Summary. The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70–80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 126.96.36.199) was approximately equivalent to that of carnitine palmitoyltransferase (EC 188.8.131.52); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.
D. E. Brooks
The mechanisms controlling the maturation of spermatozoa in the epididymis remain poorly understood although it is clear that an adequate supply of androgens is required for the normal maintenance of the organ. The technique of efferent duct ligation prevents spermatozoa from entering the epididymis and therefore enables metabolic processes to be studied in the androgen-maintained epididymis without contribution from spermatozoa. Castration enables the epididymis to be studied when spermatozoa and androgenic support are absent. Determinations of enzyme activities have indicated that, provided about 6 weeks are allowed following efferent duct ligation for the epididymis to become largely emptied of spermatozoa, the results of efferent duct ligation are indistinguishable from those after castration and later androgen replacement (Brooks, 1976b, c). These two techniques therefore enable a study to be made of the epididymis with and without androgenic support.
D. E. Brooks
Summary. α-Chlorohydrin has been examined both for its ability to act as a substrate for glycerol kinase and as an inhibitor of the reaction of glycerol with glycerol kinase. Using a purified enzyme from Candida mycoderma, it was established that α-chlorohydrin does not act as a substrate for glycerol kinase, but does act as a competitive inhibitor (K i of 30 mm) of purified glycerol kinase and the enzyme present in a sonicated preparation of ram spermatozoa. Neither α-chlorohydrin nor α-chlorohydrin phosphate acted as inhibitors of NAD- or flavin-linked glycerolphosphate dehydrogenase. It is concluded that α-chlorohydrin does not cause the impairment of sperm metabolism as a result of phosphorylation catalysed by glycerol kinase.
D. E. Brooks
Summary. The incorporation of radioactive mannose and fucose into secretory glycoproteins by rat epididymal tissue was studied using tissue pieces in vitro. The appearance of radioactive macromolecular products in the medium occurred after a lag phase of 2 h with radioactive mannose, but with radioactive fucose the lag phase was only 15 min. Preincubation of tissue for 2 h before the addition of radioactive mannose increased the subsequent rate of incorporation by reducing the lag phase from 2 to 1 h. Tunicamycin reduced the incorporation of radioactive mannose and fucose into macromolecular products to approximately 15 and 50% of normal in the caput and cauda respectively; maximum inhibition required 10 μg tunicamycin/ml in the caput and 2 μg/ml in the cauda. Reduction of radioactive sugar incorporation by tunicamycin did not result in qualitative changes in the profile of the radioactive glycoproteins that were secreted. However, immunoprecipitation of proteins D and E from incubations with radioactive methionine or mannose revealed that tunicamycin caused these proteins to be synthesized and secreted in a non-glycosylated form. Prior castration of animals reduced the incorporation of radioactive mannose and fucose, and qualitative changes in the profiles of secreted radioactive glycoproteins were apparent.
D. E. Brooks
The gross composition of the testicular excurrent duct system of the rat was examined and compared along the length of the duct and with samples of testis, bladder and liver. Changes in composition with age were examined by analysing tissue from animals at postnatal ages of 19, 36, 48, 60, 90 and 120 days.
In adult animals, testicular tissue was characterized by having the lowest dry weight, accompanied by low levels of total protein, lipid, RNA and glycogen; DNA, phospholipids and sialic acid were at levels similar to other tissues. A high proportion of the total protein was soluble. The ductuli efferentes plus initial segment of the epididymis were characterized by high levels of total lipid. The caput epididymidis contained a low level of total protein but a high level of acid-soluble phosphorus. The cauda epididymidis had a low dry weight and low levels of total protein, soluble protein, and lipid, but high levels of acid-soluble phosphorus, DNA and sialic acid. The ductus deferens contained small amounts of RNA and DNA but had a high dry weight, high total protein, soluble protein and glycogen.
