PROLACTIN-INDUCED TERMINATION OF OBLIGATE DIAPAUSE OF MINK (MUSTELA-VISON) BLASTOCYSTS IN-VITRO AND SUBSEQUENT ESTABLISHMENT OF EMBRYONIC STEM-LIKE CELLS

The mink reproductive cycle includes an obligatory period of embryonic diapause and delayed implantation, which continues in vitro and reduc es the efficiency of embryonic stem (ES) cell establishment. Blastocys ts recovered on day 7 and on days 13-16 after final mating were cultur ed in Dulbecco's modified Eagle's medium (DMEM) supplemented with vari ous concentrations of prolactin to determine optimal conditions for em bryo attachment and subsequent establishment of embryonic stem cells. Five treatments were applied to both ages of blastocyst: A, DMEM contr ol (n = 16); B, DMEM + 5 mu g prolactin ml(-1) after 10 days initial c ulture in DMEM alone (n = 17); after 1 day of initial culture: C, DMEM + 10 ng prolactin ml(-1) (n = 17); D, DMEM + 1 mu g prolactin ml(-1) (n = 19); and E, DMEM + 5 mu g prolactin ml(-1) (n = 17). Prolactin te rminated diapause of day 13-16 blastocysts at all concentrations teste d. The maximum attachment of embryos in vitro and subsequent productio n of ES-like cells occurred in medium supplemented with 5 mu g prolact in ml(-1). Prolactin did not affect attachment rates for day 7 blastoc ysts when 5 mu g prolactin ml(-1) was added, but prolactin at concentr ations of 1 mu g ml(-1) and 5 mu g ml(-1) when added on day 1 of cultu re enhanced ES-like cell line establishment. Two principal cell types were observed in the colonies: small stem cells and trophoblast-like c ells with large areas of cytoplasm. The morphological evaluation of mi nk ES-like cell colonies was confirmed by cytochemical staining for al kaline phosphatase. Mink embryonic stem-like cells were found to stain positive for alkaline phosphatase. Alkaline phosphatase activity was lost upon cellular differentiation.