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Escherichia coli -induced inflammation changes the expression of acetylcholine receptors (M2R, M3R, and α-7 nAChR) in the pig uterus

Barbara Jana

Barbara Jana

Department of Reproductive Biology, Institute of Animal Reproduction and Food Research of the Polish Academy of SciencesOlsztyn, Poland
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Jana, Barbara
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Jarosław Całka

Jarosław Całka

Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and MazuryOlsztyn, Poland
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Całka, Jarosław
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Katarzyna Palus

Katarzyna Palus

Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and MazuryOlsztyn, Poland
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Palus, Katarzyna
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Małgorzata Sikora

Małgorzata Sikora

Department of Reproductive Biology, Institute of Animal Reproduction and Food Research of the Polish Academy of SciencesOlsztyn, Poland
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Sikora, Małgorzata
  
06. Nov. 2020
Escherichia coli -induced inflammation changes the expression of acetylcholine receptors (M2R, M3R, and α-7 nAChR) in the pig uterus's Cover Image
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Online veröffentlicht: 06. Nov. 2020
Seitenbereich: 531 - 541
Eingereicht: 27. Apr. 2020
Akzeptiert: 20. Okt. 2020
DOI: https://doi.org/10.2478/jvetres-2020-0073
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© 2020 B. Jana et al. published by Sciendo
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Fig. 1

Messenger RNA levels of M2R (A and B) and M3R (C and D) in the endometrium (A and C) and myometrium (B and D) of gilts from the Ctrl, Sal, and E. coli groups. Results of quantification by real-time PCR are expressed as the mean ± SEM of ratios relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * – P < 0.05 and ** – P < 0.01 indicate differences between groups for the same type of receptor
Messenger RNA levels of M2R (A and B) and M3R (C and D) in the endometrium (A and C) and myometrium (B and D) of gilts from the Ctrl, Sal, and E. coli groups. Results of quantification by real-time PCR are expressed as the mean ± SEM of ratios relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * – P < 0.05 and ** – P < 0.01 indicate differences between groups for the same type of receptor

Fig. 2

Protein expression of M2R (A and B), M3R (C and D), and α-7 nAChR (E and F) in the endometrium (A, C and E) and myometrium (B, D and F) of gilts from the Ctrl, Sal and E. coli groups (n = 5 for each group). Representative blots are shown on upper panels. The results of quantification of protein contents are expressed as the mean ± SEM of ratios relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *** – P < 0.001 indicates differences between groups for the same type of receptor. M – marker
Protein expression of M2R (A and B), M3R (C and D), and α-7 nAChR (E and F) in the endometrium (A, C and E) and myometrium (B, D and F) of gilts from the Ctrl, Sal and E. coli groups (n = 5 for each group). Representative blots are shown on upper panels. The results of quantification of protein contents are expressed as the mean ± SEM of ratios relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *** – P < 0.001 indicates differences between groups for the same type of receptor. M – marker

