Endometrial Gland Development Research Articles

P-797 Single-cell analysis sheds light on investigation of epithelial stem/progenitor cells in human endometrial organoid

Abstract Study question How to find endometrial epithelial stem cell (eeSC) in the human endometrial gland? Summary answer Endometrial organoid culture model combined with omics techniques can be a promising platform for investigating eeSC functions and endometrial gland development. What is known already Endometrial stem/progenitor cells are thought to be involved in endometrial regeneration, with endometrial stromal stem cells having already been extensively studied. On the other hand, study of human eeSC is challenging due to the lack of specific markers to isolate and examine their functional properties. In addition, the in vivo phenotype of primary endometrial epithelial cells cannot be maintained for a long duration in two-dimensional culture. The development of three-dimensional endometrial organoid model provides suitable alternatives for studying eeSC, as they can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario of the glandular epithelial cells. Study design, size, duration This is a basic science study of endometrial organoids. Endometrial biopsies were performed on day 2 after the injection of human chorionic gonadotrophin in women undergoing in vitro fertilization treatment (IVF) but having no fresh embryo transfer in Queen Mary Hospital, The University of Hong Kong. Endometrial organoids were derived from human endometrial gland fragments. The established organoids were digested into single cells for droplet-based single-cell sequencing to investigate their cellular population and component. Participants/materials, setting, methods The filtered high-quality single-cell analysis data were clustered and assigned to specific cell types according to their expressions of classical markers. Potential stem cells cluster was identified by trajectory analysis, RNA velocity, CytoTRACE and literature search. The existence of specific eeSC markers was validated by immunofluorescence staining and flow cytometric analysis of primary endometrial tissues/endometrial organoids. The clonogenicity of the isolated eeSCs was determined by clonogenic assay. Main results and the role of chance The endometrial organoid exhibit morphology and marker expression pattern of human endometrial glands. They can also be expanded for long-term culture. The resemblance between in vitro generated organoid and the in vivo endometrial gland was further supported by single-cell RNA-seq analysis. Seurat clustering identifies 8 cell clusters in endometrial organoids. RNA velocity analysis revealed a potential eeSC cluster at the starting point of the differentiation trajectory from the immature cell cluster toward the most mature one. Besides, the eeSC cluster had relatively high expression of known stem-cell markers including Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) and Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3). Five genes including Intercellular Adhesion Molecule 1 (ICAM1), L1 Cell Adhesion Molecule (L1CAM), Annexin A2 (ANXA2), Mesothelin (MSLN), Integrin Subunit Alpha 3 (IGTA3) were subsequently identified as potential markers of the eeSC cluster. Further analysis by immunostaining demonstrated the expressions of CD54 and ANXA2 in ∼1% and ∼30% of the whole organoids cell suspensions, respectively. The expressions of CD54 and ANXA2 were also identified in the glandular tissue of human endometrium. Limitations, reasons for caution With basal out/apical in phenotype, the endometrial organoids are not exactly similar to the endometrium in vivo, hampering the exploration of their surface marker in cell interaction experiments. Like other organoid systems, it is inevitable for endometrial organoids to have heterogeneity in cells and inconsistency in expansion and biological performance. Wider implications of the findings Gene manipulation technology like CRISPR-Cas9 could be utilized to genetically alter the eeSc to study their biological properties. The construction of eeSC biobank will offer an opportunity for the development of personalized regenerative medicine with diagnosis, preventive intervention, and treatment approaches for gynecological diseases. Trial registration number Not applicableNot applicable

Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation are differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that a dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in PGR expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest specific roles between each isoform, but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

RPLP1 Is Up-Regulated in Human Adenomyosis and Endometrial Adenocarcinoma Epithelial Cells and Is Essential for Cell Survival and Migration In Vitro.

