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Summary. Plasma

The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.

(Osborne, 1992) and different studies of prognosis have yielded widely differing results (Hawkins, 1993). We have selected four putative biochemical indices of prognosis and evaluated them in a prospective study. The putative markers chosen were the oestrogen receptor (ER), the epidermal growth factor receptor (EGFR), cathepsin D (cath D) and cyclic AMP-binding protein(s) (c-AMP-b).The oestrogen receptor, a nuclear protein (Mr 66 kDa) has been shown to relate to both endocrine sensitivity (Jensen, 1975) and prognosis in multiple studies, including our own previous work (Humeniuk et al., 1982;Hawkins et al., 1987aHawkins et al., , 1991, although some consider that it is only of prognostic significance in the early years after treatment (e.g. Saez et al., 1983). The receptor for epidermal growth factor is a membrane protein (Mr 180 kDa), which has been reported to be a sign of bad prognosis, again in multiple studies (Sainsbury et al., 1987;Gasparini et al., 1992). The proteolytic enzyme, cathepsin D (Mr 52 kDa), has been suggested to be involved in metastasis formation and reported in several studies also to be a sign of bad prognosis when present in high levels (for a review see Rochefort, 1990). Lastly, work in our own laboratories and elsewhere has demonstrated that high levels of cyclic AMP-binding protein(s), the regulatory subunits of the enzyme protein kinase A, represent another sign of poor prognosis, in both retrospective (Miller et al., 1990) and prospective (Miller et al., 1993)

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The possible role of calmodulin in regulating a number of calcium-dependent functions exhibited by human spermatozoa was investigated by using the antagonists trifluoperazine and calmidazolium. At high doses both antagonists inhibited the motility of human spermatozoa and induced a concomitant rise in [Ca2+]i and a decline in cAMP. Lower doses of these antagonists, particularly calmidazolium, suppressed the ability of human spermatozoa to generate reactive oxygen species and exhibit sperm-oocyte fusion, without influencing [Ca2+]i, cAMP, or motility. This inhibition of sperm-oocyte fusion was effective even if the spermatozoa were subsequently exposed to A23187, suggesting that calmodulin may regulate this aspect of human sperm function at a point downstream from calcium influx. Both radiolabelling and affinity chromatography techniques were used to detect a number of calcium-dependent and calcium-independent calmodulin acceptor proteins in the human spermatozoon. The major calcium-dependent acceptor proteins exhibited Mr values of 32,000 and 22,000-27,000, respectively, and did not appear to be associated with the sperm plasma membrane.

The tick-borne protozoan parasite Theileria annulata causes a debilitating disease of cattle called Tropical Theileriosis. The parasite predominantly invades bovine macrophages (mϕ) and induces host cell transformation by a mechanism that has not been fully elucidated. Infection is associated with loss of characteristic mϕ functions and phenotypic markers, indicative of host cell de-differentiation. We have investigated the effect of T. annulata infection on the expression of the mϕ differentiation marker c-maf. The up-regulation of c-maf mRNA levels observed during bovine monocyte differentiation to mϕ was suppressed by T. annulata infection. Furthermore, mRNA levels for c-maf and the closely related transcription factor mafB were significantly lower in established T. annulata-infected cell-lines than in bovine monocyte-derived mϕ. Treatment of T. annulata-infected cells with the theileriacidal drug buparvaquone induced up-regulation of c-maf and mafB, which correlated with altered expression of down-stream target genes, e.g. up-regulation of integrin B7 and down-regulation of IL12A. Furthermore, T. annulata infection is associated with the suppression of the transcription factors, Pu.1 and RUNX1, and colony stimulating factor 1 receptor (CSF1R) which are also involved in the regulation of monocyte/mϕ differentiation. We believe these results provide the first direct evidence that T. annulata modulates the host mϕ differentiation state, which may diminish the defence capabilities of the infected cell and/or promote cell proliferation. Musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors play an important role in cell proliferation, differentiation and survival; therefore, regulation of these genes may be a major mechanism employed by T. annulata to survive within the infected mϕ.

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5-3.5) and molecular mass (45-85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.