Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor.

Mice were humanely treated, and surgical procedures were conducted in accordance with the guidelines described in the Guide for the Care and Use of Laboratory Animals (“The Guide”, 8th edition, 2011), published by the National Research Council of the National Academies, Washington, DC, USA ( http://www.nap.edu (accessed on 2 January 2024)). Before animal experiments were conducted, the animal protocol (AN-4203) associated with these studies was prospectively approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. In a C57BL6J background, the Plzf conditional knockout mouse ( Plzf d/d ) was generated by crossing our Pgr-cre knockin ( Pgr cre/+ ) mouse [ 36 ] with our recently generated Plzf floxed ( Plzf f/f ) mouse in which exon 2 of the murine Plzf gene is floxed [ 37 ]. Mice were housed in temperature-controlled rooms (22 ± 2 °C) operating on a 12 h light/12 h dark photocycle in a vivarium facility at Baylor College of Medicine, which is accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care). An irradiated Formulab Diet (LabDiet/Lab Supply, Fort Worth, TX, USA (#5008)) and fresh water were made available to mice ad libitum. For acute P4 treatment, six-week-old mice were ovariectomized and rested for two weeks before receiving an intrascapular subcutaneous (s.c.) injection of P4 (1 mg) dissolved in sesame oil. Ovariectomized mice injected with sesame oil served as vehicle controls. To elicit an artificial receptive state in the uterus, eight-week-old ovariectomized mice received an E2 and P4 hormone treatment regimen as previously described [ 56 ]. Briefly, mice were initially primed with a daily injection of E2 (100 ng) for two days before resting for two days. Following the two-day rest period, mice received a daily injection of P4 (1 mg) for three days. Following the three-day P4 treatment period, mice received a combination of P4 (1 mg) and E2 (50 ng); the E2 injection at this time mimics the natural E2 nidatory spike prior to embryo implantation. Mice were euthanized 15 h following the P4E2 injection to provide uterine tissue for histological and molecular analysis. An established protocol was used to induce an artificial decidual response in the uterus of an ovariectomized mouse [ 57 ]. Briefly, eight-week-old ovariectomized mice received three daily s.c. injections of E2 (100 ng). Following two days of rest, mice were administered three daily s.c. injections of E2 (6.7 ng) plus P4 (1 mg). Six hours following the third E2P4 injection, sesame oil (50 μL) was instilled into the lumen of the left uterine horn (stimulated (S)); the right horn did not receive oil (unstimulated (U)). After intraluminal instillation of the deciduogenic stimulus, mice received daily s.c. injections of E2P4 for 5 days and then weighed before euthanasia. Trimmed of mesometrial membrane and vasculature, dissected stimulated and unstimulated uterine horns from each mouse were weighed for wet-weight measurements before further analysis. To time-specific days of gestation, Plzf f/f and Plzf d/d females (8–10 weeks old) were housed overnight with fertility-proven wild-type male mice. The following morning, coitus was visually confirmed in the female by the retention of a postcoital vaginal plug; the morning of detecting the vaginal plug was assigned as the morning of gestation day 1 (GD1). Prior to euthanasia on specific GDs for the described studies below, pregnant mice were individually housed. For breeding trials, sexually mature Plzf f/f control and Plzf d/d mutant females were housed with fertility-proven C57BL/6 males. Over at least a 6-month breeding period, the date of pup delivery, the number of litters, and the number of pups per litter were recorded for each female. To elicit superovulation, 21-day-old female mice were intraperitoneally (i.p.) injected with pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, St. Louis, MO, USA (5 international units (IU)/100 μL of sterile 0.9% saline)). Forty-eight hours post-injection, mice received an i.p. injection of human chorionic gonadotropin (hCG; Sigma-Aldrich (5 IU/100 μL of sterile 0.9% saline)). Sixteen hours later, oocytes were harvested from the lumen of the fallopian tubes and counted using a dissecting microscope as previously detailed [ 84 ]. Using fallopian tube tissue that was dissected from Plzf f/f and Plzf d/d females at GD 2, two-cell stage embryos were retrieved by gently flushing the fallopian tube lumen with 100 μL of sterile phosphate-buffered saline (PBS). To visualize emerging implantation sites along both uterine horns of mice at GD5, a Chicago sky blue dye solution (1% in PBS; 100 μL per mouse) was injected into one lateral tail vein before mice were euthanized 2–5 min later [ 84 ]; stained reproductive tract tissues were removed first for the purposes of imaging and subsequent cellular and molecular analyses. Following overnight fixation in 4% paraformaldehyde, tissues were stepwise dehydrated by sequential incubation in increased