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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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Sema4D and plexin-B1 are expressed in mouse vaginal epithelia. (A) Sema4D mRNA was detected by RT-PCR in the uteri, vaginas and ovaries of WT mice, but not in those of Sema4D−/− mice. Plexin-B1 mRNA was detected by RT-PCR in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (B) Western blotting detected Sema4D in WT vaginas, but not in Sema4D−/− vaginas. Plexin-B1 protein was detected in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (C) Immunohistochemical analyses with anti-Sema4D antibodies detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (arrow). (D) IHC did not detect Sema4D in Sema4D−/− vaginas. (E) Plexin-B1 was detected in WT vaginal mucosa by immunohistochemical analysis using plexin-B1-specific antibodies (arrow). (F) Plexin-B1 was also detected in Sema4D−/− vaginal mucosa by IHC (arrow). Scale bar=50 μm. (Magnification of C-F, ×400). Sema4D, semaphorin 4D; WT, wild-type; RT-PCR, reverse transcription-polymerase chain reaction; IHC, immunohistochemistry.
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f3-mmr-11-02-0829: Sema4D and plexin-B1 are expressed in mouse vaginal epithelia. (A) Sema4D mRNA was detected by RT-PCR in the uteri, vaginas and ovaries of WT mice, but not in those of Sema4D−/− mice. Plexin-B1 mRNA was detected by RT-PCR in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (B) Western blotting detected Sema4D in WT vaginas, but not in Sema4D−/− vaginas. Plexin-B1 protein was detected in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (C) Immunohistochemical analyses with anti-Sema4D antibodies detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (arrow). (D) IHC did not detect Sema4D in Sema4D−/− vaginas. (E) Plexin-B1 was detected in WT vaginal mucosa by immunohistochemical analysis using plexin-B1-specific antibodies (arrow). (F) Plexin-B1 was also detected in Sema4D−/− vaginal mucosa by IHC (arrow). Scale bar=50 μm. (Magnification of C-F, ×400). Sema4D, semaphorin 4D; WT, wild-type; RT-PCR, reverse transcription-polymerase chain reaction; IHC, immunohistochemistry.

Mentions: In order to ascertain whether Sema4D mRNA is expressed in the mouse vagina, reverse transcription PCR analyses were performed with RNA from mouse uteri, vaginas and ovaries. Sema4D mRNA was detected in the mouse vagina; however, the transcripts were not amplified in any organs in the Sema4D−/− mice (Fig. 3A). To confirm the existence of Sema4D protein in the mouse vagina, western blotting was performed using protein extracts from WT and Sema4D−/− vaginas (Fig. 3B). The analysis detected Sema4D protein in WT vaginas, but not in Sema4D−/− vaginas (Fig. 3B). Using the same blot, the antibodies against plexin-B1, a Sema4D receptor, revealed the existence of plexin-B1 in WT and Sema4D−/− vaginas (Fig. 3B). To localize the expression of Sema4D and plexin-B1 in the mouse vagina, immunohistochemical analyses were performed on vaginal tissues from WT and Sema4D−/− mice. The antibodies against Sema4D detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (Fig. 3C), but not in Sema4D−/− mice (Fig. 3D). However, the plexin-B1 antibodies detected plexin-B1 localization in WT (Fig. 3E) and Sema4D−/− vaginal epithelia (Fig. 3F).


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)

Sema4D and plexin-B1 are expressed in mouse vaginal epithelia. (A) Sema4D mRNA was detected by RT-PCR in the uteri, vaginas and ovaries of WT mice, but not in those of Sema4D−/− mice. Plexin-B1 mRNA was detected by RT-PCR in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (B) Western blotting detected Sema4D in WT vaginas, but not in Sema4D−/− vaginas. Plexin-B1 protein was detected in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (C) Immunohistochemical analyses with anti-Sema4D antibodies detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (arrow). (D) IHC did not detect Sema4D in Sema4D−/− vaginas. (E) Plexin-B1 was detected in WT vaginal mucosa by immunohistochemical analysis using plexin-B1-specific antibodies (arrow). (F) Plexin-B1 was also detected in Sema4D−/− vaginal mucosa by IHC (arrow). Scale bar=50 μm. (Magnification of C-F, ×400). Sema4D, semaphorin 4D; WT, wild-type; RT-PCR, reverse transcription-polymerase chain reaction; IHC, immunohistochemistry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-11-02-0829: Sema4D and plexin-B1 are expressed in mouse vaginal epithelia. (A) Sema4D mRNA was detected by RT-PCR in the uteri, vaginas and ovaries of WT mice, but not in those of Sema4D−/− mice. Plexin-B1 mRNA was detected by RT-PCR in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (B) Western blotting detected Sema4D in WT vaginas, but not in Sema4D−/− vaginas. Plexin-B1 protein was detected in WT and Sema4D−/− vaginas. +/+, WT mice; −/−, Sema4D−/− mice. (C) Immunohistochemical analyses with anti-Sema4D antibodies detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (arrow). (D) IHC did not detect Sema4D in Sema4D−/− vaginas. (E) Plexin-B1 was detected in WT vaginal mucosa by immunohistochemical analysis using plexin-B1-specific antibodies (arrow). (F) Plexin-B1 was also detected in Sema4D−/− vaginal mucosa by IHC (arrow). Scale bar=50 μm. (Magnification of C-F, ×400). Sema4D, semaphorin 4D; WT, wild-type; RT-PCR, reverse transcription-polymerase chain reaction; IHC, immunohistochemistry.
Mentions: In order to ascertain whether Sema4D mRNA is expressed in the mouse vagina, reverse transcription PCR analyses were performed with RNA from mouse uteri, vaginas and ovaries. Sema4D mRNA was detected in the mouse vagina; however, the transcripts were not amplified in any organs in the Sema4D−/− mice (Fig. 3A). To confirm the existence of Sema4D protein in the mouse vagina, western blotting was performed using protein extracts from WT and Sema4D−/− vaginas (Fig. 3B). The analysis detected Sema4D protein in WT vaginas, but not in Sema4D−/− vaginas (Fig. 3B). Using the same blot, the antibodies against plexin-B1, a Sema4D receptor, revealed the existence of plexin-B1 in WT and Sema4D−/− vaginas (Fig. 3B). To localize the expression of Sema4D and plexin-B1 in the mouse vagina, immunohistochemical analyses were performed on vaginal tissues from WT and Sema4D−/− mice. The antibodies against Sema4D detected Sema4D in the suprabasal layer of the vaginal epithelia in WT mice (Fig. 3C), but not in Sema4D−/− mice (Fig. 3D). However, the plexin-B1 antibodies detected plexin-B1 localization in WT (Fig. 3E) and Sema4D−/− vaginal epithelia (Fig. 3F).

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