Uterine glands originate from islands of FOXA2-positive luminal epithelium cells that differentiate de novo and invade uterine stroma

Graphical Abstract Legend: The epithelium of the adult uterus consists of a luminal epithelium (LE) that is negative for the Forkhead box A2 (FOXA2) transcription factor and a glandular epithelium (GE) that is positive for FOXA2 and required for fertility. We discovered that gland formation (adenogenesis) within the neonatal bovine uterus was associated with the differentiation of islands of LE cells into FOXA2+ cells (LE to GE specification). This process precedes bud formation and tubulogenesis. The LE to GE specification does not involve cellular proliferation (based on MKI67 marker). The LE to GE specification may be the initial step in gland formation that is followed by rupture of the basement membrane and penetration of the underlying stroma by FOXA2+ cells.

Dear editor, The onset of bovine uterine gland development (adenogenesis) can occur in utero (late gestation) or shortly after birth making cattle distinctly different from most other farm and laboratory species where gland development is postnatal [1][2][3].Regardless of the timing, the development of uterine glands in all mammalian uteri is initiated with the differentiation of luminal epithelium (LE) into glandular epithelium (GE).The cells of the LE rupture the basement membrane and penetrate the underlying stroma forming a bud and subsequent tube-like structures lined with GE.Later, these GE-lined tubes elongate, coil and branch to form the mature glands of the endometrium [4,5].
A universal feature of the GE is the expression of Forkhead box A2 (FOXA2); a transcription factor from the fork-head transcription factor subfamily [6].Importantly, FOXA2 is expressed specifically in the developing and the adult GE but is absent in the LE.Global knock-out of Foxa2 is embryonic lethal in mice [7].Subsequent studies of conditional deletion of Foxa2 in the neonatal mouse uterus (using Pgr-Cre) inhibited formation of glands and led to infertility secondary to an aglandular endometrium [3].Conditional deletion of Foxa2 in the adult uterus (using Ltf-Cre) led to a morphologically normal uterus that failed to maintain pregnancy [3].Failure to maintain pregnancy was explained by the loss of LIF expression, an essential embryokine, within the Foxa2 knockout glands [8].These studies demonstrated the central role that FOXA2 plays in the formation and function of uterine glands.Uterine glands knocked out in neonatal ovine uteri (UGKO model) led to infertility and confirmed that uterine glands are essential for fertility in ruminants as well [9].Our lab studies the interaction between the microbiome and tissue regeneration in the postpartum bovine uterus (uterine involution).Part of this process involves the restoration of uterine glands that may arise from existing glands [10] or perhaps arise from the development of new glands de novo.We elected to use neonatal calves as a model to study the earliest phases of glandular development in the bovine.We found that the LE invaginates into the stroma to form a bud and subsequently tubules before coiling and branching (as expected).To our surprise, however, expression of FOXA2 protein occurred in patches of cells (islands) within the LE before glands formed.The first phase of glandular development in the bovine, therefore, was the formation of islands of FOXA2-positive (+) cells within the LE that then appeared to direct the formation of glands into the underlying stroma.This is distinctly different from the current conceptual model that FOXA2+ cells are found within the GE after the gland has formed, but not prior to [2,3].
We performed immunohistochemistry on 5 μm cross sections to evaluate FOXA2 and MKI67 (cellular proliferation marker) within the uteri of beef-dairy crossbred calves at postnatal days (PND) 0 (PND0, n = 4), 7 (PND7, n = 2), 14 (PND14, n = 2), and 28 (PND28, n = 3).The uterus was collected within 10 min after humane slaughter (University of Missouri Animal Care and Use Protocol number 40224; M.C.Lucy PI), fixed in 10% neutral buffer formalin, and processed using immunohistochemical methods and antibodies described previously [10].Uterine cross sections were from the midpoint of the uterine horn (halfway between the uterotubal junction and uterine body).The primary antibodies were against FOXA2 (ab108422; rabbit monoclonal; 1:500 dilution; Abcam; Waltham, MA) and MKI67 (clone RM360; rabbit monoclonal; 1:500 dilution; Bio SB; Santa Barbara, CA).Negative controls (omission of primary antibody) were negative for positive staining.Individual calves varied with respect to the timing of glandular development.In