The extent to which the early mouse embryo is able to synthesize the components required for the formation of RNA from naturally occurring exogenous energy sources which support development in vitro remains to be determined. Recent reports have described the synthesis of RNA and DNA in pre-implantation mouse embryos when cultured or incubated in vitro with labelled nucleosides (Ellem & Gwatkin, 1968; Pikó, 1970), but an exogenous supply of these nucleic acid precursors is not necessary for early development (Thomson TenBroeck, 1968). The present study was, therefore, undertaken to discover whether pyruvate and glucose, as exogenous energy sources, act as precursors for the synthesis of soluble-RNA (s-RNA) during this time. Moreover, since mouse embryos require the presence of a bicarbonate buffer system for their development in vitro, and since the embryos gain a substantial amount of carbon through the fixation of CO2 (Wales, Quinn & Murdoch, 1969; Graves & Biggers, 1970) incorporation of 14C from bicarbonate into the s-RNA of the embryos has also been examined.
R. N. Murdoch and R. C. Jones
Summary. Cooling diluted boar semen to 5°C and the addition of glycerol at 20°C and 5°C all depressed the subsequent metabolism of glucose, but had no effect on total oxygen uptake or lactate production. Ultrastructural studies showed that although glycerol had no effect on middle-piece cytology, it increased the incidence of acrosomal vesiculation at 5°C. It is concluded that acrosomal vesiculation does not affect the metabolism of spermatozoa and it is suggested that the reported detrimental effect of glycerol on the fertilizing capacity of spermatozoa is related to its membrane activity.
R. G. WALES and R. N. MURDOCH
The composition of sheep fetal fluids obtained on Days 22, 31 and 44 of pregnancy has been studied.
The osmotic pressure of allantoic fluid was low and its volume increased twofold between Days 31 and 44 of pregnancy. A seventeenfold increase in volume occurred in the amniotic fluid over the same period.
Fructose was the main reducing sugar present and increased in concentration in the allantoic fluid between Days 31 and 44 of pregnancy. During this time, the allantoic fluid also showed an increase in sorbitol, protein, amino acids, calcium and magnesium and a decrease in lactic acid, potassium and bicarbonate. Amniotic fluid was more constant in composition and had a lower total reducing sugar, fructose, protein, sorbitol, amino acid, calcium and magnesium content and higher sodium, potassium and chloride content than allantoic fluid. Phosphorus occurred in low concentration in both fluids.
Glutamic acid was the only acidic amino acid present in allantoic fluid and occurred in lower concentration than glutamine. Alanine and glycine were the major neutral amino acids, with lower concentrations of serine, valine, threonine and leucine/isoleucine being present. Basic amino acids made up about 30% of the total. Substantial increases occurred in the concentrations of alanine, glutamic acid, glutamine and serine in the allantoic fluid between Days 31 and 44 of pregnancy.
The changes in the composition of allantoic fluid suggest that major developmental changes in metabolic activity are occurring in the sheep fetus between Days 31 and 44 of pregnancy.
R. N. MURDOCH and I. G. WHITE
There was usually an increase in the manometrically determined oxygen uptake of rabbit spermatozoa which has previously been subjected to an incubation in the uterus of the doe, and also an increase in aerobic glycolytic activity as estimated by glucose utilization and lactate accumulation. Freshly ejaculated rabbit spermatozoa produced aerobically about as much labelled carbon dioxide from [6-14C]glucose as from [1-14C]glucose, whereas spermatozoa incubated in utero oxidized [1-14C]glucose at a rate distinctly above that shown by fresh spermatozoa. The time required for the development of these metabolic changes was similar to that required for the capacitation of rabbit spermatozoa.
Rabbit spermatozoa maintained in vitro for 2 hr or more at 37° C suffered a marked decline in metabolism. The yield of 14CO2 from [1-14C]glucose, however, declined less than that from [6-14C]glucose.
R. N. MURDOCH and I. G. WHITE
Ram and bull spermatozoa oxidize acetate faster than or similarly to glucose, while dog and rabbit spermatozoa oxidize glucose faster than acetate; rabbit spermatozoa, in particular, have hardly any capacity to oxidize acetate.
Glucose and fructose are oxidized at about the same rate by the spermatozoa of all four species, each sugar giving rise to similar amounts of lactate.
Lactate is readily oxidized by spermatozoa of all four species; with rabbit and dog spermatozoa the rate of lactate oxidation greatly exceeds that of glucose, fructose, pyruvate or acetate.
Pyruvate is oxidized by ram and bull spermatozoa faster than acetate. With dog and rabbit spermatozoa, the recovery of 14CO2 from [1-14C]pyruvate is 2 to 4 times that from [2-14C] or [3-14C]pyruvate; this may be due to a dismutation of pyruvate.
R. N. MURDOCH and I. G. WHITE
The metabolism of human spermatozoa has been studied using concentrated suspensions of washed cells incubated with radioactive substrates in small Warburg flasks, enabling the oxygen uptake to be measured accurately.
Oxidative metabolism was greater at pH 7·0 than at pH 8·5. Phosphate ions (20 mm) depressed oxidative metabolism at pH 7·0 but increased it at pH 8·5. Fructolysis was stimulated by phosphate ions irrespective of the pH. Phosphate ions decreased the oxidation of lactate and acetate by about the same absolute amount which suggests that it may restrict the entry of acetyl CoA into the Krebs cycle.
Potassium ions (15 mm) increased both fructolysis and oxidative metabolism, and bicarbonate increased the oxygen uptake.
Glucose and fructose were oxidized more readily than acetate, glycerol or sorbitol, but only the polyols significantly increased the oxygen uptake.
