Hwangbo, Yong
(College of Animal Life Sciences, Kangwon National University)
,
Lee, Mi-Rim
(College of Animal Life Sciences, Kangwon National University)
,
Cheong, Hee-Tae
(College of Veterinary Medicine, Kangwon National University)
,
Yang, Boo-Keun
(College of Animal Life Sciences, Kangwon National University)
,
Park, Choon-Keun
(College of Animal Life Sciences, Kangwon National University)
The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by $17{\beta}$-estradiol ($E_2$) and progesterone ($P_4$) in porcine uterine epithelial cells (pUECs). pUECs were collected from porcine uterine horn an...
The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by $17{\beta}$-estradiol ($E_2$) and progesterone ($P_4$) 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 $E_2$, and $P_4$ 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 $E_2$ compared to 0.1% DMSO treatment in serum-free group (p<0.05). Then, $E_2$ and $P_4$ 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 $E_2$ treatment (p<0.05). PAs activity was enhanced in $E_2$ 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.
The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by $17{\beta}$-estradiol ($E_2$) and progesterone ($P_4$) 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 $E_2$, and $P_4$ 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 $E_2$ compared to 0.1% DMSO treatment in serum-free group (p<0.05). Then, $E_2$ and $P_4$ 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 $E_2$ treatment (p<0.05). PAs activity was enhanced in $E_2$ 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.
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문제 정의
However, regulation of PAs activity in porcine uterus is still unclear. Therefore, this study was conducted to investigate change of PAs activity and mRNA expression by P4 and E2 according presence of absence fetal bovine serum (FBS) condition in porcine uterine epithelial cells (pUECs).
This study was conducted to confirm effect of steroid hormones on activation of PAs and PAs-related genes expression in pUECs at pre-ovulatory phase. Both of steroid hormones that were diluted with 0.
제안 방법
Extraction of RNA from pUECs was carried out using trizol reagent (Takara, Shiga, Kusatsu, Japan) and cDNA was synthesized by PrimeScriptTM 1st strand cDNA Synthesis Kit (Takara) according to manufacturer’s instructions.
All of primers used in this study were designed by Primer3 online software (Table 1). Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan) was used to real time RTPCR and cDNA amplification was performed following three steps: denaturation at 95℃ for 15 sec, annealing at 60℃ for 30 sec, extension at 72℃ for 30 sec, 40 cycles. Relative expression levels of urokinase-type PA (uPA), tissue-type PA (tPA), uPA specific receptor (uPAR), and type-1 PA inhibitor (PAI-1) mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed by 2−ΔΔCt method.
데이터처리
Data from experiment 1 were analyzed using Duncan’s multiple range test, and t-test was used for experiment 2.
성능/효과
In conclusion, we found that effect of steroid hormones is strongly in serum-free condition than serum condition and PAs activity was enhanced by estrogen, whereas P4 did not affect. The Serum-free condition was proper to measure activity of protease by steroid hormones because various components in FBS could influence to express receptors for hormones and activation of protease.
During the pre-ovulatory phase, expression of PRs and ERs in uterine cells is increased by rapidly increased estrogen from ovary and increased concentration of P4 prevent ERs expression via binding with PRs (McCracken, 1980). These studies demonstrated that E2 and P4 are closely associated with regulation of functional and physiological conditions in endometrium as a key regulatory factors.
후속연구
Therefore, because of several protease inhibitors including α2M in FBS, changes of PAs activity by both steroid hormones was not detected. However, more researches are needed whether FBS inhibits function of steroids or action of proteases such as plasmin.
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