two PND0 calves (gestation length 278 and 279 days), for example, we observed an intact LE (i.e.no breaching of the basement membrane) with diffusely distributed FOXA2 and MKI67 positive cells (Figure 1A).This appears to be the preadenogenic state of the bovine uterus.Glandular development, including LE invagination/budding, was found in other calves on PND0 (gestation length 272 and 276 days) and all calves at later PND (Figure 1B and C).Glands were confined to the intercaruncular (ICAR) endometrium and were not found in the caruncular (CAR) endometrium (as expected).Within individual calves at PND0, regions of ICAR endometrium differed with respect to the extent of glandular development (Figure 1B).In less developed (aglandular) ICAR regions, we observed patches or islands of FOXA2+ cells within the LE (Figure 1B, top middle).These islands of FOXA2+ cells were ∼50-100 μm in diameter and did not co-localize with distinct patches of MKI67+ cells (Figure 1B, bottom middle).The islands of FOXA2+ cells aligned themselves along the basement membrane in a columnar manner and were typically surrounded by a multilayer cluster of FOXA2 negative LE.In regions with well-developed tubular glands, cells within the GE were uniformly positive for FOXA2 (Figure 1B, top right).Proliferating cells (MKI67+) were localized within LE and the leading tip of the gland that had completed penetration into the endometrial stroma (Figure 1B, bottom right).Once tubulogenesis had occurred in the bovine, therefore, the pattern of FOXA2 and MKI67 expression was similar to other species [2].While not as extensive as PND0, FOXA2+ islands were observed within the LE of the developing uterus until approximately PND28 perhaps indicating this early phase of glandular development lasts for several weeks postnatally (Figure 1C).We then used FOXA2 and MKI67 staining in serial sections in an attempt to investigate the depth of the FOXA2+ islands and whether individual islands were associated with cellular proliferation (MKI67+ cells).The islands were at least 35 μm in cross section (Figure 1D, middle) and in some cases contiguous with a gland bud (Figure 1D, right) but were not positive for MKI67 throughout the serial cross section (data not shown).There appears to be the localized differentiation of a group of cells that lead to an island of FOXA2+ cells that then initiate budding and gland formation from the LE.A mechanism must exist to ensure that the islands are created and spaced appropriately to give rise to evenly spaced gland openings within the ICAR endometrium of the adult bovine.
The unexpected results from the present study define the earliest phase of adenogenesis within the bovine uterus.This phase that appears to last for several weeks postnatally has not been previously reported in any species.Forkhead transcription factors like FOXA2 have "pioneering" properties owing to their capacity to open condensed chromatin and recruit transcription factors involved in cell fate specification [6]; in this case, the differentiation of noninvasive cells of the LE into an island of invasive cells capable of forming a uterine gland.The mechanisms that lead to the spatially constrained induction of FOXA2 within the LE that precedes uterine gland formation will require further study.These additional studies will address the earliest phases of glandular development and also inform the broader question of how FOXA2, a transcription factor that is essential for fertility, is initially expressed within the LE and subsequently absent in the LE but maintained within the GE.

Figure 1 .
Figure 1.Immunohistochemistry for FOXA2 transcription factor and MKI67 cellular proliferation marker in neonatal calves at PND0, 7, 14, and 28.(A) PND0 calf without glandular development showing dispersed FOXA2+ and MKI67+ cells within the LE of ICAR endometrium at two locations (Boxes 1 and 2).(B) PND0 calf with regions of aglandular CAR and glandular ICAR endometrium immunostained for FOXA2 and MKI67.Within the ICAR endometrium there are islands of FOXA2+ cells that are confined to the LE (Box 1; middle top panel) and also dispersed MKI67+ cells (middle bottom panel).There are also regions with developing tubular glands (Box 2) that are FOXA2+ (right top panel) and MKI67+ at the gland tips (right bottom panel).(C) Uterine glandular development on PND 7 (left), 14 (middle), and 28 (right) showing islands of FOXA2+ cells in the LE (top panels) that are not associated with MKI67+ cells (bottom panels).(D) Serial sections through islands of FOXA2+ cells (Box 1 middle and Box 2 right) demonstrating FOXA2+ cells across seven serial sections (middle) and contiguous staining of FOXA2+ islands with gland buds.