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Mice were passively immunized over 6 weeks with contraceptive doses of an anti-cumulus oophorus antibody preparation. The persistence of oestrus, accompanied by complete inhibition of conception, was observed throughout treatment. After cessation of treatment, fertility was restored within 2 weeks. Histological examination did not reveal any depletion of the ovarian oocyte stock. These results warrant further research into the nature of cumulus antigens and their use in active immunization studies.

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.

The mucified cumulus oophorus represents an outer enveloping layer around ovulated mammalian oocytes. This coat in its definitive expanded form appears late in the preovulatory development as a result of intensive secretion of intercellular matrix by cumulus cells. We have shown recently that antibodies to the cumulus matrix inhibit human fertilization in vitro. This study was undertaken to assess, in an animal model, the effects of anticumulus oophorus antibodies on fertility by use of different passive immunization protocols. A purified anticumulus immunoglobulin fraction was prepared from hyperimmune rabbit serum and administered at different times before and after mating to mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A dose-dependent negative effect of this anticumulus antibody preparation on the number of fertilized eggs recovered from the oviducts of treated animals was observed when the antibodies were given before mating. High antibody doses also interfered with oocyte maturation and ovulation if applied on the day of eCG treatment, but no effects on these processes were found when the antibodies were given on the day of hCG treatment. The antifertility effect of anticumulus antibodies was reversible and the antibodies did not affect postfertilization development. These findings make cumulus oophorus antigens serious candidates for the development of a contraceptive vaccine.

Human oocytes that failed to fertilize in vitro were examined for the degree of meiotic maturity and reinseminated either in the living state or after treatment with a concentrated salt solution as used for storage of human zonae pellucidae for diagnostic tests of sperm function. No dependence of zona pellucida penetrability on the oocyte maturity status was observed in either treatment group. Zonae pellucidae of metaphase I and metaphase II oocytes were equally penetrable after the salt treatment, whereas penetration occurred only exceptionally when the oocytes were living. These results show that, after in vitro aging and subsequent salt treatment, eventual differences in oocyte maturity status will not influence results of zona pellucida penetration tests. On the other hand, successful penetration of salt-stored zonae pellucidae by spermatozoa does not necessarily imply that these spermatozoa are able to penetrate the zona pellucida of a fresh living oocyte.

The ultrastructure of human ovocytes was studied, the latter having been obtained laparoscopically following induced ovulation, using clomiphene and HCG. Optimum vacuum size for aspiration was tested on embryonated rat ova. It was found to be between 10 kPa and 30 kPa, that is between 100 and 300 mm H2O. Ultrastructural alteration was recordable neither from human nor from rat ovocytes. No change in ultrastructure was obtained either from subsequent in vitro culturing, and some of the ovocytes were embryonated with good success.

Human oocytes that failed to fertilize in a clinical in vitro fertilization (IVF) program were freed from the zona pellucida and reinseminated with spermatozoa from subfertile patients or from fertile donors. Penetration results were, in general, significantly worse when patients spermatozoa were used as compared with donor spermatozoa. When inseminated with donor spermatozoa, oocytes from cases of male infertility gave better penetration results as compared with those from cases of idiopathic infertility with no apparent sperm defect. No differences in penetration results were found between oocytes aged 1 day and 2 days in culture before zona removal and reinsemination, even though less pronuclei reached full functional maturity in the more aged group of eggs. These results indicate that human zona-free eggs prepared from oocytes after unsuccessful IVF attempts may be used to assess the fertilizing ability of human spermatozoa. Moreover, when used with donor spermatozoa, this system may help to determine the contribution of the oocyte to fertilization failure.

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona-free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nucleolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.

The present era of infertility management is characterized by an increasing interest in defects of sperm--egg interactions. When the spermatozoon meets the egg, it must first react with the enveloping coat which controls sperm access to the cell surface of the oocyte. Adequate sperm motility is a major condition for penetration through the egg investments. The ability of spermatozoa to respond to physiological stimuli by a timely acrosome reaction is another necessary prerequisite for this event, as well as for sperm fusion with the oocyte. The clinical significance of current laboratory tests evaluating different aspects of gamete function is discussed and a practical guide is given for their indication and interpretation and for the use of their results in choosing rational therapy.

The nature of oocyte cytoplasmic factors controlling the development of the male pronucleus was investigated by inseminating human, zona-free oocytes at metaphase of the 1st and 2nd meiotic division. Oocytes at metaphase of the 2nd meiotic division could support the full structural and functional development of male pronuclei, whereas the vast majority of those at metaphase of the 1st meiotic division failed to do so. This suggests that oocyte cytoplasmic factors required for male pronuclear formation do not develop fully until the oocyte reaches the 2nd meiotic metaphase. When increasing numbers of spermatozoa entered one oocyte, the transformation of sperm nuclei into pronuclei was impaired progressively. The later stages of pronuclear development were particularly sensitive to polyspermy. These factors should be taken into consideration in the development of techniques of micromanipulation-assisted insemination.