The present review examines the mechanisms involved in sperm storage in the epididymis of therian mammals in terms of the supply of energy substrate and the regulation of motility and metabolism. Lipids, glucose, lactate and glycerol are possible metabolic substrates for sperm in the epididymis, but the role of these is uncertain and it may differ between marsupials and eutherians. Sperm are normally immotile in the epididymis, but ram and rabbit sperm may have an uncoordinated motility. Sperm metabolism is suppressed but is probably not strongly coupled to motility. Work on diluted sperm indicates that cyclic adenosine monophosphate, Ca2+, and pH play roles as intracellular messengers controlling the motility and metabolism of sperm, but no first messenger has been identified. A number of mechanisms of suppressing sperm motility and metabolism in the epididymis are considered, including a collective autoregulation, oxygen tension, osmotic pressure, viscosity and the extracellular concentration of H+, Ca2+, Na+, HCO3- and carnitine. However, there is no conclusive evidence for any of the mechanisms and there is clearly some variation between species in the mechanism of suppressing sperm activity. Sperm activation stimulates motility and a 4-5-fold increase in respiration rate that has not been reversed without compromising viability. Following activation, respiration supported by endogenous and/or exogenous substrates is much higher in marsupial than eutherian sperm, and marsupial sperm do not show a large stimulation of respiration on the addition of exogenous substrate, as is characteristic of most eutherian sperm.

The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25-30 nl of CEF was stimulated by calcium ions (Ca2'), N2,2'-0-dibutyrylguanosine 3':5'-cyclic monophosphate (dibutyryl cGMP), cyclic adenosine 3'5'-monophosphate (CAMP), N6,2'-0dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl CAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8bromo CAMP), caffeine, theophylline and bicarbonate ions (HC0,-). Other agents such as magnesium ions (Mg2'), veratridine, phospholipase C (PLC), ionophore A23187, 1,2dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A2 (PLA2), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-'4C]glucose and [I-'4C]acetate as exogenous energy sources for oxidative metabolism. No detectable l4C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca2+ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA, pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway. o 1994 WileYLiss, Inc.

The biochemical assay of alkaline phosphatase in the uterus of mice revealed a 13-fold increase in the activity of the enzyme during the peri-implantation period with a relatively short-lived maximum being reached on Day 7 of pregnancy. A similar increase in the activity of the enzyme occurred between Days 5 and 7 of pseudopregnancy in uterine horns of mice receiving a deciduoma-inducing stimulus on Day 4. The presence of factors in uterine homogenates which could possibly modify the activity of the enzyme could not be detected. Low and unaltered levels of alkaline phosphatase activity were found in the blood serum of mice between Days 1 and 10 of pregnancy, suggesting that no appreciable entry of the uterine enzyme into the circulation occurs in this species. Alkaline phosphatase activity in the uterine homogenates was associated with particulate fractions sedimented during centrifucation at at 500, 10,000 and 105,000 g, and only about 29% of the activity was associated with the cytosol fraction. The subcellular distribution of the enzyme activity was the same in decidualized and non-decidualized horns. The association of the enzyme with the particulate material in uterine cells was considered to involve lipoprotein membranes because treatment of homogenates with 0.2% (v/v) Triton X-100 solubilized the enzyme without affecting its catalytic activity.

SummarySuccinate dehydrogenase (SDH), lactate dehydrogenase (LDH), glutamateoxaloacetate transaminase (GOT), and acid and alkaline phosphatase are present in ram, bull, dog, rabbit, and human semen but the concentrations differ from one species to another. Amylase is found in the semen of all these species except the ram, and glucose·6-phosphate dehydrogenase (GDH) in all except the bull.Studies of the ram and bull showed that if a particular enzyme is present in whole semen, it occurs in both the spermatozoa and seminal plasma but there was often a great difference in the activity of the two fractions, e.g.· SDH and LDH activity was greater in the spermatozoa than in the plasma. SDH appears to be very tightly bound in ram spermatozoa since, unlike LDH, it is not released by sonic disruption. LDH, GOT, GDH, and acid phosphatase are leached out of ram and bull spermatozoa by sudden cooling (cold shock) or deep freezing.The enzyme content of ram testicular fluid is low compared with epididymal and vesicular fluids and some of the enzymes found in the seminal plasma probably have their origin in the epididymis and seminal vesicles.

