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Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

Ito T, Bai T, Tanaka T, Yoshida K, Ueyama T, Miyajima M, Negishi T, Kawasaki T, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Mol Med Rep (2014)

Bottom Line: The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity.The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D.The experimental reduction of plexin‑B1 expression in vaginal epithelial cells demonstrated the integral role of plexin‑B1 in Sema4D‑induced apoptotic cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468‑8503, Japan.

ABSTRACT
The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The experimental reduction of plexin‑B1 expression in vaginal epithelial cells demonstrated the integral role of plexin‑B1 in Sema4D‑induced apoptotic cell death. These results suggest a non‑redundant role of Sema4D in the postnatal tissue remodeling process in five‑week‑old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin‑B1.

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In Sema4D−/− mice, the number of apoptotic cells in the vaginal epithelia is significantly fewer than in WT vaginal epithelia. (A) TUNEL and immunohistochemistry with anti-cleaved caspase-3 antibodies detected apoptotic cells in five-week-old WT mouse vaginal epithelia. Apoptotic cells identified by these methods were hardly detectable in Sema4D−/− vaginal epithelium. Nuclei were visualized with 4′,6-diamidino-2-phenylindole. Scale bar=50 μm. (B) Apoptotic cells detected with TUNEL and cleaved caspase-3 immunohistochemistry were significantly lower in number in Sema4D−/− vaginal mucosa compared with WT vaginal epithelium. WT, wild-type vaginal epithelium; Sema4D−/−, Sema4D−/− vaginal epithelium. *P<0.05. (C) Western blot analysis detected significantly less cleaved caspase-3 in Sema4D−/− vaginal tissue compared with WT vaginal tissue. WT, wild-type vaginal tissue. Sema4D−/−, Sema4D−/− vaginal tissue. *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
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f4-mmr-11-02-0829: In Sema4D−/− mice, the number of apoptotic cells in the vaginal epithelia is significantly fewer than in WT vaginal epithelia. (A) TUNEL and immunohistochemistry with anti-cleaved caspase-3 antibodies detected apoptotic cells in five-week-old WT mouse vaginal epithelia. Apoptotic cells identified by these methods were hardly detectable in Sema4D−/− vaginal epithelium. Nuclei were visualized with 4′,6-diamidino-2-phenylindole. Scale bar=50 μm. (B) Apoptotic cells detected with TUNEL and cleaved caspase-3 immunohistochemistry were significantly lower in number in Sema4D−/− vaginal mucosa compared with WT vaginal epithelium. WT, wild-type vaginal epithelium; Sema4D−/−, Sema4D−/− vaginal epithelium. *P<0.05. (C) Western blot analysis detected significantly less cleaved caspase-3 in Sema4D−/− vaginal tissue compared with WT vaginal tissue. WT, wild-type vaginal tissue. Sema4D−/−, Sema4D−/− vaginal tissue. *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Mentions: TUNEL assay and cleaved caspase-3 immunohistochemistry was applied to detect apoptotic cells in situ and examine apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Several TUNEL-positive and cleaved caspase-3-positive cells were observed in the WT vaginal epithelia (Fig. 4A). By contrast, there were fewer TUNEL-positive and cleaved caspase-3-positive cells in the Sema4D−/− vaginal epithelia (Fig. 4A). Statistical analyses revealed significantly fewer TUNEL-positive and cleaved caspase-3-positive cells in Sema4D−/− vaginal epithelia compared with WT epithelia (Fig. 4B). Western blotting of cleaved caspase-3 confirmed the significantly lower level of apoptosis in the Sema4D−/− vaginal tissues compared with the WT tissues (Fig. 4C).


Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

Ito T, Bai T, Tanaka T, Yoshida K, Ueyama T, Miyajima M, Negishi T, Kawasaki T, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Mol Med Rep (2014)

In Sema4D−/− mice, the number of apoptotic cells in the vaginal epithelia is significantly fewer than in WT vaginal epithelia. (A) TUNEL and immunohistochemistry with anti-cleaved caspase-3 antibodies detected apoptotic cells in five-week-old WT mouse vaginal epithelia. Apoptotic cells identified by these methods were hardly detectable in Sema4D−/− vaginal epithelium. Nuclei were visualized with 4′,6-diamidino-2-phenylindole. Scale bar=50 μm. (B) Apoptotic cells detected with TUNEL and cleaved caspase-3 immunohistochemistry were significantly lower in number in Sema4D−/− vaginal mucosa compared with WT vaginal epithelium. WT, wild-type vaginal epithelium; Sema4D−/−, Sema4D−/− vaginal epithelium. *P<0.05. (C) Western blot analysis detected significantly less cleaved caspase-3 in Sema4D−/− vaginal tissue compared with WT vaginal tissue. WT, wild-type vaginal tissue. Sema4D−/−, Sema4D−/− vaginal tissue. *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262505&req=5

f4-mmr-11-02-0829: In Sema4D−/− mice, the number of apoptotic cells in the vaginal epithelia is significantly fewer than in WT vaginal epithelia. (A) TUNEL and immunohistochemistry with anti-cleaved caspase-3 antibodies detected apoptotic cells in five-week-old WT mouse vaginal epithelia. Apoptotic cells identified by these methods were hardly detectable in Sema4D−/− vaginal epithelium. Nuclei were visualized with 4′,6-diamidino-2-phenylindole. Scale bar=50 μm. (B) Apoptotic cells detected with TUNEL and cleaved caspase-3 immunohistochemistry were significantly lower in number in Sema4D−/− vaginal mucosa compared with WT vaginal epithelium. WT, wild-type vaginal epithelium; Sema4D−/−, Sema4D−/− vaginal epithelium. *P<0.05. (C) Western blot analysis detected significantly less cleaved caspase-3 in Sema4D−/− vaginal tissue compared with WT vaginal tissue. WT, wild-type vaginal tissue. Sema4D−/−, Sema4D−/− vaginal tissue. *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Mentions: TUNEL assay and cleaved caspase-3 immunohistochemistry was applied to detect apoptotic cells in situ and examine apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Several TUNEL-positive and cleaved caspase-3-positive cells were observed in the WT vaginal epithelia (Fig. 4A). By contrast, there were fewer TUNEL-positive and cleaved caspase-3-positive cells in the Sema4D−/− vaginal epithelia (Fig. 4A). Statistical analyses revealed significantly fewer TUNEL-positive and cleaved caspase-3-positive cells in Sema4D−/− vaginal epithelia compared with WT epithelia (Fig. 4B). Western blotting of cleaved caspase-3 confirmed the significantly lower level of apoptosis in the Sema4D−/− vaginal tissues compared with the WT tissues (Fig. 4C).

Bottom Line: The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity.The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D.The experimental reduction of plexin‑B1 expression in vaginal epithelial cells demonstrated the integral role of plexin‑B1 in Sema4D‑induced apoptotic cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468‑8503, Japan.

ABSTRACT
The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The experimental reduction of plexin‑B1 expression in vaginal epithelial cells demonstrated the integral role of plexin‑B1 in Sema4D‑induced apoptotic cell death. These results suggest a non‑redundant role of Sema4D in the postnatal tissue remodeling process in five‑week‑old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin‑B1.

Show MeSH
Related in: MedlinePlus