Long-term label retaining cells localize to distinct regions within the female reproductive epithelium

Abstract

The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.

Introduction

The uterus is a highly regenerative organ that undergoes cyclical remodeling throughout the reproductive life of the female. During the menstrual cycle in species such as humans and old world primates the endometrium transitions through cycles of growth, differentiation, degeneration (menses), and regeneration of stromal (mesenchymal) and epithelial tissues. Remarkably, the luminal most two-thirds of the endometrium degenerates and is shed from the body during menses. The remaining basalis layer is then proposed to regenerate the lost tissue. Uterine remolding and tissue regeneration also occurs during and following pregnancy and these processes are elevated in invasively implanting hemochorial species such as humans and rodents. Here, uterine epithelial cells undergo apoptosis at the implantation site to facilitate embryonic invasion of the uterine wall. Concomitantly, stromal cells terminally differentiate into decidual cells forming the decidua, which is required for establishment and maintenance of early pregnancy. Following parturition and expulsion of the placenta, which consists of fetal chorionic tissue and maternal decidual tissue, the endometrium is regenerated similarly to menses. This process, termed uterine involution, occurs relatively quickly allowing the uterus to be receptive to another embryo within 3–4 d in mice or 40–45 d in humans. Proper endometrial regeneration following menses or parturition is necessary for preparation of the uterus for subsequent reproductive cycles and pregnancies.

Most adult organs exhibit some degree of plasticity and advances in stem cell biology have established a role for adult stem/progenitor cells in tissue renewal. It has been described in humans and mice that bone marrow-derived cells contribute to regeneration through engraftment into the endometrium of transplant recipients.^1-3 However, the functional contribution of the donor-derived cells to the endometrium, including the ability to colonize and repopulate the tissue, respond to steroid hormones and contribute to decidualization, has yet to be established. Furthermore, Cervellό et al.⁴ conclude that although bone marrow-derived cells do engraft in the endometrium of transplant recipients at a very low percentage they do not contribute to the side population and are therefore unlikely to contribute to endometrial tissue in a stem/progenitor cell capacity.

An alternative view is that the female reproductive tract harbors an endogenous population of stem/progenitor cells that contribute to maintenance of the tissue in postnatal life. In support of this, in vitro clonogenicity assays reveal the presence of highly clonogenic epithelial and stromal cells obtained from human endometrium.⁵ Clonogenic stromal cells were subsequently shown to differentiate in vitro into various mesenchymal cell lineages, thereby demonstrating the multipotent nature of these cells.^6,7 Side-population cells have been isolated from human endometrium and are shown to display phenotypic characteristics of somatic stem cells.⁸ Furthermore, epithelial and mesenchymal cell lines were derived from primary human endometrial side-population cells that appear to retain stem/progenitor cell activity following transplantation under the kidney capsule.⁹

Adult stem cells self-renew, generate lineage-differentiating progeny and are relatively quiescent compared with more differentiated cells within the same tissue such as transit amplifying cells. Because stem cells are thought to divide relatively infrequently, they often retain label while other more frequently dividing cells dilute out and eventually loose label through cell division. Bromodeoxyuridine (BrdU) label retention has been used to identify slow cycling label-retaining cells (LRC) as potential stem/progenitor cells in mouse endometrium and myometrium.^10-13 Although BrdU label retention has been used for many years in various tissues to identify presumptive stem/progenitor cells, the validity of this method has recently been questioned,¹⁴ and the immortal strand hypothesis¹⁵ continues to be debated in several fields of stem cell research.^14,16-18 Additionally, use of BrdU limits further assessment of stem/progenitor cells because the nucleotide label is a mutagen and because no reliable system has been developed to collect viable BrdU LRCs. Therefore, transgenic approaches have been developed to circumvent caveats of nucleotide labeling. For instance, by crossing mice that express histone H2B-green fluorescent protein (H2B-GFP) under the control of a tetracycline (Tet)-responsive regulatory element with mice that express a tetracycline transactivator (tTA) or reverse tetracycline transactivator (rtTA), the expression of H2B-GFP (fusion protein) can be temporally controlled by administration of doxycycline (dox).^19,20 H2B-GFP is stably incorporated into nucleosomes during DNA replication allowing for identification and characterization of LRCs. Furthermore, this transgenic mouse system allows for embryonic labeling when the majority of cells present in developing tissues are undifferentiated, and this cannot be accomplished with nucleotide labeling due to genotoxicity. The LRC transgenic mouse model was used recently to identify LRCs in the ovarian surface epithelium (OSE)²¹ and the oviduct epithelium.²² LRCs in the OSE were proposed to contribute to wound repair following ovulation, while it was speculated that oviductal LRCs might be stem/progenitor cells for the endometrium.

