Thaddeus Mann

Lipid peroxidation in spermatozoa.

Ram spermatozoa produce aerobically an organic peroxide which can be determined quantitatively by the thiobarbituric acid reaction. The reaction is more intense in spermatozoa that have been stored at 5°C. Cold shock and homogenization release from spermatozoa a substance, presumably a lipid, which provides the substrate for peroxidation. Lipid peroxidation is probably linked with the decline in motility and respiratory activity, and the structural damage to sperm membranes, which characterize ageing or degenerating spermatozoa.

Passage of Chemicals into Human and Animal Semen: Mechanisms and Significance

This review will begin with a brief account of the secretory mechanisms operating in the male reproductive tract. The entry of the different chemicals will be dealt with; with separate reference to testicular fluids, epididymal plasma, prostatic and vesicular secretions, and whole semen. Examples will be given of a number of chemical substances capable of passing into the secretions of the male reproductive tract and semen, in man and in animals--antispermatogenic and antiandrogenic agents, industrial chemicals, chemotherapeutic drugs, food additives, etc. The review will end with a critical appraisal of the methods involved and the results obtained from analyses of various chemicals in semen.

Sorbitol metabolism in spermatozoa

Sorbitol was identified as a chemical constituent of the seminal plasma in the ram, rabbit, bull, boar, stallion, cock and man. Its concentration was determined by an enzymic-spectrophotometric method based on the use of purified sorbitol dehydrogenase from liver. Ram spermatozoa oxidize sorbitol to fructose, and they are also capable of reducing fructose, and to a smaller extent glucose, to sorbitol. The oxidative conversion of sorbitol to fructose makes it probable that sorbitol contributes to the lactic acid which accumulates in semen incubated in vitro. Triphenyl tetrazonium chloride was used as an electron acceptor instead of molecular oxygen, to study the oxidative behavior of ram spermatozoa towards sorbitol, fructose, and glucose. Cold shock’ treatment, which rapidly immobilizes spermatozoa, produced little or no effect on the rate of triphenyl tetrazonium chloride reduction, either in the absence or in the presence of sorbitol, fructose, or glucose. Prolonged aerobic incubation of a washed sperm suspension which depletes the spermatozoa of ‘endogenous’ substrate, did not abolish the ability of the‘exhausted’ sperm cells to reduce triphenyl tetrazonium chloride in the presence of sorbitol, fructose, or glucose. Sorbitol dehydrogenase, the enzyme responsible for the reversible oxidoreduction of sorbitol to fructose in ram spermatozoa, was prepared in a soluble and highly active form, by grinding sperm with lavigated aluminium oxide and extraction with phosphate buffer. The soluble enzyme preparation requires diphosphopyridine nucleotide (DPN) as coenzyme, and catalyzes the reaction. sorbitol + DPN+ ⇌ fructose + DPNH+ H+. The oxidizing activity of sperm sorbitol dehydrogenase is highest towards D-sorbitol and L-iditol, and much lower towards D-xylitol; D-ribitol, D-mannitol, and D-arabitol are only poorly oxidized. In the reverse reaction, which involves the reduction of sugars to polyols, only fructose, and to a smaller extent, sorbose, are reduced. The Michaelis constants (Km) obtained were 9.8 x 10-3 for serbitol, and 9.0 x 10-3 M for iditol. In intact spermatozoa the steady state of the reversible enzymic conversion of sorbitol to fructose depends upon the actual ratio between oxidized and reduced DPN. It is suggested that upon the respective levels of fructose and sorbitol in seminal plasma may depend the ratio between the oxidized and reduced form of DPN within the sperm cells themselves.

Biochemistry of Seminal Plasma and Male Accessory Fluids; Application to Andrological Problems

Many years were to pass after this pronouncement upon the uniqueness of the seminal plasma by Louis Nicolas Vauquelin (author of ‘Experiences sur le sperme humain’, the earliest treatise on the chemistry of semen), before its origin and site of formation were properly established and the complex chemical nature of this fluid began to be inquired into.

Adenosine 3´:5´-cyclic monophosphate in relation to motility and senescence of spermatozoa

Spermatozoa of the ram, bull, boar, stallion and cock contain 37 to 177 pmol cyclic AMP/109 cells. Slow cooling of spermatozoa followed by storage causes only a minor decrease but cold shock leads to a rapid and nearly total loss of cyclic AMP, some of which passes into the external medium. Fructose prevents the loss of cyclic AMP in anaerobically incubated spermatozoa. Motility and synthesis of cyclic AMP are abolished by fluoride, but both are restored after removal of fluoride by washing. Determinations of cyclic AMP represent a sensitive indicator of the onset of senescence changes in spermatozoa.

Secretory Function of the Prostate, Seminal Vesicle, Cowper’s Gland and Other Accessory Organs of Reproduction

The complicated make-up of the seminal plasma, and the role which the male accessory secretions play in the elaboration of this body fluid did not escape the notice of Littre and other anatomists of his day, but over two centuries passed before the properties of the individual secretions were recognized and their physicochemical characteristics elucidated. It took some time again before primitive examination of accessory glands by palpation or at laparotomy as the sole criterion of their functional condition was replaced by quantifiable biochemical methods. The rationale for the adoption of the modern approach rests upon the fact that these glands secrete a number of characteristic substances that at ejaculation enter the semen, in which they can be determined accurately in very small amounts of material. The chemical methods have stood the test of time well and have been discussed at length (Mann 1974b, 1975a; Mann and Lutwak-Mann 1976). Only relatively recently, as a result of chemical investigations, has the extent of the remarkable species differences in the secretory activity of the accessory glands received due attention. At the same time it became clear that species variations apart, there are considerable individual fluctuations in the secretory output, arising from differences in the size of accessory organs and conditioned by hormonal influences in the prostate and seminal vesicles in particular.