concentrations of ethanol; dehydrated and fixed tissues were cleared with xylene before paraffin embedding. Paraffin-embedded tissues were sectioned to 5 μm thickness onto slides before tissue sections were stained with hematoxylin and eosin (H&E) for general histological analysis [ 84 , 85 ]. Immunohistochemical visualization of PLZF protein was achieved using an anti-PLZF primary mouse monoclonal antibody (D-9 [ 86 ]; Santa Cruz Biotechnology Inc., Dallas, TX, USA, #sc-28319, 1:150 dilution) in combination with the Mouse on Mouse (M.O.M.) Elite Immunodetection kit, Peroxidase (Vector Laboratories Inc., Burlingame, CA, PK-2200). Using the BrdU in situ detection kit (BD Biosciences, San Jose CA, USA, #551321), cells in the S-phase of the cell cycle were visualized by immunohistochemical detection of 5-bromo-2′-deoxyuridine (BrdU). After immunostaining, tissue sections were counterstained with hematoxylin prior to the application of Permount solution to aid the placement of coverslips. For cell counting in general, immunopositive cells were counted within a field of 300 cells to obtain the mean number of immunopositive cells per 300 cells counted. For the majority of studies, at least 3–4 uterine fields of 300 cells were counted per mouse. Unless otherwise specified, the mean number of immunopositive cells was calculated from at least 3 mice per genotype and treatment group. Recent versions of the Photoshop and Illustrator programs within the Adobe Creative Suite software package (Adobe Systems Inc. San Jose, CA, USA ( https://www.adobe.com/creativecloud/plans.html )) were used for raw image processing, compiling of image composites, and the annotation of the final figures for manuscript preparation. Experimental conditions for murine PLZF immunoblot analyses have been reported previously [ 33 , 37 ]. Briefly, protein concentration was obtained using the Bradford reagent (ThermoFisher Scientific Inc., Waltham, MA, USA, #23225) before protein extracts (20 μg/lane) were resolved on a 4–15% polyacrylamide-SDS gel. Resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking PVDF membranes with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with an anti-PLZF primary mouse monoclonal antibody (D-9; Santa Cruz Biotechnology Inc. # sc-28319) or a mouse monoclonal anti-β-actin (AC-74, Sigma-Aldrich; #A2228) overnight at 4 °C. Following the primary antibody incubation step, the immunoblots were washed and then incubated with an anti-mouse IgG 1 k horseradish peroxidase (HRP)-conjugated secondary antibody (m-IgG Fc BP-HRP; Santa Cruz Biotechnology Inc., sc-525409) for 1 h at room temperature. Resultant chemiluminescence signals were detected using the SuperSignal West Pico PLUS Chemiluminescent substrate kit (ThermoFisher Scientific Inc., #34580). For quantitative real-time PCR (qRT-PCR) analyses, total RNA was prepared from uterine tissue using the RNeasy Plus Mini kit (Qiagen Inc., Germantown, MD #74134). The NanoDrop 2000 UV/Visual spectrophotometer (ThermoFisher Scientific Inc.) was used to quantitate RNA before the reverse transcription step using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific Inc. #4368814). Following reverse transcription, amplified cDNA was diluted to 10 ng/μL before qRT-PCR was conducted using the Fast TaqMan 2X Mastermix (Applied Biosystems/Life Technologies, Grand Island, NY, USA, #4352042); the TaqMan assays used in these experiments are listed in Table S1 . All qRT-PCR experiments were performed on the 7500 Fast Real-Time PCR system (Applied Biosystems/Life Technologies); the delta-delta cycle threshold was used to normalize expression to the internal 18S reference. Using microtainer tubes containing a serum separator microguard (Becton, Dickinson and Company, Franklin Lakes, NJ, USA (#365967)), whole blood was collected from virgin and pregnant mice at GD5 as previously described [ 85 ]. To ensure complete blood coagulation, drawn blood was held at room temperature for 30 min before serum samples were separated by centrifugation at 2000 rpm for 10 min at 4 °C; serum was stored at −80 °C until analysis. Serum P4 and E2 levels were measured by the Ligand Assay and Analysis Core of the Center for Research in Reproduction at the University of Virginia (Charlottesville, VA, USA); assay details are available at https://med.virginia.edu/research-in-reproduction/ligand-assay-analysis-core/assay-methods/ (accessed on 15 October 2023). The two-tailed unpaired Student t-test (Welch-corrected) was used to estimate the statistical significance of differences between the two groups. Unless otherwise stated, data were graphically displayed as the mean ± standard error of the mean (s.e.m.). Differences between means with a p -value < 0.05 were considered statistically significant, with the number of asterisks indicating the level of significance: * p < 0.05; ** p < 0.01; and *** p < 0.001. Version 9 of the Prism software package from GraphPad Software Inc. (San Diego, CA, USA) was used for the statistical analyses in these studies.

