Epididymal compounds and their influence on the metabolism and survival of spermatozoa.код для вставкиСкачать
American Journal of Primatology 1:143 - 155 (1981) Epididymal Compounds and Their Influence on the Metabolism and Survival of Spermatozoa I.G. WHITE Department of Veterinary Physiology,Uniuersity of Sydney, N.S.W., 2006,Australia Epididymal fluid, which is derived from testicular fluid, contains several unusual compounds. Little information is available on the composition of the testicular fluid of primates, but the fluid of the ram, bull, boar, and rat contains high concentrations of inositol and certain amino acids. Analyses have been made of epididymal fluid collected from the cauda epididymis of the Rhesus monkey and several nonprimate species (e.g., ram, bull, dog, stallion, rabbit, guinea pig, rat, and hamster), but similar information on the human is lacking. Cauda epididymal fluid appears to be similar in composition from one mammalian species to another. However, the epididymal plasma differs considerably from blood, lymph, and other extracellular fluids. The environment of spermatozoa in the epididymis is, therefore, highly specialized, and presumably in some way contributes to the prolonged survival of spermatozoa in this organ, and provides substrates for the metabolism of the spermatozoa. The chief characteristics of the cauda epididymal plasma are the low concentration of inorganic ions and the high levels of several unusual organic constituents namely, glycerylphosphorylcholine, carnitine, sialic acid, amino acids, glycosidases, and phosphatases. At least one antifertility compound, namely, orally administered achlorohydrin, appears to be concentrated in the epididymis. Studies on laboratory animals, domestic species, and man, suggest that it inhibits enzymes of the glycyolytic pathway in spermatozoa, and this may be the basis for its antifertility activity. Key words: testis, epididymis, spermatozoa, monkey, ram, bull, glycerylphosphorylcholine,carnitine, a-chlorohydrin INTRODUCTION The primary function of the epididymis is to bring about the ripening or maturation of the spermatozoa and to provide a receptacle for their storage. In the epididymis, spermatozoa are bathed in the fluid or epididymal plasma that fills the lumen of the duct. Epididymal fluid contains several unusual compounds and is derived from the rete testis fluid, which is modified by the absorptive and secretory activity of the epididymal epithelium. Received December 11, 1980; accepted December 12, 1980 Address reprint requests to I.G. White, Department of Veterinary Physiology, University of Sydney, N.S.W., 2006, Australia. 0275-2565/81/0102-0143$04.00 0 1981 Alan R. Liss. Inc. 144 White In this paper, the chemical composition of the testicular and epididymal fluid of mammalian species is briefly reviewed. Where possible, information on primates has been included, but the great gaps in knowledge for such species points to the need for increased research in this area. TESTICULAR FLUID Spermatozoa are swept out ofthe seminiferous tubules, through the rete testis, and into the epididymis in fluid that is believed to be actively secreted by the Sertoli cells. Studies on a number of species, but only to a very limited extent on nonhuman primates and not yet on man, show that the composition is different from that of blood and lymph draining the testis due to a blood-testis barrier. The selectively permeable barrier restricts the passage of some substances from the blood into the testicular fluid, even though they pass into the lymph. Although some work has been done in this area using the rat and ram as models Isetchell & Waites, 1975;Waites, 19761,amore exhaustive study ofthe passage of pharmacologically active substances from the blood into the testicular fluid is clearly desirable. Such a study, especially if conducted in primates, could suggest new approaches to arresting the development of human spermatozoa in the epididymis. Information on the composition of the testicular fluid first came from analyses of material obtained from slaughtered bulls and rams by severing the junction of the testis and epididymis. Since then, a more elegant technique has been evolved which enables the rete testis to be cannulated and fluid to be obtained from living animals, eg, ram, bull, rabbit, rat, and even monkey. [White, 1973; Setchell, 1974; Setchell & Waites, 1975; Voglmayr, 1975; Waites, 19761. Testicular fluid has a low spermatozoa1concentration and the composition is shown in Table I. Although the data available for primates is very limited, there is a considerable amount of work on other mammalian species. Perhaps the most striking feature of the testicular fluid is the low glucose and high inositol content. TABLE I. Chemical Composition of Testicular Fluid Constituent Sodium Potassium Calcium Magnesium Chloride Bicarbonate Protein Urea Alanine (mM) Glycine (mM) Glutamic acid (mM) Aspartic acid (mM) Serine (mM) Glucose Inositol Lactic acid Ascorbic acid Glycerylphosphorylcholine Testosterone (p.g/100 ml) Monkey 313 29 Ham 27 1 48 4.1 0.8 454 49 280 28 0.33 1.87 2.03 0.33 0.36 <2 131 5.9 3.0 80 0.3-3.0 2.8-8.0 Bull Boar Rat 308 35 1.9 1.0 433 42 540-1300 34 1.04 1.09 2.19 0.43 0.73 <2 347 21 4.8 329 54 476 497 128 510 165 0.48 1.19 1.96 0.29 <2 126 6.6 2.8 9-19 2.3 Values are mgilO0 ml unless otherwise stated. Data are from White, 1979. 