Effect of nutrition on the onset of male sex hormone activity and sperm formation in monozygous bull-calves.

The effect of a reduced food intake on the onset of androgenic activity and the appearance of spermatozoa was studied in maturing bull-calves. Three pairs of identical twin-calves were used. In each instance, one twin was reared on a ‘high plane’ of nutrition, consisting of normal feeding, and the other on a ‘low plane’ of reduced food intake. Semen was collected from the twin-calves by the electric stimulation method, and analyzed for sperm density, fructose and citric acid. The appearance of fructose and citric acid in semen was taken as an indicator of the onset of secretory function in the seminal vesicles, which depends on the presence of the male sex hormone. Fructose and citric acid appeared in electrically-discharged semen from the normally fed bull-calves several months before the first spermatozoa. This suggested that the male sex hormone began to act in the young animal several months before the appearance of the first spermatozoa. Restriction of food intake had a marked delaying influence on the onset of fructose and citric acid secretion, and a smaller delaying effect on the appearance of spermatozoa. The delaying effect of underfeeding on the secretory function of the bull seminal vesicles as reflected in the diminished output of fructose and citric acid, appeared to be the result of an inadequate stimulation of the gonads by the gonadotrophic hormone. Injections of gonadotrophin were found to elicit a prompt appearance of both fructose and citric acid in the semen. Alterations in the composition of semen caused by underfeeding were shown to run parallel to histological changes in the testes and male accessory organs. Low-plane feeding retarded the differentiation of the seminiferous tubules and of the interstitial tissue in the male gonads. The histological changes in the seminal vesicles induced by underfeeding corresponded closely to the diminished secretory output of fructose and citric acid in these glands.

Morphology of Bull Testes and Seminal Vesicles in Relation to Testicular Androgens

Correlations were studied between various morphological and functional characteristics of the testis and seminal vesicle in bull calves and bulls, ranging in age from 28 days to 17½ years. Data obtained by the analysis of testosterone and androstenedione in the testes, and of fructose and citric acid in the seminal vesicles, were related to results of gross anatomical and histological measurements of these organs. The mean diameter of the seminiferous tubules was adopted as a means of assessing testicular function, since it was shown that this diameter is related to the weight of the testis as a whole (r = 0.92, P < 0.001), to the arrangement of the interstitial tissue, and to definite stages in the differentiation of the seminiferous epithelium . At 28 days, when the bull testis is relatively inactive, both as regards spermiogenesis and the endocrine function, the mean diameter of the seminiferous tubules was 48 μ. At the age of 90 days, coinciding with the appearance of spermatogonia in the testes, and just prior to the marked increase in fructose and citric acid formation in the seminal vesicles, it was 66 μ. At about 4½ months, when the androstenedione/testosterone ratio falls abruptly, the tubular diameter increased to more than 90 μ. By 7½ months, when the first spermatozoa appear in the tubules, it had risen to 177 μ. A significant correlation was established between the growth and secretory activity of the seminal vesicles and the diameter of the seminiferous tubules in bull testes. Correlation coefficients were established between the tubular diameter and the following: the weight of the seminal vesicles, r = 0.95, P < 0.001; the contents o f fructose and citric acid in the seminal vesicles, r = 0.81, P = 0.01 to 0.001; and the concentrations of fructose and citric acid in the seminal vesicles, r = 0.82 and r = 0.76, respectively, P = 0.01 to 0.001.

On the presence and role of inositol and certain other substances in the seminal vesicle secretion of the boar.

meso-Inositol was identified as a major chemical constituent of the seminal vesicle secretion in the boar, and isolated in pure crystalline state. The concentration of inositol in the vesicular secretion exceeds considerably the amounts hitherto recorded for material of either plant or animal origin. The fluid secreted by boar seminal vesicles contains 2 to 3 g inositol in 100 ml. The dry matter of the fluid is about 15 g/100 ml., one-third of which is dialyzable; of this between 40 and 70% is composed of inositol. Two methods are described by means of which it is possible to obtain several grams of pure crystalline inositol from the seminal vesicle secretion of a single animal. Certain other characteristic features of the boar vesicular secretion are described, including the occurrence of substances such as fructose as well as an hitherto unidentified carbohydrate, also of ergothioneine and potassium citrate; and an almost complete absence of sodium chloride. One of the functions of inositol and citrate appears to be the maintenance of the osmotic pressure in the seminal vesicle secretion, and probably also in the semen of the boar. The secretion of inositol, ergothioneine, fructose and citric acid, by the seminal vesicles, sets in at an early stage of the development of the boar, a considerable time before the maturation of spermatozoa.

Male Reproductive Function and the Composition of Semen: General Considerations

The modern era of the physiology of reproduction in the male is commonly, and rightly so, accepted as having been ushered in by Leeuwenhoek’s sensational letter to the Royal Society, dated November 1677, reporting the first-ever demonstration in semen of motile spermatozoa. From another of his famous communications, cited above and submitted 8 years later, it is evident that Leeuwenhoek intuitively associated the existence of spermatozoa with male fertilizing ability, by recognizing that even though a man may be keenly interested in the opposite sex, this alone is not enough to guarantee the birth of offspring. He anticipated, moreover, yet another basic concept of male reproductive biology, by proclaiming that for the act of procreation to be fulfilled, the spermatozoa, as well as being motile, must also be sufficiently energetic to survive in the female tract for a certain period, presumably to attain their full potential. The precise duration of that critical timespan he was, of course, unable to back up with experimental evidence.