Although embryo abnormalities are a major cause of preclinical pregnancy loss [ 1 , 2 , 3 , 4 , 5 ], an increasing number of studies implicate the endometrium as an additional contributing factor in embryo implantation failure and early embryo miscarriage, reviewed in [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Given that assisted reproductive technologies (ARTs) depend on the transfer of healthy embryos into a receptive endometrium, a non-receptive endometrium at the time of embryo transfer is thought to also undercut the full potential of ART-conceived pregnancies [ 13 , 14 , 15 , 16 , 17 ]. In addition, a non-receptive endometrium is implicated as one of a number of factors leading to recurrent pregnancy loss [ 18 , 19 ], defined in the United States as two or more consecutive pregnancy losses that are diagnosed by ultrasound and/or histopathology [ 20 ]. Termed the adverse ripple effect [ 21 , 22 ], incomplete progression of the normal cellular and molecular changes in the endometrium during the peri-implantation period has also been linked with initiating adverse outcomes that symptomatically manifest in the subsequent trimesters of pregnancy; these include pre-eclampsia, placental insufficiency, intrauterine fetal restriction, and preterm birth [ 21 ]. From primate to rodent, the process of embryo implantation sequentially progresses through defined interdependent developmental stages, ostensibly beginning with blastocyst apposition and attachment to the luminal epithelium of the receptive endometrium, then embryo invasion into the subepithelial stroma, followed by decidualization of a surrounding zone of stromal fibroblasts into specialized epithelioid decidual cells; reviewed in [ 21 , 23 ]. Surrounding the conceptus, decidual cells support embryonic development and invasion as well as furnish protection against an adverse cytotoxic microenvironment until placentation [ 9 ]. Progesterone (P4), through the progesterone receptor (PGR), is indispensable for the majority of these early endometrial cellular changes that are required for embryo implantation, stromal decidualization, and subsequent development of the maternofetal interface [ 24 ]. Although our understanding of endometrial P4 responsiveness at the cellular level is significantly advanced, the molecular underpinnings of these cellular responses remain incomplete. A member of the evolutionary conserved POK (POZ and Kruppel) family of C 2 H 2 -type zinc finger transcription factors [ 25 , 26 ], the promyelocytic leukemia zinc finger (PLZF; also known as ZBTB16 or ZNF 145) can act as a transcriptional activator or repressor depending on cell and signaling context [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. The PLZF transcription factor mediates a wide spectrum of early developmental programs as well as physiological effects in the adult, ranging from skeletal patterning, innate immune cell development, and hematopoiesis to spermatogenesis, reviewed in [ 26 ]. Supporting these various physiologies, PLZF controls the expansion of the stem cell pool and its differentiation, regulates cellular crosstalk, as well as governs cell proliferation, differentiation and programmed cell death, reviewed in [ 26 ]. Through an integrative approach using ChIP-seq (chromatin immunoprecipitation followed by deep sequencing) and RNA-seq datasets derived from human endometrial stromal cells (HESCs), PLZF was previously identified as a direct target of the PGR [ 33 ]. Subsequent cell-based studies demonstrated that progestin-induction of PLZF not only occurs in HESCs but was required for progestin-dependent HESC decidualization in cell culture [ 33 , 35 ]. This clinically significant in vitro result, along with immunohistological data showing PLZF expression occurs in human endometrial tissue, specifically during the P4-dominant secretory phase of the menstrual cycle [ 33 ], indicated that PLZF exerts an important P4 mediator role in the mammalian endometrium in vivo. To test this proposal in vivo, a new bigenic mouse model ( Plzf d/d ) was generated in which PLZF function is conditionally ablated in PGR-positive cells by crossing our previously reported Pgr cre knockin mouse [ 36 ] with our recently generated Plzf floxed ( Plzf f/f ) mouse in which exon 2 of the Plzf gene is floxed [ 37 ]. Comparative histomorphometric, immunohistochemical, cellular, and molecular studies demonstrate that PLZF is essential for P4-dependent endometrial stromal decidualization in vivo. Furthermore, our studies on the Plzf d/d bigenic strongly support an important role for PLZF in P4-dependent endometrial receptivity, which is essential for embryo implantation and the early establishment of the maternofetal interface. Together, our mouse studies described here, along with our previously published HESC investigations [ 33 , 35 ], strongly support the hypothesis that the evolutionally conserved endometrial PLZF transcription factor is critical in mediating both P4-dependent uterine receptivity and decidualization.

