into non-glycogen macromolecules. Amino acids influenced the metabolism of glucose by limiting the entry of glucose carbon into the non-glycogen macromolecular pool and directing more glucose into the synthesis of acid-soluble glycogen. Increasing the K+ concentration to 60 mM in the culture medium caused a small but significant increase in the number of eight-cell embryos degenerating during culture for 24 h but the metabolism of glucose was unaffected over this time. At the time of morula transformation to the blastocyst this level of potassium ions suppressed glycogen synthesis by 50% over 5 h but did not affect its turnover during chase culture. It is concluded that factors other than those studied here contribute to the maintenance of the low glycogen levels found in uterine embryos.

Amino Acids↗

PubMed1985

Metabolism of glucose by preimplantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine.

Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development.

Animals↗

PubMed1984

Synthesis and degradation of labelled glycogen pools in preimplantation mouse embryos during short periods of in vitro culture.

The incorporation and turnover of glucose carbon by mouse embryos during short periods of in vitro culture were studied using [U-14C]glucose as marker. Particular attention was given to the synthesis and degradation of the acid-soluble and acid-insoluble glycogen pools. During a 5-h culture period, incorporation into all fractions isolated increased during development from the 1-cell to the early blastocyst stage. During maturation of the blastocyst, incorporation into acid-insoluble glycogen fell whilst that into acid-soluble glycogen continued to rise linearly over this 24 h period. At the late blastocyst stage over 80% of total glucose carbon accumulated was in this latter fraction. The decrease in acid-insoluble glycogen observed above was found to occur as a sudden change between 96 and 104 h post-hCG. The turnover of glucose carbon incorporated into the glycogen pools was studied using pulse-chase culture techniques. The results indicate that the considerable amounts of glycogen accumulated at the later stages of preimplantation development cannot act as a major energy source in vitro except in the absence of energy substrates. Under these conditions of energy starvation, however, the breakdown of acid-insoluble glycogen is inhibited. The findings show that the late preimplantation embryo accumulates large amounts of acid-soluble glycogen in vitro due to increased rate of synthesis and low rate of degradation. It is suggested that the falling levels of glycogen reported in the uterine embryo are most likely a result of environment factors other than the availability of energy substrates.

Animals↗

PubMed1984

Studies of the distribution of glycogen between the inner cell mass and trophoblast cells of mouse embryos.

Autoradiographic and histochemical techniques were used to determine the localization of glycogen synthesized during in-vitro culture of preimplantation mouse embryos. During early cleavage embryos accumulated little glycogen and that which was synthesized was spread evenly in the blastomere cytoplasm. However, morula and early blastocyst stages accumulated relatively large amounts of glycogen, especially in the peripheral or trophoblastic cells in comparison to the inner cells or inner-cell-mass cells. Immunosurgical techniques were used to study the incorporation of radiolabelled glucose into the biochemical pools of inner-cell-mass and trophoblastic cells during culture for 24 h. In general, trophoblastic cells incorporated considerably more isotope than did inner-cell-mass cells, especially into the acid-soluble glycogen fraction. However, inner cell masses isolated on Day 4 of pregnancy incorporated more glucose into acid-soluble glycogen than did inner cells isolated from blastocysts at the end of culture for 24 h in isotope.

Animals↗

PubMed1984

Degradation of biochemical pools labelled with [14C]glucose during culture of 8-cell and morula--early blastocyst-stage mouse embryos in vitro and in vivo.

When 8-cell mouse embryos were chase cultured for 24 h in vitro or in vivo (in uteri of pseudopregnant mice) there was no indication of utilization of the small amount of acid-soluble glycogen synthesized during the pulse. At the morula-early blastocyst stage of development almost 50% of the label incorporated during the pulse was found in the acid-soluble glycogen fraction. The biochemical pools at this stage were relatively stable in vitro and in vivo during a short (5 h) chase period. However, marked degradation of the acid-soluble glycogen pool occurred during long periods of exposure to the uterine environment and, over 48 h in utero, almost all of the label was lost from this pool. By contrast, embryos cultured in vitro for the same period retained greater than 60% of their acid-soluble glycogen. Utilization of glucose carbon in the acid-insoluble glycogen fraction occurred during in-vitro and in-vivo chase but there was a suggestion that the change in vivo was less than that in vitro. The non-glycogen macromolecular pool was relatively stable except during extended chase culture of morulae-early blastocysts when some utilization occurred. Under these conditions utilization was less in utero than in vitro. The experiments show that the uterine environment has a marked influence on the metabolism, particularly of glycogen, of the embryo and indicate that some factor in the uterus causes net degradation of acid-soluble glycogen by the embryo at the late preimplantation stage of development.

Animals↗