52 0.90 1.87 0.08 0.17 (2 32-88 2.6 Epididymal Compounds and Effect on Sperm 145 Glucose is not, therefore, available as a n energy source to spermatozoa during the whole of the 2 or 3 hr that it takes them to pass from the seminiferous tubules to the head of the epididymis. Inositol does not stimulate the respiration of washed sperm [Voglmayr & White, 19711and is not utilized to any extent as a substrate [Voglmayr & Amman, 19731. Lactic acid, however, would be available to spermatozoa from the testicular fluid, if the oxygen tension were sufficiently high for its oxidation, as it is readily oxidized by testicular spermatozoa in vitro. As might be expected from the existence of a blood-testis barrier, the total protein content of the testicular fluid is less than that of the blood plasma and their nature is different [Waites, 19761. Three interesting proteins have been described in ram testicular fluid. The androgen-binding protein (ABP)of ram testicular fluid is produced by the Sertoli cells, and may be concerned with the transport of androgens to the caput epididymis [Hansson et al, 19761. “Inhibin” if absorbed from the caput epididymidis, might regulate spermatogenesis via suppression of FSH production by the anterior pituitary. Testicular fluid of the ram also contains a peptide inhibitor of sperm acrosin which may inactivate any proteases that leak out of the acrosome. A number of amino acids, eg, glutamic acid, glycine, alanine, aspartic acid, and lysine, occur in a higher concentration in testicular fluid than in the blood plasma of the bull, ram, and rat due presumably to synthesis within the seminiferous tubules. The slow rate of oxidation indicates that amino acids are unlikely to form a significant metabolic fuel for spermatozoa in the epididymis. Testosterone, dehydroepiandrosterone, and also estrogens have been found in the rete testis fluid of the monkey, bull, ram, boar, rat, and rabbit in concentrations a t least as high as in the peripheral blood plasma [Waites, 19761.Transfer studies indicate that the steroids arise from the interstitial cells; the seminiferous tubules are exposed to a high concentration of testosterone, which plays a part in the maintenance of spermatogenesis and perhaps also directly influences spermatozoa in the epididymis. EPIDIDYMAL FLUID The fluid in the lumen of the epididymis constitutes the environment not only for the maturation but also for the storage of spermatozoa, and it is not unreasonable to suppose that the composition of the fluid may play some part in both processes [Bedford, 1975; Glover, 1976; Orgebin-Crist et al, 19761. Better understanding of the mechanisms involved in maintaining the epididymal environment may provide means of manipulating it to the disadvantage of the sperm. Information on the secretory function of the epididymis would seem particularly relevant to the possibility of introducing systematically or locally administered spermicides into the lumen. One such antifertility agent, u-chlorohydrin, is concentrated in the cauda epididymis and will there attack the stored sperm [Waites, 19761. Fluid has been obtained from the epididymis of animals after slaughter and fluid collected from fistula or cannula in the vas deferens of live animals. The volume of fluid flowing through the epididymis of the ram is less than 1mliday, which contrasts sharply with a volume of40 mliday produced from a cannula inserted into the rete testis. Clearly, much of the fluid produced by the testis is absorbed in the efferent ducts and caput epididymis and leads to a much greater concentration of spermatozoa in the tail. It must be remembered, of course, that the cells ofthe epididymis also secrete substances into the lumen so that the composition of the fluid a t any point along the duct will represent a balance between absorption ofwater and sodium chloride and secretion of more complex organic components. Analyses have been made of epididymal fluid collected from the cauda epididymis of the Rhesus monkey and several nonprimate species (eg, ram, bull, dog, stallion, rabbit, guinea pig, rat, and hamster), but similar extensive published information on the human is lacking (Table 11). The data ofJones  would indicate that cauda epididymal fluid 10.5 t 0.2 - - 18.2 ? 1.5 49.2 2 5.2 4.0 i 0.8 Monkey Nonenzymatic (mg%1 7366 i 1321 Total protein Glycerylphosphoryl2396 ? 187 choline 1290 ? 134 Carnitine 349 ? 27.8 Total sialic acid Urea nitrogen Ammonia 238 t 46 Total phosphorus Inorganic 5.2 ? 0.9 phosphorus Ionic (MEQ) Na+ KMg+ Ca2Zn2+ C1- Constituent 13.2 i- 2.5 459 9.5 46.3 2.4 1.1 18.1 3168 ? 634 27.9 t 3.2 221 1274 2 175 t 213 T 17.2 ? 4.8 ? 368 t i 41.3 f 1.3 32.1 ? 1.6 2.4 2 0.8 1.1 i 0.2 0.1 t 0.2 12.0 ? 0.1 Stallion 2677 i 344 313 ? 30.5 179 t 55.6 17.3 i 0.9 4.5 ? 0.6 339 ? 31 2743 22.1 i 3.8 23.7 ? 3.0 0.6 ? 0.2 0.8 t 0.2 0.05 -+ 0.1 9.4 ? 0.3 Ram 6.7 2 0.5 1320 ? 310 98 t 4.2 105 t 9.2 16.2 k 1.5 3.5 ? 0.4 393 2 12.9 2483 t 152 30.1 i 2.5 36.3 T 1.1 1.5 2 0.1 1.8 t 0.2 0.2 t 0.0 7.8 ? 0.3 Boar 22.4 -+ 1.8 924 t 239 320 t 73.3 265 2 12.3 9.3 2 0.2 2.5 ? 0.2 590 ? 43 3016 i 335 22.0 i 2.8 37.6 ? 