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Summary. The mechanism by which a horse conceptus-derived immunosuppressive factor (HCS) of Mr > 100 000 inhibits lymphocyte proliferation was investigated. The factor was obtained from the culture supernatants of 20-day-old horse conceptuses; activity, identified by reduced uptake of [3H]thymidine by mitogen-stimulated lymphocytes, was greatest (P < 0\m=.\01) in cultures stimulated by mitogen from pokeweed. HCS also suppressed cell proliferation stimulated by phytohaemagglutinin (P < 0\m=.\01), but had no effect on lipopolysaccharide-stimulated cells (P > 0\m=.\05). Data from a fluorescence\x=req-\ activated cell sorter indicated that supplementation with HCS reduced the number of T cells in phytohaemagglutinin-stimulated cultures and suppressed proliferation of T and B cells in pokeweed-mitogen-stimulated cultures compared with controls. Cell proliferation was greater (P < 0\m=.\01) in cultures supplemented with HCS 24 h after stimulation than in those treated at the start of stimulation, and was even greater (P < 0\m=.\01) when cells were treated 48 h after stimulation. The removal of HCS from treated lymphocyte cultures resulted in complete recovery of cell responsiveness, and stimulated proliferation of treated cells did not differ (P > 0\m=.\05) from that of control cells. The addition of stimulated equine lymphocyte supernatant to cultures supplemented with HCS did not significantly increase (P > 0\m=.\05) cell proliferation in response to pokeweed mitogen.Addition of recombinant human interleukin 2 (rIL-2) to HCS-treated cultures did not alter the suppressive activity of HCS, although cell proliferation was greater in cultures supplemented with rIL-2 than in controls (P < 0\m=.\01). HCS inhibition of IL-2 receptor (IL-2R) function was investigated using an IL-2-dependent murine cytolytic T lymphocyte cell line; the fraction of HCS of Mr > 100 000 had no effect (P > 0\m=.\05) on proliferation of IL-2-dependent murine cytolytic T lymphocyte cells induced by rIL-2. Together, these data suggest that HCS suppresses proliferation of T lymphocytes during the early stages of cell activation by inhibiting IL-2R interaction and that this suppression interferes with interactions between T cells and B cells, thereby also indirectly inhibiting proliferation of B cells. The potent immunosuppressive capacity of HCS may be one factor responsible for inhibiting cell-mediated fetal allograft rejection during pregnancy.

Golden Syrian hamster embryos are difficult to cryopreserve due to their high sensitivity to cryoprotectants and in vitro handling. The objective of this study is to develop a robust open pulled straw (OPS) vitrification technique for cryopreserving hamster embryos at various developmental stages. We first systematically tested the concentrations of cryoprotectants and the exposure times of two-cell embryos to various vitrification solutions. We identified pretreatment of two-cell embryos with 10% (v/v) ethylene glycol (EG) þ 10% (v/v) dimethylsulfoxide (DMSO) for 30 s followed by exposure in the vitrification solution, EDFS30 (containing 15% EG þ 15% DMSO), for 30 s before plunging into liquid nitrogen (two-step exposure method) as the optimal OPS vitrification protocol. We then investigated the resourcefulness of this protocol for vitrifying hamster embryos at different developmental stages. The results showed that high blastocyst rates from embryos vitrified at two-cell, four-cell, eight-cell, or morula stage (62%, 78%, 80%, or 72%, respectively), but not those verified at pronuclear (0%) or blastocyst stage (24%; P < 0.05), were achieved by this protocol. When embryos vitrified at the two-cell stage were recovered and then directly transferred to recipient females, 29% of them developed to term, a development rate not significantly different (P > 0.05) from the 40% birth rate of the unvitrified controls. In conclusion, we have developed an effective two-step OPS vitrification protocol for hamster embryos.

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