At present, the location of epithelial stem/progenitor cells within the endometrium has not been established. As such, in this study we employed a transgenic mouse model to identify LRCs throughout the female reproductive tract with particular interest on identifying LRCs in the endometrium. We hypothesize that the female reproductive tract contains a population of long-term LRCs that persist even following dramatic tissue remodeling during pregnancy and following endometrial regeneration that accompanies uterine involution.

Results

Epithelial LRCs are located in the distal oviduct and endocervix transition zone following embryonic through postnatal labeling

We used the LRC transgenic mouse model initially to assess label retention in the uterus in an attempt to identify and characterize uterine epithelial stem cells. To accomplish this, H2B-GFP; Rosa-rtTA double transgenic (Tg) females were treated transplacentally/translactionally with doxycycline (dox, pulse period) from embryonic day (E) 13.5 to postnatal day (PND) 21 to induce expression of the H2B-GFP fusion protein. By pulsing embryonically, there is a greater likelihood of labeling stem cells as opposed to only labeling transit amplifying or progenitor cells which can occur with pulsing adult animals. Additionally pulsing from E 13.5-PND 21 allowed for labeling during Müllerian duct development and postnatal maturation of the uterus. At birth (PND 0.5) the entire luminal epithelium (LE) was labeled, as was the majority of the stroma and myometrium (Fig. 1A and B). Dox was withdrawn at PND 21, initiating the chase period. At chase day zero (i.e., PND 21; Fig. 1C and D) most of the cells throughout the uterus were labeled however, surprisingly regions within the LE and glandular epithelium (GE) were devoid of GFP label. By six wk of age (WOA; 3 wk chase) the majority of the label was lost in the epithelium. The brightest LRCs in the uterus were concentrated to the outer stroma and myometrium (Fig. 1E and F). By eight WOA (5 wk chase; Fig. 1G and H) label retention was indistinguishable from untreated controls (i.e., double Tg females not treated with dox; Fig. 1I and J).

Figure 1. Temporal label retention of H2B-GFP in the uterus after pulsing with doxycycline from embryonic day 13.5 to postnatal day 21. At postnatal day (PND) 0.5, just after birth during the pulse period, nearly all of the cells throughout the uterus contained H2B-GFP label demonstrating the efficiency of labeling cells in utero (A and B; n = 2). (C and D) Representative uterine cross-section from PND 21 at the end of the pulse period showing that the majority of the cells were labeled (n = 3). (E and F) At six wk of age (WOA; 3 wk chase) the epithelium contained only dim label-retaining cells (LRCs) where as bright LRCs were located in the stroma and myometrium (n = 3). (G and H) By eight WOA (5 wk chase), only dim label retaining cells were observed throughout the uterus (n = 3) and were indistinguishable from controls (I and J). (I and J) PND 21 control uterus from a double transgenic female (H2B-GFP; Rosa-rtTA) that was not treated with doxycycline demonstrating baseline, leaky expression of H2B-GFP.

