Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts.

The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.

Transplantation into genetically alymphoid mice as an approach to dissect the roles of uterine natural killer cells during pregnancy--a review.

Mice genetically deficient in the natural killer (NK) cell lineage lack uterine (uNK cells) and demonstrate morphometrically-quantifiable histopathology within their implantation sites. Two particular mouse strains, tg(epsilon),26 and RAG-2 null x gamma(c) null, have been used successfully as transplant recipients to address questions relating to the biology of uNK cells. uNK cells did not differentiate within decidualized uterine graft segments from normal mice, which were anastomosed orthotopically into immunodeficient hosts. uNK cells did appear in similar grafts placed into immunocompetent hosts, indicating that uNK cells or their progenitors must home to the uterus. This was confirmed by splenocyte transplantation into pregnant uNK cell deficient recipients. Only splenocytes from pregnant donors, not those from non-pregnant donors, homed to the uterus. Homing in this in vivo assay was independent of the CC-chemokine receptors, CCR-2 and CCR-5. Longer-term bone marrow cell reconstitution of neonatal or virgin adult uNK cell-deficient mice has identified a functional role for uNK cells in modification of the decidual arterioles which is mediated by IFN-gamma. By utilizing mutant and gene-ablated mice as donors for tissue or haematopoietic cell transplants to uNK cell deficient mice, it should be possible to fully characterize the in vivo regulation and functions of these pregnancy-specific uterine lymphocytes.

Functional analysis of murine uterine natural killer cells genetically devoid of oestrogen receptors.

Uterine Natural Killer (uNK) cell differentiation in vivo requires oestrogen (E) priming prior to progesterone (P). Hybridomas between uNK precursor and SP2/0 cells express message for E receptor (ER)alpha but nor PR. However, mature, rodent and human uNK cells lack these receptors. To functionally assess requirements for uNK cell expression of ERalpha or ERbeta during precursor differentiation, marrow was transplanted from either ERalpha(o/o) (alphaERKO) or ERbeta(o/o) (betaERKO) mice into alymphoid RAG-2(o/o)/gammac(o/o) females. Recipients were mated and their implantation sites were examined by light microscopy, morphometry and ultrastructure. High numbers of uNK cells were established from each donor strain. Graft-derived uNK cells were similar in number and morphology to uNK cells of normal mice, suggesting that neither alpha- nor beta-ER is required for uNK precursor cell differentiation. Induction of spiral artery modification in the transplant recipients indicated that graft-derived uNK cells had functional properties. A novel technique for rapid isolation of highly purified uNK cells from normal mice using Dolichos biflorus agglutinin (DBA) lectin-conjugated magnetic beads was employed to obtain RNA. Expression of alpha- and beta-ER was absent by RT-PCR from NK cells isolated from the uterus, supporting the conclusions from the in vivo study.

A study on the density and distribution of uterine Natural Killer cells at mid pregnancy in mice genetically-ablated for CCR2, CCR 5 and the CCR5 receptor ligand, MIP-1 alpha.

Several chemoattractants mediate Natural Killer (NK) cell migration. The CC-chemokines, monocyte inflammatory protein (MIP)-1 alpha, regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage chemotactic protein (MCP)-1 are the most potent. Peripheral NK cells express the CC-chemokine receptor CCR2, for MCP-1 and CCR5, for MIP-1 alpha and RANTES. These chemokines are detected in the uterus during the estrous cycle and become elevated during pregnancy. To assess the roles of CCR2, CCR5 and MIP-1 alpha in NK cell migration to the uterus and localization within implantation sites, histological analysis was conducted on implantation sites from mice genetically-ablated for CCR2, CCR5, MIP-1 alpha or CCR2 and MIP-1 alpha. Uterine NK (uNK) cell densities in both the decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp) of all mutant strains matched wildtype controls. Ratios of vascular: non-vascular uNK cell position were identical in mutants and controls. In the decidua basalis, 25-35% and in the MLAp, 15-20% of uNK cells were perivascular. Intravascular uNK cells were observed in the decidua basalis but not in the MLAp and were more numerous at gestation day 10 than 12. Two measures of uNK cell activation, cell diameter and cytoplasmic granule number, were similar in the mutants and controls. Thus, migration, distribution and activation of NK cells within the pregnant uterus are independent of CCR2, CCR5 and MIP-1 alpha.

Prolonged gestation does not extend survival of uterine natural killer lymphocytes in mice deleted in the receptor for prostaglandin F2alpha.

During decidualization in mice and women, expansion of the Natural Killer (NK) cell lineage occurs within the uterus. In rodents, peak numbers of uterine (u)NK cells are reached at mid-gestation. The population then declines and residual cells are shed with the placenta. Decidualization, but not a fetus, is required to induce division and maturation of uNK cells. Mechanisms regulating the decline in uNK cells are unknown. To determine if the conceptus or its products have regulatory roles on uNK cell survival during normal gestation, a histological time course study was undertaken of implantation sites in mice ablated in the gene for the Prostaglandin F2alpha receptor (PGF2alphaR). These females experience normal gestation but fail to initiate labour and delivery. Their pregnancies extend a further 4-7 days before onset of maternal compromise. Large numbers of uNK cells were present in PGF2alphaR null mice by gestational day (gd) 10 and numbers had begun to decline at gd 14. By gd 18, very few uNK cells remained and no uNK cells were found at day 22 of extended gestation. Thus, the population history of uNK cells in PGF2alphaR null mice resembles that of uNK cells in normal mice, suggesting that the placenta, its products, the fetus and PGF2alpha are not factors that influence the rate of uNK cell decline in late gestation.