Adenomyosis is defined as the development of endometrial epithelial glands and stroma within the myometrial layer of the uterus. These "ectopic" lesions share many cellular characteristics with endometriotic epithelial cells as well as endometrial adenocarcinoma cells, including enhanced proliferation, migration, invasion and progesterone resistance. We recently reported that the 60S acidic ribosomal protein P1, RPLP1, is up-regulated in endometriotic epithelial cells and lesion tissue where it plays a role in cell survival. To evaluate if a similar pattern of expression and function for RPLP1 exists in adenomyosis and endometrial cancer, we examined RPLP1 expression in adenomyosis and endometrial cancer tissue specimens and assessed its function in vitro using well-characterized cell lines. A total of 12 control endometrial biopsies and 20 eutopic endometrial and matched adenomyosis biopsies as well as 103 endometrial adenocarcinoma biopsies were evaluated for RPLP1 localization by immunohistochemistry. Endometrial adenocarcinoma cell lines, Ishikawa, HEC1A, HEC1B and AN3 were evaluated for RPLP1 protein and transcript expression, while in vitro function was evaluated by knocking down RPLP1 expression and assessing cell survival and migration. RPLP1 protein was up-regulated in eutopic epithelia as well as in adenomyosis lesions compared to eutopic endometria from control subjects. RPLP1 was also significantly up-regulated in endometrial adenocarcinoma tissue. Knockdown of RPLP1 in endometrial adenocarcinoma cell lines was associated with reduced cell survival and migration. RPLP1 expression is up-regulated in eutopic and ectopic adenomyotic epithelia as well as in the epithelia of endometrial cancer specimens. In vitro studies support an essential role for RPLP1 in mediating cell survival and migration, processes which are all involved in pathophysiology associated with both diseases.

Sex bias in utero alters ovarian reserve but not uterine capacity in female offspring†.

Environmental stressors to which a fetus is exposed affect a range of physiological functions in postnatal offspring. We aimed to determine the in utero effect of steroid hormones on the reproductive potential of female offspring using a porcine model. Reproductive tracts of pigs from female-biased (>65% female, n = 15), non-biased (45-54.9% female, n = 15), and male-biased litters (<35% females, n = 9) were collected at slaughter (95-115kg). Ovaries and uterine horns were processed for H&E or immunohistochemistry. Variability of data within groups was analyzed with a Levene's test, while data were analyzed using mixed linear models in R. In the ovarian reserve, there was a significant birth weight by sex ratio interaction (P = 0.015), with low birth weight pigs from male-biased litters having higher numbers of primordial follicles with opposite trends seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower birth weight decreased the proportion of glands found in the endometrium (P = 0.045) and was more variable in both male-biased and female-biased litters (P = 0.026). The variability of primordial follicles from male-biased litters was greater than non- and female-biased litters (P = 0.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (P = 0.028). A crucial finding was the greater variability in primordial follicles in the ovaries from females derived from male-biased litters and stromal cell count in the endometrium of females from male- and female-biased litters. This could be inflating the variability of reproductive success seen in females from male-biased litters.

Melatonin in Endometriosis: Mechanistic Understanding and Clinical Insight.

Endometriosis is defined as the development of endometrial glands and stroma outside the uterine cavity. Pathophysiology of this disease includes abnormal hormone profiles, cell survival, migration, invasion, angiogenesis, oxidative stress, immunology, and inflammation. Melatonin is a neuroendocrine hormone that is synthesized and released primarily at night from the mammalian pineal gland. Increasing evidence has revealed that melatonin can be synthesized and secreted from multiple extra-pineal tissues where it regulates immune response, inflammation, and angiogenesis locally. Melatonin receptors are expressed in the uterus, and the therapeutic effects of melatonin on endometriosis and other reproductive disorders have been reported. In this review, key information related to the metabolism of melatonin and its biological effects is summarized. Furthermore, the latest in vitro and in vivo findings are highlighted to evaluate the pleiotropic functions of melatonin, as well as to summarize its physiological and pathological effects and treatment potential in endometriosis. Moreover, the pharmacological and therapeutic benefits derived from the administration of exogenous melatonin on reproductive system-related disease are discussed to support the potential of melatonin supplements toward the development of endometriosis. More clinical trials are needed to confirm its therapeutic effects and safety.

Epidural or intrathecal morphine for post-operative analgesia following laparoscopic endometriosis surgery

Background: Endometriosis is the development of endometrial glands or stroma outside the uterine cavity, in the pelvis, ovaries, and fallopian tubes. Laproscopy is the most common method used in management of endometriosis and it also eliminates endometriosis-related scar tissue, implants, and cysts. Epidural or intrathecal morphine is an effective way to alleviate post-operative pain after laparoscopic endometriosis surgery.Methods: Literature search was conducted in three electronic databases-Google Scholar, Cochrane (Controlled Trials Register), and PubMed (MEDLINE/Index Medicus). Postoperative pain, epidural and intrathecal morphine, laparoscopy surgery, patient-controlled analgesia (PCA), opioids, and morphine were the search criteria used.Results: After removing duplicate studies, the search yielded 106 results. Out of which50 full-text publications that matched the inclusion criterion were retrieved.Conclusion: Single intrathecal morphine injection is safe and effective in managing post-operative pain, however continuous epidural analgesia is risky and difficult to manage after laparoscopic endometriosis surgery.