Recently, epithelial transition zones (TZ) including the anorectal,²³ esophagogastric,²⁴ and corneal-limbus junctions²⁵ have been proposed as the location of epithelial stem cells for the corresponding tissues. Additionally, in humans the endo-ectocervix junction contains reserve/basal cells that are postulated to serve as progenitors for squamous and/or columnar epithelium in the endocervix.²⁶ We therefore, looked at other tissues in the reproductive tract for the presence of LRCs. To allow for easier identification of bright epithelial LRCs (i.e., cells that are more quiescent) we attempted to reduce the number of dim epithelial LRCs by assessing label-retention following one pregnancy during which substantial epithelial proliferation occurs to repair the damaged tissue. At 3 wk postpartum (WPP; 9–13-wk chase), bright epithelial LRCs were concentrated at the distal oviduct (Fig. 2A and B) and the endocervical TZ (Fig. 2E and F). The proximal oviduct (Fig. 2C) contained very few LRCs, the majority of which were dim. Similarly the utero-tubal junction (Fig. 2D) was devoid of bright LRCs and only rarely contained dim LRCs. The presence of LRCs in the distal oviduct corroborates findings by Wang et al.²² in which LRCs were identified using a brief pulse/chase strategy in sexually mature adult mice. Furthermore, using the transplacental/translactational labeling approach, we identified bright LRCs within the endocervical TZ where the stratified epithelium of the cervix abuts the single columnar epithelium of the uterus. The majority of bright label retention was seen in cervical basal cells throughout the length of the cervix from the uterus to the vagina (Fig. 2E).

Figure 2. Label retention in the female reproductive tract three wk postpartum. (A–F) Reproductive tracts from 3-wk postpartum double transgenic (H2B-GFP; Rosa-rtTA) female mice (n = 6) pulsed with doxycycline from embryonic day 13.5 to postnatal day 21. (A) Bright label-retaining cells (LRCs) were observed in the distal oviduct epithelium and ovarian surface epithelium (white arrowheads). (B) Magnified image of boxed area in (A) showing bright epithelial LRCs in the distal oviduct. (C) LRCs in the proximal oviduct epithelium were dim and sporadic. (D) The utero-tubal junction did not contain LRCs in the epithelium. (E) Bright LRCs were present in the endocervical transition zone (TZ) where stratified cervical epithelium meets single columnar uterine epithelium. (F) In the endocervical TZ the majority of label-retention was observed in basal cells at higher magnification (E). Baseline H2B-GFP expression in distal oviduct (G) and endocervical TZ (H) from 5 mo old double transgenic (H2B-GFP; Rosa-rtTA) control mouse that did not receive doxycycline treatment.

Characterization of label-retaining cells

We began characterizing LRCs in the oviduct and endocervical TZ by assessing expression of estrogen receptor α (ERα) and progesterone receptor (PGR). Epithelial LRCs within the oviduct were not found to express ERα however they were often found located next to ERα expressing cells (Fig. 3A–C). Within the endocervical region, ERα was not expressed in the epithelium and therefore LRCs were devoid of ERα (Fig. 3D–F). However, it should be noted that different stages of the estrous cycle might impact expression patterns of ERα within the cervix as they do in the uterus.^27,28 With regard to PGR, expression was only found in the proximal oviduct (data not shown) but was absent from the distal oviduct, and accordingly, LRCs in this region did not show PGR expression (Fig. 3G–I). PGR was expressed in the epithelium of the endocervical TZ and some dim LRCs did co-localize with PGR (Fig. 3J–L). However, there appeared to be an inverse relationship between bright, basal LRCs and PGR expression as the majority of cells that co-localized with PGR were located suprabasally and were either dim LRCs or did not retain label. These data suggest that LRCs do not directly respond to estrogen and progesterone via classical receptors; however, they may be regulated by these hormones through indirect actions of neighboring cells.

