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  • Author: D. W. RICHARDSON x
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D. W. RICHARDSON and R. ANSBACHER

Summary.

The number of conceptuses occurring after placement of an IUD in the right uterine horn of twenty golden hamsters was compared to the number present in ten hamsters subjected to sham operation on Days 7, 12 and 16 of gestation. A unilaterally placed IUD prevented pregnancy in the containing horn, but exerted no significant effect upon implantation sites in the contralateral horn. Sham operations did not prevent implantation.

Histological examination revealed inflammatory reaction only in those uteri containing a silk suture. No communication between uterine lumina could be demonstrated.

The presence of an IUD did not alter the oestrous cycles of hamsters.

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D. M. Woolley and D. W. Richardson

Summary. The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.

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R. J. Aitken and D. W. Richardson

Summary. Conditions have been defined under which the sperm binding capacity of the mouse zona pellucida can be measured. As a measure of the concentration of anti-zona antibodies the inhibition of sperm binding appears to be both repeatable and sensitive. Using this technique anti-zona antibody titres were monitored in 6 rats actively immunized with extracts of mouse ovarian tissue. These animals exhibited a rapid rise in anti-zona antibody titre following the induction of immunity, and an associated significant (P < 0·05) decline in their fertility, both in relation to the proportions of animals exhibiting fertile matings and of matings resulting in conception. Three of the animals exhibited permanent sterility despite repeated exposure to fertile males.

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D. W. Richardson, R. J. Aitken and N. B. Loudon

Summary. Progresssively motile spermatozoa were present in 42/47 (89%) of post-vasectomy specimens, and in 16 of 24 samples (66%) demonstrable fertilizing potential could be detected in the zona-free hamster egg assay. The mean ± s.e.m. post-vasectomy penetration rate of 20·6 ± 6·0% was, however, significantly lower (P < 0·001) than the pre-vasectomy value of 49·7 ± 6·4%. After vasectomy the concentration of motile spermatozoa in the ejaculates declined in a manner that was significantly correlated with the post-operative time interval. The spermatozoa also lost their potential for fertilization, although functionally competent spermatozoa were still being recovered from ejaculates produced 8 days after vasectomy.

These results emphasize the ability of human spermatozoa to retain some residual fertilizing potential when stored at core body temperature in sites distal to the epididymis, and suggest a role for functional tests in the management of the vasectomized male.

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D. W. RICHARDSON and R. M. F. S. SADLEIR

Summary.

At concentrations varying from 2·5% to 10·0%, glycerol, dimethyl sulphoxide, ethylene glycol, methyl formamide and methyl acetamide had approximately equally toxic effects on human spermatozoa; whereas dimethyl formamide and dimethyl acetamide were considerably more toxic at the same concentrations.

Glycerol and ethylene glycol afforded an equal protective action to human spermatozoa during freezing and thawing, approximately 50% of the original motility being retained, but negligible protection was exhibited by methyl formamide, dimethyl formamide, methyl acetamide and dimethyl acetamide. Only 34% of the original motility was retained when human spermatozoa were frozen in the diluent containing dimethyl sulphoxide.

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R. J. Aitken, E. Holme, D. W. Richardson and M. Hulme

Summary. Intact and univalent antibodies were prepared against mechanically isolated mouse zonae pellucidae solubilized in a variety of ways (heat, low pH, SDS, urea and trypsin). The antisera bound avidly and specifically to solubilized iodinated zona antigens and the intact zona structure.

When the concentrations of immunoreactive Fab material in the intact and univalent antibody preparations were equalized and compared for their ability to block the sperm-binding stage of fertilization, only the intact γ-globulin preparations possessed antifertility activity. These results indicate that antibodies raised against intact solubilized zonae pellucidae block fertilization by cross-linking antigens on the outer zona surface, thereby indirectly masking the sperm receptor sites. The integrity of these surface components did not appear to be affected by solubilization procedures that disrupt non-covalent bonds (heating, low pH, SDS and urea) although they did appear to be adversely affected by trypsin treatment.

None of the antisera tested contained antibodies directed against the sperm receptor site indicating that these critical components lack immunogenicity.