0.4 2.2 t 0.2 1.7 i 0.1 0.1 2 0.0 15.5 ? 1.6 Dog ? 1.8 0.1 t 0.1 t 0.8 2 t 2.1 -c 0.8 12.2 -e 1.1 1609 I 266 671 t 93.1 157 2 24.7 23.4 t 2.9 2.1 ? 0.4 231 L 6.8 4440 t- 38 20.2 23.1 14.4 0.94 0.21 9.8 Rabbit 1.5 t 1.2 i 0.1 i 0.1 i 0.1 t 1.3 i 16.8 ?1.3 1406 2 176 1085 t 108 234 i 37.1 26.4 i 0.7 1.4 i 0.1 333 2 7.2 4200 i 154 17.0 18.2 4.4 0.2 0.05 10.0 Guinea-pig 29.5 976 1318 142 14.0 1.7 190 4093 20.0 - 24.3 43.1 2.6 0.22 Rat TABLE 11. The Concentration of Some Ionic, Enzymatic, and Nonenzymatic Constituents of Epididymal Plasma From the Cauda Epididymis (Jones, 1978) 5 *. s s Q h Y Epididymal Compounds and Effect on Sperm 0 2 m In t: 9 tl m m 1 d 4 m 0 ?+ 2m m 0 rl ! Iy i 2 ti 0, 2 1 0 ti t- (0 I x 2 tl 3 In 0 $ OD 0.1 tl 3 N 9 m (0 g .. 222 m N tI 0 % t7Y tl a0 a rl , 2 8 ti tl m 2 m 2 m tl cuW 0 9 2 3 tl NC91 y t-do $1 tl t i I 2 2 I I I 2 rl Lo m OD W 0 0 a! o? * tl W cuW 1 2 2 W 0 m N tl $1 W m rl cu m e ". OD 0.1 m 2m 2 ". 0 5 E: t- tI $1 $1 tl +I t: Lu t? y! W W * Lu (0 ?+ 1 3 t- x 2 cn P4 0.1 + 2 8 tl +I tl m y! m (0 m m rl 8 N m m 0 tI e m m us 0 3 2 2 t' tl tl tl $1 m m m m m m 2 I tI cn m fl t0 "'! a, m 01 t- m 3 m (0 0 tI W tl 0 1 ; tl tl rl N W tl t- 2 "'! a, 2 2 2m _1 h m h 0 3 cn tI d m 147 148 White is essentially similar in composition from one mammalian species to another, although there may be differences in the concentrations of some constituents. However, the epididymal plasma differs considerably from blood, lymph, and other extracellular fluids. The environment of spermatozoa in the epididymis is, therefore, highly specialized, and presumably in some way contributes to the prolonged survival of spermatozoa in this organ, and provides substrates for the metabolism of the spermatozoa. The chief characteristics of the cauda epididymal plasma are the low concentration of inorganic ions and the high levels of several unusual organic constituents, namely, glycerylphosphylcholine,carnitine, sialic acid, hypotaurine, glycosidases, and phosphatases. The concentrations of Na' ions are about 20.0 mEqiliter which is much less than in blood plasma [Jones, 19781.The levels ofK+ions generally equal or exceed that of Na+; in the monkey, in particular, K ' levels are twice those of Na' . It is generally agreed that pH values vary along the length of the epididymis, but are usually within the range of 6.5 to 7.0 [Levine & Marsh, 1971; Jones & Glover, 1973; Wales et al, 1966; Levine & Kelly, 19783, although occasionally higher values have been obtained [White & Wales, 19611. Glycerylphosphorylcholine(GPC), Glycerol, and a-Chlorohydrin One of the quantitatively most important constitutents of epididymal fluid is the andogen-dependent glycerylphosphorylcholine.GPC is a specific secretory product of the epididymis. It is found principally in the caput [Wallace et al, 19661, and very high concentrations accumulate in cauda epididymal plasma. That it is, in fact, the epididymis and not the testis that is responsible for the production of GPC is shown by the progressive increase in its concentration in fluids taken from the rete testis, the head and tail of the epididymis, and the vas deferens of the ram and bull [White, 19731. Concurrent analyses indicate that the concentration of sodium and chloride ions decreases, while the potassium concentration remains about the same. It would appear, therefore, that at least some sodium chloride may be replaced by GPC as fluid passes from the rete testis through to the vas deferens, and the osmotic pressure of the lumen fluid tends to be maintained, in the face of loss of sodium chloride, by the secretion of GPC from the epithelial cells lining the lumen. When labeled 32Pis injected into guinea pigs, rats, and rabbits, it is incorporated into the GPC of the epididymis within several hours. Phospholipid has been suggested as a possible precursor of GPC; the specific activity of phospholipid was always less than GPC in experiments on the rabbit, however, and this suggests a pathway of GPC synthesis in the epididymis that may not proceed via lecithin [White, 19723. The epididymal epithelium oxidizes lipids and some GPC may also accummulate in the epididymal lumen as a result of this activity and perhaps to a lesser extent as the result of oxidation of phospholipids by sperm. Clearly, as fluid passes through the epididymis, tremendous quantities of GPC are added to it and this raises the question of the function, if any, of GPC in that organ, and a possible interrelation with the orally active male antifertility agent, a-chlorohydrin, which is concentrated in the epididymis and bears a striking structural resemblance to GPC. It is possible that GPC might be in part responsible for the fact that spermatozoa can survive for long periods in the epididymis; GPC might even play some part in the maturation process that occurs as spermatozoa pass through the epididymis. GPC, as such, is not metabolized by spermatozoa, but the female tract secretions of many species possess a diesterase capable of splitting off the choline leaving phosphorylglycerol, which spermatozoa can metabolize [White & Wallace, 19611. The physiological significance of this reaction is uncertain, but it is at least theoretically possible that GPC can act as a substrate for sperm