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  • Author: B. E. MORTON x
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The colloid mill was found capable of disrupting bovine epididymal spermatozoa in such a way that even the very resistant sperm middle piece was opened. The homogenate thus produced was fractionated by both differential centrifugation and isopycnic density gradient centrifugation. The latter method was found very useful in producing homogenous fractions of cell components including in order of increasing density : mitochondrial fragments, acrosomes, membranes, tail principal pieces, middle pieces, middle piece exteriors, contractile fibres and denuded heads. The mitochondrial particles consumed oxygen while the cell membranes had hexokinase-like activity.

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The release of acrosomal hyaluronidase from hamster spermatozoa capacitating in test-tubes was measured. In order to do this, a colorimetric hyaluronidase assay of increased sensitivity was developed, based upon the finding that an albumin—substrate complex was hydrolysed much more rapidly than hyaluronic acid alone. To estimate the degree of morbidity of the sperm samples, cytoplasmic lactic dehydrogenase loss into the media was monitored. This loss did not correlate with the percentage of motile spermatozoa, but was almost identical to the amount of hyaluronidase appearing in the medium when the spermatozoa were incubated in non-capacitating Tyrode's solution. Sperm hyaluronidase release, but not lactic dehydrogenase loss, was elevated by the addition of serum albumin in the presence or absence of human serum dialysate. This hyaluronidase release was magnified when heat-detoxified human serum was used to capacitate the spermatozoa. The relationship of these findings to capacitation-associated acrosome vesiculation and detachment is discussed.

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A. C. Cavanagh, B. E. Rolfe, S. Athanasas-Platsis, K. A. Quinn and H. Morton

Summary. The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.

Keywords: early pregnancy factor; platelet-activating factor; rosette inhibition test; leukotriene; mouse

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A. C. Cavanagh, H. Morton, S. Athanasas-Platsis, K. A. Quinn and B. E. Rolfe

Summary. Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.

Keywords: early pregnancy factor, rosette inhibition test; inhibitor; mouse

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S. Athanasas-Platsis, K. A. Quinn, T-Y. Wong, B. E. Rolfe, A. C. Cavanagh and H. Morton

Summary. Early pregnancy factor (EPF) is a monitor of the incidence of fertilization and the progress of the early embryo. To determine whether, as well as being a marker of embryonic viability, EPF is also necessary for embryonic survival, passive immunization studies with monoclonal and polyclonal antibodies to EPF were carried out on pregnant mice. In the preparation of monoclonal antibodies, it was noted that most anti-EPF producing hybridomas failed to grow in vitro, while those that did grow produced only low yields of specific IgM antibodies.

Two stable hybridoma cell lines were established both producing low affinity anti-EPF IgM; polyclonal anti-EPF IgG was prepared in rabbits. Mice were passively immunized with 500 μg monoclonal anti-EPF IgM at 32 and 56 h post coitum (total dose 1 mg) or with 500 μg polyclonal anti-EPF IgG at 8, 16, 32 and 40 h post coitum (total dose 2 mg). At 10 days, only 6/18 and 3/6 mice receiving monoclonal antibodies and 2/7 and 1/6 mice receiving polyclonal antibodies had maintained their pregnancies. In contrast, all mice receiving control IgM (N = 14) or control IgG (N = 4) and 22/23 receiving saline were still pregnant at Day 10.

Keywords: pregnancy; early pregnancy factor; monoclonal antibodies; passive immunization; embryonic viability; mice