Figure 3. Expression of estrogen receptor α (ERα) and progesterone receptor (PGR) by label-retaining cells (LRC) in the distal oviduct and endocervical transition zone (TZ). (A–C) LRCs (orange arrowheads) in the distal oviduct epithelium do not express ERα but are located near non-LRCs (white arrowheads) that do express ERα. (D–F) ERα is not expressed in the endocervical TZ epithelium and subsequently LRCs (orange arrowheads) do not express ERα, however stromal non-LRCs (white arrowheads) do express the receptor. (G–I) PGR is not expressed in the distal oviduct and accordingly LRCs do not express the receptor in this region. (J–L) In the endocervix TZ, PGR is predominantly expressed by suprabasal cells that do not retain label or are dim LRCs (white arrowheads), but the receptor is not expressed by bright basal LRCs (orange arrowheads).

We further characterized LRCs by assessing expression of the stem-cell markers c-Kit and p63. Most of the LRCs in both the distal oviduct (Fig. 4A–C) and endocervical TZ (Fig. 4D–F) expressed c-Kit as did many non-LRCs, however c-Kit was not expressed in the luminal epithelium of the uterus (data not shown). p63 is a transcription factor expressed in basal cells of stratified epithelium including the cervix, vagina, skin and prostate²⁹ and serves as a stem/progenitor cell marker for these tissues. Here we characterized expression of phospho-p63 (P-p63) in basal-like LRCs. As anticipated, LRCs in the endocervical TZ that appeared to be basal cells based on location, expressed P-p63 confirming their basal cell identity (Fig. 5D–F). Expression of P-p63 was not observed in the oviduct (Fig. 5A–C) or uterus (data not shown). Together these data suggest that LRCs located in the distal oviduct and endocervical TZ are less differentiated based on expression of stem cell markers and lack of expression of ERα and PGR.

Figure 4. C-Kit expression by label retainting cells (LRC) in distal oviduct and endocervical transtition zone (TZ) epithelium. (A–I) Representative uterine cross sections from 3-wk postpartum double transgenic (H2B-GFP; Rosa-rtTA) female mice (n = 6) pulsed with doxycycline from embryonic day 13.5 to postnatal day 21. Some LRCs (orange arrowheads) as well as some non-LRCs (white arrowheads) express c-Kit in both the distal oviduct (A–C) and endocervix TZ (D–F) epithelia. (G–I) Uterine cross section with primary antibody omitted from immunofluorescence protocol.

Figure 5. Label-retaining cell (LRC) expression of phospho-p63 in the distal oviduct and endocervical transition zone (TZ) in uteri three wk postpartum. (A–C) Phospho-p63 (P-p63) is not expressed in the oviduct and therefore expression is not observed by label-retaining cells (LRCs) in this region. (D–F) Basal LRCs (orange arrowheads) in the endocervical TZ express P-p63.

Proliferation during endometrial regeneration

To determine if LRCs proliferate during endometrial regeneration we used a menses-like mouse model of endometrial breakdown and repair. Decidualization was mechanically induced in pseudopregnant female mice resulting in near-complete loss of the luminal epithelium (LE) and retention of only a small number of epithelial glands. Following ovariectomy (ovex) and subsequent loss of progesterone stimulus, the decidual tissue degenerates and the endometrium, including the LE and GE, regenerates to a fully repaired state by 72 h post-ovex. Phospho-histone H3 (P-HH3) expression was used as a marker of mitosis to assess epithelial cell proliferation during endometrial regeneration. At 12 h (data not shown) and 24 h post-ovex, P-HH3 was not expressed in the distal (Fig. 6A–C) or proximal oviduct (data not shown) indicating the absence of mitotically active cells in the oviduct at either of these time points. In the endocervix, P-HH3 was not expressed by any of the basal LRCs, but rather was expressed by the suprabasal cells (Fig. 6G–I) as well as the uterine LE (Fig. 6D–F) at both 12- and 24 h post-ovex. These results suggest that LRCs situated in the distal oviduct and endocervix do not participate in re-epithelialization of the endometrium. However it remains to be determined if LRCs of the distal oviduct and endocervical TZ proliferate at other times during endometrial regeneration or if they are reserved for long-term maintenance of the tissue and replenishment of progenitor-like cells in the uterus at a time apart from endometrial regeneration.

