Modulation of the inflammatory environment by spermatozoa through regulation of transforming growth factor beta in porcine uterine epithelial cells.

This study investigated the changes in the mRNA expression of transforming growth factor beta (TGF-ß), plasminogen activators (PAs), and interleukin (IL) caused by sperm, as well as the regulatory mechanism of PA activity through TGF-ß, in porcine uterine epithelial cells. The cells were isolated from the uterine horn of pig and co-incubated with Percoll-separated boar sperm (45% or 90%), or TGF-ß for 24 h. The mRNA expression of TGF-ß isoforms (TGF-ß1, 2 and 3) and their receptors (TGF-ß R1 and R2), PAs (urokinase-type, uPA; tissue-type, tPA; uPA receptor, uPAR; type 1 PA inhibitor, PAI-1), IL-6 and IL-8 was analyzed using real-time PCR. Supernatant was used to measure PA activity. Co-incubation with sperm from the 90% Percoll layer increased TGF-ß1 mRNA, whereas TGF-ß2 and TGF-ß3 were decreased (P < 0.05). However, both TGF-ßRs were not changed by the presence of the spermatozoa. Expression of tPA, PAI-1, IL-6, and IL-8 mRNA was down-regulated by 90% Percoll-separated sperm (P < 0.05), and sperm from 45% Percoll increased uPA expression (P < 0.05). TGF-ß decreased tPA and IL-8 mRNA expression, and increased uPAR and PAI-1 mRNA (P < 0.05). The suppressive effect of TGF-ß on PA activity was blocked by Smad2/3 and JNK1/2 signaling inhibitors (P < 0.05). In conclusion, sperm separated in 90% in porcine uterus could suppressed inflammation via modulation of TGF-ß and down-regulation of PAs and ILs. Therefore, the regulatory mechanism of inflammation by sperm in the porcine uterus could be associated with interactions between numerous cytokines including TGF-ß.

Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs.

The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3-6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles 3-6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

Effects of 17ß-estradiol, Interleukin-1ß, and Human Chorionic Gonadotropin on Activity and mRNA Expression of Plasminogen Activators in Porcine Endometrial Cells.

This study aimed to investigate changes in the activity and mRNA expression of plasminogen activators (PAs) induced by 17ß-estradiol (E2), human chorionic gonadotropin (hCG), and interleukin-1ß (IL-1ß) in porcine endometrial cells. Endometrial cells were isolated from the epithelium and cultured to 80% confluence. They were then treated for 24 h with E2 (0.2, 2, 20, and 200 ng/mL), IL-1ß (0.1, 1, 10, and 100 ng/mL), and hCG (0.5, 1, 1.5 and 2 IU/mL). mRNA expressions of urokinase-type (uPA) and tissue-type (tPA) PAs were analyzed using reverse transcription PCR, and activities were measured using a PA activity assay. mRNA expressions of uPA and tPA increased with E2 treatment; however, this was not significant. Similarly, treatment with hCG did not influence the mRNA expressions of PAs. Interestingly, treatment with 0.1 ng/mL IL-1ß significantly reduced the mRNA expression of uPA, but did not affect that of tPA. Treatment with 2, 20, and 200 ng/mL E2 increased PA activity compared with the control group; treatment with 0.1 and 1 ng/mL IL-1ß significantly increased PA activity compared with the other IL-1ß treatment groups, whereas treatment with 10 and 100 ng/mL IL-1ß decreased. Treatment with 2 IU/mL hCG increased PA activity compared with the other treatment groups, although there were no significant differences between the hCG and control groups. In conclusion, the activity and mRNA expression of PAs were differently regulated by the hormone/cytokine and its concentration in porcine endometrial cells. Therefore, understanding PA regulatory mechanisms may help to improve the reproductive potential of domestic animals.

Alpha-Linolenic Acid: It Contribute Regulation of Fertilization Capacity and Subsequent Development by Promoting of Cumulus Expansion during Maturation.

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and 100 µM ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in 50 µM ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by 50 µM ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and 50 µM ALA groups (p<0.05), especially, treatment of 50 µM ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and 50 µM ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

Effects of Progesterone and 17ß-Estradiol under Presence or Absence of FBS on Plasminogen Activators Activity in Porcine Uterine Epithelial Cells.

The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by 17ß-estradiol (E2) and progesterone (P4) in porcine uterine epithelial cells (pUECs). pUECs were collected from porcine uterine horn and cultured at 80% confluence. Then, 0.1% (v/v) DMSO, 20 ng/mL E2, and P4 with or without fetal bovine serum (FBS) treated to cultured cells for 24 hours. The supernatants were used for measurement of PAs activity and expression of urokinase-type PA (uPA), tissue-type PA (tPA), uPA specific receptor (uPAR), and type-1 PA inhibitor (PAI-1) mRNA were analyzed by real-time PCR. The expression of PAs-related genes was not affect by steroid hormones in both of serum treatment groups. However, PAs activity was increased by treatment of E2 compared to 0.1% DMSO treatment in serum-free group (p<0.05). Then, E2 and P4 were diluted with 0.002% (v/v) DMSO for reduction of its effect and treated to cultured cells without FBS. Only tPA mRNA was significantly increased by E2 treatment (p<0.05). PAs activity was enhanced in E2 treated group compared to control groups (p<0.05). These results indicate that serum-free condition is more proper to evaluate effect of steroid hormones and activation of PAs in pUECs was mainly regulated by estrogen. These regulation of PAs activation may be associated with uterine remodeling during pre-ovulatory phase in pigs, however, further studies are needed to investigate precise regulatory mechanism.

Change of uterine histroph proteins during follicular and luteal phase in pigs.

The aim of this study was to examine protein expression patterns of uterine histroph (UH) during the follicular phase (FP) and luteal phase (LP) in pigs. Forty-nine common proteins were identified from FP and LP samples; five were significantly down-regulated (>1.5-fold), while 15 were significantly up-regulated (>1.5-fold) in LPUH compared with FPUH (P<0.05). The 20 differentially-expressed proteins are involved in cell proliferation, cell responses, translation, transport, and metabolism and their molecular functions include nucleic acid binding, oxygen activity, enzymatic activity, growth activity, iron binding, and redox binding. Protein expression of vascular endothelial growth factor D (VEGFD), coatomer subunit gamma-2 (G2COP), collagen alpha 4 chain (COL4), cysteine rich protein 2 (CRP2), myoglobin (MYG), and galactoside 3-L-fucosyltransferase 4 (FUT4) was analyzed by Western blotting. These proteins were significantly higher in LPUH compared to FPUH (P<0.05). These data expand our understanding of changes in the intrauterine environment during the pre-implantation period in pigs.