Ion-Channel Proteins in the Prepubertal Bitch Reproductive System: The Immunolocalization of ASIC2, ASIC4, and PIEZO2

Abstract

Ion channels play a crucial role in various physiological processes, yet their functions in the reproductive system remain underexplored. This study investigates the expression and the localization of ASIC2, ASIC4, and PIEZO2 ion channels in the reproductive tracts of prepubertal bitches. Western blotting on samples from eight prepubertal bitches confirmed the presence of these ion channels in ovarian, uterine, and uterine tubes tissues, and validated antibody specificity. Immunohistochemistry revealed that all primordial follicles expressed these ion channels, while only some developing follicles showed immunolabeling. These findings suggest ion channels' potential involvement in oocyte differentiation and maturation. The localization of these channels in uterine tubes, uterine lining, and glandular epithelium suggests a role in tissue maintenance, oocyte transport, and embryo implantation. Additionally, their expression in the tunica media of reproductive vasculature points to a potential role in vascular regulation. Future studies are needed to elucidate the specific mechanisms underlying the role of these channels in reproductive physiology.

Keywords: ASIC; PIEZO; bitches; ion channels; prepubertal stage; reproductive system.

Figure 1

Photomicrographs of (a) H&E- and (b) Masson’s trichrome-stained cross-section of the prepubertal bitch’s ovary (low magnification). The overall organization of the ovary in cortex and medulla can be observed beside the arrangement of the capsule of ovary (blue arrows), and subsurface epithelial structures. Interstitial cells (green arrow); ovarian medullary blood vessels (MBV); rete ovarii (yellow arow). Magnification 4× (a); 10× (b).

Figure 2

Photomicrographs of a H&E-stained ovary of a prepubertal bitch. (a,b) Normal primordial (black arrows), primary (red arrow), and early secondary (yellow arrow) follicles. (c,d) Secondary follicles (yellow arrow), with central oocyte and the surrounding layer of granulosa cells, within the ovarian stroma. Magnification 20× (a,c); 40× (b,d).

Figure 3

Photomicrographs of H&E-stained sections of prepubertal bitches’ uterine tubes. (a–c) depict the fimbriae, (d–f) show the isthmus, and (g–i) represent the uterus. The uterine cuboidal epithelium is indicated by the red arrow, while the uterine glands are marked by the yellow arrow. Magnification 10× (a,d,g); 20× (b,e,h); 40× (c,f,i).

Figure 4

Western blot analyses showing bands corresponding to the molecular weights of the bitches’ ASIC2 (a), ASIC4 (b), and PIEZO2 (c) proteins.

Figure 5

Photomicrographs of ASIC2, ASIC4, and PIEZO2 immunoreactivity in the ovary of prepubertal bitches. (a) ASIC 2 immunoreactive primordial follicles; (b) ASIC4 immunopositive primordial follicles; (c) PIEZO2 immunolabeled primordial follicles. Magnification 40×.

Figure 6

Photomicrographs of ASIC2, ASIC4, and PIEZO2 immunoreactivity in the ovary of a prepubertal bitch. (a) ASIC2 immunolabelled oocyte of primary (arrowheads) and granulosa cells of secondary (chevron arrow) follicles beside the ASIC2 non-reactive primary follicles (red arrowhead); (b) ASIC2 immunopositive oocytes and granulosa cells of primary follicles; (c) ASIC2 non-immunolabelled primary follicles; (d) ASIC2 immunoreactive granulosa cells of secondary follicles; (e) ASIC4 immunoreactive granulosa cells of primary (arrowheads) and secondary (chevron arrow) follicles beside the ASIC4 non-immunolabelled primary follicles (red arrowheads), (f) ASIC4 immunopositive oocyte and granulosa cells of primary follicles; (g) ASIC4 non-immunoreactive secondary follicles; (h) ASIC4 immunolabeled granulosa of secondary follicle; (i) PIEZO2 immunoreactive oocyte and granulosa cells (arrowheads) in primary follicles and non-immunoreactive (red arrowhead) primary follicle; (j) PIEZO2 immunolabelled granulosa cells of primary follicles; (k) PIEZO2 non-immunoreactive primary follicles; (l) PIEZO2 immunolabelled granulosa cells of secondary follicles (arrowhead). Magnification 20× (a,e,i); 40× (b–d,f–h,j–l).