Evaluation of the potential role of diethylstilbestrol on the induction of endometriosis in a rat model – An alternative approach

Endometriosis is known to be a gynaecological condition characterised by persistent inflammation and abnormal development of endometrial stroma and glands. Researchers require a rodent model to analyse the disease environment. Animal models are the best option for investigating the etiology and effective treatment of debilitating illnesses in women since rodents, like humans, menstruate. In order to develop the model system, diethylstilbestrol (DES) was examined for its ability to induce endometriosis in rats by investigating its effect on the estrus cycle, hormones, and key markers. The results demonstrated that animals given DES had an erratic estrus cycle and aberrant hormone levels. Histomorphology revealed the development of an endometriosis environment with degenerative epithelium and enlarged glandular cells after DES induction. The higher levels of estrogen, progesterone, and MCP-1 were shown in the endometriosis induced animals. Endometriosis-induced groups had decreased levels of HOXA10 and HOXA11 and increased levels of VEGF and COX-2. Finally, the DES demonstrated endometriosis induction efficacy, implying that it might be a viable replacement for endometriosis induction.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

Regulation of Inflammatory and Proliferative Pathways by Fotemustine and Dexamethasone in Endometriosis.

Endometriosis is a common disease. Its pathogenesis still remains uncertain, but it is clear that cell proliferation, apoptosis and chronic inflammation play an important role in its development. This paper aimed to investigate the anti-proliferative and anti-inflammatory effects of a combined therapy with fotemustine and dexamethasone. Endometriosis was induced by intraperitoneal injections of uterine fragments from donor animals to recipient animals. Next, the pathology was allowed to develop for 7 days. On the seventh day, fotemustine was administered once and dexamethasone was administered daily for the next 7 days. On Day 14, the animals were sacrificed, and peritoneal fluids and lesions were explanted. In order to evaluate the gastrointestinal side effects of the drugs, stomachs were harvested as well. The combined therapy of fotemustine and dexamethasone reduced the proinflammatory mediator levels in the peritoneal fluid and reduced the lesions’ area and diameter. In particular, fotemustine and dexamethasone administration reduced the heterogeneous development of endometrial stroma and glands (histological analysis of lesions) and hyperproliferation of endometriotic cells (immunohistochemical analysis of Ki67 and Western blot analysis of PCNA) through the mitogen-activated protein kinase (MAPK) signaling pathway. Combined fotemustine and dexamethasone therapy showed anti-inflammatory effects by inducing the synthesis of anti-inflammatory mediators at the transcriptional and post-transcriptional levels (Western blot analysis of NFκB, COX-2 and PGE2 expression). Fotemustine and dexamethasone administration had anti-apoptotic activity, restoring the impaired mechanism (TUNEL assay and Western blot analysis of Bax and Bcl-2). Moreover, no gastric disfunction was detected (histological analysis of stomachs). Thus, our data showed that the combined therapy of fotemustine and dexamethasone reduced endometriosis-induced inflammation, hyperproliferation and apoptosis resistance.

Generation and analysis of Prss28 and Prss29 deficient mice using CRISPR-Cas9 genome-editing.

Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands and a critical regulator of their differentiation, development and function. Progesterone and FOXA2 regulate members of a serine proteinase gene family (Prss28 and Prss29). Here, CRISPR-Cas9 genome-editing was used to create mice with a heterozygous or homozygous deletion of Prss28 or/and Prss29 to determine their biological roles in uterine function. Female mice lacking Prss28 and Prss29 or both developed normally and were fertile without alterations in uterine histoarchitecture, uterine gland number, or and gene expression. Thus, Prss28 and Prss29 are dispensable for female fertility and do not impact endometrial gland development or uterine function mice.