To determine the expression pattern of PLZF in the endometrium of the pregnant mouse, immunohistochemical analysis was performed ( Figure 1 A–C). On the morning of gestation day 6 (GD6), the majority of decidual cells are immunopositive for PLZF expression within the murine decidua ( Figure 1 A). Immunopositivity for PLZF is located exclusively in the nucleus of decidual cells ( Figure 1 A), which follows a similar cellular spatial staining pattern in HESCs [ 33 ]. Outside the decidua, endometrial stromal cells within the inter-implantation sites (IIS) express significantly lower levels of PLZF, whereas a subset of glandular epithelial cells display strong PLZF expression ( Figure 1 B). Similar to the virgin uterus [ 33 ], PLZF expression is not detected in the murine endometrium at GD1 ( Figure 1 D), whereas PLZF expression is clearly evident in pre-decidual stromal fibroblasts at GD5 ( Figure 1 E,F). Interestingly, lower levels of PLZF are also detected in a subset of luminal and glandular epithelial cells within this tissue ( Figure 1 E,F). Immunohistochemical data shown in Supplementary Figure S1A–E further confirms that PLZF is expressed not only in fully differentiated decidual cells at GD6 in the mouse but also in pre-decidual stromal fibroblasts and at low levels in the luminal and glandular epithelial cellular compartments at GD5. The specificity of the PLZF antibody was confirmed using a positive control (testicular tissue derived from two-week-old male mice [ 38 ]), Supplementary Figure S1F,G . To determine PLZF’s role in murine endometrial biology, a new bigenic mouse model was generated in which PLZF is specifically ablated in cells that express the PGR. By crossing our previously reported Pgr cre mouse [ 36 ] with a new mouse model in which exon 2 of the Plzf gene is floxed ( Plzf f/f ) [ 37 ], the early skeletal developmental abnormalities of the Plzf KO mouse can be avoided in the resultant conditional bigenic, termed the Plzf d/d mouse hereon. Encoding greater than 50% of the PLZF protein [ 39 ] that encompasses the initiating ATG, the N-terminal BTB/POZ domain, and the first two C 2 H 2 zinc fingers of the PLZF DNA binding domain, the exon 2 deletion effectively abrogates PLZF expression in the Plzf KO mouse [ 39 ] and in a mouse model in which Plzf f/f is crossed with the global cytomegalovirus promoter-driven cre ( CMV cre ) mouse [ 37 ]. To confirm that PLZF is not expressed in the uterus of the P4-treated Plzf d/d mouse, an established P4 treatment regimen on ovariectomized mice was used ( Figure 2 A) [ 33 , 37 ]. Western immunoblot analysis clearly shows that the PLZF protein is not induced in the Plzf d/d uterus in response to acute P4 exposure ( Figure 2 B), a result which is also confirmed by immunohistochemistry ( Figure 2 C,D). While the P4-treated Plzf f/f uterus shows PLZF expression in endometrial stromal cells with low-level PLZF expression in a subset of luminal epithelial cells ( Figure 2 C), the Plzf d/d endometrium is devoid of PLZF immunoreactivity ( Figure 2 D). Because PLZF is not expressed in the P4-treated Plzf d/d endometrium ( Figure 2 B,D), we asked whether the expression of other P4 target genes is compromised in the absence of PLZF. Using quantitative real-time PCR (qRT-PCR), we demonstrate that the expression of the majority of the P4 responsive genes tested ( Figure 2 E) is either not induced or minimally induced by P4 in the Plzf d/d endometrium ( Figure 2 E). Together, these results confirm that the Plzf d/d mouse operates as designed and that the artificial induction of a significant number of P4 responsive genes in the ovariectomized mouse model is compromised in the Plzf d/d endometrium. To determine whether endometrial PLZF has a functional role during the early stages of pregnancy establishment, a standard breeding trial for at least a 6-month period was conducted using age-matched Plzf f/f and Plzf d/d females housed with fertility-proven stud males. The results clearly show that while Plzf f/f mice produce the expected number of litters with normal numbers of pups, the Plzf d/d female does not produce pups during this time period ( Table 1 ). The infertility phenotype displayed by the Plzf d/d female mouse occurs despite exhibiting normal ovarian activity, producing pre-implantation blastocysts similar in number to the Plzf f/f mouse, and having P4 and estradiol (E2) serum levels that are equivalent to levels detected in the Plzf f/f mouse at GD 5 ( Supplementary Figure S2 ). Using a timed natural pregnancy model [ 48 ], implantation sites are clearly visible following tail vein injection of Chicago Sky Blue stain along both uterine