metabolism in the female genital tract although not in the epididymis. Spermatozoa can metabolize glycerol [Mann & Whate, Epididymal Compounds and Effect on Sperm 149 1957; White, 19571, and Brooks 119791 has suggested that it might serve as an energy substrate for spermatozoa in the epididymis, although reliable estimates of glycerol in the epididymis are not yet available. Glycerol could pass to the epididymal lumen, either as a result of lipid metabolism in the epididymal epithelium, or by transport from the blood stream in a manner analagous to the accumulation of radioactivity by the epididymis following the administration of radioactive a-chlorohydrin [Crabo & Appelgren, 1972; Edwards et al, 19751. Glycerol enters the glycolytic pathway of the spermatozoon through a phosphorylation step followed by oxidization to dihydroxyacetone phosphate catalyzed by mitochondrial glycerol phosphate dehydrogenase. Studies on ram spermatozoa indicate that the glycerol analogue, a-chlorohydrin exerts its detrimental effect on spermatozoa by blocking the glycolytic pathway a t the glyceraldhyde phosphate stage. However, a-chlorohydrin does not itself appear to be the actual active compound causing disruption of sperm metabolism. Preincubation of sperm with a-chlorohydrin is required before inhibition of the metabolism of glycerol or glucose occurs [Edwards et al, 19761 and a-chlorohydrin does not itself inhibit the glycolytic enzymes in ram sperm [Mohri et al, 1975; Brown-Woodman et al, 19781. It is probably a metabolic product of a-chlorohydrin that inhibits the enzymes ofthe glycolytic sequence in sperm [Mohri et al, 19751 and, in particular, glyceraldehyde phosphate dehydrogenase. Carnitine The rat cauda epididymis has the highest carnitine and acetylcarnitine content of any tissue [Marquis & Fritz, 19651.Carnitine has been found in epididymal plasma of the bull [Casillas, 19731, in the epididymal tissue of monkeys and boars [Casillas, 19721, and in fluid from the epididymis and seminal vesicles of humans [Lewin, 19761. Accumulation of carnitine by the epididymis is similar to GPC in being dependent on this androgen. However, carnitine is not synthesized by the epididymis[Casillas & Erickson, 1975al but is concentrated from the blood, so that in humans, for example, the epididymal carnitine concentration is 10-50 times higher than blood plasma [Bohner et al, 19741. The regions involved in carnitine transport from the blood are the distal caput, corpus, and proximal cauda with the consequence that the concentration of carnitine progressively increases throughout the length of the epididymis [Brooks et al, 19741. Its high concentration must contribute to the osmotic pressure of the fluid and at least partly redress the loss of sodium chloride in the proximal region of the duct. The role of carnitine as a carrier of fatty acids across mitochondrial membranes is well established in other tissues and acetylcarnitine may serve as an energy reservoir and buffer against rapid changes in the concentration of acetyl-CoA in spermatozoa. It has also been suggested that the maturation of sperm in the epididymis may be related to accumulation of carnitine in the sperm during epididymal transit. Due to membrane changes during maturation, only caput spermatozoa are permeable to carnitine [Casillas, 19731, although both cauda and caput spermatozoa are apparently permeable to acetylcarnitine. It is now clear that the acetylation state of carnitine can be profoundly influenced by the presence of exogenous substrates [Casillas & Erickson, 1975b; Milkowski et al, 1976; Van Dop et al, 19771 and that acetylcarnitine can act as a ready source of oxidizable acetyl units for spermatozoa [Storey & Keyhani, 1974;Hutson et al, 1977a1,in addition to serving as a buffer to acetylCoA levels. It is possible that acetylcarnitine represents an important energy store in spermatozoa particularly as bovine epididymal spermatozoa incubated with substrates, eg, fructose, glucose, pyruvate, lactate, and acetate, have a higher acetylcarnitine content than spermatozoa incubated without substrate [Casillas & Erickson, 1975131. 150 White Inositol The epididymis contains significant amounts of inositol [Eisenberg & Bolden, 19641, and although it is carried to the epididymis in the rete testis fluid [Setchell et al, 19683, inositol may also be accumulated from the blood stream [Lewin et al, 19761. Lactic Acid Lactic acid occurs in epididymal plasma as well as testicular fluid [Scott et al, 1963b; Setchell et al, 1969; Setchell, 1970; White, 19731, and the presence of this lactic acid probably accounts for the slightly acidic nature of caudal plasma. Values for lactic acid were substantially higher in epididymal plasma which had been collected from rabbits postmortem compared with material obtained from anaesthetized animals [Jones, 19781, presumably due to tissue anoxia. The production of lactate by the rat epididymis would seem sufficient to meet the needs of spermatozoa. However, in addition to such lactate resulting from epididymal glycolysis, it might also be transported from the blood stream to the epididymal lumen. The oxidation of lactate by spermatozoa requires oxygen and from the measurements of Cross and Silver ,epididymal tissue