Fig. 3

Representative images of M2R (A–H) and M3R (I–P) in the uteri of gilts from the Ctrl, Sal, and E. coli groupsImage A – weak immunoexpression of M2R in the luminal epithelium of the Ctrl groupImage B – weak immunoexpression of M2R in the luminal epithelium of the E. coli groupImage C – moderate and weak immunoexpression of M2R in the glandular epithelium of the Sal groupImage D – moderate and weak immunoexpression of M2R in the glandular epithelium of the E. coli groupImage E – moderate immunoexpression of M2R in the arteries of the endometrium and in the endothelium, and weak or absent immunoexpression in the muscle layer of the Ctrl groupImage F – moderate immunoexpression of M2R in the arteries of the endometrium and in the endothelium, and weak or absentimmunoexpression in the muscle layer of the E. coli groupImage G – weak or absent immunoexpression of M2R in muscle cells of the myometrium of the Sal groupImage H – weak or absent immunoexpression of M2R in muscle cells of the myometrium of the E. coli groupImage I – strong immunoexpression of M3R in the luminal epithelium and moderate immunoexpression in the glandular epithelium of the Ctrl groupImage J – weak immunoexpression of M3R in the luminal epithelium and glandular epithelium of the E. coli groupImage K – strong immunoexpression of M3R in the glandular epithelium of the Sal groupImage L – strong immunoexpression of M3R in the glandular epithelium of the E. coli groupImage M – moderate immunoexpression of M3R in the arteries of the endometrium, endothelium, and muscle layer of the Ctrl groupImage N – weak immunoexpression of M3R in the arteries of the endometrium, endothelium, and muscle layer of the E. coli groupImage O – moderate and weak immunoexpression of M3R in the muscle cells of the myometrium of the Sal groupImage P – strong immunoexpression of M3R in the muscle cells of the myometrium of the E. coli groupImage Q – negative control for M2R after replacing the primary antibody with rabbit normal IgGImage R – negative control for M3R after replacing the primary antibody with rabbit normal IgGA – artery, E – endothelium, GE – glandular epithelium, LE – luminal epithelium, MCM – muscle cells of myometrium, ML – muscle layer, NC – negative control
Representative images of M2R (A–H) and M3R (I–P) in the uteri of gilts from the Ctrl, Sal, and E. coli groupsImage A – weak immunoexpression of M2R in the luminal epithelium of the Ctrl groupImage B – weak immunoexpression of M2R in the luminal epithelium of the E. coli groupImage C – moderate and weak immunoexpression of M2R in the glandular epithelium of the Sal groupImage D – moderate and weak immunoexpression of M2R in the glandular epithelium of the E. coli groupImage E – moderate immunoexpression of M2R in the arteries of the endometrium and in the endothelium, and weak or absent immunoexpression in the muscle layer of the Ctrl groupImage F – moderate immunoexpression of M2R in the arteries of the endometrium and in the endothelium, and weak or absentimmunoexpression in the muscle layer of the E. coli groupImage G – weak or absent immunoexpression of M2R in muscle cells of the myometrium of the Sal groupImage H – weak or absent immunoexpression of M2R in muscle cells of the myometrium of the E. coli groupImage I – strong immunoexpression of M3R in the luminal epithelium and moderate immunoexpression in the glandular epithelium of the Ctrl groupImage J – weak immunoexpression of M3R in the luminal epithelium and glandular epithelium of the E. coli groupImage K – strong immunoexpression of M3R in the glandular epithelium of the Sal groupImage L – strong immunoexpression of M3R in the glandular epithelium of the E. coli groupImage M – moderate immunoexpression of M3R in the arteries of the endometrium, endothelium, and muscle layer of the Ctrl groupImage N – weak immunoexpression of M3R in the arteries of the endometrium, endothelium, and muscle layer of the E. coli groupImage O – moderate and weak immunoexpression of M3R in the muscle cells of the myometrium of the Sal groupImage P – strong immunoexpression of M3R in the muscle cells of the myometrium of the E. coli groupImage Q – negative control for M2R after replacing the primary antibody with rabbit normal IgGImage R – negative control for M3R after replacing the primary antibody with rabbit normal IgGA – artery, E – endothelium, GE – glandular epithelium, LE – luminal epithelium, MCM – muscle cells of myometrium, ML – muscle layer, NC – negative control