No abstract

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\m=.\0than at pH 8\m=.\5. Phosphate ions (20 mm) depressed oxidative metabolism at pH 7\m=.\0but increased it at pH 8\m=.\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.

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0·8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO± lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.Some metalloenzyme properties of the phosphatase were also studied and it was demonstrated that both Mg2+ and Zn 2 + ions are necessary for hydrolytic activity.Treatment with neuraminidase retarded the anodal migration of the enzyme during electrophoresis on cellulose acetate membranes but did not influence its activity or catalytic properties. These results suggest that the uterine alkaline phosphatase is a sialoglycoprotein. The sialic acid residues, however, do not appear to constitute part of the active centre of the enzyme.In addition, the optimum pH for activity depended on substrate concentration and decreased with decreasing substrate concentration. Apparent Km values also depended on variations in pH and decreased with decreasing pH. Plots of pKm versus pH revealed a functional group with a pK value of 9 ·45. The enzyme also hydrolysed a variety of compounds having either phosphomonoester or pyrophosphate linkages and was inactivated after heating at 60°C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50·1 kJmol-'.

Mouse embryos require the presence of a bicarbonate buffer system for their development in vitro. The energy requirements for oocyte maturation and early zygote development prompted Biggers, Whittingham & Donahue (1967) to suggest that the fixation of carbon dioxide may play a r\l=o^\le in the energy metabolism of the early embryo. The present study was undertaken in an attempt to demonstrate this reaction in the eight-cell mouse embryo. For each experiment, 400 eight-cell mouse embryos were flushed from the reproductive tracts of superovulated female albino mice. The basic medium was Krebs-Ringer bicarbonate containing 25 mm-sodium dl lactate, 0\m=.\25mm\ x=req-\ sodium pyruvate, 0\m=.\01 mm-sodium ketoglutarate, 0\m=.\01 mm-sodium malate, 0\m=.\01 mm-sodium citrate, 1 mg/ml bovine serum albumin, 60 \g=m\g/mlpenicillin, 50 \g=m\g/mlstreptomycin. After washing by transfer through two changes (2 ml each) of this medium, the embryos were cultured in 20 \ g=m\ l droplets of medium (forty embryos/drop) containing NaH14CO3 (specific activity 10 mCi/m-mole) in a 35-mm diameter plastic culture dish (Falcon Plastics) containing 3 ml light paraffin oil. The culture dish was cemented in a glass petri dish of approxi¬ mately 27-ml volume. Sufficient radio-active bicarbonate, of the same specific activity as that in the culture medium, was pipetted into one side of the glass petri dish to produce a 5% C02 atmosphere in the dish after acidification and 0-2 ml of 6 n-H2S04 was pipetted into the opposite side of the dish. The lid of the petri dish was sealed and the dish tipped slightly to mix the acid with the bicarbonate to release carbon dioxide.After a 24-hr period of culture at 37°C , all the developed embryos were separated from the medium as previously described (Wales & Biggers, 1968). The medium was also collected and both embryos and medium were acidified with 0-1 ml 2 n-H2S04 and placed in a closed container containing 2 N-NaOH for 24 hr to absorb liberated carbon dioxide. After neutralization with NaOH, a small aliquot was taken for radio-assay and the remainder was fractionated to determine the amount of label in the acid-soluble, protein and lipid fractions as described below.50-µ1 sample of sheep serum was added to both embryos and medium to act as carrier for the labelled compounds. Protein was precipitated with 2-5% perchloric acid. After washing the precipitate four times with 20% trichloroacetic acid, it was extracted with chloroform-ether (1:1). The acid-soluble 541