appearsto be reasonably aerobic; this has been confirmed by direct intraluminal measurements of oxygen tension by Free et a1 [ 19761. Amino Acids A number of amino acids occur in cauda epididymal fluid in concentrations greater than in rete testis fluid, due presumably to selective resorption of fluid in the head of the epididymis. Quantitatively, glutamate (10-30 mM) is the most important amino acid in epididymal plasma of the ram and bull [Setchell et al, 1967; Sexton et al, 1971;Johnson et al, 1972; Brown-Woodman & White, 19741. In boar and rabbit epididymal plasma hypotaurine (50 mM) is the principal amino acid. Some of the 12 or so amino acids present in epididymal plasma [Neumark & Schindler, 19671 are potential metabolites for both the spermatozoa and epididymal epithelium, although they do not increase the oxygen uptake of the washed spermatozoa and are only oxidized a t a slow rate. Lipids Fatty acids could be made available for sperm metabolism as a result of metabolic activity of the epididymal epithelium or by the transport of fatty acids from the blood stream into the epididymal lumen. Free fatty acids could not, however, be detected in epididymal plasma from the ram, boar, or rabbit [Jones, 19783, but palmitic, stearic, and oleic acids have been found in cauda epididymal plasma of the rat at about five times the concentration of the blood plasma [Brooks et al, 19741. In addition to substrate supplied from the epididymal plasma, it is possible that the spermatozoa themselves are endowed with intracellular fuel. The traditional view is that fatty acids derived from the side chains of the phospholipid components of the spermatozoa constitute this substrate during epididymal transit and storage. Decreases in the total phospholipid content of spermatozoa during epididymal transit have been reported for the ram [Scott et al, 19671, bull [Poulos et al, 19731, boar [Grogan et al, 19661, rat [Terner et al, 19751,and monkey [Arora et al, 19751.The total loss of lipid in bovine sperm during epididymal transit would be sufficient to support sperm respiration for 28 hr. However, as epididymal transit takes about 14 days, it is clear that endogenous lipid is not sufficient on its own to maintain sperm metabolism. Some workers have even questioned the ability of a t least ejaculated ram and human spermatozoa to utilize endogenous phospholipids [Poulos & White, 1973; Darin-Bennett, Poulos 8.z White, 19731. Epididymal Compounds and Effect on Sperm 151 Steroids Several steroids occur in epididymal as well as testicular fluid. Ganjam and Amann [ 1973, 19761 report the presence of high concentrations of testosterone, dihydrotestosterone, 3-P-androstanediol, dehyroepiandiosterone, and progesterone in cauda epididymal fluid of the bull. The spermatozoa bind, in a nonsaturable fashion, various steroids with different affinities [Amann & Hammerstedt, 19761, but there are few, if any, specific, saturable, high-affinity, steroid-binding sites on bull spermatozoa. Spermatozoa can also interconvert certain steroids to some extent [Hammerstedt & Amann, 19761. However, in vitro, very high concentrations of steroids are required to change the metabolic parameters of spermatozoa, and even then the effects are not very spectacular [Hammerstedt & Amann, 19761. Enzymes The presence of a wide variety of hydrolytic enzymes in epididymal plasma of the Rhesus monkey and all species investigated might at first sight seem paradoxical, since these enzymes might be expected to have an adverse effect on the survival of spermatoma. It has been suggested that in the normal animal they may help remove dead or effete spermatozoa from the epididymis, thereby ensuring that only normal fertile spermatozoa are ejaculated [Mann, 1964; Waldschmidt et al, 1964; Mennela & Jones, 19771.They may also have some role during sperm maturation, such as modification of membrane-bound surface glycoproteins. Sialic Acid and Glycoprotein Another androgen-dependent constituent of the cauda epididymis is the amino-sugar sialic acid which has been shown to occur in particularly high concentrations bound to protein in the cauda epididymis of the rat. The degree of polymerization of these sialomucoproteins influences the phy sico-chemical properties of the epididymal plasma, particularly its viscosity, and they may serve as lubricants to assist in sperm transport by reducing the friction between the densely packed sperm in the cauda [Hamilton, 19751. There have been at least two recent reports of an interaction between the glycoprotein in epididymal fluid and epididymal sperm suggesting that glycoproteinsmay be involved in sperm maturation. Thus Brandt et a1 [19781 have shown that motility may be induced in immotile sperm taken from the caput epididymis of the bull by exposing them to a mixture of cyclic AMP phosphodiesterase inhibitor, eg, theophylline and a glycoprotein factor in the seminal plasma. Zimmermann et a1 L19791working with the boar have found that as sperm mature in the epididymis they become less permeable to Na+ and K+ and that the factor responsible is probably a glycoprotein in the cauda plasma. SURVIVAL OF SPERM IN EPIDIDYMIS Experiments on the ligated epididymis of the rabbit, bull, and ram show that conditions in the epididymis are peculiarly conducive to the survival of spermatozoa. The ability of spermatozoa to survive for long periods in the epididymis may be linked with either physical or chemical conditions in the organ, and it is generally believed that spermatozoa are immotile, or nearly so, in the epididymis and that their metabolism is in a quiescent state. Although there is no direct evidence that sperm are, in fact, in an inactive state in the cauda epididymis, there has been no lack of theories as to why this may be so. These involve (1)low oxygen tension, (2) lack of substrate, (3) low pH, (4) high carbon dioxide tension, (5)high potassium, (6) hypertonity, (7) dense packing of the sperm, and (8) the presence of a metabolic regulator in the sperm. 