J. A. Sakoff and R. N. Murdoch
The present study exploited the deciduogenic actions of concanavalin A (Con A) in pseudopregnant Quackenbush strain mice to assess whether alterations in uterine Ca2+ status accompany stromal cell transformations that culminate in the decidual cell reaction and the establishment of pregnancy. It was found that the introduction of 15 blastocyst-size Con-A-coated Sepharose beads, but not lectin-free Sepharose beads, into the lumen of the left uterine horn of pseudopregnant mice on day 3 or 4 induced a decidual response that was virtually indistinguishable from that produced by blastocysts during normal pregnancy. The right uterine horn received no treatment and served as a control. Although the simultaneous administration of 45CaCl2 (1 mmol l−1, 200 mCi mmol−1) on day 3 of pseudopregnancy impeded this response, the deciduogenic potential of the Con-A-coated beads was not disrupted when these agents were simultaneously administered on day 4. This experiment with 45CaCl2 produced discrete areas of decidualization, as shown by the pontamine sky blue reaction on day 5 of pseudopregnancy. The decidualized areas contained significantly greater amounts of 45Ca2+ than did the non-decidualized interjacent areas, indicating that the beads preferentially stimulated the rate of influx of the cation into these sites. Much of this cellular 45Ca2+ content was lost in the early afternoon of day 5 of pseudopregnancy at a time when the luminal epithelium undergoes autolytic breakdown. The intraluminal introduction of 125 μg of Con A in solution (5 μl) was also deciduogenic but stimulated a larger uterine response than did the Con-A-coated beads and promoted a significantly greater uptake and retention of 45Ca2+ than did the intraluminal injection of other agents. The results suggest that the embryonic signal responsible for induction of the decidual cell reaction in mice involves surface interactions between the embryo and uterine luminal epithelium to promote the influx of Ca2+ from the lumen into the epithelial cells and to modify their metabolism to stimulate the stromal cell transformations.
R. N. MURDOCH and I. G. WHITE
The metabolism of washed ram spermatozoa has been studied in the presence and absence of low levels of bicarbonate (6 mm) and CO2 (2%). Although bicarbonate stimulated the O2 consumption of ram spermatozoa in the presence of glucose, acetate or lactate, its most profound effect was on glycolysis and it greatly stimulated the rate of glucose breakdown to lactic acid under both aerobic and anaerobic conditions. Low concentrations of potassium (5 mm) enhanced the stimulating effect of bicarbonate on glycolysis but, in its absence, bicarbonate greatly depressed O2 uptake.
Bicarbonate failed to stimulate the conversion of glucose to 6-phospho-glucose when sonicated spermatozoa were incubated in the presence of ATP and sodium fluoride, or the conversion of pyruvate to lactate in intact spermatozoa. This suggests that reactions catalysed by glucokinase and lactic dehydrogenase are not sites at which bicarbonate acts to enhance glycolysis. However, bicarbonate did stimulate the conversion of phosphoenol pyruvate to lactic acid when spermatozoa were incubated under anaerobic conditions in the absence of added ADP; this indicates that the reaction catalysed by pyruvate kinase in ram spermatozoa may be at least one site at which bicarbonate acts to stimulate glycolysis. Although the addition of ADP (5 mm) greatly increased the conversion of phosphoenol pyruvate to lactic acid, it obliterated the stimulating effect of bicarbonate.
R. V. Hyne, R. N. Murdoch and B. Boettcher
Summary. The metabolism and motility of human ejaculated spermatozoa incubated in vitro with steroids were studied. Progesterone and norethynodrel depressed the respiration, glycolytic metabolism and the motility of washed spermatozoa. Lynoestrenol did not affect the respiration or glycolysis of the spermatozoa, but did inhibit motility. Oestradiol did not cause any consistent alteration of the sperm metabolism, and did not affect the motility. Progesterone and norethynodrel appear to act on the plasma membrane of human spermatozoa to increase its permeability and hence to facilitate the loss of essential cofactors required for the glycolytic and oxidative processes.
J. Clulow, R. C. Jones and R. N. Murdoch
Summary. Demembranated spermatozoa from the rete testis developed vigorous flagellation when reactivated with ATP, but showed no forward progression such as that seen in samples from the cauda epididymidis. The proportion of spermatozoa that were reactivated was smaller for samples from the rete testis than from the cauda epididymidis.
Studies in vitro of undiluted micropuncture samples from the epididymis indicated that the activity of spermatozoa is suppressed as they develop the capacity for motility. However, as spermatozoa spontaneously became activated during the collection or subsequent incubation of undiluted samples, it was concluded that the suppressive action is labile.
The activity of spermatozoa in vitro was examined in diluted samples from the cauda epididymidis. A concentration of 2·5 mmol extracellular calcium/1 was better than lower concentrations. Diluents at pH 5·5 completely inhibited sperm motility when they contained 20 mmol lactate/1 (but not glutamate) and the effect was reversed by readjusting the diluent to pH 7·4. However, lactate was not considered to suppress sperm motility in situ, as the plasma from the cauda epididymidis contained only 2·7 ± 0·5 mmol lactate/1. There was no effect of sodium concentration (1 and 115 mmol/1), pH (5·5 and 7·4) or amiloride (0 and 1 mmol/1) on sperm motility, indicating that motility is not dependent on the concentration of sodium above 1 mmol/1 or on a sodium–proton exchange system.
The relative viscosity of plasma from the cauda epididymidis did not affect the motility of spermatozoa.
Keywords: sperm maturation; sperm activation; sperm motility; marsupial spermatozoa; tammar wallaby