Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes

Abstract

Background: Recent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed.

Results: 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca²⁺ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells.

Conclusion: Collectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.

Keywords: Calcium; Endometrium; Ovine; Phosphate; Vitamin D.

Fig. 1

Quantification of phosphate, calcium, and 25(OH)D in uterine flushings, endometrial homogenates, and plasma. Phosphate was quantified in uterine flushings (A), plasma (C), and endometrial homogenates (E) using a colorimetric assay. Similarly, calcium was quantified in uterine flushings (B), and endometrial homogenates (F) using a colorimetric assay. 25(OH)D was quantified in plasma (D) and endometrial homogenates (G) by ELISA. Error bars represent SEM. Different letters indicate that means differ from one another (P < 0.05). n = 4–5 per day

Fig. 2

Quantification of mRNAs with roles in phosphate signaling and transport. The mRNA transcripts for FGFR1 (A), FGFR2 (B), FGF23 (C), KL (D), ADAM10 (E), ADAM17 (F), and SLC20A1 (G) were quantified in endometria using qPCR. Different letters indicate that group means differ (P < 0.05). Error bars represent SEM. n = 4–5 per day. *P < 0.05. **P < 0.01

Fig. 3

Quantification of mRNAs with roles in calcium and vitamin D signaling, transport, and metabolism. The mRNA transcripts for ATP2B4 (A), PTHrP (B), S100G (C), S100A9 (D), TRPV6 (E), CYP2R1 (F), CYP24 (G), and VDR (H) were quantified in endometria using qPCR. Different letters indicate that group means differ (P < 0.05). Error bars represent SEM. n = 4–5 per day. *P < 0.05. **P < 0.01

Fig. 4

Representative immunolocalization of FGF23, KL, and ADAM17 proteins in ovine endometria. Representative images of immunohistochemical localization of FGF23 (A-G), KL (H-N), and ADAM17 (O-U) proteins in ovine endometria on Days 1, 9, and 14 of the estrous cycle. Rabbit IgG (rIgG) controls were included at equivalent protein concentrations to the antibody of interest as a negative control (D, K, R). BV = blood vessels, LE = luminal epithelium, GE = glandular epithelium, sGE = superficial glandular epithelium, Myo = myometrium. Scale bars represent 100 μm

Fig. 5

Representative immunolocalization of S100A9, S100A12, and S100G proteins in ovine endometria. Representative images of immunohistochemical localization of the calcium binding proteins S100A9 (A-G), S100A12 (H-N), and S100G (O-U) in ovine endometria on Days 1, 9, and 14 of the estrous cycle. Rabbit IgG (rIgG) controls were included at equivalent protein concentrations to the antibody of interest as a negative control (D, K, R). BV = blood vessels, LE = luminal epithelium, GE = glandular epithelium, sGE = superficial glandular epithelium, Myo = myometrium. Scale bars represent 100 μm

Fig. 6

Representative immunolocalization of CYP2R1, CYP27B1, and VDR proteins in ovine endometria. Representative images of immunohistochemical localization of CYP2R1 (A-G), CYP27B1 (H-N), and VDR (O-U) proteins in ovine endometria on Days 1, 9, and 14 of the estrous cycle. Rabbit IgG (rIgG) controls were included at equivalent protein concentrations to the antibody of interest as a negative control (D, K, R). BV = blood vessels, LE = luminal epithelium, GE = glandular epithelium, sGE = superficial glandular epithelium, Myo = myometrium. Scale bars represent 100 μm