152 White The question of what constitutes the substrate for the metabolism of spermatozoa stored in the epididymis is an intriguing one and might constitute a point of attack for decreasing their viability. Conditions in the tubules are probably aerobic, at least immediately next to the epithelium and even if, as is generally supposed, spermatozoa are in a quiescent state, at least some basal metabolism might be expected to maintain the integrity of the cells, The relatively high concentration of lactic acid and acetyl carnitine in the epididymal lumen may provide substrate for spermatozoal metabolism. Although GPC occurs in tremendous quantities in the luminal fluid, it cannot be used directly by spermatozoa; glutamic acid is only oxidized extremely slowly and there is little fructose, glucose, or acetate present. If, of course, there were a constant interchange between epididymal fluid and the blood, a substrate such as glucose could still be of importance in the economy of the spermatozoa, despite a low level at any one time. Some recent studies on the ram and rabbit are of interest in this regard, in that they show that intravenously infused 14C glucose appears in the fluid of the cauda epididymis. CONCLUSIONS Detailed knowledge of the nature of testicular fluid, which forms the basis of epididymal fluid, is available for the ram, bull, boar, and rat but information on primates is very meager. More information is available on the composition of epididymal fluid of the rhesus monkey, but there are still many gaps in knowledge compared to the situation for domestic species. The significance of the high concentration of unusual compounds eg, glycerylphosphorylcholine, carnitine, amino acids, and inositol, in epididymal fluid is unknown. The fact that a-chlorohydrin is concentrated in the epididymis and inhibits the metabolism of sperm gives hope that a satisfactory orally active male contraceptive may eventually be found that interferes with sperm in the epididymis. REFERENCES Amann, R.P.; Hammerstedt, R.H. Binding of steroids by intact sperm. BIOLOGY OF REPRODUCTION 15: 670-677, 1976. Arora, R.; D i n a k a r , N.; Prasad, M.R.N. Biochemical changes in the spermatozoa and luminal contents of different regions of the epididymis of the rhesus monkey (Macaca rnulatta). CONTRACEPTION 11: 689-700, 1975. Bedford, J.M. Maturation, transport, and fate of spermatozoa in the epididymis. pp 303-317, Section 7, Vol. V i n HANDBOOK O F PHYSIOLOGY. American Physiological Society. D.W. Hamilton; R.O. Greep, eds. Washington, D.C., 1975. Bohmer, T.; Rydning, A,; Solberg, H.E. Carnitine levels in human serum in health and disease. CLINICAL CHEMISTRY ACTA 57: 55-61, 1974. Johnson, D.J.; Hoskins, Brandt, H.; Ascott, T.S.; D.D. Evidence for a n epididymal origin of bovine sperm forward motility protein. BIOLOGY OF REPRODUCTION 19: 830-835, 1978. Brooks, D.E. Biochemical environment of sperm maturation. pp. 23-34 in THE SPERMATO- ZOON. D.W. Fawcett; J.M. Bedford, eds. Baltimore, Urban and Schwanenberg, 1979. Brooks, D.E.; Hamilton, D.W.; Mallek, A.H. Carnitine and glycerylphosphorylcholine in the reproductive tract of the male rat. JOURNAL OF REPRODUCTION AND FERTILITY 36: 141-160, 1974. Brown-Woodman, P.D.G.; Mohri, H.; Mohri, T.; Suter, D.; White, I.G. Mode of action of achlorhydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by a-chlorohydrin and location of block i n glycolysis. BIOCHEMICAL JOURNAL 170: 27-37, 1978. Brown-Woodman, P.D.C.; White, I.G. Amino acid composition of semen and the secretions of the male reproductive tract. AUSTRALIAN JOURNAL OF BIOLOGICAL SCIENCES 27: 415-422, 1974. Casillas, E.R. Accumulation of carnitine by bovine spermatozoa during maturation in the epididymis. JOURNAL OF BIOLOGICAL CHEMISTRY 248: 8227-8232, 1973. Casillas, E.R. The distribution of carnitine in male reproductive tissues and its effect on palmitate oxidation by spermatozoa1 particles. Epididymal Compounds and Effect on Sperm BIOCHIMICA BIOPHYSICA ACTA 280: 545-551,1972. Casillas, E.R.; Erickson, B.J. Studies on carnitine synthesis in the rat epididymis. JOIJRNAL OF REPRODUCTION AND FERTILITY 44: 287-291, 1975a. Casillas, E.R.; Erickson, B.J. The role of carnitine in spermatozoan metabolism: Substrate induced elevations in the acetylation state of carnitine and coenzyme A in bovine and monkey spermatozoa. BIOLOGY OF REPRODUCTION 12: 275-283, 197510. Crabo, B.; Appelgren, L-E. Distribution of ['4C]a-chlorohydrin in mice and rats. JOURNAL OF REPRODUCTION AND