Gut microbiota contributes to the development of endometrial glands in gilts during the ovary-dependent period

BackgroundThe hyper-prolificacy Meishan gilts achieved a superior endometrial gland development (EGD) than white crossbred gilts during the ovary-independent period (before 60 d of age). Then, the EGD continues under the management of ovary-derived steroid hormones that regulated by gut microbiota (after 60 d of age). However, whether Meishan gilts’ superiority in EGD lasting to the ovary-dependent period (after 60 d of age) and the role of gut microbiota in this period both remain unclear.MethodsMeishan gilts and Landrace x Yorkshire (LxY) gilts were raised under the same housing and feeding conditions until sexual maturity and then we compared their EGD and gut microbiota. Meanwhile, we transplanted fecal microbiota from Meishan gilts to L×Y gilts to explore the role of gut microbiota in EGD. We sampled plasma every 3 weeks and collected the uterus, ovary, liver, and rectal feces after the sacrifice. We then determined the hormone concentrations and expressions of the EGD-related genes. We also profiled the gut microbiota using 16S rDNA sequencing and metabolites of plasma and liver tissue using untargeted metabolomics. Finally, the correlation analysis and significant test was conducted between FMT-shifted gut microbes and EGD-related indices.ResultsMeishan gilts have larger endometrial gland area (P < 0.001), longer uterine horn length (P < 0.01) but lighter uterine horn weight (P < 0.05), a distinctive gut microbiota compared with L×Y gilts. Fecal microbiota transplantation (FMT) increased endometrial gland area (P < 0.01). FMT markedly shifted the metabolite profiles of both liver and plasma, and these differential metabolites enriched in steroid hormone biosynthesis pathway. FMT increased estradiol and insulin-like growth factor 1 but decreased progesterone dynamically. FMT also increased the expression of the EGD-related genes estrogen receptor 1 gene, epithelial cadherin, and forkhead box protein A2. There is a significant correlation between FMT-shifted gut microbes and EGD-related indices.ConclusionSexually matured Meishan gilts achieved a superior EGD than LxY gilts. Meanwhile, gut microbiota contribute to the EGD potentially via regulating of steroid hormones during the ovary-dependent period.

Diagnosis and treatment of adenomyosis.

Adenomyosis is a benign gynecological disorder characterized by aberrant development of endometrial glands and stroma within the myometrium, causing inflammation and neuroangiogenesis.[1][1],[2][2] Adenomyosis often coexists with other gynecological conditions and may cloud the clinical presentation

Uterine function in Meishan pigs

The Meishan pig provides a biological model with the genetic capacity to express a high prolificacy. This prolificacy can be partially attributed to a higher ovulation rate and a higher rate of prenatal survival at a given ovulation rate throughout gestation than in European breeds. Both early embryonic survival (factors inherent to the ovum and uterus, which occur before day 25 of gestation) and uterine capacity (factors inherent to uterine limitation, which occur from 30 days of gestation to parturition) may contribute to prenatal survival. Crossbreeding studies show that the prolificacy of Meishan pigs is primarily of maternal origin, but not whether the effects occur via the ovum, uterus or systemic factors. Although there have been few comparative studies on uterine function for prolific pigs, experiments relating to three general areas have been reported. During neonatal uterine development, endometrial gland development occurs shortly after birth in Meishan and European breeds. Tissue culture for 24 h showed that secretion of at least three uterine proteins (one identified as retinol-binding protein) increased in clear temporal association with development of the endometrial gland. The secretion of two additional proteins appears to change in a breed-specific manner. Although regulatory roles for uterine proteins remain to be defined, biochemical events associated with neonatal endometrial gland development may affect subsequent adult uterine function. During the time of maternal recognition of pregnancy or early gestation, reports suggest that uterine secretions are enhanced in Meishan pigs, which may partially explain their increased prolificacy. However, other studies show smaller embryo and placental weights at day 30 of gestation, suggesting that prolificacy of Meishan females is related to a maternal ability to control embryo/fetal and placental growth. During the later fetal period (uterine capacity) in European pigs, endometrial response to a crowded uterine environment is minimal, but the conceptus responds to a crowded uterine environment by altering placental function; similar experiments using Meishan pigs are in progress. Once the regulatory determinants of uterine function are understood, improvements can possibly be made in the reproductive efficiency of all swine.

