Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.

Olvanil activates sensory nerve fibers, increases T cell response and decreases metastasis of breast carcinoma.

BACKGROUND: Inactivation of sensory neurons expressing transient receptor potential vanilloid 1 (TRPV1) enhances breast cancer metastasis. Sensory neurons have profound effects on immune response to a wide range of diseases including cancer. Hence, activation of sensory nerves using feasible approaches such as specific TRPV1 agonists may inhibit breast cancer metastasis through neuroimmune pathways. TRPV1 agonists are considered for the treatment of pain and inflammatory diseases. METHODS: We here first determined the effects of four different TRPV1 agonists on proliferation of three different metastatic breast carcinoma cells since TRPV1 is also expressed in cancer cells. Based on the results obtained under in-vitro conditions, brain metastatic breast carcinoma cells (4TBM) implanted orthotopically into the mammary-pad of Balb-c mice followed by olvanil treatment (i.p.). Changes in tumor growth, metastasis and immune response to cancer cells were determined. RESULTS: Olvanil dose-dependently activated sensory nerve fibers and markedly suppressed lung and liver metastasis without altering the growth of primary tumors. Olvanil (5 mg/kg) systemically increased T cell count, enhanced intra-tumoral recruitment of CD8+ T cells and increased IFN-γ response to irradiated cancer cells and Con-A. Anti-inflammatory changes such as increased IL-10 and decrease IL-6 as well as S100A8+ cells were observed following olvanil treatment. CONCLUSIONS: Our results show that anti-metastatic effects of olvanil is mainly due to activation of neuro-immune pathways since olvanil dose used here is not high enough to directly activate immune cells. Furthermore, olvanil effectively depletes sensory neuropeptides; hence, olvanil is a good non-pungent alternative to capsaicin.

Presence of S100A8/Gr1-Positive Myeloid-Derived Suppressor Cells in Primary Tumors and Visceral Organs Invaded by Breast Carcinoma Cells.

BACKGROUND: Increased S100A8/A9 expression in Gr1-positive cells has been shown in myeloid-derived suppressor cells and may play a role in the formation of a metastatic milieu. We aimed to determine S100A8/A9 expression alone and with coexpression of Gr1 (a myeloid marker) in primary tumor and visceral tissues invaded by metastatic breast carcinoma. MATERIALS AND METHODS: Female BALB/c mice were injected with 4TLM, 4THM, and 67NR orthotopically. Confluent cells (75%-80%) were used. Primary tumor, lung, liver, and spleen tissue samples were removed 26 days after injection. Peripheral blood smears and metastasis assay were performed, as was immunohistochemistry and staining. RESULTS: S100A8/A9 immunoreactivity alone or coexpressed with Gr1 was found in primary tumors formed by 4TLM and 4THM cells, which was markedly higher than in primary tumors formed by nonmetastatic 67NR cells. Similarly, liver and lung tissues obtained from mice injected with 4TLM or 4THM cells were invaded by S100A8/A9-positive and Gr1-positive cells. Double-positive cells were markedly fewer in liver and lung tissues of animals injected with 67NR cells. S100A8/A9-positive cells were mostly localized in red pulp of spleens. We observed an increased number of neutrophils in the peripheral blood of mice injected with metastatic breast carcinoma cells. CONCLUSION: Tumor-derived factors may increase S100A8/A9-positive cells locally and systemically, and S100A8/A9-positive cells may provide an appropriate milieu for the formation of metastasis.