TRPV6 and Calbindin-D9k-expression and localization in the bovine uterus and placenta during pregnancy

Abstract

Background: Transient receptor potential channel type 6 (TRPV6) and Calbindin-D9k (CaBP-9k) are involved in the active calcium (Ca2+) transport mechanism in many tissues including placenta and uterus, suggesting a role in the establishment and maintenance of pregnancy. Moreover, TRPV6 and CaBP-9k seem to support the materno-fetal Ca2+ transport that is crucial for fetal Ca2+ homeostasis, bone growth and development. However, it is unknown if these proteins are also involved in the aetiology of pathologies associated with parturition in cows, such as retained fetal membranes (RFM). The aim of the current study was to create an expression profile of uterine and placentomal TRPV6 and CaBP-9k mRNAs and proteins during pregnancy and postpartum in cows with and without fetal membrane release.

Methods: Uteri and placentomes of 27 cows in different stages of pregnancy and placentomes of cows with and without RFM were collected. Protein and mRNA expression of TRPV6 and CaBP-9k was investigated by real-time PCR, immunohistochemistry and Western blot.

Results: In the uterine endometrium, highest TRPV6 and CaBP-9k expression was found in the last trimester of pregnancy, with a particular increase of protein in the glandular epithelium. In the placentomes, a gradual increase in TRPV6 mRNA was detectable towards parturition, while protein expression did not change significantly. Placentomal CaBP-9k expression did not change significantly throughout pregnancy but immunohistochemistry revealed an increase in staining intensity in the maternal crypt epithelium. Immunohistochemical, stronger placental CaBP-9k signals were seen in animals with RFM compared to animals with an undisturbed fetal membrane release, while protein levels, measured by Western blot analyses did not change significantly.

Conclusions: The results of the present study demonstrate a dynamic expression of TRPV6 and CaBP-9k during pregnancy in the bovine uterine endometrium and placentomes, suggesting a functional role for these proteins in Ca2+ metabolism during pregnancy. The temporal and spatial expression patterns indicate that TRPV6 and CaBP-9k may be involved in materno-fetal Ca2+ transport, mainly through an interplacentomal transport, and that both proteins may participate in physiological processes that are crucial for fetal and placental development. However, neither TRPV6 nor CaBP-9k seem to be causative in the retention of fetal membranes.

Figure 1

Uterine and placentomal TRPV6 and CaBP-9k mRNA expression. TRPV6 and CaBP-9k mRNA expression in the intercaruncular wall (Figure 1A, C) and in the placenta (n ≥ 3 animals/month) (Figure 1B, D) from the 2^nd to 9^th months of pregnancy, determined by real-time PCR. Bars with ** differ significantly (p < 0.05) compared to the second month (*). Data were analysed using a parametric one-way analysis of variance (ANOVA), followed by Dunnett’s comparison test and are presented as means ± SD. The nonparametric dataset of placental CaBP-9k was analysed using the Kruskal-Wallis test, followed by Dunn`s post test.

Figure 2

Immunolabelling of TRPV6 in the uterine endometrium. Localisation of uterine TRPV6 during pregnancy with strong labelling in the glandular (GE) and luminal epithelia (LE). Figure 2F shows a negative control, without anti-TRPV6 treatment. Scale bars indicate 200 μm.

Figure 3

Immunolabelling of TRPV6 in the placentomes and adherent fetal membranes. Localisation of TRPV6 during pregnancy in the placenta and adherent membranes with strong staining in the inner layer (IL) of the place tome (A, D) and the adherent fetal membranes (FM) (A, C). Considerably weaker staining in the middle (ML) and outer labyrinth (OL) layers (A, B). Scale bars indicate 200 μm.

Figure 4

Immunolabelling of CaBP-9k protein in the uterine endometrium. Localisation of CaBP-9k in the glandular (GE) and luminal epithelia (LE) during pregnancy (A-D). Strongest staining was detected in the fetal membranes (FM) (A, B). Scale bars indicate 200 μm.