FERTILITY 30: 161-163, 1972. Cross, B.A.; Silver, LA. Neurovascular control of oxygen tension in the testis and epididymis. JOURNAL OF REPRODUCTION AND FERTILITY 3: 377-395, 1962. Darin-Bennett, Annabelle; Poulos, A,; White, I.G. A re-examination of the role of phospholipids as energy substrates during incubation of ram spermatozoa. JOURNAL O F REPRODUCTION AND FERTILITY 34: 543546, 1973. Edwards, E.M.; Dacheux, J-L.; Waites, G.M.H. Effects of a-chlorohydrin on the metabolism of testicular and epididymal spermatozoa of rams. JOURNAL O F REPRODUCTION AND FERTILITY 48: 265-270, 1976. Edwards,E.M.;Jones,A.R.; Waites,G.M.H.The entry of a-chlorohydrininto body fluids of male rats and its effect upon the incorporation of glycerol into lipids. JOURNAL OF REPRODUCTION AND FERTILITY 43: 225-232, 1975. Eisenberg, F.; Bolden, A.H. Reproductive tract as site of synthesis and secretion of inositol in the male rat. NATURE (LONDON) 202: 599600, 1964. Free, M.J.; Schluntz, G.A.; Jaffe, R.A. Respiratory gas tensions in tissues and fluids of the male rat reproductive tract. BIOLOGY OF REPRODUCTION 14: 481-488, 1976. Ganjam, V.K.; Amann, R.P. Testosterone and dehydrotestosterone concentration in the fluid milieu of spermatozoa in the reproductive tract of the bull. ACTA ENDOCRINOLOGICA 7 4 186-200, 1973. Ganjam, V.I.C.; Amann, R.P. Steroids in fluids and sperm entering and leaving the bovine epididymis, epididymal tissue, and accessory sex gland secretions. ENDOCRINOLOGY 99: 1618-1630, 1976. Glover, T.D. Investigations into the physiology of the epididymis in relation tomale contraception. JOURNAL OF REPRODUCTION AND FERTILITY, SUPPLEMENT 24: 95-114, 1976. Grogan, D.E.; Mayer, D.T.; Sikes, J.D. Quantitative differences in phospholipids of ejaculated spermatozoa and spermatozoa from three levelsof the epididymis ofthe boar. JOURNAL 153 OF REPRODUCTION AND FERTILITY 12: 431-436,1966. Hamilton, U.W. Structure and function of the epithelium lining the ductuli efferentes, ductus epididymidis, and ductus deferens in the rat. pp 259-301, Sect. 7, Vol. V in HANDBOOK OF PHYSIOLOGY. D.W. Hamilton; R.O. Greep, eds. Washington, D.C. American Physiological Society, 1975. Hammerstedt, R.H.; Amann, R.P. Interconversion of steroids by intact bovine sperm and epididymal tissue. BIOLOGY OF REPRODUCTION 15: 686-694, 1976. Hammerstedt, R.H.; Amann, R.P. Effect of physiological levels of exogenous steroids on metabolism of' testicular, cauda epididymal and ejaculated bovine spermatozoa. BIOLOGY OF REPRODUCTION 15: 678-685, 1976. Hansson, F.; Weddington, S.C.; French, F.S.; McLean, W.; Smith, A,; Nayfeh, S.N.; Ritzen, E.M.; Hagenas, L. Secretion and role of androgen binding proteins i n t h e testis and epididymis. JOURNAL OF REPRODUCTION AND FERTILITY, SUPPLEMENT 24: 17-23, 1976. Hutson, S.M.; V a n Dop, C.; Lardy, H.A. Mitochondria1 metabolism of pyruvate i n bovine spermatozoa. JOURNAL OF BIOLOGICAL CHEMISTRY 252: 1309-1315, 1977a. Johnson, L.A.; Pursel, V.G.; Gerrits, R.J.; Thomas, C.H. Free amino acid composition of porcine seminal, epididymal and seminal vesicle fluids. JOURNAL OF ANIMAL SCIENCES 34: 430-434, 1972. Jones, R. Comparative biochemistry of mammalian epididymal plasma. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY 61B: 365-370,1978. Jones, R.; Glover, T.D. The collection and composition of epididymal plasma from the cauda epididymis of the rabbit. JOURNAL OF REPRODUCTION AND FERTILITY 34: 395403, 1973. Levine, N.; Kelly, H. Measurement of pH in the rat epididymis in uiuo. JOURNAL OF REPRODUCTION AND FERTILITY 52: 333335, 1978. Levin, N.; Marsh, D.J. Micropuncture studies on the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, and epididymis, and the vas deferens in rats. JOURNAL OF PHYSIOLOGY 213: 557-570, 1971. Lewin, L.M.; Beer, R.; Lunenfeld, B. Epididymis and seminal vesicles as sources of carnitine in human seminal fluid: The clinical significance of the carnitine concentration in human seminal fluid. FERTILITY AND STERILITY 27: 9-13, 1976. Lewin, L.M.; Yannai, Y.; Sulimovici, S.; Kraicer, P.F. Studies on the metabolic role of myo-inositol. Distribution of radioactive myoinositol in the male rat. BIOCHEMICAL JOURNAL 156: 375380, 1976. 154 White Mann, T. THE BIOCHEMISTRY OF SEMEN AND OF THE MALE REPRODUCTIVE TRACT. Methuen, London, 1964. Mann, T.; White, I.G. Glycerol metabolism by spermatozoa. BIOCHEMICAL JOURNAL 65: 634-639, 1957. Marquis, N.R.; Fritz, I.B. Effects of testosterone on the distribution of carnitine, acetylcarnitine and carnitine acetyltransferase in tissues of the reproductive system of the male rat. JOURNAL OF BIOLOGICAL CHEMISTRY 240: 2197-2200, 1963. Mennella, M.R.F.; Jones, R. The activity of some nucleolytic enzymes in semen and in the secretions of the male reproductive tract. ANDROMGIA 9: 15-22, 1977. Milkowski,A.L.; Babcock,D.F.;Lardy, H.A.Activation of bovine epididymal sperm respiration by caffeine. Its transient nature and relationship to the utilization of acetyl carnitine. ARCHIVES O F BIOCHEMISTRY AND BIOPHYSICS 176: 250-256, 1976. Mohri, H.; Suter, D.A.I.; Brown-Woodman, P.D.C.; White, I.G.; Ridley, D.D. Identification of the biochemical lesion produced by achlorohydrin i n spermatozoa. NATURE (LONDON) 255: 75-77, 1975. Neumark, H.; Schindler, H. Amino acids, m