Figure 7

Photomicrographs of ASIC2, ASIC4, and PIEZO2 immunolabelling, counterstained with hematoxylin, in bitches’ ovaries. (a) ASIC2 immunolabelled oocyte of primary follicles (arrowheads); (b) ASIC2 non-reactive oocyte in primary follicles (red arrowhead); (c) ASIC4 immunopositive oocytes (arrowhead) of primary follicles; (d) ASIC4 non-immunolabelled oocyte (red arrowhead) in primary follicles; (e) PIEZO2 immunoreactive oocyte (arrowhead) of primary follicles; (f) PIEZO2 non-immunoreactive oocyte (red arrowhead) in primary follicles. Magnification 40×.

Figure 8

Transmitted light and immunofluorescence photomicrographs of ASIC2, ASIC4, and PIEZO2 immunoreactivity in the ovary of a prepubertal bitch. (a) ASIC2 immunolabelled oocyte (arrowheads) and theca cells (arrows) of primary follicles; (b) corresponding transmitted light and immunofluorescence merged confocal micrographs of section (a); (c) ASIC4 immunolabelled oocyte (arrowheads) and theca cells (arrows) of primary follicles; (d) corresponding transmitted light and immunofluorescence merged confocal micrographs of section (c); (e) PIEZO2 immunoreactive theca cells (arrows) of primary follicles; (f) corresponding transmitted light and immunofluorescence merged confocal micrograph of section (e); magnification 40×.

Figure 9

Photomicrographs of ASIC2, ASIC4, and PIEZO2 immunoreactivity in the fimbriae of a prepubertal bitch (a–c). (b) ASIC2, (e) ASIC4, and (h) PIEZO2 immunolabeled fimbrial lining epithelium (arrowheads). (c) ASIC2, (f) ASIC4, and (i) PIEZO2 immunoreactive fimbrial glandular epithelium (arrows). Magnification 20× (a,d,g); 40× (b,c,e,f,h,i).

Figure 10

ASIC2, ASIC4, and PIEZO2 immunoreactivity in the isthmus of a prepubertal bitch. (a) ASIC2, (b) ASIC4, (c) PIEZO2 immunolocalization in the lining (arrowheads) and glandular (arrows) epithelium. Magnification 20×.

Figure 11

ASIC2 (a), ASIC4 (d), and PIEZO2 (g) immunoreactivity in the uterine lining, epithelium (arrowhead), and glands (arrow) of a prepubertal bitch. (b) ASIC2 immunopositive lining of the epithelium (arrowheads); (c) high magnification of ASIC2 immunoreactive uterine glands; (e) ASIC4 immunoreactive lining of the epithelium (arrowheads); (f) ASIC4 immunoreactivity in uterine glands, (h) PIEZO2 immunopositive lining of the epithelium (arrowheads); (i) PIEZO2 immunolabelled uterine glands. Magnification 20× (a,d,g); 40× (b,c,e,f,h,i).

Figure 12

ASIC2, ASIC 4, and PIEZO2 immunoreactivity in the ovarian and endometrium blood vessels of a prepubertal bitch. (a) ASIC2, (b) ASIC4, and (c) PIEZO2 immunoreactivity in the tunica media (arrows) of the prepubertal bitch’s ovarian blood vessel. (d) ASIC2, (e) ASIC4, and (f) PIEZO2 immunoreactivity in the tunica media (arrows) of the prepubertal bitch’s endometrium blood vessel. Magnification 20×.