A simple technique for the purification of plasma membranes from ejaculated boar spermatozoa.

Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.

Developmental expression of cathepsin L and c-rasHa in the mouse placenta.

An investigation is described of the expression of the cysteine proteinase cathepsin L during placental development. In addition, whether cathepsin L expression is linked to c-rasHa expression in development, as it is in metastatic cells, is examined. Large amounts of cathepsin L and its transcript are present in the mouse placenta, more than six times more than in adult kidney and liver. Throughout gestation, cathepsin L and its transcript are located in the giant cells and spongiotrophoblasts of the placenta. Several forms of different mobility on denaturing gels are found in the placenta. Their apparent molecular weights, as determined from the gels, are 43,000, 39,000, 29,000, and 20,000. The 39-kDa form is procathepsin L. The 29-kDa and 20-kDa forms are lysosomal cathepsin Ls. The 39-kDa procathepsin L and the 20-kDa mature cathepsin L are the most abundant species in the placenta and are present in about equal amounts throughout gestation. At any time during gestation, placental minces synthesize and secrete only procathepsin L. The amniotic fluid of the fetus contains the 43-kDa form of cathepsin L and procathepsin L, but no detectable amounts of mature cathepsin L. By contrast, serum from nonpregnant or pregnant mice contains three forms of cathepsin L (i.e., the 43-kDa form, procathepsin L, and mature cathepsin L). Cathepsin L and the rasHa oncogene are expressed in two coincident waves corresponding to periods during which the placenta is invasive and just before parturition. The presence of large amounts of cathepsin L in the placenta suggests that the proteinase has a significant function there. Expression of cathepsin L in the placenta is potentially under the control of the ras gene product p21; both are under developmental control.

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa.

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.

Correlation of immunological studies and disease progression in chronic progressive multiple sclerosis.

Thirty untreated patients with clinically definite chronic progressive multiple sclerosis were matched with 10 patients with clinically stable definite multiple sclerosis and 16 patients with other neurological diseases. A group of 12 normal control (NC) volunteers was matched to these groups. All patients with chronic progressive multiple sclerosis and normal control subjects were analyzed for the concanavalin A suppressor assay, mitogen stimulation, and phenotyping of peripheral blood mononuclear cells. In addition, serum was analyzed for interleukin-2 levels. Results of mitogen stimulation studies did not distinguish the groups. Concanavalin A-induced suppression was significantly decreased in the patients with chronic progressive multiple sclerosis (p less than 0.01). Phenotyping of fresh cells showed an elevated CD4: CD8 ratio in the patients with chronic progressive multiple sclerosis. Neither phenotyping nor concanavalin A-induced suppression correlated with or predicted the degree of disability, but the serum levels of interleukin-2 correlated inversely with disability (p less than 0.01) and directly with a poor prognosis after 18 months of observation (p less than 0.05). Serum levels of interleukin-2 decreased as the disease progressed.

Thymectomy in multiple sclerosis. Two preliminary trials.

In two preliminary trials, thymectomy was performed on 35 multiple sclerosis (MS) patients, with (Group 1) or without (Group II) azathioprine therapy for 1 year. Formal studies of clinical neurologic status were conducted at yearly intervals after operation. Each group was compared with carefully matched control patients. Group I patients showed significant improvement in total functional groups (Kurtzke scale) and pyramidal functions 1 and 2 years following thymectomy. Other individual functions showed no significant difference. There was a statistical improvement in disability status (Kurtzke) for patients with relapsing-remitting MS 1 and 2 years following thymectomy. This subset also had a significant decrease in MS exacerbations. Group II patients showed none of the favorable trends seen in Group I. Functional groups, disability status scale, and exacerbations were similar to those of the control patients. Several studies indicate azathioprine therapy alone is of questionable benefit in MS. In myasthenia gravis (MG), the full immunosuppressive effect of thymectomy may not be realized for several years. Therefore, it is possible that in Group I patients a dual mechanism of immunosuppression--azathioprine and thymectomy--is yielding a favorable response not yet apparent in the Group II patients having thymectomy alone. If our data still appear favorable after a third follow-up year, a formal pilot study will be undertaken.