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. 2010 Sep 9:8:109.
doi: 10.1186/1477-7827-8-109.

Distribution and quantitative changes in amounts of aquaporin 1, 5 and 9 in the pig uterus during the estrous cycle and early pregnancy

Affiliations

Distribution and quantitative changes in amounts of aquaporin 1, 5 and 9 in the pig uterus during the estrous cycle and early pregnancy

Mariusz T Skowronski. Reprod Biol Endocrinol. .

Abstract

Background: Aquaporins (AQPs) are a family of membrane channel proteins that facilitate bulk water transport. To date, 11 isoforms of AQPs have been reported to be expressed in the female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig uterus during different stages of the estrous cycle and early pregnancy.

Methods: Immunoperoxidase and semi-quantitative immunoblotting techniques were used to examine the distribution and changes in amounts of AQP1, AQP5 and AQP9 in uteral cells of pigs at the early (Days 2-4), middle (10-12), late (14-16) stage of the luteal phase and late (18-20) stage of the follicular phase of the estrous cycle as well as on Days 14-16 and 30-32 of gestation (the onset and the end of implantation process).

Results: The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within uterine blood vessels. In cyclic gilts, endometrial and myometrial expression of AQP1 protein did not change significantly but increased during gestation. AQP5 was localized in smooth muscle cells and uterine epithelial cells. Endometrial expression of AQP5 protein did not change significantly between Days 2-4 and 10-12 of the estrous cycle but increased on Days 14-16 and 18-20 as well as during early pregnancy. Myometrial expression of AQP5 did not differ significantly during the estrous cycle but increased in the pregnancy. The anti-AQP9 antibody labeled uterine epithelial cells of uterus. Endometrial expression of AQP9 did not change significantly between Days 2-4 and 10-12 of the estrous cycle but increased on Days 14-16 and 18-20 as well as during early pregnancy.

Conclusions: The results suggest that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different uterine phases in the estrous cycle and early pregnancy.

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Figures

Figure 1
Figure 1
Western blot analysis of AQP1 expression. Expression of AQP1 assessed by Western blotting in the endometrium and myometrium of porcine uterus on Days 2-4 (A), 10-12 (B), 14-16 (C) and 18-20 (D) of estrous cycle as well as on Days 14-16 (E) and 30-32 (F) of pregnancy. Densitometric analyses of AQP1 protein levels normalized against β-actin (A-F). The data are summarized in (G) for endometrial and myometrial (H) expression of AQP1 during studied stages of the cycle. Comparison of AQP1 expression determined by Western blotting in porcine endometrium (I) and myometrium (J), between two stages of the cycle and two periods of pregnancy. Immunoblotting for AQP1 protein (K) in the uterus. The blots were probed with anti-AQP1 (Lane 2) or anti-AQP1 preadsorbed with the excess synthetic peptide (Lane 1). RT-PCR was performed on total RNA (L) from uteral tissue. Values are the mean ± SEM (n = 5). a,bMeans with different superscripts are significantly different.
Figure 2
Figure 2
Western blot analysis of AQP5 expression. Expression of AQP5 assessed by Western blotting in the endometrium and myometrium of porcine uterus on Days 2-4 (A), 10-12 (B), 14-16 (C) and 18-20 (D) of estrous cycle as well as on Days 14-16 (E) and 30-32 (F) of pregnancy. Densitometric analyses of AQP5 protein levels normalized against β-actin (A-F). The data are summarized in (G) for endometrial and myometrial (H) expression of AQP5 during studied stages of the cycle. Comparison of AQP5 expression determined by Western blotting in porcine endometrium (I) and myometrium (J), between two stages of the cycle and two periods of pregnancy. Immunoblotting for AQP5 protein (K) in the uterus. The blots were probed with anti-AQP5 (Lane 2) or anti-AQP5 preadsorbed with the excess synthetic peptide (Lane 1). RT-PCR was performed on total RNA (L) from uteral tissue. Values are the mean ± SEM (n = 5). a,bMeans with different superscripts are significantly different.
Figure 3
Figure 3
Western blot analysis of AQP9 expression. Expression of AQP9 assessed by Western blotting in the endometrium of porcine uterus on Days 2-4, 10-12, 14-16 and 18-20 of estrous cycle (A) as well as on Days 14-16 and 30-32 of pregnancy (B). Densitometric analyses of AQP9 protein levels normalized against β-actin (A and B). The data are summarized for endometrial expression of AQP9 during studied stages of the cycle and pregnancy (A and B). Comparison of AQP9 expression determined by Western blotting in porcine endometrium between two stages of the cycle and two periods of pregnancy (C). Immunoblotting for AQP5 protein (D) in the uterus. The blots were probed with anti-AQP5 (Lane 2) or anti-AQP5 preadsorbed with the excess synthetic peptide (Lane 1). RT-PCR was performed on total RNA (E) from uteral tissue. Values are the mean ± SEM (n = 5). a,bMeans with different superscripts are significantly different.
Figure 4
Figure 4
Immunolocalization of AQP1, 5 and 9. Immunohistochemical staining of AQP1 in paraffin-embedded sections of the uterus from pigs. Anti-AQP1 antibody labels capillary endothelium of the uterus (A-F). Immunoperoxidase labeling of AQP1 from the pig kidney cortex (positive control) (S). The labeling is seen in both of the apical cell membrane and in the basolateral cell membrane in proximal tubule cells. AQP5 antibody stains luminal and glandular epithelial cells (arrows) and smooth muscle cells (G-L). The anti-AQP5 labels apical membrane of type I pulmonary epithelial cells of the pig (positive control) (U). AQP9 labels luminal and glandular epithelial cells of the uterus (arrows) (M-R). Immunoperoxidase labeling of AQP9 from the pig liver (positive control) (X). No staining was observed with using non-immune immunoglobulins (negative controls) (T, W and Y). L, lumen; CV, central vein; T, trophoblast. Bar = 50 μm.

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