Biology subjects

Spencer, T. E.

Publications and source records attributed to Spencer, T. E..

9 recordsLinked to original sources

bioRxiv2026

ENDOMETRIAL GLANDULAR EPITHELIAL CELL PLASTICITY DURING POSTPARTUM REGENERATION AND ESTROUS CYCLICITY

Several different mechanisms of regeneration have been proposed to explain repair of the endometrial luminal epithelium (LE) following menstrual/estrous cyclicity, parturition or other damaging insults to the uterine mucosa. These include activity of an as-yet unidentified epithelial stem cell population, mesenchymal transdifferentiation, or integration of bone marrow-derived cells. Here, we used a novel Cag-Flox-STOP-Flox-Pgrmc2-3XFlag (Pgrmc2-3XFlag) reporter mouse coupled with a recently developed GE-specific cre recombinase mouse (Prss29-Cre) to determine if and to what extent the GE contributes to regeneration of the LE following parturition and during aging in nulliparous mice. To initiate regeneration in the endometrium, control (Pgrmc2-3XFlag) and reporter (Prss29-Cre;Pgrmc2-3XFlag) mice were placed with males for one pregnancy. Postpartum (pp) uterine tissues were then collected 5 days (1x5dpp), 3-weeks (1x3wpp) and 7-weeks (1x7wpp) later. A fourth set of uterine tissues was collected 3-weeks after three pregnancies (3x3wpp). The number of glands did not change at any time point. Indelibly marked FLAG+ GE cells were detected by immunohistochemistry and immunofluorescence in GE as expected. As demonstrated in serial uterine sections of uteri from all four treatment groups, GE cells migrated toward the LE and formed junctional points (JP) where GE cells transitioned from cuboidal GE cells to pseudostratified columnar LE cells. Transitioning cells at junctional points expressed markers of both GE (i.e., FOXA2) and LE (i.e., CALB1) tissues. Following transition, GE-derived LE cells expressed PGR and ESR1 consistent with surrounding developmentally-derived LE cells. About 17 FLAG+ LE cell islands were also observed in 1x5dpp 4 mm longitudinal sections at sites distant from GE-LE junctional points. Interestingly, the number of islands did not change from 1x5dpp to 1x7wpp. However, the total number of GE-derived FLAG+ LE cells observed in each island decreased from 1x5dpp to 1x3wpp, but then tended (p=0.07) to increase by 1x7wpp. There was no difference in the number of GE-LE junctional points or GE-derived FLAG+ LE islands between 1x3wpp and 3x3wpp tissues. However, a significant increase in the total (p=0.02) and average (p=0.02) number of FLAG+ LE cells was observed in 3x3wpp LE islands highlighting the proliferative expansion of these cells over the intervening ~80 days. GE-LE junctional points were also observed in 5-month old nulliparous Prss29-Cre;Pgrmc2-3XFlag mice at diestrus, but not in younger 6-week old mice. Collectively, these findings establish the GE as a quantifiable cellular source for LE regeneration during postpartum involution and as part of normal LE cell turnover during the estrous cycle in nulliparous mice. We propose a mechanism whereby GE cells within glandular structures are not terminally differentiated, but rather are stably differentiated to perform glandular function. However, when needed and based on the level of endometrial damage, GE cells can dedifferentiate, become integrated in to LE tissue and then reprogram to take on an LE phenotype as part of the regenerative process following parturition and during estrous cyclicity over time.

physiology↗

bioRxiv2026

A transient epithelial plasticity state defines the developmental window for uterine gland specification

Uterine gland development and function is essential for reproduction and womens health, yet the epithelial cell states and signaling interactions that govern gland fate specification are not well understood. Here, integration of single cell and spatial transcriptomics with organoid culture, lineage tracing, and genetic and hormonal perturbation models were used to define mechanisms regulating postnatal uterine epithelial differentiation. A developmentally restricted epithelial plasticity state was identified that precedes luminal and glandular cell lineage segregation and is accompanied by dynamic reorganization of stromal-epithelial communication during uterine differentiation. Pseudotime analysis revealed progressive acquisition of gland-associated programs, including forkhead box A2 (Foxa2), retinoic acid metabolic genes, and epithelial estrogen receptor alpha (Esr1) expression. Functional studies revealed that ESR1 acquisition and retinoic acid signaling suppress the multilayered organoid phenotype associated with epithelial plasticity, thereby promoting epithelial specification and lineage commitment. Moreover, neonatal hormonal perturbation of adenogenesis and conditional deletion of Foxa2 abolished this organoid phenotype. Together, these findings demonstrate that ESR1 acquisition, retinoic acid signaling and FOXA2-dependent glandular differentiation each restrict a transient epithelial plasticity state, coupling the loss of developmental plasticity to the emergence of the glandular lineage.

