Bo Li

Progesterone is essential for establishing uterine receptivity and sustaining early pregnancy. However, how the uterus acquires the competence to respond to progesterone at the appropriate time remains poorly understood. Here we show that ERBB2 governs the timing of progesterone responsiveness required for uterine receptivity. In endometrial samples from women with recurrent spontaneous abortion (RSA), ERBB2 is downregulated specifically in the stromal compartment, accompanied by a parallel reduction in stromal progesterone receptor (PGR). Uterine deletion of Erbb2 in mice blunts the rise of stromal PGR at the onset of the receptive window, deferring implantation and impairing decidualization despite normal circulating progesterone and estrogen. Epithelial deletion has no such effect, placing the requirement in the stroma. High-dose progesterone fails to rescue the phenotype; advancing progesterone priming by 24 hours restores stromal PGR, HAND2, Bmp2 , and on-time implantation. Collectively, the present study provides evidence that ERBB2 directs the temporal window of stromal progesterone priming by setting the threshold for PGR induction, thereby synchronizing the maternal-fetal interface and safeguarding pregnancy. Abstract Figure:

This protocol is a version of mFISH3D-2.0 V.2 (Murakami et al.; DOI 10.17504/protocols.io.kqdg3qn21v25/v2; see also the associated manuscript, Neuron, DOI 10.1016/j.neuron.2025.12.027), adapted for whole-mount 3D fluorescence in situ hybridization (HCR-based) in mouse uterine tissue. Modifications from the original brain protocol include a shortened fixation step, an abbreviated MeOH/delipidation series, and omission of the Proteinase K and photobleaching steps. This protocol enables researchers to visualize their gene(s) of interest in three dimensions within intact, whole-mount mouse uterine tissue, preserving spatial context and tissue architecture that is lost with conventional 2D section-based in situ hybridization. It performs particularly well for epithelial genes, where signal localizes to a defined cell layer against a dark stromal background. In our hands, we routinely image three genes per tissue, one probe set per HCR amplifier channel.

Pregnancy requires expanded maternal tolerance to semiallogeneic fetal-expressed antigens to protect against fetal loss and other pregnancy complications. Our understanding of how this biological imperative works remains uncertain. Here we show pregnancy primes expansion of antigen-experienced Krüppel-like factor 2 (KLF2)+ CD4 T cells with fetal specificity, and that these maternal cells play an essential role in optimal fertility and protecting against fetal resorption. Antigen-experienced KLF2+ CD4 cells purified from pregnant dams are poised for IL10 production and suppress responder T cell proliferation in coculture. Mice with conditional loss of KLF2 in T cells become pregnant less efficiently after allogeneic mating with genetically discordant males, but not after syngeneic mating with genetically identical males. Impaired fertility is associated with diminished and variable early pregnancy progesterone levels, whereas progression from mating to pregnancy is restored with exogenous progesterone. To bypass fertility defects, complementary experiments show that initiating induced KLF2 deletion in CD4 T cells midgestation causes fetal resorption associated with expansion of activated fetal-specific CD8 effector T cells across maternal tissues, particularly in the uterine draining lymph node and at the maternal–fetal interface. Reciprocally, these phenotypes are overturned with CD8 T cell depletion or in mice reconstituted with CD4 cells from wild-type donors which upregulate KLF2 expression in pregnant recipients. These findings demonstrate that maternal KLF2+ CD4 T cells promote fetal tolerance and fertility, particularly during allogeneic pregnancy.

Background: Decidualization requires stromal reprogramming to establish pregnancy, yet the upstream mechanisms governing cytoskeletal remodeling remain obscure. While primary cilia direct this transformation, how stromal cells integrate post-translational modifications to regulate the mechanics of ciliogenesis remains unknown. This study aims to investigate the role of neddylation, a ubiquitin-like modification, in coordinating the structural and transcriptional plasticity required for mouse decidualization. Methods: We utilized the NAE inhibitor MLN4924 in an in vivo mouse model of artificial decidualization. To decipher the underlying molecular mechanism, we combined pharmacological blockade with genetic silencing of Uba3 and Cul3 in mouse uterine stromal cells. We employed RNA-sequencing to map global transcriptomic landscapes, complemented by real-time PCR, immunofluorescence, and Western blotting to assess ciliary dynamics and cytoskeletal architecture. Additionally, the ciliogenesis inhibitor Ciliobrevin A (CBA) was used to study the functional link between primary cilia and decidualization. Finally, we performed rescue experiments with small-molecule inhibitors of RhoA (Rhosin) and ROCK (Y27632), alongside the microtubule stabilizer Tubastatin A (TubA), to dissect the specific mechanotransduction pathways driving the observed phenotypes during mouse decidualization. Results: We identified neddylation as a key regulator of stromal remodeling during decidualization. Pharmacological inhibition and genetic silencing significantly suppressed primary cilia formation and impaired the decidual response. Transcriptomic profiling revealed that neddylation inhibition aberrantly reactivates hormone-suppressed actomyosin contractile genes, particularly Myl9 . Mechanistically, we demonstrate that neddylation selectively activates the E3 ligase Cullin 3 (Cul3) to target RhoA for degradation. Disruption of this axis leads to RhoA accumulation and excessive actomyosin contractility, creating a rigid cytoskeletal environment that restrains ciliogenesis. Remarkably, relieving cytoskeletal tension (via Rhosin or Y27632) or stabilizing microtubules (via TubA) functionally rescued the decidualization defects, overcoming the upstream transcriptional dysregulation. Conclusions: These findings establish the Cul3-RhoA axis as a critical mechanical checkpoint linking protein modification to the plasticity required for pregnancy success. By maintaining a low-tension state permissive for ciliogenesis, neddylation ensures the execution of the decidual program. Furthermore, our data suggest potential reproductive risks and highlight the neddylation-ciliary axis as a novel theoretical target for disorders of impaired decidualization.

