Development of Immortalized Endometrial Epithelial and Stromal Cell Lines from the Mink (Mustela Vison) Uterus and their Effects on the Survival in Vitro of Mink Blastocysts in Obligate Diapause1

Abstract

Mink endometrial cell lines were established by stable transfection of a plasmid vector encoding the SV40 large T antigen driven by the human beta-actin promoter. A second plasmid vector, pSV2neo, was employed for selection of transfected cells. Specificity and homogeneity of consequent cell lines were evaluated by immunocytochemistry employing antibodies against cytokeratin, desmin, and vimentin. Cytokeratin was found exclusively in epithelial cells, whereas vimentin appeared primarily in stromal cells. Neither cell line showed detectable desmin activity. These cell lines along with Buffalo rat liver (BRL) cells were employed in coculture with mink embryos in obligate diapause. Mink stromal and BRL cell lines were most effective in enhancing embryo survival in vitro. The percentages of cocultured embryos that survived for 72 h or more were 65% with epithelial cells, 75% with stromal cells, 68% with the combination of stromal and epithelial cells, and 93% with BRL cells. Only 23% of the embryos cultured without cells survived beyond 48 h. Embryo growth was also observed; some embryos in coculture showed trophoblastic outgrowth and adhesion to the cell surfaces. These results demonstrate that mink embryos in obligate delay can survive and develop in culture and that coculture with uterine or BRL cells increases the length and frequency of survival.

Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

“…Cell Culture-An immortalized mink uterine stromal cell line (36) was used for the in vitro experiments described herein. Cells were cultured in Dulbecco's modified Eagle's medium/ F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), containing 1% of penicillin/streptomycin (Invitrogen) and 0.5% of Fungizone (Invitrogen).…”

Embryonic diapause: development on hold

“…Similarly, mink blastocysts in diapause resumed development when co-cultured with conspecific uterine cell lines, and wallaby blastocysts in diapause are able to reactivate and develop normally when transferred to a reactivated uterus (Tyndale-Biscoe, 1970;Moreau et al, 1995). Wallaby blastocysts displaying delay in their metabolic reactivation were found to be from mothers with a low endometrial metabolism, indicating a failure of the maternal system to reactivate (Spindler et al, 1998).…”

“…In the wallaby during diapause, uterine secretions are low and the uterus remains quiescent (Renfree, 1973;Shaw and Renfree, 1986;Tyndale-Biscoe and Renfree, 1987;Spindler et al, 1998). Furthermore, dormant blastocysts from both the wallaby and the mink do not reactivate when cultured in vitro (Moreau et al, 1995;Renfree and Shaw, 2000). One potential candidate for a missing factor is LIF, which is essential for embryonic stem cell proliferation (Zhao et al, 2012) and for implantation in the mouse (Hondo and Stewart, 2005).…”

Cadence of procreation: Orchestrating embryo–uterine interactions

“…Interestingly, mink embryos in diapause co-cultured with uterine cell lines displayed the capacity to reinitiate embryonic development in vitro, providing evidence that the uterus maintains diapause in this species [59]. Maternal factors responsible for pausing embryonic development across species have not been clearly defined.…”