Response of xenografts of developing human female reproductive tracts to the synthetic estrogen, diethylstilbestrol

Abstract

Human female fetal reproductive tracts 9.5-22 weeks of gestation were grown for 1 month in ovariectomized athymic adult female mouse hosts that were either untreated or treated continuously with diethylstilbestrol (DES) via subcutaneous pellet. Normal morphogenesis and normal patterns of differentiation marker expression (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19, ESR1, PGR, TP63, RUNX1, ISL1, HOXA11 and α-ACT2) were observed in xenografts grown in untreated hosts and mimicked observations of previously reported (Cunha et al., 2017) non-grafted specimens of comparable age. DES elicited several notable morphological affects: (a) induction of endometrial/cervical glands, (b) increased plication (folding) of tubal epithelium, (c) stratified squamous maturation of vaginal epithelium and (d) vaginal adenosis. DES also induced ESR1 in epithelia of the uterine corpus, cervix and globally induced PGR in most cells of the developing human female reproductive tract. Keratin expression (KRT6, KRT7, KRT8, KRT14 and KRT19) was minimally affected by DES. Simple columnar adenotic epithelium was devoid of TP63 and RUNX1, while DES-induced mature vaginal epithelium was positive for both transcription factors. Another striking effect of DES was observed in grafts of human uterine tube, in which DES perturbed smooth muscle patterning. These results define for the first time IHC protein markers of DES action on the developing human reproductive tract, which provide bio-endpoints of estrogen-induced teratogenesis in the developing human female reproductive tract for future testing of estrogenic endocrine disruptors.

Keywords: Diethylstilbestrol; Epithelium; Estrogenic response; Human female fetal reproductive tract; Mesenchyme; Tissue interactions.

Figure 1.

KI67 immunostaining in 13-week intact female fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. The developing uterine tube (A), vaginal plate (B) and uterus (C) all exhibit considerable numbers of proliferating MKI67-reactive epithelial and mesenchymal cells. Scale bar in (A) also applies to (B).

Figure 2.

ESR1 immunostaining of a 13-week intact female fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. The epithelia of the uterine tube and vaginal plate (UGS-derived) are reactive for ESR1, while endometrial/cervical epithelia are not (B). The red arrowhead marks the uterotubal junction remarkable for the sharp transition in ESR1 staining. Detail of ESR1-negative endometrial epithelium (C). Note that the inner layer of mesenchymal cells of the uterine corpus (presumed endometrial stroma) is strongly ESR1-positive in contrast to peripheral mesenchyme (presumptive myometrium), which is weakly ESR1-positive. The stroma of the uterine tube is also ESR1 reactive but to a far lesser degree than in the corresponding area of uterine corpus.

Figure 3.

KRT8 (A-C) and KRT7 (D-F) immunostaining in an intact human fetal reproductive tract grown for 4 weeks in an untreated ovariectomized female athymic mouse host. The specimens come from 13-week (A-C & F [AC121]) and 14-week fetuses (D-E [AC159]). In truly stratified epithelium (presumptive exocervix and vagina) the basal epithelial cells (arrowheads) are unstained. KRT7 & KRT8 is expressed in single layered epithelium or apical epithelial cells of multilayered epithelia. Vg. Plate=vaginal plate.

Figure 4.

KRT19 immunostaining of an intact 13-week human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. KRT19 was detected in simple columnar epithelia of the uterine tube (E), uterine corpus (A-B), endocervix (not illustrated), as well as in stratified epithelia of the presumed exocervix, vagina and vaginal plate (A, C & D) in basal through apical epithelial cells.

Figure 5.

KRT6 immunostaining of an intact 13-week (D) human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host and immunostained for KRT6. Stratified squamous epithelia of the presumed exocervix, vagina and vaginal plate (A, C & D) express KRT6 in basal through apical epithelial cells. Simple columnar epithelia of the uterine tube (not illustrated), uterine corpus and presumed endocervix do not express KRT6 (A-B).

Figure 6.

KRT14 immunostaining in an intact 13-week human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. KRT14 was detected only in the vagina (A & C)/vaginal plate (A-B). The vaginal plate is FOXA1-positive and derived from UGS epithelium (Robboy et al., 2017), while the stratified PAX2-positive Müllerian-derived vaginal epithelium (Robboy et al., 2017) (A & C) is mostly KRT14-negative. Stratified squamous epithelia of the presumed exocervix (A) as well as the simple columnar epithelia of the endocervix, uterine corpus and uterine tube lack KRT14 reactivity (A).

