Next, intracellular calcium ions are transported by calcium transport proteins, calbindinD9k (S100G) and calbindin-D28k (CALB1), to the b a solateral side of epithelial cells.
S100G, an intracellular calcium ion transport protein, is expressed in the uterine endometrium during the estrous cycle and pregnancy in mice (Warembourg et al., 1987), rats (Tatsumi et al., 1999) and pigs (Krisinger et al., 1995).
In our previous study, it has been shown that endometrial TRPV6 expression is increased by estrogen during the implantation period (Choi et al., 2009), but effect of estrogen on endometrial S100G expression has been not determined.
Therefore, this study was to determine i) regulation of the uterine endometrial S100G expression by estrogen and IL1B during the implantation period; ii) expression of S100G in conceptuses during early pregnancy; and iii) expression of S100G in the uterine endometrium from gilts with SCNT-derived conceptuses compared to that from gilts with conceptuses derived from natural mating on day (D) 12 of pregnancy.
To determine the effects of cytokines on S100G expression, explant tissues were treated with 0, 1, 10, 100 ng/ml IL1B (catalog number 19401; Sigma) in the presence of both [E.sub.2] (50 ng/ml) and [P.sub.4] (3 ng/ml) at 37[degrees]C for 24 h.
Total RNA extraction, cloning of porcine S100G and RT-PCR
The cDNA templates were then diluted 1:4 with sterile water and amplified by PCR using Taq polymerase (Takara Bio, Shiga, Japan) and specific primers (forward, 5'-TCC TGC AGA ACT GAA GAG CA-3'; reverse, 5'-GCA GAG ACA TGG GTG GTT TT-3') based on mRNA sequences of porcine S100G (GenBank accession no.
To analyze levels of S100G mRNAs in the uterine endometrium, real-time RT-PCR was performed using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA, USA) using the SYBR Green method.
Data from real-time RT-PCR analysis for S100G mRNA levels were subjected to least squares ANOVA using the General Linear Models procedures of SAS (Cary, NC, USA).
Regulation of S100G expression by steroid hormones in the uterine endometrium pregnancy in pigs