Primary cilia are solitary, non-motile, microtubule-based organelles that protrude from the surface of most vertebrate cells, functioning as highly special

Endometrial regeneration is essential for reproductive cycles and pregnancies, allowing the endometrium to undergo estrogen-driven repair, growth, and renewal after menstruation and parturition. Epithelial cells lining the uterine cavity undergo apoptosis during estrous cycles, and remnant cells can quickly restore this lining through a process known as re-epithelialization. It is presumed that adult stem/progenitor cells in the uterine stroma also contribute to re-epithelialization. However, the specific cell type(s) and the underlying mechanisms have not been determined. Herein, we use genetic lineage tracing assays in mice to identify Nestin⁺ perivascular cells as active contributors to re-epithelialization. Notch signaling maintains Nestin⁺ perivascular cells in a quiescent state, but these cells re-enter the cell cycle and differentiate into epithelial cells via estrogen-stimulated suppression of Notch signaling dependent on estrogen receptor alpha (ESR1). These findings demonstrate that perivascular cells support re-epithelialization and reveal a mechanism regulating the quiescence and activation of uterine perivascular cells.

Preimplantation uterine gland differentiation, involving glandular branching, is a prerequisite for secretion of leukemia inhibitory factor (LIF), thereby establishing uterine receptivity to embryo implantation. This differentiation process is disrupted by the absence of FOXA2, a transcription factor specifically present in uterine glands. It remains unclear whether preimplantation glandular differentiation is regulated exclusively by gland-intrinsic mechanisms or is influenced by the surrounding microenvironment through mesenchymal-epithelial interactions. We show here that uterine deletion of Erbb3 leads to impaired gland branching and FOXA2 expression, as well as reduced female fertility. In contrast, females with uterine epithelial-specific deletion of Erbb3 show normal fertility, suggesting a critical role of stromal ERBB3 signaling. A transcriptomic profiling of day 3 stromal cells identified Igf1 as a candidate paracrine factor mediating the stromal-epithelial interaction. Thus, the uterine deletion of Igf1r leads to a reduced gland branching phenotype similar to that observed in Erbb3 ^d/d uteri. These results demonstrate that ERBB3 regulates glandular branching and embryo implantation via the ERBB3-IGF1-IGF1R signaling pathway.

Primary cilia are antenna-like organelles that sense extracellular signals and function as signaling hubs essential for vertebrate development and homeostasis. Decidualization is crucial for pregnancy establishment and maintenance in both humans and mice. While primary cilia are present in endometrial stromal cells, their role in pregnancy remains unknown. Here, we identify TMEM67, a key component of the ciliary transition zone, as a critical regulator of mouse decidualization. Loss of primary cilia triggers RhoA-MLC2-dependent actomyosin contraction, which transmits mechanical forces to the nuclear lamina, leading to micronuclei formation. Within these micronuclei, double-stranded DNA (dsDNA) can directly bind to cyclic GMP-AMP synthase (cGAS) in situ, initiating downstream signaling. This activation of the cGAS-STING pathway reduces CCL6 production and impairs decidualization. Furthermore, pharmacological inhibition of actin polymerization or RhoA-ROCK signaling alleviates mechanical forces surrounding stromal cells, restores ciliogenesis, maintains nuclear integrity, suppresses the cGAS-STING pathway activation, and ultimately rescues decidualization. Our findings reveal a previously unrecognized mechanism by which primary cilia regulate the actin cytoskeleton to maintain nuclear integrity and prevent DNA leakage. This safeguards against aberrant activation of the cGAS-STING pathway, which would otherwise trigger detrimental immune signaling and impair decidualization.

Uterine receptivity is essential for successful implantation. In mice, uterine receptivity begins with the secretion of LIF from uterine glands stimulated by estrogen on the morning of day 4 pregnancy. We hypothesize that gland readiness for estrogen stimulation is indispensable for uterine receptivity. The current study reveals that uterine glands undergo a differentiation process with expanded branching during the preimplantation period. The single-cell RNA profiling of glandular cells identifies that LIF is expressed exclusively in a Prss29+ subgroup of glandular cells on day 4 of pregnancy. Interestingly, Foxa2-deficient glands lacking LIF production fail to develop branches and the functional Prss29+ subgroup. This Prss29+ subgroup develops prior to estrogen secretion. Collectively, our findings show that uterine glands undergo a FOXA2-dependent maturation process to acquire the competence, named “transitional phase”, for entering the receptive phase. The “transitional phase”, predicting uterine receptivity one day before implantation, is a landmark concept in uterine receptivity.