Figure 7.

TP63 immunostaining of an intact 13-week human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. TP63 was strongly expressed in the stratified epithelium of the vaginal plate (D), vagina (B & D), but not in the simple columnar epithelia of the uterine tube (C), uterotubal junction (F) and upper uterine. Transitions between stratified and simple columnar epithelium (D-E) exhibited a sharp reduction in TP63 to patchy single cell expression, which was also seen in the presumed endocervical region (A).

Figure 8.

RUNX1 immunostaining in an intact 13-week human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host and immunostained for RUNX1. RUNX1 was strongly expressed in the stratified epithelium of the vaginal plate (B & F), vagina (B & F), and also in the simple columnar epithelia of the presumed endocervix (B-C) and the uterine corpus (A & D). RUNX1 expression diminished to undetectable at the uterotubal junction (E) and was not expressed in the uterine tube (D).

Figure 9.

ISL1 (A) and HOXA11 (B) immunostaining in an intact 13-week human fetal reproductive tract (AC121) grown for 4 weeks in an untreated ovariectomized female athymic mouse host. ISL1 is expressed in the mesenchyme associated with the vaginal plate and vagina, whereas HOXA11 is expressed in the mesenchyme associated with the uterine corpus.

Figure 10.

ESR1 immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. Tubal epithelium (A) expresses ESR1 but at a level reduced relative to non-grafted and specimens grafted into untreated hosts. ESR1 expression in the uterine tube, glands of the uterine corpus and cervix (B) (low magnification}, (C) (high magnification) of uterine glands. In the vaginal region ESR1 reactivity is seen in both mature squamous epithelium and adenosis, while the mesenchyme is largely unreactive (D).

Figure 11.

PGR immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. PGR is expressed strongly in epithelial and inner mesenchymal cells of the female reproductive tract (A). In vagina, PGR is expressed principally in the basal/suprabasal cells and in the adenosis (B).

Figure 12.

KRT7 immunostaining in a 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. KRT7 expression is reduced in the endometrial glands (C) and uterine tube (D), but prominent in the vaginal adenosis (B).

Figure 13.

KRT19 immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. KRT19 is expressed in the adenosis, other glandular epithelia, and in the suprabasal cells in the vaginal squamous epithelium (B).

Figure 14.

KRT6 immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. The vaginal squamous epithelial cells were highly reactive, while adenosis was not (B). The glandular epithelial cells of the cervix, uterine corpus and uterine tube were non-reactive except for small patches (A, arrowheads).

Figure 15.

KRT14 immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. KRT14 is reactive in the basal epithelial cells of the vagina (A-B), but not in adenosis (B), nor epithelium of the cervix, uterine corpus and uterine tube (A).

Figure 16.

TP63 immunostaining in an intact 13- and 14-week human fetal reproductive tract grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. TP63 immunostaining (AC159) is absent in the uterine tube and uterine corpus (A,, [AC159]). Early and maturing squamous epithelia in the cervix (B, [AC159]) and mature vaginal epithelium are TP63-positive but not adenotic epithelium in the vagina (C, [AC122]). Foci of epithelium in the region of the endocervix/uterine corpus (D, [AC159]) exhibit patchy TP63 expression (arrowheads).

Figure 17.

RUNX1 immunostaining in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. Mature vaginal epithelium (A-B) expresses TP63, but not adenotic epithelium (B). RUNX1 was not detected in the uterine tube, uterine corpus (A) and uterine glands (C).

Figure 18.

ISL1 (A) and HOXA11 immunostaining (B) in an intact 13-week human fetal reproductive tract (AC122) grown for 4 weeks in a DES-treated ovariectomized female athymic mouse host. ISL1-reactive mesenchyme (A) surrounds vaginal squamous/transitional epithelium. The adenotic epithelium is unreactive. The mesenchyme of the endometrial stroma is highly and diffusely HOXA11 reactive (B).

Figure 19.

αACT2 immunostaining of 13-week human uterine tubes grown for 4 weeks in (A, [AC121]) an untreated ovariectomized host and (B, [AC122]) in a DES-treated ovariectomized host stained for αACT2. Note that in the control (A), a substantial mesenchymal layer encompasses the tubal epithelium, which DES treatment (B) has obliterated, resulting in a misplaced αACT2 smooth muscle layer, which now comes into a intimate association with the epithelium.