Summary. The value of Poisson distribution theory in describing and predicting the nature of sperm—egg interaction in vitro has been investigated using an interspecific in-vitro fertilization system, incorporating zona-free hamster oocytes and human spermatozoa. The frequency distribution of polyspermic oocyte penetrations in 72 experiments exhibited good agreement with the Poisson distribution at all levels of fertilization, indicating that each oocyte must be of equal penetrability and that there can be no block to polyspermy in this interspecific system. Poisson distribution theory also accurately described the relationship between oocyte penetration and sperm motility in 50 out of 54 separate experiments spread across 10 serial dilution curves. For each dilution series the shape of the fitted curve was fixed but its location along the x-axis varied from donor to donor. The fixed nature of the relationship between sperm motility and egg penetration enables the results of such in-vitro fertilization experiments to be corrected for the number of motile spermatozoa in the incubation media. On the basis of these findings a protocol is described for assessing the results of the zona-free hamster oocyte penetration assay, which involves analysis of the degree of polyspermy followed by the application of Poisson distribution theory to correct the results to a standard concentration of motile spermatozoa. Changes in the penetrating ability of human spermatozoa after vasectomy and characterization of the degree of inter-ejaculate variation in penetrating potential are two clinical examples of such analyses given in the text. The statistical methods described in this paper should also be of general relevance to the study of fertilization mechanisms, in providing a rationale by which to analyse the quantitative nature of sperm—egg interaction in vitro.
R. J. Aitken and R. A. Elton
Summary. A Poisson–gamma distribution model has been developed for studying sperm–oocyte interaction in the zona-free hamster egg penetration test. With the aid of this model we have analysed the relationship between the penetration results achieved with semen of normal fertile men and the concentration of spermatozoa and oocytes used in the assay. From this analysis we have drawn up reference tables estimating the number of spermatozoa and occytes which must be present in the assay for the result to represent a significant (P < 0·05) decline in sperm function relative to the normal fertile population.
Since there is, as yet, no fixed protocol for the penetration assay, these tables have been constructed to fit a range of methodologies varying with respect to the nature of the preincubation phase and including (a) a 7-h preincubation in Medium BWW, (b) a 3-h preincubation in hyperosmotic Medium BWW or (c) a 3-h preincubation in the presence of A23187.
R. J. Aitken and J. B. Maathuis
Summary. Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 μg protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.
J. B. Maathuis and R. J. Aitken
Summary. Uterine flushings were obtained from fertile women at various stages of the menstrual cycle. A technique was used which excluded contamination with cervical mucus and significantly lowered contamination with blood in comparison with an established technique. Contamination of the uterine flushings with seminal plasma and tubal fluid was also prevented.
The concentrations of protein and hexose in the uterine flushings, corrected for contamination with plasma, were significantly lower in the secretory stages than in the proliferative stages of the cycle. It is concluded that proteins and carbohydrates are present within the uterine lumen not only after ovulation but also during the preovulatory period.
J. B. Maathuis and R. J. Aitken
Summary. Uterine flushings and plasma collected from normal, parous women at various stages of the menstrual cycle were subjected to gel electrophoresis. The protein profiles of the flushings differed in many instances from the plasma pattern by the presence of between one and eleven non-plasma proteins. The distribution of the major non-plasma proteins during the menstrual cycle was not statistically significant but that of a pretransferrin, observed in uterine flushings and in peritoneal fluid, was significant. However, the appearance of this protein band could not be related to a particular phase of the cycle, and hence to a specific hormonal condition.
R. J. Aitken and J. S. Clarkson
Summary. Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3–5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon.
The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm–oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.
D. W. Cooper and R. J. Aitken
Summary. Six anti-lymphocyte antisera and 6 anti-lymphocyte globulin preparations were tested on serum from women in the first and second trimester of pregnancy and on appropriate sera from non-pregnant controls. There were no differences and the results do not confirm the claim that the serum of pregnant women contains a factor that will prevent human lymphocytes forming spontaneous rosettes with sheep red blood cells, and that this serum factor makes its appearance early in pregnancy.
R. J. Aitken, A. Mattei and S. Irvine
Summary. Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2·5 mm. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa.
The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1·7 mm) typical of media used for in-vitro fertilization.
The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1–10 mm. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine.
We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway.
B Houston, B Curry and R J Aitken
Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis.
R. J. Aitken, D. Harkiss and D. Buckingham
The relationship between lipid peroxidation and the functional competence of human spermatozoa has been investigated in a cohort of 31 infertility patients. Lipid peroxidation was assessed using a sensitive fluorometric assay for the generation of malondialdehyde in response to the presence of a ferrous ion promoter. Sperm function was evaluated by monitoring the movement characteristics of these cells and their capacity for sperm–oocyte fusion. Each sample was separated into high- and low-density sperm populations on discontinuous, two-step (40%:80%), Percoll gradients prior to analysis. The way in which individual ejaculates fractionated on these gradients was highly positively correlated (P < 0.001) with the lipoperoxidation status of the spermatozoa; the greater the potential for malondialdehyde generation, the higher the proportion of cells entering the low density region of the gradients. The lipoperoxidation potential of the freshly prepared spermatozoa was also highly predictive (P = 0.0001) of their capacity for movement at 3 and 24 h and their ability to exhibit sperm–oocyte fusion in response to the ionophore A23187. The potential for malondialdehyde generation in the 40% and 80% Percoll fractions was positively associated with midpiece abnormalities in the spermatozoa. These results emphasize the importance of lipid peroxidation in the pathophysiology of male infertility and suggest a mechanism by which such damage might arise.