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The mink reproductive cycle includes an obligatory period of embryonic diapause and delayed implantation, which continues in vitro and reduces the efficiency of embryonic stem (ES) cell establishment. Blastocysts recovered on day 7 and on days 13-16 after final mating were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with various concentrations of prolactin to determine optimal conditions for embryo attachment and subsequent establishment of embryonic stem cells. Five treatments were applied to both ages of blastocyst: A, DMEM control (n = 16); B, DMEM + 5 micrograms prolactin ml-1 after 10 days initial culture in DMEM alone (n = 17); after 1 day of initial culture: C, DMEM + 10 ng prolactin ml-1 (n = 17); D, DMEM + 1 microgram prolactin ml-1 (n = 19); and E, DMEM + 5 micrograms prolactin ml-1 (n = 17). Prolactin terminated diapause of day 13-16 blastocysts at all concentrations tested. The maximum attachment of embryos in vitro and subsequent production of ES-like cells occurred in medium supplemented with 5 micrograms prolactin ml-1. Prolactin did not affect attachment rates for day 7 blastocysts when 5 micrograms prolactin ml-1 was added, but prolactin at concentrations of 1 microgram ml-1 and 5 micrograms ml-1 when added on day 1 of culture enhanced ES-like cell line establishment. Two principal cell types were observed in the colonies: small stem cells and trophoblast-like cells with large areas of cytoplasm. The morphological evaluation of mink ES-like cell colonies was confirmed by cytochemical staining for alkaline phosphatase. Mink embryonic stem-like cells were found to stain positive for alkaline phosphatase. Alkaline phosphatase activity was lost upon cellular differentiation.

reproduction

Prolactin-induced termination of obligate diapause of mink (Mustela vison) blastocysts in vitro and subsequent establishment of embryonic stem-like cells