Figure 6. Expression of the mitotic marker, phospho-histone H3, by label-retaining cells (LRC) during endometrial regeneration. (A–I) Representative sections from mice at 24 h post-ovariectomy (ovex) during endometrial regeneration using the menses-like model (n = 3). (A–C) No cells within the distal oviduct show positive staining for phospho-histone H3 (P-HH3). (D–F) Uterine luminal epithelial cells do express P-HH3 during regeneration. (G–I) Non-label retaining suprabasal cells (white arrowheads) in the endocervix transition zone express P-HH3 however, basal LRCs (orange arrowheads) do not.

Altering the pulse-chase period results in epithelial label retention in the endometrium

At birth, the murine Müllerian ducts consist of undifferentiated tubal epithelium surrounded by undifferentiated mesenchyme. Maturation of the Müllerian ducts to form the oviducts, uterus and upper vagina involves region-sp.ecific luminal epithelial (LE) differentiation, mesenchymal differentiation to form the stromal and myometrial layers and formation of epithelial glands. By PND 15, the basic uterine architecture consisting of LE, GE, stroma, and multi-layered myometrium, is established.^30-32 Although the uterine histoarchitecture is complete the uterus does not reach its adult size or become fully hormonally competent until sexual maturity around 6 wk of age. Chan et al.¹² reported that BrdU-LRCs play a more important role than non-LRCs in estrogen-induced epithelial and stromal cell proliferation in prepubertal mice compared with adult cycling mice. In our initial pulse-chase model (Fig. 7A, blue box), we stopped the pulse period and began the chase just prior to growth and expansion of the postnatal/prepubertal uterus. If LRCs participate in expansion, this could account for the complete loss of label from the uterus using this model. To circumvent this labeling limitation, the pulse period was adjusted to begin at PND 21 and continue through uterine expansion on PND 42 when mice reach sexual maturity (Fig. 7A, green box). Using the revised model, mice were chased through nine MOA and long-term-LRCs were identifiable in GE even in mice that had undergone multiple pregnancies (Fig. 7B–D). Further experiments are needed to characterize these LRCs and determine to what extent they contribute to repair of the endometrium.

Figure 7. Long-term H2B-GFP label retention in the uterus after peripubertal doxycycline pulse. (A) Diagram of the 2 pulse periods used in the study. (A, label 1) Pulsing with doxycycline (dox) from embryonic day (E) 13.5 to postnatal day (PND) 21 during Müllerian duct formation, adenogenesis and establishment to of basic adult uterine histoarchitecture results in loss of label in the uterus by eight wk of age. (A, label 2) Pulsing with dox PND 21–42 during growth and expansion of the uterus to adult size results in long-term label retention in the glandular epithelium at nine months of age (B–D; n = 3).

Discussion

Endometrial regeneration is a complex process that likely involves multiple mechanisms. For instance, mesenchymal-to-epithelial transition (MET) was recently shown to contribute to endometrial regeneration.^33,34 In these studies, we identified a unique population of transitional cells that co-expressed the epithelial cell marker pan-cytokeratin and the stromal cell marker vimentin in the stromal compartment during endometrial regeneration. In the early stages of regeneration these transitional cells were located near the stromal-myometrial boarder, but then migrated to the regeneration zone near the lumen as repair progressed.³³ Additionally, using a fate-mapping technique, MET-derived epithelial cells were found in the LE and GE following parturition and completed regeneration.³³ A second mechanism of re-epithelialization is migration and proliferation of residual glands. Using scanning electron microscopy to observe human menstrual endometrium, investigators concluded that glandular stumps protruding from the denuded surface contributed cells to the LE during menstrual regeneration.^35-37 Based on epithelial mitosis in primate endometrium, Padykula et al.^38,39 proposed that GE in the lower basalis harbors the stem/progenitor cells responsible for re-epithelialization of the lumen. Using BrdU incorporation in a mouse model Kaitu’u-Lino et al.⁴⁰ similarly suggested that GE participates in re-epithelialization of the LE in a mouse model of endometrial breakdown and repair that is similar to our menses-like model. Furthermore, the authors proposed that epithelial stem/progenitor cells are located in the glands as evidenced by constant label-retention in GE, whereas no label was retained in the LE following regeneration. Finally, as already alluded to, stem cells are thought to exist in the female reproductive tract that may participate in endometrial regeneration. BrdU-LRCs have been identified in the endometrium^10,12,13,40 and myometrium,¹¹ however isolation of the LRCs for further characterization and assessment of functional contribution to the tissue by transplantation is not possible due to the labeling agent. More recently, Wang et al.²² used H2B-GFP; Rosa-rtTA double Tg females to label cells in adult cycling mice (8–12-wk-old) for seven days. However, following 2–4 wk of chase, label was completely lost in the uterus, but persisted in the distal oviduct for 47 wk. Loss of label in the uterus was likely due to lack of stable labeling of resident stem/progenitor cells. Given the short seven-day pulse period in adult mice, it is possible that stem/progenitor cells were not proliferating to ensure stable incorporation of H2B-GFP into nucleosomes resulting in rapid loss of label.