Several trends were apparent with increasing age. Dry weight increased in the ductuli efferentes plus initial segment, whilst total protein decreased in the caput and cauda epididymidis. Total lipid increased in the ductuli efferentes plus initial segment and acid-soluble phosphorus and sialic acid increased in all other segments of the excurrent duct system. In all segments the content of RNA and DNA decreased as the animals matured.
The concentration of calcium and magnesium in the excurrent duct system was not significantly different from those levels found in the liver.
High levels of spermine and spermidine were confirmed in the prostate, and were also detected in the testis, caput epididymidis and cauda epididymidis, but at a much lower concentration.
D. E. BROOKS
The development of a method to measure small amounts of nucleotides would be of considerable value in experiments concerned with metabolism in mammalian spermatozoa. As the adenine nucleotides have been shown to be the predominant nucleotides in mammalian spermatozoa (Brooks, 1970), measurement of ATP and ADP by the luciferin-luciferase system appeared to be a method ideally suited to this purpose.
Ejaculated bull semen, collected by artificial vagina, was used in the present work. The measurement of ATP was carried out in a Packard Model 3380 liquid scintillation spectrometer calibrated according to Stanley & Williams (1969). The phosphate buffer described by these workers was altered to include 2 mm-EDTA and 2 mm-2-mercaptoethanol. In addition, the luciferin-luciferase preparation made up in arsenate buffer was incubated with apyrase to reduce background luminescence
D. E. BROOKS
There has been considerable controversy regarding the existence of phosphagens, such as creatine phosphate and arginine phosphate, in mammalian spermatozoa, though it is well documented that creatine phosphate forms a normal constituent of the testis (Treskin, 1872; Eggleton & Eggleton, 1929; Ennor & Rosenberg, 1952; White & Griffiths, 1958). Wajzer & Brochart (1947) were the first to claim that creatine phosphate and arginine phosphate were present in the 'sperme' of the boar. Later, Newton & Rothschild (1961) reported that bull semen contains labile phosphorus and attributed this, at least in part, to creatine phosphate, though White & Griffiths (1958), in a study involving both the spermatozoa and seminal plasma of a number of mammaliann species (including the bull), were unable either to isolate phosphagens by a barium fractionation procedure or to demonstrate the
D. E. BROOKS
The testes of mammals which have a scrotum are maintained at a temperature several degrees below that of the abdomen; the mechanisms by which this temperature differential is maintained have recently been reviewed by Waites (1970). In contrast to the large amount of information concerning testicular temperature and its control, there have been no reported measurements of temperatures in the epididymis. The present work was carried out in order to measure epididymal temperature and to compare temperature control in this organ with that in the testis.
Thermocouples were made by joining iron and constantin wires (36 cm × 0·16 mm) end to end, the length of the junction being no longer than 2 mm. The wires were then coated with Teflon by American Durafilm Co., Inc., Newton Lower Falls, Massachusetts, U.S.A. The thermocouples were implanted in
D. E. BROOKS and T. MANN
Boar spermatozoa utilize pyruvate efficiently both anaerobically and aerobically. Pyruvate metabolism, in marked contrast to fructolysis, continues in spermatozoa immobilized by cold shock. Sperm cytoplasmic droplets alone do not metabolize pyruvate. Carbon dioxide, lactate, acetate, succinate and acetoacetate were identified as anaerobic products of pyruvate metabolism; the ratio between pyruvate utilized and lactate and acetate formed did not fit into the stoichiometry of a simple dismutation. Carbon dioxide and lactate were formed from pyruvate aerobically. The rate of pyruvate disappearance, as well as the percentage of pyruvate converted to lactate, were higher aerobically than anaerobically. Very little acetoacetate was utilized by boar spermatozoa aerobically. Lactate was oxidized but at a much lower rate than pyruvate. Acetate (0·18 μmol/109 cells) was extracted from exhaustively washed spermatozoa; this acetate is assumed to be derived from acetyl compounds of the sperm cells.
D. E. Brooks and K. Tiver
Summary. Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.