horns of the Plzf f/f mouse at GD5 ( Figure 3 A). In contrast, implantation sites are not detected in the Plzf d/d uterus at GD5 using the same staining procedure ( Figure 3 A). Follow-up immunohistochemical approaches show that while the embryo attaches to the apical surface of the luminal epithelium of the Plzf f/f endometrium at GD 5 ( Figure 3 B), embryo attachment does not occur in the Plzf d/d endometrium but instead floats in the open lumen ( Figure 3 B). Interestingly, the expression levels of a number of P4-responsive genes (Bone morphogenetic protein 2 ( Bmp2 ) [ 49 ] and Heart and neural crest derivatives expressed 2 ( Hand2 ) [ 43 ]), which are linked with the endometrium’s ability to support embryo implantation, are significantly attenuated in the Plzf d/d endometrium as compared with the Plzf f/f endometrium at GD 5 ( Figure 3 C). Conversely, genes associated with E2-signaling (i.e., lactoferrin ( Ltf ) [ 51 ] and early growth response 1 ( Egr1 ) [ 33 , 52 , 53 , 54 , 55 ]) in the endometrium are markedly elevated in the Plzf d/d endometrium as compared with the Plzf f/f endometrium ( Figure 3 C). These perturbations in the expression of P4 and E2 responsive genes in the Plzf d/d endometrium occur in the absence of significant changes in the expression of the estrogen receptor-α ( Esr 1 ) and Pgr ( Figure 3 C). Collectively, these data support a critical role for endometrial PLZF in the early stages of embryo implantation and that the absence of PLZF derails normal E2 and P4 responsiveness of the endometrium at the molecular level. Because of the impaired responsiveness of the Plzf d/d endometrium to both P4 and E2 ( Figure 3 C) and because sequential E2 and P4 exposure of the murine endometrium is critical for the development of the transient receptive state at GD4 [ 21 ], the cellular proliferative status of the Plzf f/f and Plzf d/d endometrium at GD4 was examined ( Figure 4 ). At GD4, pre-ovulatory E2-induced epithelial proliferation is significantly suppressed by rising levels of post-ovulatory P4 hormone in the normal murine endometrium [ 21 ]. As expected at GD4, the endometrium of the Plzf f/f control mouse displays a low number of epithelial cells that score positive for BrdU incorporation ( Figure 4 A (left panels)), whereas numerous fibroblastic cells are BrdU positive in the stromal compartment. In contrast, the Plzf d/d endometrium still retains a subset of epithelial cells that score BrdU positive as well as displaying a stromal compartment that is less proliferative than the Plzf f/f stroma at this time of gestation ( Figure 4 A (right panels)). These results were also confirmed using an established E2 and P4 hormone treatment protocol on ovariectomized mice that elicits an artificial receptive state [ 56 ], albeit underpinned by an exaggerated cellular proliferative response ( Supplementary Figure S3 ). Taken together, these results reveal an unexpected role for pre-decidual PLZF in the development of the receptive endometrium that occurs prior to the development of the endometrial decidua. Because the endometrial phenotype of the Plzf d/d mouse manifests during the receptive period prior to full decidualization of the stroma, the artificial decidual response assay had to be used ( Figure 5 A). Using an established E2 and P4 hormone treatment regimen on ovariectomized mice that prepares the uterine horn to undergo a decidual response [ 57 ], the Plzf d/d uterus is shown to be incapable of launching a decidual response, unlike the uterus of a similarly treated Plzf f/f mouse ( Figure 5 A–D). At the gross morphological level, the Plzf d/d uterus fails to generate the typical deciduomata in response to the artificial deciduogenic stimulus ( Figure 5 A–D), in this case, sesame oil instillation into one uterine horn. The absence of a decidual response in the Plzf d/d endometrium is also accompanied by a significant decrease in the levels of established markers of murine decidualization, such as Bmp2 , Hand2 , Prl8a2 , and Prl3c1 ( Figure 5 E) [ 43 , 49 , 50 , 58 , 59 , 60 , 61 , 62 ]. At the cellular level, there is a conspicuous absence of large epithelioid stromal decidual cells in the oil-treated Plzf d/d uterine horn, but instead, the retention of proliferative cells in the epithelium is clear ( Figure 5 F,H). Apart from furnishing long sought-after in vivo support for our previous human cell-based findings showing a critical role for PLZF in HESC decidualization [ 33 , 35 ], these results—in combination with our findings reported in Figure 4 —provide strong support for PLZF’s importance not only in endometrial decidualization but also endometrial receptivity.