In the current study we identified LRCs throughout the female reproductive tract using two different pulse/chase periods. In addition to corroborating the results of label-retention in the distal oviduct reported by Wang et al.,²² our data also provides novel evidence of LRCs in the endocervical TZ using transplacental/translactational labeling, as well as in the GE of the uterus using a 3-wk peripubertal (PND21–42) labeling protocol. Following transplacental/translactational labeling, we were able to show the persistence of LRCs in the distal oviduct and endocervical TZ through four months of age and following pregnancy, parturition and completed endometrial regeneration. Oviductal and endocervical TZ LRCs expressed stem cell markers. Furthermore, an inverse correlation between bright label retention and ERα and PGR expression was observed suggesting that LRCs are less differentiated and unable to respond directly to sex steroid hormones. The stem-like activity of distal oviductal cells has been demonstrated in vitro through sphere formation assays using cells isolated from both human⁴¹ and mouse oviducts.²² Furthermore, it was suggested that LRCs in the distal oviduct might provide progenitor and differentiated cells to the endometrium.²² However, in the current study bright LRCs in the distal oviduct and endocervical TZ did not express P-HH3. Consequently it appears that they do not participate in re-epithelialization of the endometrium. Therefore, it is unclear at this time whether oviductal or endocervical TZ LRCs contribute any cells to the endometrium and to what extent they exhibit stem/progenitor cell activity in vivo. Importantly, recent studies on the origins of serous ovarian cancer have pointed to the distal oviduct as the source of the primary tumor in both humans⁴¹ and mouse models.⁴² Similarily, Herfs et al.⁴³ provide evidence that a unique population of squamocolumnar cells at the ecto-endocervical junction in humans may be the source of cervical cancer. With increasing popularity of the cancer stem cell theory, our results, demonstrating the presence of LRCs in the distal oviduct and endocervical TZ that appear to be less differentiated, lend evidence to the existence of stem/progenitor cells in these cancer-prone regions. Additionally, investigators have proposed cancer stem cells as the source of endometrial cancer.⁴⁴ Identification of LRCs in the female reproductive tract epithelium provides a means for assessing the stem-like qualities of these cells with the potential for studying abnormal stem/progenitor cell activity associated with cancer or other hyper/hypo-proliferative diseases.

Our study also identified the presence of LRCs in the GE of the endometrium following peripubertal labeling. These cells persisted out to nine months of age, thus supporting the concept that the endometrium possesses an endogenous population of stem/progenitor cells. However, it is important to emphasize that LRCs cannot at this point be equated with stemness. Although many investigators have used label-retention to enrich for stem/progenitor cells in various other tissues, the validity of this technique must be ascertained specifically for the female reproductive tract. The “gold standard” for assessing stem cell activity of a population of cells is through transplantation to determine their functional contribution to the tissue of interest. Therefore future studies using the LRC transgenic mouse model are directed toward isolation of LRCs from the oviduct, uterus and endocervical TZ by fluorescence activated cell sorting followed by in-depth assays of stem cell activity in vitro and through transplantation in vivo.

