Fig 1.
Expression of CYP2R1 (A), CYP27B1 (B), CYP24A1 (C), GC (D), and VDR (E) mRNAs in the endometrium during the estrous cycle and pregnancy in pigs.
Endometrial tissue samples from cyclic and pregnant gilts were analyzed by real-time RT-PCR, and data are reported as expression relative to that detected on Day 12 of the estrous cycle after normalization of the transcript amount to the endogenous RPL7 control. Data are presented as mean with standard error.
Fig 2.
Localization of CYP2R1, CYP27B1, CYP24A1, GC, and VDR mRNAs in the endometrium during the estrous cycle and pregnancy by in situ hybridization analysis.
Tissue sections from skin, small intestine, or duodenum hybridized with DIG-labeled anti-sense CYP2R1, CYP27B1, CYP24A1, GC, or VDR cDNA probes served as the positive control, and representative uterine sections from the indicated day of pregnancy hybridized with DIG-labeled sense CYP2R1, CYP27B1, CYP24A1, GC, or VDR cDNA probes (Sense) served as the negative control. D, Day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; GE, glandular epithelium; St, stroma; CM, chorionic membrane. Scale bar = 100 μm.
Fig 3.
Immunohistochemical analysis of vitamin D receptor (VDR) protein in the endometrium during the estrous cycle and pregnancy in pigs.
Immunoreactive VDR protein was detected in the endometrial epithelial and stromal cells during the estrous cycle and pregnancy and in chorioallantoic tissues during pregnancy. A representative uterine section from Day 90 of pregnancy immunostained with normal rabbit IgG as a negative control (D90P IgG) is shown. D, Day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; GE, glandular epithelium; St, stroma; CM, chorionic epithelium; AM, allantoic membrane. Bars = 100 μm and 50 μm in inset.
Fig 4.
Expression of CYP2R1, CYP27B1, CYP24A1, GC, and VDR by conceptuses from Day 12 and Day 15 of pregnancy (A) and by chorioallantoic tissues during later stages of pregnancy (B).
A. RT-PCR analysis of CYP2R1, CYP27B1, CYP24A1, GC, and VDR mRNA in conceptuses on Day 12 and Day 15 of pregnancy as well as in liver and kidney tissues as positive controls was performed using total RNA preparations. RPL7 was used as a positive control. RTase +/-, with (+) or without (-) reverse transcriptase; M, molecular marker; D12 Endo, endometrium on Day 12 of pregnancy; D12 Con, Day 12 conceptus; D15 Con, Day 15 conceptus. B. Real-time RT-PCR analysis of the expression of CYP2R1, CYP27B1, CYP24A1, GC, and VDR mRNAs in chorioallantoic tissue samples on Days 30, 60, 90, and 114 of pregnancy. Data are reported as expression relative to that detected on Day 30 of pregnancy after normalization of the transcript amount to the endogenous RPL7 control, and data are presented as mean with standard error.
Fig 5.
Levels of calcitriol [1,25(OH)2D3] in plasma blood during pregnancy (A), endometrial tissues (B), and uterine flushings (C) during the estrous cycle and pregnancy in pigs.
Levels of calcitriol in plasma blood, endometrial tissue lysate, and uterine flushings were determined by ELISA. Calcitriol levels in the endometrial tissues were standardized per total tissue weight, and the amounts of calcitriol in the uterine flushings were total recoverable amounts of calcitriol. Data are presented as means with standard errors.
Fig 6.
Effects of calcitriol [1,25(OH)2D3] on the expression of implantation-related genes (A), vitamin D-related genes (B), calcium-regulatory genes (C), prostaglandin-related genes (D), and calcium-binding protein genes (E) in endometrial tissue explants.
Endometrial explants from gilts on Day 12 of the estrous cycle were cultured in the presence of 0, 2, 20, and 200 μM calcitriol. Levels of mRNAs for each endometrial gene were analyzed by real-time RT-PCR, and abundance of mRNAs is presented as expression relative to that for mRNAs in the control group of endometrial explants (0 ng/ml calcitriol) after normalization of transcript amounts to RPL7 mRNA. Data are presented as means with standard errors. For each treatment, all experiments were repeated in triplicate using endometrial tissue from each of three gilts.