QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 March 2004
doi: 10.1242/dev.01038

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kurita, T. Articles by Cunha, G. R. Search for Related Content PubMed PubMed Citation Articles by Kurita, T. Articles by Cunha, G. R. Social Bookmarking                         What's this?

Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis

¹ Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA
² Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA

View larger version (138K): [in a new window]   Fig. 1. Ontogeny of p63 in mouse female reproductive tract. P63, K14 and PR proteins were detected by IHC in mouse female reproductive tract. At E18, the sinus vagina (SVG) was uniformly positive for p63 (A) and K14 (B). By contrast, p63 was detected only in a small subset of columnar epithelial cells in the Müllerian vagina (MVG) (A,C,D) and the cervix (CVX) (A). Uterus (UT) was negative for p63 and K14. Red arrows indicate p63-positive epithelial cells. The black arrow (C) indicates boundary between sinus and Müllerian vaginal epithelia. At postnatal day 1 (P1), although p63-positive epithelial cells increased (E, red arrows), K14 was still undetectable in CVX and MVG (F). In the P2 CVX, p63-positive squamous basal epithelial cells were detected (G, red arrows) and some basal epithelial cells were weakly positive for K14 (H, red arrow). Some luminal columnar epithelial cells were also positive for p63 (G, black arrows). Note change in nuclear polarity in the emerging basal cells (G). By P14, the SCJ (red arrowheads) was formed, and p63 (I), K14 (J) and PR (K) expression abruptly changed at the SCJ. To test the effect of steroid hormones, ovariectomized adult female BALB/c mice were treated with oil (L), E2+P4 (M) or E2 alone (N). Hormone treatments did not change the boundary of p63 expression at the SCJ. E2 induced stratification but not p63 expression in UtE (N, blue arrows).

View larger version (123K): [in a new window]   Fig. 2. Induction of p63 expression by vaginal mesenchyme. UtE and VgE isolated from newborn (A-D) or adult (2-months-old, E and F) BALB/c mice were recombined with newborn UtM or VgM. The tissue recombinants were grown as renal grafts in nude mice for 4 weeks. When newborn VgE (A) or UtE (C) was recombined with UtM, the epithelium was p63-negative and columnar. By contrast, when newborn UtE (B) or VgE (D) was recombined with VgM, epithelium was p63-positive and squamous. Thus, mesenchyme determines expression of p63 in the newborn UtE and VgE. However, the original expression of p63 in adult-VgE (positive) (E) and UtE (negative) (F) was not altered by heterotypic mesenchyme.

View larger version (133K): [in a new window]   Fig. 3. Phenotype of p63–/–cervicovaginal epithelium (CVE). I. Epithelial morphology. The p63–/– CVE was columnar and developed deep inclusions or glands (black arrows). The p63–/–cervicovaginal grafts in the ovariectomized hosts showed uterine-like epithelial morphology (A). When the hosts were treated with E2, the glandular p63–/– CVE remained columnar but became hyperplastic (B). II. Cervicovaginal epithelial markers. Markers for squamous differentiation [p63 (C,D) and K14 (E,F)] and keratinization [K10 (G,H) and involucrin (I,J)] were assessed in p63+ (C,E,G,I) and p63–/– (D,F,H,J) cervicovaginal grafts by IHC. The p63–/– CVE failed to express squamous and keratinization markers. III. Common markers for uterine and cervicovaginal epithelia. Markers common for UtE and CVE [ER (K,L) and p130 (M,N)] were examined in p63+ (K,M) and p63–/– (L,N) cervicovaginal grafts by IHC. Both p63+ and p63–/– CVE strongly expressed ER and p130. IV. Regulation of PR by E2. Expression of PR was assessed by IHC in p63+ (O,Q) and p63–/– (P,R) cervicovaginal grafts in the ovariectomized (–E2, O and P) or E2-treated ovariectomized (+E2, Q and R) hosts. In the p63+ CVE, PR was detectable only when the host was treated with E2 (compare O with Q). By contrast, p63–/– CVE expressed a high level of PR in the absence of E2 (P), which is a unique phenotype of uterine epithelium. V. VgM/UtE tissue recombination. Tissue recombinants were made with UtE and VgM from E17 p63–/– and p63+ embryos. Expression of p63 was examined in the tissue recombinants. The p63+ UtE developed into normal vaginal epithelium and expressed p63 when it was recombined with p63–/– VgM (S). By contrast, p63–/– UtE recombined p63+ VgM failed to undergo squamous differentiation (T). Scale bar: 50 µm.