In summary, we employed a transgenic mouse model to identify LRCs throughout the female reproductive tract using two different pulse/chase periods. When mice were pulsed transplacentally/translactionally to label cells during Müllerian duct development and postnatal uterine maturation, label was not retained in the uterus; however, LRCs were concentrated in the distal oviduct and endocervical TZ. Alternatively when mice were pulsed peripubertally during further uterine maturation and expansion, LRCs were identified in the endometrial GE at nine months of age. These cells will be further examined as candidate endometrial epithelial stem/progenitor cells. LRCs in the distal oviduct and endovervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. It is unlikely that oviduct and endocervical TZ LRCs contribute to endometrial re-epithelialization, but may be implicated in carcinoma formation in these cancer prone areas.

Materials and Methods

Label-retaining transgenic mouse model

All protocols involving animal experiments were approved by the Institutional Animal Care and Use Committee at Washington State University. H2B-GFP (tetO-HIST1H2BJ/GFP) mice¹⁹ and Rosa-rtTA (B6.Cg-Gt[ROSA]26Sortm1[rtTA*M2]Jae/J) mice²⁰ were purchased from The Jackson Laboratory. H2B-GFP mice were mated to Rosa-rtTA mice to generate double transgenic offspring (H2B-GFP; Rosa-rtTA). In the absence of doxycycline (dox), the reverse tetracycline transactivator (rtTA) protein, which is constitutively expressed, is incapable of binding the tetracycline response element (TRE) located up-stream of the cytomegalovirus minimal promoter (mCMV) which is likewise up-stream of the H2B-GFP gene. Therefore, in the absence of dox the H2B-GFP fusion protein is not expressed. To induce H2B-GFP expression using the first pulse/chase model (Fig. 7A, label 1), females pregnant with double transgenic (Tg) offspring (H2B-GFP; Rosa-rtTA) were administered dox (pulse period) in the drinking water (1 mg/ml; replenished at least once per wk). Fetuses were therefore exposed to dox in utero beginning on embryonic day (E) 13.5, allowing rtTA to bind the TRE and induce expression of H2B-GFP, thereby labeling cells during Müllerian duct development. Dox treatment was continued to post-natal day (PND) 21 at which time treatment was withdrawn initiating the chase period. During the chase period GFP expression was diluted out in rapidly proliferating cells but was retained in infrequently dividing cells allowing for identification of the label-retaining cells (LRC). In the second pulse/chase model (Fig. 7A, label 2), double Tg (H2B-GFP; Rosa-rtTA) females were pulsed peripubertally (PND 21–42) by treatment with dox in the drinking water (1 mg/ml; replenished at least once per wk) and the chase period was initiated on PND 42.

For the first pulse/chase model uteri were collected from H2B-GFP; Rosa-rtTA females at PND 0.5 (n = 2), PND 21 (n = 3), 6 wk of age (WOA; n = 3) and 8 WOA (n = 3) to assess label-retention in the uterus. To determine the location of LRCs and the extent of label-retention within the entire reproductive tract following pregnancy ovaries, oviducts, uteri, cervices, and vaginas were collected from H2B-GFP; Rosa-rtTA females (n = 6) at least 3 wk postpartum (WPP) following one pregnancy and resumption of estrous cyclicity. H2B-GFP; Rosa-rtTA females were mechanically induced to undergo decidualization following the menses-like model described below to assess the proliferative potential of LRCs. Oviducts, uteri and cervices were collected at 12- and 24 h post-ovariectomy (ovex; n = 3 per time point). Using the second pulse/chase model, uteri were collected from H2B-GFP; Rosa-rtTA females at nine MOA (n = 3). Controls were double Tg (H2B-GFP; Rosa-rtTA) females that did not receive dox treatment and were collected to assess baseline H2B-GFP expression (n = 1–2 per time point corresponding to treated mice). All tissues collected were fixed in 4% paraformaldehyde for 10–15 min on ice and processed for gelatin embedding.

