Sphingosine kinases negatively regulate the expression of matrix metalloproteases (MMP1 and MMP3) and their inhibitor TIMP3 genes via sphingosine 1-phosphate in extravillous trophoblasts.

PURPOSE: Extracellular matrix remodeling is essential for extravillous trophoblast (EVT) cell migration and invasion during placental development and regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs). Sphingosine kinases (SPHK1 and SPHK2) synthesize sphingosine-1-phosphate (S1P), which works either intracellularly or extracellularly via its receptors S1PR1-5 in an autocrine or paracrine manner. The role of SPHKs/S1P in regulating the expression of MMPs and TIMPs in EVT is mostly unknown and forms the primary objective of the study. METHODS: HTR-8/SVneo cells were used as a model of EVT. To inhibit the expression of SPHKs, cells were treated with specific inhibitors, SK1-I and SKI-II, or gene-specific siRNAs. The expressions of MMPs and TIMPs were estimated by qPCR. RESULTS: We demonstrated that SPHK1, MMP1-3, and TIMP1-3 were highly expressed in HTR-8/SVneo cells. We found that treatment of cells with SK1-I, SKI-II, and knockdown of SPHK1 or SPHK2 increased the expression of MMP1, MMP3, and TIMP3. The addition of extracellular S1P inhibits the upregulation of MMPs and TIMPs in treated cells. CONCLUSIONS: SPHKs negatively regulate the expression of MMP1, MMP3, and TIMP3. The level of intracellular S1P acts as a negative feedback switch for MMP1, MMP3, and TIMP3 expression in EVT cells.

Thrombin stimulates gene expression and secretion of IL-11 via protease-activated receptor-1 and regulates extravillous trophoblast cell migration.

Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times. Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.

Combined effect of heat and nutritional stress on superovulation of Malpura ewes in a semi-arid region.

Sheep reared in hot semi-arid environments are generally exposed to heat and nutritional stress in some seasons of the year, which affects both production and reproduction. To assess the effect of high ambient temperature and feed scarcity on superovulation, 16 adult Malpura ewes were randomly divided into two groups of 8 animals each. G1 (control) was kept under a shed and offered a maintenance diet, and G2 (combined stress) was subjected to both nutritional (30% less of maintenance diet) and heat (38-44 °C for 6 h/day) stress. Ewes were superovulated without estrus synchronization by a combination of single injection of 200 IU eCG and 8 injections of FSH (Folltropin-V) at 12-h intervals in tapering doses of 5 mg/kg body weight, starting from the day 7 of natural estrus. eCG was given with the first injection and PGF2α (10 mg) was given with the second last FSH injection. G2 increased respiration rate and rectal temperature (P < 0.01), and blood urea level (P < 0.05), whereas it decreased average daily gain, plasma T4 concentration (P < 0.01) and body weight (P < 0.05). Plasma estradiol level was lower (P < 0.05) in G2 ewes as compared to control (G1) ewes. However, the number of ewes showed a superovulatory response (88 vs 66% ewes ≥ 3 corpus luteum), ovulation rate (8.75 vs 5.88) and embryo production (5.5 vs 3.9) decreased, and the number of large follicles (anovulation) increased (1.0 vs 2.14) in G2 ewes. G2 had a comparable effect on the superovulatory response compared to control ewes although physiological changes occurred as an adaptive mechanism to stress. Therefore, the well-adapted cyclic sheep of the semi-arid region may be used for superovulation despite the stressful condition of heat exposure and nutritional insufficiency.