Fig.4

Representative images of α-7 nAChR in the uteri of gilts from the control (Ctrl), saline (Sal) and E. coli (E. coli) groups Image A – moderate immunoexpression of α-7 nAChR in the luminal epithelium and weak immunoexpression in the glandular epithelium of the Ctrl groupImage B – weak immunoexpression of α-7 nAChR in the luminal epithelium of the E. coli groupImage C – weak immunoexpression of α-7 nAChR in the glandular epithelium of the Sal groupImage D – weak immunoexpression of α-7 nAChR in the glandular epithelium of the E. coli groupImage E – weak or absent immunoexpression of α-7 nAChR in the arteries of the endometrium, endothelium, and muscle layer of the Ctrl groupImage F – weak or absent immunoexpression of α-7 nAChR in the arteries of the endometrium, endothelium, and muscle layer of the E. coli groupImage G – weak immunoexpression of α-7 nAChR in the muscle cells of the myometrium of the Sal groupImage H – weak immunoexpression of α-7 nAChR in the muscle cells of the myometrium of the E. coli groupImage I – negative control for α-7 nAChR after replacing the primary antibody with rabbit normal IgGA – artery, E – endothelium, GE – glandular epithelium, LE – luminal epithelium, MCM – muscle cells of myometrium, ML – muscle layer, NC – negative control
Representative images of α-7 nAChR in the uteri of gilts from the control (Ctrl), saline (Sal) and E. coli (E. coli) groups Image A – moderate immunoexpression of α-7 nAChR in the luminal epithelium and weak immunoexpression in the glandular epithelium of the Ctrl groupImage B – weak immunoexpression of α-7 nAChR in the luminal epithelium of the E. coli groupImage C – weak immunoexpression of α-7 nAChR in the glandular epithelium of the Sal groupImage D – weak immunoexpression of α-7 nAChR in the glandular epithelium of the E. coli groupImage E – weak or absent immunoexpression of α-7 nAChR in the arteries of the endometrium, endothelium, and muscle layer of the Ctrl groupImage F – weak or absent immunoexpression of α-7 nAChR in the arteries of the endometrium, endothelium, and muscle layer of the E. coli groupImage G – weak immunoexpression of α-7 nAChR in the muscle cells of the myometrium of the Sal groupImage H – weak immunoexpression of α-7 nAChR in the muscle cells of the myometrium of the E. coli groupImage I – negative control for α-7 nAChR after replacing the primary antibody with rabbit normal IgGA – artery, E – endothelium, GE – glandular epithelium, LE – luminal epithelium, MCM – muscle cells of myometrium, ML – muscle layer, NC – negative control

TaqMan assays used in this study

SymbolNameAssay No.
CHRM2cholinergic receptor muscarinic 21Ss03383697_u1
CHRM3cholinergic receptor muscarinic 3Ss03387661_u1
GAPDHglyceraldehyde-3-phosphate dehydrogenaseSs03375435_u1
ACTBβ-actinSs03376081_u1
HPRThypoxanthine guanine phosphoribosyl transferaseSs03388274_m1

Distribution and intensity of M2R, M3R, and α-7 nAChR staining in the gilt uteri of the Ctrl, Sal and E_ coli groups

ReceptorM2RM3Rα-7 nAChR
Uterine structureCtrlSalE. coliCtrlSalE. coliCtrlSalE. coli
Endometrium
Luminal epithelium−/+−/+−/+−/+/++/+++−/+/++/+++−/+/++−/+/++−/+/++−/+
         Glandular epithelium−/+/++−/+/++−/+/++−/+/++/+++−/+/++/++−/+/++/+++−/+−/+−/+
         Arterial endothelium−/+/++−/+/++−/+/++−/+/++−/+/++−/+/++−/+−/+−/+
         Arterial muscle layer−/+−/+−/+−/+/++−/+/++−/+−/+−/+−/+
         Venous wall−/+−/+−/+−/+/++−/+/++−/+/++−/+−/+−/+
         Stroma−/+−/+−/+−/+−/+−/+/++−/+−/+−/+
Myometrium
         Muscle circular layer−/+/++−/+/++−/+/++−/+/++−/+/++−/+/++/+++−/+−/+−/+
         Muscle longitudinal layer−/+−/+−/+/+−/+/++−/+/++−/+/++/+++−/+−/+−/+
         Arterial endothelium−/+/++−/+/++−/+/++−/+−/+−/+−/+−/+−/+
         Arterial muscle layer−/+−/+−/+−/+−/+−/+−/+−/+−/+
         Venous wall−/+−/+−/+−/+−/+−/+−/+−/+−/+
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