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Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta.

Hayashi KG, Hosoe M, Sakumoto R, Takahashi T - Reprod. Biol. Endocrinol. (2013)

Bottom Line: A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells.Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period.

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Affiliation: Animal Physiology Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.

ABSTRACT

Background: Adrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome.

Methods: For real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation.

Results: AM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.

Conclusions: Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.

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Protein localization of AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on day 56 of gestation. Protein localization of (A) AM, (D) CRLR, (G) RAMP2 and (J) RAMP3 was detected by immunohistochemistry. (B, E, H and K) enlarged images of frames in A, D, G and J, respectively. Sections (7 μm) of the bovine placentome on Day 56 of gestation were incubated with anti-human AM, anti-human CRLR, anti-human RAMP2 and anti-human RAMP3 polyclonal antibodies. A distinct AM signal was found only in BNCs. Protein localization of CRLR, RAMP2 and RAMP3 was detected in cotyledonary villous and CEs. (C, F, I and L) no signal was detected in the negative control sections using normal rabbit IgG. BNC, trophoblast binucleate cell; CE, caruncular epithelial cell; CV, cotyledonary villous. Scale bars = 20 μm.
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Figure 7: Protein localization of AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on day 56 of gestation. Protein localization of (A) AM, (D) CRLR, (G) RAMP2 and (J) RAMP3 was detected by immunohistochemistry. (B, E, H and K) enlarged images of frames in A, D, G and J, respectively. Sections (7 μm) of the bovine placentome on Day 56 of gestation were incubated with anti-human AM, anti-human CRLR, anti-human RAMP2 and anti-human RAMP3 polyclonal antibodies. A distinct AM signal was found only in BNCs. Protein localization of CRLR, RAMP2 and RAMP3 was detected in cotyledonary villous and CEs. (C, F, I and L) no signal was detected in the negative control sections using normal rabbit IgG. BNC, trophoblast binucleate cell; CE, caruncular epithelial cell; CV, cotyledonary villous. Scale bars = 20 μm.

Mentions: The results of immunohistochemistry for AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on Day 56, 150 and 230 of gestation are shown in Figures 7, 8 and 9, respectively. Throughout gestation, a distinct AM signal was found only in BNCs (Figure 7A and B, Figure 8A and B and Figure 9A and B). CRLR, RAMP2 and RAMP3 proteins were detected in cotyledonary villous, including BNCs, and caruncular epithelial cells (Figure 7D,E,G,H,J and K, Figure 8D,E,G,H,J and K and Figure 9D,E,G,H,J and K).


Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta.

Hayashi KG, Hosoe M, Sakumoto R, Takahashi T - Reprod. Biol. Endocrinol. (2013)

Protein localization of AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on day 56 of gestation. Protein localization of (A) AM, (D) CRLR, (G) RAMP2 and (J) RAMP3 was detected by immunohistochemistry. (B, E, H and K) enlarged images of frames in A, D, G and J, respectively. Sections (7 μm) of the bovine placentome on Day 56 of gestation were incubated with anti-human AM, anti-human CRLR, anti-human RAMP2 and anti-human RAMP3 polyclonal antibodies. A distinct AM signal was found only in BNCs. Protein localization of CRLR, RAMP2 and RAMP3 was detected in cotyledonary villous and CEs. (C, F, I and L) no signal was detected in the negative control sections using normal rabbit IgG. BNC, trophoblast binucleate cell; CE, caruncular epithelial cell; CV, cotyledonary villous. Scale bars = 20 μm.
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Figure 7: Protein localization of AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on day 56 of gestation. Protein localization of (A) AM, (D) CRLR, (G) RAMP2 and (J) RAMP3 was detected by immunohistochemistry. (B, E, H and K) enlarged images of frames in A, D, G and J, respectively. Sections (7 μm) of the bovine placentome on Day 56 of gestation were incubated with anti-human AM, anti-human CRLR, anti-human RAMP2 and anti-human RAMP3 polyclonal antibodies. A distinct AM signal was found only in BNCs. Protein localization of CRLR, RAMP2 and RAMP3 was detected in cotyledonary villous and CEs. (C, F, I and L) no signal was detected in the negative control sections using normal rabbit IgG. BNC, trophoblast binucleate cell; CE, caruncular epithelial cell; CV, cotyledonary villous. Scale bars = 20 μm.
Mentions: The results of immunohistochemistry for AM, CRLR, RAMP2 and RAMP3 in the bovine placentome on Day 56, 150 and 230 of gestation are shown in Figures 7, 8 and 9, respectively. Throughout gestation, a distinct AM signal was found only in BNCs (Figure 7A and B, Figure 8A and B and Figure 9A and B). CRLR, RAMP2 and RAMP3 proteins were detected in cotyledonary villous, including BNCs, and caruncular epithelial cells (Figure 7D,E,G,H,J and K, Figure 8D,E,G,H,J and K and Figure 9D,E,G,H,J and K).

Bottom Line: A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells.Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period.

View Article: PubMed Central - HTML - PubMed

Affiliation: Animal Physiology Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.

ABSTRACT

Background: Adrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome.

Methods: For real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation.

Results: AM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.

Conclusions: Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.

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