View larger version (94K): [in a new window]   Fig. 4. Role of mesenchyme in the induction of uterine epithelial phenotype. ECM, extracellular matrix. Expression of PR (left column) and ER (right column) in UtE was examined in vivo (A-D, I-L) and in vitro (E-H). In the E18 uterine anlage, both PR (A) and ER (B) were undetectable in the epithelium. By P5, UtE in situ became strongly positive for PR (C) but was negative for ER (D). By contrast, when isolated E18-UtE was cultured in vitro for 7 days, PR was detected in only a subset (<20%) of epithelial cells (E), while ER was detected in more than a half of epithelial cells. By contrast, adult-UtE maintained a high level of PR (G) and ER (H) under the same culture conditions. When E18-UtE was recombined with UtM, the epithelium expressed PR (I) and ER (J) after 1 month of in vivo growth. In the same host, E18-UtE recombined with BLM did not express PR (K), while ER (L) was strongly expressed.

View larger version (162K): [in a new window]   Fig. 5. Expression of differentiation markers in the DES-induced uterine and cervicovaginal epithelial lesions. Differentiation marker expression was examined in the serial sections of vaginal adenosis (A-D), cervical adenosis (E-G) and uterine SQM (H-J) in the OVX adult (P60) mice neonatally treated with DES. The openings of adenotic glands in the vagina are indicated by black arrows (D). Green and red arrows indicate columnar and squamous cells, respectively. Epithelial cells in cervicovaginal adenosis were negative for p63 (A,E) and K14 (B,F) and expressed PR in the uterine pattern (C,G). Thus, adenotic epithelium was strongly positive for PR in the absence of E2 (in ovariectomized host, C and G). In the cervix, coexisting squamous cervical epithelium retained the normal cervicovaginal phenotype and was negative for PR in the absence of E2 (G). In the vaginal epithelium of neonatally DES-exposed mice, PR (C), C/EBP-ß, involucrin and K10 (not shown) were constitutively activated. Accordingly, in the vagina PR was detected not only in the adenotic but in the entire vaginal epithelium (C) in the absence of E2. In the adenotic epithelium, ER was also strongly expressed (D). Metaplastic squamous cells in the uterus were positive for ER (not shown), p63 and K14 (H and I). In the absence of E2, PR (J) was undetectable in the SQM (red arrows), while coexisting columnar UtE was strongly positive for PR (green arrows). In p63+ uterine grafts, DES induced squamous cells, which were positive for p63 (K) and K14 (L). By contrast, K14 positive squamous epithelial cells were never detected in the p63–/– uterine grafts (M). Scale bar: 50 µm.

View larger version (165K): [in a new window]   Fig. 6. Ontogeny of p63 in female reproductive tract of DES-exposed mice. P63 was detected by IHC in developing [postnatal days 5 (A-C), 7 (D,E) and 10 (F)] and adult (P60, G) mouse female reproductive tract treated with oil (A) or DES (B-G). Green arrows indicate p63-negative Müllerian-vaginal and cervical epithelial cells. DES disrupted p63 expression in Müllerian vagina and cervix via ERa [compare oil-treated ER+/+ (A), DES-treated ER+/+ (B) and DES-treated ER–/–groups (C)]. Although most cervicovaginal epithelial cells expressed p63 by 2 days after the last DES-treatment (D), a small number of p63-negative columnar epithelium persisted at P7 (D) and P10 (F) (green arrows) in the Müllerian vagina and cervix, and developed into adenosis in adulthood (G). When DES-treatment was extended from birth to P7 (E), formation of p63-positive basal layer was largely inhibited at P7 (compare D with E). Scale bar: 50 µm.

View larger version (106K): [in a new window]   Fig. 7. Role of epithelial and mesenchymal ER in the disruption of p63. Ep, epithelium. St, stroma. BV, blood vessel. (A) Four types of tissue recombinants were constructed with UtE and VgM from P2 ER+/+ and ER–/– mice (ER+/+ UtE+ER+/+ VgM, ER+/+ UtE+ER–/– VgM, ER–/– UtE+ER+/+ VgM and ER–/– UtE+ER–/– VgM) and grafted into ovariectomized female nude mice hosts without (a,b; control) or with (c-f; +DES) DES-treatment. Without the DES-treatment, p63-positive basal layer was induced in all 4 types of tissue recombinants (a,b). When ER was expressed in the epithelial cells [ER+/+ UtE+ER+/+ VgM (c) and ER+/+ UtE+ER–/– VgM (e)], induction of p63 in the UtE was blocked by DES. By contrast, induction of p63 and formation of squamous basal cells were not affected by DES in ER–/– UtE+ER+/+ VgM (d) and ER–/– UtE+ER–/– VgM (f) tissue recombinants. The results were quantitated by measuring the length of basement membrane associating with p63-positive and -negative epithelial cells (B). The result was statistically analyzed with ANOVA test. Error bars represent s.e.m. The bar marked with b is significantly higher than bars marked with c and d (P<0.05). The bars marked with a, b and c are significantly higher than bars marked with d (P<0.01). Scale bar: 100 µm.

View larger version (61K): [in a new window]   Fig. 8. Models for Müllerian duct epithelial differentiation. See the detail in the Discussion. (A) Model 1: p63 is the identity switch for the cervicovaginal epithelial fate determination. (B) Model 2: role of p63 and epithelial-mesenchymal tissue interaction in normal and abnormal Müllerian duct development.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter