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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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Precocious vaginal opening is not induced by β-estradiol injection in Sema4D−/− mice. (A) Daily subcutaneous injection of β-estradiol (0.1 μg/kg body weight) into WT mice for five consecutive days (between 12 and 16-days-old) induced precocious vaginal opening at 17 days old (vaginal opening: four out of four mice). Identical injection into Sema4D−/− mice did not induce precocious vaginal opening at 17 days old, which was further indicated by histological analysis (vaginal opening: zero out of four mice). (Magnification, ×40). uc, uterogenital canal; o, obstruction; v, external vaginal entrance; H&E, hematoxylin and eosin staining. (B) TUNEL-positive and cleaved caspase-3-positive apoptotic cells (green) were significantly less numerous in the vaginal epithelium of β-estradiol-injected Sema4D−/− mice than in β-estradiol-injected WT mice. Cellular nuclei were visualized with DAPI (blue). (Magnification, ×400). Scale bar=50 μm. (C) Graphs show the rate of TUNEL- or cleaved caspase-3-positive cells, respectively, among nucleated cells in vaginal epithelia. Each column represents the mean ± standard error of the mean (WT, n=4; Sema4D−/−, n=4). *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.
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f2-mmr-11-02-0829: Precocious vaginal opening is not induced by β-estradiol injection in Sema4D−/− mice. (A) Daily subcutaneous injection of β-estradiol (0.1 μg/kg body weight) into WT mice for five consecutive days (between 12 and 16-days-old) induced precocious vaginal opening at 17 days old (vaginal opening: four out of four mice). Identical injection into Sema4D−/− mice did not induce precocious vaginal opening at 17 days old, which was further indicated by histological analysis (vaginal opening: zero out of four mice). (Magnification, ×40). uc, uterogenital canal; o, obstruction; v, external vaginal entrance; H&E, hematoxylin and eosin staining. (B) TUNEL-positive and cleaved caspase-3-positive apoptotic cells (green) were significantly less numerous in the vaginal epithelium of β-estradiol-injected Sema4D−/− mice than in β-estradiol-injected WT mice. Cellular nuclei were visualized with DAPI (blue). (Magnification, ×400). Scale bar=50 μm. (C) Graphs show the rate of TUNEL- or cleaved caspase-3-positive cells, respectively, among nucleated cells in vaginal epithelia. Each column represents the mean ± standard error of the mean (WT, n=4; Sema4D−/−, n=4). *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

Mentions: The mouse vaginal opening process is modulated by the level of estrogen (1). An enzyme immunoassay was thus conducted to evaluate the serum estrogen level corresponding to the time of vaginal opening of five-week-old WT and Sema4D−/− mice, and no significant difference was identified between the two genotypes (WT: 19.89±6.80 pg/ml, n=5; Sema4D−/−: 23.20±3.92 pg/ml, n=5; Student’s t-test, P>0.05). To further exclude the possibility of insufficient estrogen secretion during the critical period of vaginal opening, β-estradiol was injected into infant mice for five consecutive days beginning at 12 days of age to induce precocious vaginal opening in the 17-day-old specimens (1). Administration of β-estradiol to WT mice induced premature vaginal opening in 17-day-old mice and induce apoptosis in vaginal tissue, which was detected by TUNEL assay and activated caspase-3 immunohistochemistry (Fig. 2). By contrast, administration of β-estradiol to Sema4D−/− mice did not induce premature vaginal opening, and the apoptotic level in Sema4D−/− mouse vaginal tissue was significantly lower than that in the vaginal tissue of WT mice treated with β-estradiol (Fig. 2).


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)

Precocious vaginal opening is not induced by β-estradiol injection in Sema4D−/− mice. (A) Daily subcutaneous injection of β-estradiol (0.1 μg/kg body weight) into WT mice for five consecutive days (between 12 and 16-days-old) induced precocious vaginal opening at 17 days old (vaginal opening: four out of four mice). Identical injection into Sema4D−/− mice did not induce precocious vaginal opening at 17 days old, which was further indicated by histological analysis (vaginal opening: zero out of four mice). (Magnification, ×40). uc, uterogenital canal; o, obstruction; v, external vaginal entrance; H&E, hematoxylin and eosin staining. (B) TUNEL-positive and cleaved caspase-3-positive apoptotic cells (green) were significantly less numerous in the vaginal epithelium of β-estradiol-injected Sema4D−/− mice than in β-estradiol-injected WT mice. Cellular nuclei were visualized with DAPI (blue). (Magnification, ×400). Scale bar=50 μm. (C) Graphs show the rate of TUNEL- or cleaved caspase-3-positive cells, respectively, among nucleated cells in vaginal epithelia. Each column represents the mean ± standard error of the mean (WT, n=4; Sema4D−/−, n=4). *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-11-02-0829: Precocious vaginal opening is not induced by β-estradiol injection in Sema4D−/− mice. (A) Daily subcutaneous injection of β-estradiol (0.1 μg/kg body weight) into WT mice for five consecutive days (between 12 and 16-days-old) induced precocious vaginal opening at 17 days old (vaginal opening: four out of four mice). Identical injection into Sema4D−/− mice did not induce precocious vaginal opening at 17 days old, which was further indicated by histological analysis (vaginal opening: zero out of four mice). (Magnification, ×40). uc, uterogenital canal; o, obstruction; v, external vaginal entrance; H&E, hematoxylin and eosin staining. (B) TUNEL-positive and cleaved caspase-3-positive apoptotic cells (green) were significantly less numerous in the vaginal epithelium of β-estradiol-injected Sema4D−/− mice than in β-estradiol-injected WT mice. Cellular nuclei were visualized with DAPI (blue). (Magnification, ×400). Scale bar=50 μm. (C) Graphs show the rate of TUNEL- or cleaved caspase-3-positive cells, respectively, among nucleated cells in vaginal epithelia. Each column represents the mean ± standard error of the mean (WT, n=4; Sema4D−/−, n=4). *P<0.05. Sema4D, semaphorin 4D; WT, wild-type; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.
Mentions: The mouse vaginal opening process is modulated by the level of estrogen (1). An enzyme immunoassay was thus conducted to evaluate the serum estrogen level corresponding to the time of vaginal opening of five-week-old WT and Sema4D−/− mice, and no significant difference was identified between the two genotypes (WT: 19.89±6.80 pg/ml, n=5; Sema4D−/−: 23.20±3.92 pg/ml, n=5; Student’s t-test, P>0.05). To further exclude the possibility of insufficient estrogen secretion during the critical period of vaginal opening, β-estradiol was injected into infant mice for five consecutive days beginning at 12 days of age to induce precocious vaginal opening in the 17-day-old specimens (1). Administration of β-estradiol to WT mice induced premature vaginal opening in 17-day-old mice and induce apoptosis in vaginal tissue, which was detected by TUNEL assay and activated caspase-3 immunohistochemistry (Fig. 2). By contrast, administration of β-estradiol to Sema4D−/− mice did not induce premature vaginal opening, and the apoptotic level in Sema4D−/− mouse vaginal tissue was significantly lower than that in the vaginal tissue of WT mice treated with β-estradiol (Fig. 2).

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