Immunoglobulins and monoclonal anti-blastocyst antibodies in the mouse uterine secretion during the early pre-implantation period.

A Sepharose bead blot technique was used to study immunoglobulins in the uterine secretion of mice during the pre-implantation stage. Secretion collected by Sepharose beads contained IgA, IgG, and IgM. The method could be made ten-fold more sensitive by using anti-mouse IgG or IgM conjugated to Sepharose beads. It has also been demonstrated that when injected intravenously, biotinylated purified immunoglobulins, both non-specific mouse myeloma IgG and IgM and specific anti-blastocyst IgG and IgM, is able to pass into the uterine cavity of the mouse. It was further shown that when injected systemically, anti-blastocyst antibodies can reach the blastocyst. Functionally active specific antibodies against morulae and/or blastocysts may, therefore, be able to influence the pre- and peri-implantation development of the embryo and could serve as a useful model for experiments directed towards the identification of immunological contraceptive procedures.

Production and determination of specificity of monoclonal IgG anti-mouse-blastocyst antibodies.

Five monoclonal anti-mouse-blastocyst IgG antibodies were raised by intrasplenic immunization of three mice with adhesive-stage mouse blastocysts. Each mouse received a total of 60-70 blastocysts which were either nitrocellulose-immobilized or living but irradiated. Tests for pre-implantation stage-specificity showed that the antibodies differed in specificity. None were specific for surface epitopes. One antibody recognized epitopes only on blastocysts. Other antibodies were able to discriminate between unfertilized and fertilized oocytes, uncompacted and compacted morulae, or delayed and adhesive blastocysts. By applying reduced SDS-PAGE and Western blotting to blastocysts the blastocyst-specific antibody was seen to be bound to a peptide of M(r) 34.

Monoclonal antibodies against human blastocysts.

A panel of monoclonal antibodies against the implanting mouse blastocyst was tested for cross-reactivity to human blastocysts. The monoclonal antibodies were produced by intrasplenic immunization with living mouse blastocysts or egg-cylinders. Among the 13 anti-mouse blastocyst antibodies checked, 6 ones detected antigen epitopes also in human blastocysts. If the mouse-human cross-reacting antibodies detect similar antigens in blastocysts of the two species, the results imply that anti-mouse blastocyst antibodies with specific properties could be used for studying human blastocysts. This procedure opens for a way to obtain monoclonal antibodies for analysing human blastocysts.

Experimental antigen modulation of blastocysts for mimicing dormant and invasive micrometastases.

The mouse preimplantation period can be prolonged experimentally by a delayed implantation, and this will keep the blastocyst in an inactive state. The dormancy can be interrupted by an oestrogen injection, which will make the blastocyst invasive. Thus the blastocyst mimics both dormant and invasive micrometastasis. Considering the similarities between blastocysts and micrometastases in cellular activation, we have developed methods for examining antigen modulations using the blastocyst as a model. Evaluating the total amount of antibody in the blastocyst and also the trophoblast antigen synthesis, shedding, and endocytosis, we found that dormant cells exposed to a surface-specific antibody kept the immune complexes on the cell surface for a longer time than did the invasive cells. The rate of internalization of immune complexes was low in both dormant and invasive cells, but since the shedding activity was less active in the dormant cells they contained more antibodies totally than did the invasive ones under the same conditions. When exposing the cells to an anti-cytoplamic antibody or to non-specific antibodies, the amount of antibodies in the cytoplasm of invasive cells was higher than in dormant cells. The methods used for examining the antigen modulation by the blastocyst should be useful also for studying the handling of antibodies by micrometastases.

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation.

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.

Confocal laser fluorescence scanning microscopy of the oolemma distribution of monoclonal antibodies against mouse and human egg surface membrane.

Monoclonal antibodies were raised against zona-free mouse oocytes by intrasplenic immunizations. Antibodies, which detected antigen epitopes in unfertilized but not in fertilized mouse eggs (fertilization-specific antibodies) and which in addition recognized epitopes in unfertilized human oocytes (mouse-human cross-reacting antibodies) were used to visualize the distribution of their corresponding antigens by confocal laser fluoresence scanning microscopy (CLFSM). We found that among 26 antibodies tested, two ones showed a patch pattern. Since a patchy distribution of cell membrane molecules indicates the presence of integral proteins with ability to transduce signals to the cytoplasm, we suggest that the method here applied will select for those oolemma molecules that could be involved in the fusion of sperm cells.