Menses-like mouse model

A menses-like state was induced in double transgenic females as previously described³³ to assess proliferation during endometrial regeneration. Briefly females were placed with vasectomized (i.e., sterile) male CD1 mice and upon the observation of a vaginal plug were designated day of pseudopregnancy (DOPP) 0.5. On DOPP 4, sesame oil (20 μl) was injected into the uterine lumen to mechanically induce endometrial decidualization. At 72 h post-oil induced decidualization, progesterone (P₄) stimulus was removed by ovariectomy (ovex) to allow the deciduoma to degenerate and the endometrium to regenerate. Mice were euthanized and oviducts, uteri and cervices were collected at 12- and 24 h post-ovex (n = 3 per time point) during endometrial regeneration. Tissues were fixed in 4% PFA on ice for 15 min and processed for gelatin embedding and freezing.

Gelatin embedding and frozen tissue preparation

Following PFA fixation all tissues were washed three times in ice cold PBS and then incubated overnight at 4 °C in 15% sucrose buffered in PBS. The following day tissues were incubated at 37 °C for 1 h in gelatin (15% sucrose, 7.5% gelatin in PBS), embedded in gelatin on a cold block and allowed to solidify and cool to 4 °C. Tissue blocks were frozen at −50 °C to −65 °C in isopentane cooled by liquid nitrogen and stored at −80 °C until sectioning. Tissues were cryo-sectioned at 5 μm and thaw mounted. Gelatin was removed from the tissue sections by incubating slides in 37 °C PBS. Following gelatin removal slides were either counterstained with 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories) mounting medium and viewed directly using fluorescence microscopy or used for Immunofluorescence.

Immunofluorescence

Immunofluorescence was conducted on all postpartum samples (n = 6) and all 12- and 24-h post-ovex samples (n = 3 per time point). The following primary antibodies were used: ERα (rabbit polyclonal 1:500; Santa Cruz Biotechnology cat # sc-542), PGR (rabbit monoclonal 1:50; Thermo Scientific cat # RM-9102-S0), c-Kit (goat polyclonal 1:50; R&D Systems cat # AF1356), P-p63 (rabbit polyclonal 1:50; Cell Signaling cat # 4981S), and P-HH3 (rabbit polyclonal 1:500; Millipore cat # 06-570). Secondary antibodies used were AlexaFluor 546 donkey anti-rabbit (1:500) or donkey anti-goat (1:1000). Following removal of gelatin (described above), tissues were incubated for 1 h at room temperature (RT) in blocking solution (0.1% triton X 100, 0.1% BSA and 10% normal donkey serum in PBS), followed by incubation with primary antibody diluted in blocking reagent for 1 h at RT (for PGR and C-Kit) or overnight at 4 °C (for ERα, Phospho-p63 and Phospho-HH3). Tissues were then washed three times with PBS and secondary antibody diluted in blocking solution was applied in the dark for 45 min at RT. Tissues were washed in PBS followed by water, then counter stained with DAPI mounting medium and coverslipped. Omission of primary antibodies served as a negative control.

Glossary

Abbreviations:

BrdU

bromodeoxyuridine

LRC

label-retaining cell

H2B-GFP

histone H2B-green fluorescent protein

rtTA

reverse tetracycline transactivator

dox

doxycycline

OSE

ovarian surface epithelium

Tg

transgenic

LE

luminal epithelium

GE

glandular epithelium

PND

postnatal day

WOA

wk of age

MOA

months of age

WPP

week post-partum

ovex

ovariectomy

ERα

estrogen receptor alpha

PGR

progesterone receptor

P-p63

phospho-p63

P-HH3

phospho-histone H3

TZ

transition zone

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported in part by NIH HD066297