Figure 5

Immunolabelling of CaBP-9k in placentomes and adherent fetal membranes. Localisation of CaBP-9k during pregnancy with strong signals in the inner layer (IL) of the place tome and in the fetal membranes (FM) compared to the outer labyrinth layer (OL) (A, C). Strong staining in the binucleate trophoblast cells (BNC) and in the maternal epithelium (ME) inside the place tome (B, C). Figure 2F shows a negative control, without anti-TRPV6 treatment. Scale bars indicate 200 μm.

Figure 6

Western blot analyses of endometrial and placentomal TRPV6 protein during pregnancy. A) Representative Western blot of TRPV6 protein expression in the uterine wall (n = 3 animals/month). Bands of approximately 75 kDa were detected. An additional band of approximately 90 kDa was seen in the 2^nd and 3^rd months. After incubation with N-glycosidase F (PNGase F), only the smaller band of 75 kDa was seen, demonstrating that the other band is the glycosylated variant of TRPV6. As a negative control, samples were preincubated with a control antigen. B) Relative expression of TRPV6 to GAPDH was determined by densitometry using ImageJ software. No significant changes were detected (p > 0.05). C) Representative Western blot analyses of TRPV6 in the placenta from 2^nd to 9^th months of pregnancy (n = 3 animals/month). D) The expression of TRPV6 relative to GAPDH was determined by measuring the optical density using ImageJ software. No significant changes (p > 0.05) were detected. Data were analysed using a parametric one-way analysis of variance (ANOVA), followed by Dunnett’s comparison test, where the second month was taken as the control group, and are presented as means ± SD.

Figure 7

Western blot analyses of endometrial and placental CaBP-9k protein during pregnancy. A) Representative Western blot of CaBP-9k in the uterine wall (n = 3 animals/month). Bands of approximately 9 kDa were detected. B) The highest expression relative to GAPDH was found in the 7^th month of pregnancy, determined by measuring the optical density using ImageJ software. Bars with ** differ significantly (p < 0.05) compared to the 2^nd month (*). C) Representative Western blot of CaBP-9k in the placenta (n = 3 animals/month). D) Relative expression levels of CaBP-9k to GAPDH were determined by densitometry using ImageJ software. Significant changes in the expression levels were not detected (p > 0.05). Data were analysed using a parametric one-way analysis of variance (ANOVA), followed by Dunnett’s comparison test, where the second month was taken as the control group, and are presented as means ± SD.

Figure 8

Placental TRPV6 and CaBP-9k mRNA expression post partum in cows with and without retained fetal membranes. Detection of TRPV6 (A) and Calbindin-D9k (CaBP-9k) (B) mRNA levels in the placenta of postpartum cows that retained the fetal membranes for more than 12 hours (RFM; n = 5) and cows that discharged the fetal membranes (DFM; n = 6), measured by real-time PCR (Taqman). No significant changes were observed (p > 0.05). Data were analyzed by an unpaired one-tailed t-test and are presented as means ± SD.

Figure 9

Localisation of TRPV6 protein in the placentomes and fetal membranes in cows with (RFM) (C, D) and without retained fetal membranes (DFM) (A, B). Strongest labeling of TRPV6 protein was seen in the fetal membranes (FM) of all animals and in the maternal crypt epithelium (ME) of cows with RFM (D).

Figure 10

Localisation of placental CaBP-9k protein post partum in cows with (RFM) (C, D) and without retained fetal membranes (DFM) (A, B). Strong staining was detected in the fetal membranes (FM) of all investigated animals and in the maternal crypt epithelium (ME) of animals with retained fetal membranes (D) and in the binucleate trophoblast cells (BNC) of all animals.

Figure 11

Western blot analyses of placental TRPV6 and CaBP-9k postpartum in cows with (RFM) and without retained fetal membranes (DFM). A) Representative Western blot of TRPV6 in the placenta. Bands of approximately 75 kDa were detected in all animals. B) Relative expression of TRPV6 was determined by measuring the optical density relative to GAPDH using ImageJ software. Significant changes in expression levels were not detected (p > 0.05). C) Representative Western blot of CaBP-9k. D) The relative expression levels to GAPDH were determined by densitometry using ImageJ software. No significant changes were detected (p > 0.05). Data were analyzed by an unpaired one-tailed t-test and are presented as means ± SD.