developmental biology↗

bioRxiv2026

Luminal epithelium remodeling underlies endometrial regeneration during menstruation and pregnancy

Menstruation and pregnancy impose an immense regenerative burden on the endometrium. These events breach the luminal epithelium lining the uterine cavity, which is proposed to be replenished by cells in adjoining epithelial glands. In contrast to this gland-centric model, we find that luminal and glandular epithelia are maintained by separate progenitor populations during homeostasis, induced menstruation, pregnancy, and postpartum repair in mice. Although our data indicate that gland cells do not contribute substantially to luminal epithelium regeneration under physiological conditions, we find that they can serve as facultative progenitors that resurface the tissue after chemical ablation. During menstruation, the luminal epithelium bypasses the need for gland contributions by undergoing extensive expansion and morphogenesis to re-epithelialize stromal surfaces concurrently with tissue breakdown. Analogous morphogenesis occurs during gestation, revealing luminal epithelial expansion as a unifying mechanism enabling simultaneous stromal disruption and re-epithelialization, which may underlie the endometrium's remarkable regenerative capacity.

developmental biology↗

bioRxiv2023

Development of Polarity-Reversed Endometrial Epithelial Organoids

The uterine epithelium is composed of a single layer of hormone responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a major advance for facilitating mechanistic studies in vitro. However, a major limitation for the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of apical membrane, thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. Here, a straightforward strategy was developed that successfully reverses the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Our investigations highlight the utility of polarity-reversed EEO to study interactions with E. coli and blastocysts. This method of generating apical-out EEO lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

cell biology↗

bioRxiv2023

Extensive rewiring of the gene regulatory interactions between in vitro-produced conceptuses and endometrium during attachment

Pregnancy loss is a significant problem when embryos produced in vitro are transferred to a synchronized uterus. Currently, mechanisms that underlie losses of in vitro-produced embryos during implantation are largely unknown. We investigated this problem using cattle as a model of conceptus attachment by analyzing transcriptome data of paired extraembryonic membrane and endometrial samples collected on gestation days 18 and 25, which spans the attachment window in cattle. We identified that the transfer of an in vitro-produced embryo caused a significant alteration in transcript abundance of hundreds of genes in extraembryonic and endometrial tissues on gestation days 18 and 25, when compared to pregnancies initiated by artificial insemination. Many of the genes with altered transcript abundance are associated with biological processes that are relevant to the establishment of pregnancy. An integrative analysis of transcriptome data from the conceptus and endometrium identified hundreds of putative ligand-receptor pairs. There was a limited variation of ligand-receptor pairs in pregnancies initiated by in vitro-produced embryos on gestation day 18, and no alteration was observed on gestation day 25. In parallel, we identified that in vitro production of embryos caused an extensive alteration in the co-expression of genes expressed in the extraembryonic membranes and the corresponding endometrium on both gestation days. Both the transcriptional dysregulation that exists in the conceptus or endometrium independently, and the rewiring of gene transcription between the conceptus and endometrium are a potential component of the mechanisms that contribute to pregnancy losses caused by in vitro production of embryos. SIGNIFICANCE STATEMENTThe successful establishment of pregnancies following the transfer of an in vitro produced embryo is essential for cattle production and assisted human reproduction. Most of the pregnancies initiated by the transfer of an in vitro produced embryo fail, in part because of dysfunctional interaction between the embryo and endometrium during pregnancy establishment. Our study identified that conceptuses produced in vitro and their corresponding endometrium have massive dysregulation in gene activity during the peri-implantation window, which affects crucial biological functions necessary for pregnancy. These gene expression alterations are a major contributor to the high rates of pregnancy loss following the transfer of an in vitro produced embryo. Our findings have implications for improving assisted reproduction in both agriculture and biomedicine.

