An approach to uncover the relationship between 17b-estradiol and ESR1/ESR2 ratio in the regulation of canine corpus luteum

Abstract

The canine corpus luteum (CL) is able to synthetise, activate and deactivate 17b-estradiol (E2) and also expresses nuclear estrogen receptors in a time-dependent manner during diestrus. Nevertheless, we are still missing a better comprehension of E2 functions in the canine CL, especially regarding the specific roles of estrogen receptor alpha (ERa) and ERb, encoded by ESR1 and 2, respectively. For that purpose, we analyzed transcriptomic data of canine non-pregnant CL collected on days 10, 20, 30, 40, 50 and 60 of diestrus and searched for differentially expressed genes (DEG) containing predicted transcription factor binding sites (TFBS) for ESR1 or ESR2. Based on biological functions of DEG presenting TFBS, expression of select transcripts and corresponding proteins was assessed. Additionally, luteal cells were collected across specific time points during diestrus and specificity of E2 responses was tested using ERa and/or ERb inhibitors. Bioinformatic analyses revealed 517 DEGs containing TFBS, from which 67 for both receptors. In general, abundance of predicted ESR1 targets was greater in the beginning, while abundance of ESR2 targets was greater in the end of diestrus. ESR1/ESR2 ratio shifted from an increasing to a decreasing pattern from day 30 to 40 post ovulation. Specific receptor inhibition suggested an ERa-mediated positive regulation of CL function at the beginning of diestrus and an ERb-mediated effect contributing to luteal regression. In conclusion, our data points toward a broad spectrum of action of E2 and its nuclear receptors, which can also act as transcription factors for other genes regulating canine CL function.

Keywords: 17b-estradiol; ESR1; ESR2; diestrus; dog; progesterone.

Figure 1

Gene expression of ESR1 (A), ESR2 (B) and ratio of ESR1/ESR2 (C) in canine CL during diestrus (10 to 60 days p.o.). Data are presented as mean ± standard error of relative gene expression (n = 4 animals/group). Bars with different letters indicate significant differences among groups (P < 0.05).

Figure 2

Gene expression of CTNNB1 (A), LEF-1 (B), CCND1 (C), NDGR2 (D), and ATP1A1 (E) in canine CL during diestrus (10 to 60 days p.o.). Data are presented as mean ± standard error of relative gene expression (n = 4 or 5 animals/group). No difference among groups were observed (P > 0.05).

Figure 3

Gene expression of, MKI67 (A), FAS (B), BAX (C), CASP8 (D), CASP9 (E) and CASP3 (F) in canine CL during diestrus (10 to 60 days p.o.). Data are presented as mean ± standard error of relative gene expression (n = 4 or 5 animals/group). Bars with different letters indicate significant differences among groups (P < 0.05).

Figure 4

Immunolocalization of CASPASE 3, CASPASE 8, CASPASE 9 and BAX in canine CL during diestrus. Lines 10, 20, 30, 40, 50, 60, days after ovulation. NC, negative control. Black arrows indicate the cytoplasmic and white arrows indicate nuclear staining. Scale bar 50 μm.

Figure 5

Protein expression of Caspase 3 (A), Caspase 8 (B), Caspase 9 (C) and BAX (D) in canine corpus luteum. Representative blots on top of each column, which represent mean ± standard error (n = 4/group). Different letters indicate significant differences among groups (P ≤ 0.05).

Figure 6

Gene expression MKI67, cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1), 3-β-hydroxysteroid dehydrogenase/Δ-5-4 (HSD3B1), solute carrier family 2 (facilitated glucose transporter), and member 4 (SLC2A4) in luteal cells collected at 20, 40, and 60 days after ovulation in diestrous bitches. Bars indicate six different groups: Control (no treatment), E2 (treated with 100 nM E2), ERa block (treated with 10 nM methyl-piperidone-pyrazole [MPP]), ERb block (treated with 10 uM (1,5-a) pyrimidine [PHTPP]), E2 + ERa block (treated with E2 + MPP), and E2 + ERb block (treated with E2 + PHTPP). Data represent mean ± standard error of relative gene expression (n = 4 animals/group). Bars with different letters indicate significant differences among groups (P < 0.05).

Figure 7

Gene expression of CASP3, CASP8, CASP9, BAX, and FAS in canine luteal cells collected on days 20, 40, and 60 after ovulation and cultivated for 10 days until confluence was reached. Bars indicate six different groups: Control, E2 (treated with E2), ERa block (treated with methyl-piperidino-pyrazole [MPP]), ERb block (treated with pyrazolo (1,5-a) pyrimidine [PHTPP]), E2 + ERa block (treated with E2 + MPP), and E2 + ERb block (treated with E2 + PHTPP). Data represent mean ± standard error of relative gene expression (n = 4 animals/group). Bars with different letters indicate significant differences among groups (P < 0.05).