Depending on tissue type and signaling context, PLZF has been shown to be rapidly induced by the following steroid hormones: aldosterone, cortisone, glucocorticoid, and testosterone [ 32 , 63 , 64 , 65 ]. These findings are significant because, like P4, these steroids exert their physiological effects through their closely related nuclear receptors of the subfamily 3 (group C) of the nuclear receptor superfamily [ 66 ]. A recent retrospective analysis of past microarray datasets revealed that support already existed as early as 2002 for the rapid induction of Plzf transcripts by P4 in the uterus of the ovariectomized mouse (Gene Expression Omnibus (GEO) profiles: Zbtb16 -Progesterone effects on uterus time course ( ncbi.nlm.nih.gov/geoprofiles/913691 ) accessed on 12 July 2023). Moreover, PLZF induction during the P4-dominant early- to mid-secretory phase of the human menstrual cycle was also previously documented in a separate microarray study (GEO profiles: ZBTB16 -Endometrium throughout the menstrual cycle ( ncbi.nlm.nih.gov/geoprofiles/?term=24460831 ) accessed on 12 July 2023). Together, these early microarray datasets provided the first indication that PLZF is an early molecular target of P4 signaling in the murine uterus and that the levels of endometrial PLZF expression are significantly elevated during the P4-dependent secretory phase of the menstrual cycle, raising the question of whether P4 induction of endometrial PLZF is essential for early pregnancy establishment. More recently, our group provided in vitro functional support for this proposal using a primary HESC culture model [ 33 ], which demonstrated that rapid progestin-induction of PLZF from basal levels in pre-decidual HESCs is essential for progestin-dependent HESC decidualization. Our subsequent in vitro studies also revealed that basal levels of PLZF in pre-decidual HESCs are essential for their proliferation, migration, and invasion [ 35 ], cellular properties known to be displayed by HESCs as they decidualize [ 67 ]. Although studies on cultured primary HESCs provided essential cellular and molecular insights into the role of PLZF in HESC decidualization, whether these in vitro functional findings translated to the in vivo situation remained an open question. Because the Plzf KO mouse exhibits a severe skeletal patterning defect [ 39 , 68 ], which is also observed in humans [ 69 ], we took advantage of our recently generated conditional Plzf d/d mouse that circumvents these confounding phenotypes [ 37 ]. While the initial objective in using this mouse model was to confirm our in vitro findings that PLZF is crucial for endometrial stromal cell decidualization, our first in vivo results here show that PLZF expression in pre-decidual stromal fibroblasts is critical for the development of endometrial receptivity for embryo implantation, a developmental step that precedes decidualization. Our initial molecular analysis reveals that the induction of the majority of P4-responsive molecular targets tested in this study is significantly attenuated when PLZF is absent during the peri-implantation period. Therefore, this molecular result further supports PLZF as an apex P4 mediator that is positioned high in the hierarchy of immediate P4-responsive target genes. At the cellular level, the attenuated P4 responsiveness of the Plzf d/d endometrium manifests as an incomplete suppression of E2-induced epithelial proliferation; such an unchecked heightened E2 response has been linked to implantation failure in a number of mouse models [ 70 , 71 , 72 ] and implicated in human implantation failure [ 73 , 74 , 75 , 76 ]. Moreover, the significant decrease in the number of proliferating pre-decidual stromal cells in the Plzf d/d endometrium at this time parallels similar findings from our previous HESC studies [ 33 , 35 ], where PLZF is required for P4-dependent expansion of the pre-decidual stromal cell population prior to its decidualization. The above aberrant cellular responses are underscored at the molecular level by a marked increase in the levels of E2-dependent molecular targets in Plzf d/d endometrium. In particular, the detection of elevated Egr1 levels in the Plzf d/d uterus