developmental biology↗

bioRxiv2022

The estrogen receptor α cistrome in human endometrium and epithelial organoids

ContextEndometrial health is impacted by molecular processes that underlie estrogen responses. ObjectiveTo define estrogen regulation of endometrial function by integrating the estrogen receptor alpha (ESR1) cistrome and transcriptome of endometrial biopsies taken from the proliferative and midsecretory phases of the menstrual cycle and hormonally stimulated endometrial epithelial organoids. DesignESR1 ChIPseq and RNAseq were performed on proliferative or mid-secretory endometrial biopsies and on hormone treated organoid cultures. SettingEndometrial samples were obtained from volunteers at outpatient research clinics for ChIPseq and for organoid culture. Patients or Other ParticipantsParticipants were fertile, reproductive aged women with normal cycle length, and without any history of infertility or irregular cycles. In total, 5 new endometrial biopsies obtained from 5 women were used in this study and were analyzed together with previously published cycle stage endometrial RNAseq data. Intervention(s)There were no interventions in this study. Main Outcome Measure(s)The cycle stage specific ESR1 binding sites and gene expression identification of human endometrium and organoid cultures were integrated with changes in gene expression. ResultsGenes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids was more distal to the gene promoter when compared to primary endometrium. Organoid estrogen/ESR1 candidate target genes impacted formation of cellular protrusions, and chromatin modification, ConclusionsAnalysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen impacts endometrial health and function.

genomics↗

bioRxiv2022

Inserting Cre Recombinase into the Prolactin 8a2 gene for Decidua-Specific recombination in Mice

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here we describe a new decidua specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

genetics↗

bioRxiv2022

Sperm exposure to accessory gland secretions alters the transcriptomic response of the endometrium in cattle

In a recent study from our group, mating to intact, but not vasectomised, bulls modified the endometrial transcriptome, suggesting an important role of sperm in the modulation of the uterine environment in this species. However, it is not clear whether these changes are driven by intrinsic sperm factors, or by factors of accessory gland (AG) origin that bind to sperm at ejaculation. Thus, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the AGs, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to AG factors. To this end, bovine endometrial explants collected from heifers in oestrus were incubated alone (control), or with epididymal or ejaculated sperm. RNA-sequencing revealed 1912 differentially expressed genes (DEGs) between in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs genes detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. In both cases, the top pathways associated with these genes included T cell regulation and NF-KB and IL17 signalling. To confirm whether AG factors were directly responsible for the dampening of the endometrial response elicited by ejaculated sperm, endometrial explants were incubated with epididymal sperm previously exposed, or not, to seminal plasma (SP). Exposure to SP abrogated the downregulation of SQSTM1 by epididymal sperm, and partially inhibited the upregulation of MYL4 and CHRM3 and downregulation of SCRIB. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.

genomics↗

bioRxiv2019

Transcriptional and Progesterone Receptor Binding Profiles of the Human Endometrium Reveal Important Pathways and Regulators in the Epithelium During the Window of Implantation

ContextPoor uterine receptivity is one major factor leading to pregnancy loss and infertility. Understanding the molecular events governing successful implantation is hence critical in combating infertility.\n\nObjectiveTo define PGR-regulated molecular mechanisms and epithelial roles in receptivity.\n\nDesignRNA-seq and PGR-ChIP-seq were conducted in parallel to identify PGR-regulated pathways during the WOI in endometrium of fertile women.\n\nSettingEndometrial biopsies from the proliferative and mid-secretory phases were analyzed.\n\nPatients or Other ParticipantsParticipants were fertile, reproductive aged (18-37) women with normal cycle length; and without any history of dysmenorrhea, infertility, or irregular cycles. In total, 42 endometrial biopsies obtained from 42 women were analyzed in this study.\n\nInterventionsThere were no interventions during this study.\n\nMain Outcome MeasuresHere we measured the alterations in gene expression and PGR occupancy in the genome during the WOI, based on the hypothesis that PGR binds uterine chromatin cycle-dependently to regulate genes involved in uterine cell differentiation and function.\n\nResults653 genes were identified with regulated PGR binding and differential expression during the WOI. These were involved in regulating inflammatory response, xenobiotic metabolism, EMT, cell death, interleukin/STAT signaling, estrogen response, and MTORC1 response. Transcriptome of the epithelium identified 3,052 DEGs, of which 658 were uniquely regulated. Transcription factors IRF8 and MEF2C were found to be regulated in the epithelium during the WOI at the protein level, suggesting potentially important functions that are previously unrecognized.\n\nConclusionPGR binds the genomic regions of genes regulating critical processes in uterine receptivity and function.\n\nPrecisUsing a combination of RNA-seq and PGR ChIP-seq, novel signaling pathways and epithelial regulators were identified in the endometrium of fertile women during the window of implantation.

developmental biology↗