Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

Abstract

Background: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct.

Methods: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR.

Results: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla.

Conclusions: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.

Figure 1

Expression of PR, PGRMC1 and PGRMC2 mRNAs in the bovine oviduct. Messenger RNA transcripts for PR (A), PGRMC1 (B) and PGRMC2 (C) detected by conventional RT-PCR in bovine whole oviduct sections at Day 1.5, Day 4 and Day 5 post-ovulation. Experiments were repeated twice and one representative assay is shown. Arrows indicate the size of the amplified product. I-A: ipsilateral ampulla; I-I: ipsilateral isthmus; C-A: contralateral ampulla; C-I: contralateral isthmus.

Figure 2

Western blots for PR, PGRMC1 and PGRMC2 in the bovine oviduct. Western blot analysis of PR, PGRMC1 and PGRMC2 in one bovine whole ampulla section at Day 5 post-ovulation. Molecular weight markers are indicated on the left of each blot and molecular weights of the bands are indicated on the right. Experiments were repeated three times and one representative blot is shown.

Figure 3

Localisation of PR in the bovine oviduct by immunohistochemistry. Representative images of PR immunohistochemical localisation in the ampulla (A, B) and the isthmus (C, D) of bovine oviducts at Day 1.5 (A, C) and Day 5 (B, D) post-AI. A inset: magnification of the luminal epithelium showing a nuclear and cytoplasmic staining in the epithelial cells and a nuclear staining in the stromal cells. D inset: control section incubated with mouse IgG in place of primary antibody. Black arrows indicate nuclear staining and arrow heads indicate cytoplasmic staining. LE: luminal epithelium; S: stroma; M: muscle. Original magnification A-C x200 and D x100. Scale bars: 100 μm.

Figure 4

Localisation of PGRMC1 in the bovine oviduct by immunohistochemistry. Representative images of PGRMC1 immunohistochemical localisation in the ampulla (A, B) and the isthmus (C, D) of bovine oviducts at Day 1.5 (A, C) and Day 5 (B, D) post-AI. C inset: magnification of the luminal epithelium showing a cytoplasmic staining mainly localised in the epithelial cells. D inset: control section incubated with rabbit serum in place of primary antibody. Arrow heads indicate cytoplasmic staining. LE: luminal epithelium; S: stroma; M: muscle. Original magnification A-C x200, D x100 and C inset x1000. Scale bars: 100 μm.

Figure 5

Localisation of PGRMC2 in the bovine oviduct by immunohistochemistry. Representative images of PGRMC2 immunohistochemical localisation in the ampulla (A, B) and the isthmus (C, D) of bovine oviducts at Day 1.5 (A, C) and Day 5 (B, D) post-AI. A inset: magnification of the luminal epithelium showing a nuclear and cytoplasmic staining of epithelial cells. D inset: control section incubated with mouse IgG1 in place of primary antibody. Black arrows indicate nuclear staining and arrow heads indicate cytoplasmic staining. LE: luminal epithelium; S: stroma; M: muscle. Original magnification A-D x200 and A inset x1000. Scale bars: 100 μm.

Figure 6

Quantitative expression of PR, PGRMC1 and PGRMC2 mRNAs in the bovine oviduct. Differential mRNA expression of PR (A), PGRMC1 (B) and PGRMC2 (C) between the ampulla and the isthmus in bovine oviduct epithelial cells at Day 3.5 post-ovulation in either cyclic or pregnant cows. Data are means ± SEM of 4 cows for each status (ipsilateral and contralateral oviducts were grouped). Concentrations of mRNAs were measured in duplicate. Means with designations (*) and (**) are significantly different at p < 0.05 and p < 0.01, respectively.