i n e s and peptides of ram epididymal semen. JOURNAL O F REPRODUCTION AND FERTILITY 14: 469-471, 1967. Orgebin-Crist, M-C.; Danzo, B.J.; Cooper, T.G. Re-examination of the dependence of t h e epididymal sperm viability on the epididymal environment. JOURNAL OF REPRODUCTION AND FERTILITY, SUPPLEMENT 24: 115-128, 1976. Poulos, A.; Voglmayr, J.K.; White, I.G. Phospholipid changes in spermatozoa during passage through the genital tract of the bull. BIOCHIMICA BIOPHYSICA ACTA 306: 194-202,1973. Poulos. A,; White, I.G. Phospholipids of human spermatozoa and seminal plasma. JOURNAL OF REPRODUCTION AND FERTILITY 35: 265-272, 1973. Scott, T.W.; Voglmayr, J.K.; Setchell, B.P. Lipid composition and metabolism in testicular and ejaculated ram spermatozoa. BIOCHEMICAL JOURNAL 102: 456-461, 1967. Scott, T.W.; Wales, R.G.; Wallace, J.C.; White, I.G. Composition of ram epididymal and testicular fluid and the biosynthesis of glycerylphosphorylcholine by the rabbit epididymis. JOURNAL OF REPRODUCTION AND FERTILITY, SUPPLEMENT 6: 49-59,1963b. Setchell, B.P. Testicular blood supply, lymphatic drainage and secretion of fluid pp 101239, Vol. 1in THE TEST1SA.D. Johnson; W.R. Gomes; N.L. Van Denmark, eds. New York and London, Academic Press, 1970. Setchell, B.P. Secretions of the testis and epididymis. JOURNAL OF REPRODUCTION AND FERTILITY 07: 165-177, 1974. Setchell, B.P.; Dawson, R.M.C.; White, R.W. The high concentration of free myo-inositol in rete testis fluid from rams. JOURNAL O F REPRODUCTION AND FERTILITY 17: 219220, 1968. Setchell, B.P.; Hinks, N.T.; Voglmayr, J.K.; Scott, T.W. Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa. BIOCHEMICAL JOURNAL 105: 1061-1065, 1967. Setchell, B.P.; Scott, T.W.; Voglmayr, J.K.; Waites, G.M.H. Characteristics of testicular spermatozoa and the fluid which transports them into the epididymis. BIOLOGY O F REPRODUCTION, SUPPLEMENT 1: 40-66, 1969. Setchell, B.P.; Waites, G.M.H. The blood testis barrier. pp 143-172 in Section 7, Vol. V in HANDBOOK O F PHYSIOLOGY. D.W. Hamilton; R.O. Greep, eds. Washington, D.C., American Physiological Society, 1975. Sexton, T.J.; Amann, R.P.; Flipse, R.J. Free amino acids and protein in rete testis fluid, vas deferens plasma, accessory sex gland fluid and seminal plasma of the conscious bull. JOURNALOFDAIRY SCIENCE 54: 412-416,1971. Storey, B.T.; Keyhani, E. Energy metabolism of spermatozoa I1 comparison of pyruvate and fatty acid oxidation by mitochondria of rabbit epididymal spermatozoa. FERTILITY AND STERILITY 25: 857-864, 1974. Terner, C.; Mallaughlin, J . ; Smith, B.R. Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymis. JOURNAL OF REPRODUCTION AND FERTILITY 45: 1-8, 1975. Van Dop, C.; Hutson, S.M.; L a d y , H.A. Pyruvate metabolism in bovine epididymal spermatozoa. JOURNAL O F BIOLOGICAL CHEMISTRY 252: 1303-1308,1977. Voglmayr, J.K. Metabolic changes i n spermatozoa during epididymal transit. pp 437460, Sect. 7, Vol. V in HANDBOOK O F PHYSIOLOGY. D.W. Hamilton; R.O. Greep, eds. Washington, D.C. American Physiological Society, 1975. Voglmayr, J.K.; Amann, R.P. The distribution of free myo-inositol in fluids, spermatozoa and tissues of the bull genital tract and observations on its uptake by the rabbit epididymis. BIOLOGY OF REPRODUCTION 8: 504-513, 1973. Voglmayr, J.K.; White, I.G. Synthesis and metabolism of myo-insitol in testicular and ejaculated spermatozoa of the ram. JOURNAL OF REPRODUCTION AND FERTILITY 24: 29-37, 1971. Waites, G.M.H. Permeability of the seminiferous tubules and the rete testis to natural and synthetic compounds. JOURNAL O F REPRODUCTION AND FERTILITY, SUPPLEMENT 24: 49-69, 1976. Waldschmidt, M.; Karg, H.; Kinzler, M. Ver- Epididymal Compounds and Effect on Sperm kommen von Desoxyribonuklease in mannlichen Geschlectssekneten beim Rind. NATURWISSENSCHAFTEN 51: 364, 1964. Wales, R.G.; Wallace, J.C.; White, I.G. Composition of bull epididymal and testicular fluid. JOURNAL OF REPRODUCTION AND FERTILlTY 12: 139-144, 1966. Wallace, J.C.: Wales, R.G.; White, I.G. The respiration of the rabbit epididymis and its synthesis of glycerylphosphorylcholine. AUSTRALIAN JOURNAL O F BIOLOGICAL SCIENCES 19: 849-856, 1966. White, I.G. Metabolism of glycerol and similar compounds by bull spermotozoa. AMERICAN JOURNAL OF PHYSIOLOGY 189: 307-310, 1957. White I.G. Metabolism of sperm with particular reference to the epididymis. ADVANCES IN BIOSCIENCES 10: 157-168,1972. White, I.G. Biochemical aspects of spermatozoa and their environment in the male reproductive tract. JOURNAL OF REPRODUCTION 155 AND FERTILITY, SUPPLEMENT 18: 223235,1973. White, I.G. Accessory sex organs and fluids of the male reproductive tract. pp 105-123 in ANIMAL MODELS FOR RESEARCH ON CONTRACEPTION AND FERTILITY. N.G. Alexander, ed. New York, Harper and Row, 1979. White, I.G.; Wales, R.G. Comparison of epididymal and ejaculated semen of the ram. JOURNAL OF REPRODUCTION AND FERTILITY 21: 225-237, 1961. White, I.G.; Wallace, J.C. Breakdownofseminal glycerylphosphorylcholineby secretions of the female reproductive tract. NATURE (LONDON) 189: 843-844, 1961. Zimmerman, K.J.; Crabo, B.G.; Moore, K.; Weisberg, S.; Deihel, F.C.; Graham, E.F. Movements of sodium and potassium into epididymal boar spermatozoa. BIOLOGY O F REPRO1)UCTION 21: 173-179, 1979.