is interesting as P4-induction of PLZF has been shown to directly suppress Egr1 expression during HESC decidualization [ 33 ]. Our past studies also demonstrate that while EGR1 expression in pre-decidual stromal fibroblasts is essential for these cells to later decidualize [ 35 ], the suppression of EGR1 expression by P4-induction of PLZF is a prerequisite if these pre-decidual stromal fibroblasts are to develop into decidual cells. As reported for HESCs [ 33 ], the levels of ESR1 and PGR are not altered in the Plzf d/d endometrium to account for these molecular changes. Because of our past human studies [ 33 , 35 ], our initial objective in using the Plzf d/d mouse was to determine the in vivo importance of PLZF in murine endometrial stromal cell decidualization, which normally occurs at GD6 onwards [ 77 ]. To bypass the earlier Plzf d/d endometrial receptivity phenotype, the well-described murine artificial decidual response assay was used to determine PLZF’s endometrial intrinsic role in the decidualization of this tissue [ 48 ]. Supporting our previous human cell culture studies [ 33 , 35 ], the artificial decidual response assay confirmed a critical in vivo role for endometrial PLZF in the terminal differentiation of endometrial stromal fibroblasts into decidual cells. Taken together, these Plzf d/d mouse findings here underscore at least two important roles for PLZF during the peri-implantation period: (i) development of uterine receptivity to allow embryo attachment to the apical surface of the luminal epithelium and (ii) endometrial stromal fibroblast decidualization, occurring a day later in the pregnant mouse [ 21 ]. Apart from PLZF expression in pre-decidual and terminally differentiated decidual stromal cells of the murine uterus during early pregnancy, our immunohistochemical data reveal that PLZF is expressed in a subset of cells both in the luminal and glandular epithelial compartments. These observational findings indicate that PLZF may exert a previously unsuspected uterine cell-type specific role in the establishment of the maternofetal interface. With the availability of the Plzf f/f model [ 37 ], addressing the cell-type specific involvement of PLZF in uterine receptivity and decidualization is now feasible due to the recent generation of uterine cell-type specific cre mouse models. Indeed, these cre-driver models are designed not only to study the selective gene functions in the glandular and luminal epithelial compartments but also to further resolve gene functionality between two stromal cell states: the pre-decidual and decidual cell states. While we have provided initial molecular data to support PLZF as an early mediator of P4/PGR action in the murine uterus during the peri-implantation period, genome-scale omics analysis will be essential to furnish a more comprehensive understanding of the molecular mechanisms by which PLZF exerts its physiological roles in murine uterine function. With the N-terminal BTB/POZ domain of PLZF known to be responsible for homo- and hetero-dimerization, chromatin remodeling, epigenetic transcriptional control, and formation of high-molecular-weight DNA-protein complexes [ 26 ], while PLZF’s nine C 2 H 2 zinc finger motifs in its C-terminus are responsible for direct sequence-specific DNA binding to target genes [ 26 ], endometrial PLZF is predicted to orchestrate crucial evolutionary conserved signaling programs in response to P4. Given that we [ 33 , 35 ] and others [ 78 ] have shown that progestins (i.e., medroxy progesterone acetate (MPA)) induce PLZF in HESCs [ 33 , 35 , 78 ], coupled with the fact that MPA is frequently used in female reproductive medicine for the treatment of endometriosis [ 79 ], gynecological malignancies [ 80 ], postmenopausal hormone therapy [ 81 ], uterine fibroids [ 82 ], and contraceptives [ 83 ], future investigations into the molecular mechanisms that underlie this endometrial C 2 -H 2 zinc finger transcription factor are warranted. In summary, conclusions from these studies are that PLZF is indispensable for P4-dependent endometrial receptivity and decidualization and that PLZF may mediate these endometrial responses in a cell-type-specific manner.