The Function And Pathways Of Leptin In Porcine Implantation

Litter size is the most crucial reproduction trait in swine. Implantation is a key period which can affect litter size of sow. There is accumulating evidence that leptin may be directly involved in mammalian reproduction, especially in embryo implantation. The study was designed to explore the spatio-temporal expression pattern of ob/ob-Rb in porcine endometrium, to verify the function of leptin in cellular level, to study the pathways induced by leptin in porcine implantation.Experiment1, twelve multiparous Yorkshire sows (3-5parity) were assigned to four groups (n=3/group) as follows:non-pregnant group (13-18day of the cycle), early, middle and later phase of implantation groups (day13,18and24of pregnancy). The mRNA and protein expressions of ob/ob-Rb in porcine endometrium during implantation were compared using RT-PCR and WB. Immunohistochemistry was also performed to study the localization of leptin and ob-Rb protein in porcine endometrium. Ob mRNA level went up with the advance of pregnancy and was higher at implantation site than inter-implantation site (P<0.05). But ob-Rb mRNA went down with the advance of pregnancy and lessened in implantation site compared with inter-implantation site (P<0.05). Localization of leptin in porcine uterus was in luminal epithelium and glandular epithelium in pregnant pigs, but distinct immune-staining of leptin also detected in stroma in non-pregnancy porcine uterus except for luminal epithelium and glandular epithelium.Experiment2, establishment of porcine endometrial epithelial cell (PEEC) model in vitro. A randomly selected uterine horn of multiparous Large White sows (Day18of pregnancy) was ligatured and washed with sterile PBS. Endometrium tissue was cultured at37℃,5%CO2. Endometrial epithelial cell (EEC) was acquired by trypsin digestion for2-3times. Then the morphology, immunochemistry and growth character of EEC were assessed. EEC growth curve was drawn by MTT method. In conclusions, PEEC was successfully established by tissue explants culture method.Experiment3, the function of leptin was verified at cell level. PEECs were treated with basal medium containing leptin (0,0.01,0.1,1,10nM) for24/48h. The effects of leptin on PPECs proliferation, the expression of factors associated with implantation and antioxidant capability of PEECs were tested using MTT method, RT-PCR, WB and antioxidant assay kits. The result manifested that leptin did not affect the PPECs proliferation after treating cell with leptin for24h and48h compared with control group (P>0.05). Leptin can upregulate the expression of factors associated with implantation in dose-dependent manner(P<0.05). Leptin decreased the concentration of ROS and MDA, upregulated the activity of GSH-PX and SOD in dose-dependent manner (P<0.05), and enhanced the T-AOC in PEECs.Experiment4studied the effects of pathways inhibitors (AG490, LY294002and U0126) on leptin-regulation in the expression of factors associated with implantation and antioxidant capability of PEECs. The results showed that blocking JAK2, PI(3)K/Akt participated the regulation of leptin on the expression of all factors associated with implantation of PEECs. But MAPK pathway was partly involved in the expression regulation by leptin. And MAPK was found as an important pathway in the process of leptin regulated PEECs antioxidation.In conclusion, ob/ob-Rb expressed in porcine endometrium spatio-temporal specifically. leptin played action in porcine implantion through regulating expressions of factors associated with implantation and improving the antioxidant capability of PEECs. JAK2-STAT3, PI(3)K-Akt and MAPK pathways inordinately participated in the process of leptin-regulated in the expression of factors associated with implantation and the antioxidant capability of PEECs. Thereinto, PI(3)K may be the key pathway which took part in the process of leptin regulated the implatation fators expression, and MAPK may be an important pathway involved in improvement of PEECs antioxidant capability induced by leptin.