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R. J. Aitken, M. Sutton, P. Warner and D. W. Richardson

Summary. In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0–100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6·8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations.

A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0·874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, amplitude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate amplitude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm—cervical mucus interaction (R 2 = 0·53).

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R. G. Gosden, D. W. Richardson, N. Brown and D. W. Davidson

Summary. The gametogenic potential of young male CBA/Ca mice aged 4–5 months was compared with that of animals 20–23 months of age which had recently ceased to sire offspring. The older mice had substantially lower circulating levels of testosterone and smaller testes. Testicular and epididymal spermatozoa were reduced in number, had more abnormal forms and fewer were progressively motile. Ageing seminiferous tubules varied in their degree of functional atrophy from complete depletion of germ cells to maintenance of sperm production. They were enclosed by a thickened basal lamina and more collagenous connective tissue and occasionally allowed the penetration of lanthanum between Sertoli cells into the ad-luminal compartment. There was no evidence of autosensitization to sperm antigens.

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R. J. Aitken, D. Buckingham, K. West, F. C. Wu, K. Zikopoulos and D. W. Richardson

Summary. Cells isolated from the ejaculates of a high proportion of patients exhibiting oligozoospermia are characterized by generation rates of reactive oxygen species that considerably exceed those obtained for the normal fertile population. The purpose of this study was to resolve the cellular source of this enhanced activity. Semen samples from a cohort of oligozoospermic patients and a group of fertile controls were fractionated on discontinuous Percoll gradients to generate three cell populations (0, 50 and 100%) of differing density. For each fraction, both the steady-state and the phorbolester-induced chemiluminescent signals were significantly (P < 0·001) greater for the oligozoospermic samples than for the fertile controls. In the fertile donors, leucocytes comprised the major source of reactive oxygen species, particularly in the low-density Percoll fractions; in oligozoospermic patients, however, spermatozoa were identified as a second major source of reactive oxygen species. Particularly striking was an intense phorbol-ester-induced chemiluminescent signal generated by oligozoospermic spermatozoa, purified by passage through isotonic Percoll and free of leucocyte contamination, which was 167 times greater than the median signal generated by the corresponding fraction from the fertile controls (P < 0·001). These results emphasize the importance of spermatozoa as a major source of reactive oxygen species in oligozoospermia and have implications for the diagnosis and treatment of this condition, as well as for the design of appropriate diagnostic strategies.

Keywords: oligozoospermia; reactive oxygen species; spermatozoa; human

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R. J. Aitken, E. A. Rudak, D. W. Richardson, J. Dor, O. Djahanbahkch and A. A. Templeton

Summary. Anti-zona antibodies are effective inhibitors of fertilization in vitro and, regardless of whether passive or active immunization techniques are used, in vivo. Antibodies raised against unfractionated zona pellucida antigens are chiefly directed against a group of carbohydrate-rich components localized on the outer surface of the zona. The interaction of anti-zona antibodies with these sites induces the formation of a surface precipitate which occludes the sperm binding sites by a process of steric hindrance, and stabilizes the zona structure against digestion by the proteolytic enzymes of the sperm head. Active immunization studies indicate that the long-term induction of infertility without adverse side effects is feasible in both laboratory rodents and primates when the zona pellucida is used as a target.

Anti-sperm antibodies also exhibit a capacity for inhibiting fertilization in vivo and in vitro. To determine the most appropriate detection method to screen patients for anti-sperm antibodies several homologous and heterologous antisera were analysed by 5 different agglutination and immobilization techniques and then compared for their ability to inhibit the fertilizing capacity of human spermatozoa using the zona-free hamster egg penetration test. The results obtained with the Franklin-Dukes tube—slide test exhibited the closest correlation with the anti-fertility activity of a given antiserum; this activity could be amplified by the addition of complement to the medium. It is concluded that antibodies directed against the sperm head are responsible for limiting the fertilizing capacity of human spermatozoa in vitro and that it is these antibodies on which attention should be focused to unravel the role that immunological factors play in the aetiology of infertility in vivo.