Immature female mink, 8 weeks of age in July, were treated with implants releasing melatonin. Mating, which induced ovulation, took place during the normal breeding season in the following March. Circulating prolactin and progesterone concentrations did not undergo the expected gestational increases, and no embryos implanted. A similar absence of gestational changes in prolactin and progesterone values ensued in primiparous mink treated with the melatonin implant 2-3 days after the second of 2 matings. Administration of exogenous sheep prolactin (0.5 mg/day) by minipump induced precocious elevation of progesterone concentrations in mated mink. Prolactin administration overcame the effects of melatonin, in that the corpora lutea were activated and embryos implanted, but exogenous prolactin resulted in degeneration of implanted embryos both in the presence and absence of chronic melatonin. The results suggest that melatonin has a single effect in alteration of gestation in mink; i.e. the prevention of prolactin secretion. Hyperprolactinaemia may inhibit embryo development in this species.

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.

The mink corpus luteum (CL) involutes after ovulation and remains dormant, synthesizing low amounts of progesterone until reactivated to terminate embryonic diapause. We examined the mitotic and steroid synthetic capacity of luteal cells from the diapause and postimplantation phases of mink gestation. Cells from diapause divided in vitro, reaching confluence in 7-8 days. Three phenotypes were distinguishable: a fusiform cell in whorls, a hypertrophied epithelioid cell, and a small epithelioid cell. The first and second cell types divided in vitro after confluence, evidenced by localization of proliferating cell nuclear antigen (PCNA) in their nuclei. The small epithelioid cells were present in cell nests and showed no PCNA activity. Cells derived from reactivated CL did not reach confluence and had no PCNA activity. Progesterone accumulation was enhanced in luteal cells from diapause by LH, FSH, and dibutyryl (Bu2)cAMP, and by LH and (Bu2)cAMP in cells from reactivated CL. In luteal cells from the diapause phase of gestation, LH and (Bu2)cAMP induced increases in mRNA coding for steroidogenic acute regulatory protein, while cytochrome P450 side-chain cleavage enzyme mRNA was increased by prolactin, LH and (Bu2)cAMP. Cellular concentrations of 3beta-hydroxysteroid dehydrogenase-delta5-4-isomerase mRNA were increased by prolactin and (Bu2)cAMP. Thus, luteinization in the mink CL does not engender exit from the cell cycle, as both fusiform and hypertrophied cells from diapause divide in vitro. Reactivation appears to represent terminal differentiation. LH is capable of stimulating steroidogenesis in vitro in luteal cells from diapause, and prolactin and LH appear to have both specific and overlapping stimulatory effects on the CL of this species.

The reproductive cycle of the mink displays rigid seasonality and obligate embryonic diapause. After ovulation, the corpus luteum (CL) involutes, and it secretes basal progesterone until activated prior to implantation. To study changes in the relative abundance of luteal prolactin and LH receptor mRNA through gestation, ovaries and serum were collected from pregnant female mink at 2-day intervals (n = 3 per date) through embryonic diapause and CL activation (March 19-31) and at 5-day intervals through implantation and early-postimplantation gestation (March 31-April 15). To determine the effect of endogenous prolactin, mink received Alzet osmotic minipumps releasing 2 mg/day 2-bromo-alpha-ergocryptine (bromocriptine) or saline on March 19. Ovaries and serum were taken from 3 animals every 2 days until March 31. Prolactin receptor mRNA in ovaries was low during CL activation but increased 3-fold through embryo implantation. Its abundance correlated with prolactin binding to ovarian membranes and with circulating prolactin. Bromocriptine suppressed endogenous prolactin levels and prevented the increase in prolactin receptor mRNA. There was a transient peak in LH receptor mRNA in the ovaries at March 19-23, which declined to basal levels by March 25 and remained constant through midgestation. Bromocriptine prevented the preimplantation peak in LH receptor mRNA and reduced its abundance below pretreatment levels. The results suggest that prolactin up-regulates its receptor and maintains the LH receptor in the mink CL. The pattern of LH receptor mRNA argues for a role for LH in CL reactivation and termination of embryonic diapause in mink.

Ovarian follicular dynamics were studied during the breeding season, before and after ovulation in mink. Nulliparous female mink were stimulated to ovulate with an injection of 4 \g=m\g GnRH. Ovaries from three animals were collected on days 0, 2, 3, 4, 5, 6