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

Background:Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects.Objectives:Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects.Methods:Female mice were exposed to GEN on postnatal days (PND)1–5 and uterine tissues collected on PND5, PND22–26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators.Results:GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice.Discussion:Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336

Uterine secretory activity and histology of prepubertal female dogs in an animal model of partial ablation of endometrial glands development

ABSTRACT: Inadequate exposure of the female reproductive system to steroids in uterine developmental periods can partially inhibit the development of endometrial glands in dogs. However, the effects of steroids on the formed glands functionality remain unknown, as well as the possible occurrence of endometrial fibrosis. This study aimed to evaluate the secretory activity of endometrial glands in prebubertal female dogs submitted to a protocol of partial ablation of the uterine adenogenesis. Sixteen females of non-specific breed were distributed into two groups; MPA (n=8), females that received applications of medroxyprogesterone acetate every 3 weeks; and C (n=8) untreated control females. Ovariohysterectomy was performed in all animals at the age of 6 months and evaluated the uterine horns by histological and histochemistry exams. The secretion intensity (degrees 1-4) was evaluated using periodic acid-Schiff (PAS) and alcian blue (AB) pH 2.5. Histological evaluation was performed using Masson’s trichrome and toluidine blue. Only degree 1 and 2 marks for PAS were observed in both groups, with no difference of uterine secretion intensity between the groups regarding the degrees found. However, the MPA group revealed higher intensity of uterine secretion compared to group C (p&lt;0.05). Staining with AB pH 2.5 also revealed only degree 1 and 2 marks in both groups, with no statistically significance between them. Masson’s trichrome staining revealed no marks in the periglandular region in both groups. A higher among of mast cells was observed in the myometrial region of the uterus in both groups. Prepubertal female dogs with partial ablation of the uterine adenogenesis present minimal uterine secretory activity, absence of periglandular fibrosis and increased presence of mast cells in the myometrium compared to endometrium.

43 Effect of early life nutrition on endometrial gland development and endometrial gene expression in heifers

In cattle, uterine gland development (adenogenesis) occurs postnatally, beginning immediately after birth, and is normally complete by approximately 2 months of age. Normal development of uterine glands is essential for subsequent fertility. Early life plane of nutrition can impact age at puberty, but the impact of early postnatal nutrition on adenogenesis is unclear. The aim of this study was to examine the effect of enhanced nutrition during the first 21 weeks of life on heifer reproductive organ growth, endometrial gland development, and endometrial gene expression. To this end, Angus×Holstein-Friesian heifer calves with a mean (±s.d.) age and body weight of 19 (±5) days and 51.2 (±7.8) kg, respectively, were assigned to either a high (HIGH; n=16; targeted average daily gain, ADG, 1.2 kg/d) or a moderate plane of nutrition (MOD; n=16; targeted ADG 0.5 kg/d). Calves were slaughtered at five months of age, and their reproductive tracts were collected and dissected for analysis. To assess endometrial gland number, a cross-section of the midpoint of one horn (n=8 per group) was fixed in 10% neutral buffered formalin, embedded, and processed for histology. Slides were stained with haematoxylin-eosin. Five pictures were randomly taken on each slide using a stereomicroscope at 40× magnification and the number of glands was counted using ImageJ (https://imagej.nih.gov/ij/) software. In addition, endometrial tissue samples were collected and snap frozen for subsequent quantitative RT-PCR analysis. At the time of slaughter, calves on the HIGH and MOD diets presented a body weight of 189.6 and 110.2kg, respectively (P&amp;lt;0.001), indicating an ADG of 1.18 kg/d for the HIGH group and 0.50 kg/d for the MOD group (P&amp;lt;0.001). Calves in the HIGH group had a greater (P&amp;lt;0.001) total reproductive tract (mean±s.d.; 114.01±35.56g v. 52.67±14.29g), isolated cervix (33.17±9.35g v. 16.14±5.27g), uterus (59.35±25.78g v. 27.46±10.61g), and ovarian (14.80±11.38g v. 5.40±1.94g) tissue weights compared with those in the MOD group. This difference was also present when organ weight was expressed on a per unit of body weight basis (P&amp;lt;0.05). However, no differences were found in the number of endometrial glands between groups (HIGH: 453.50±94.18v. MOD: 482.38±124.69). In accordance with this result, transcript abundance of six paracrine mediators of gland development (ESR1, IGF1, IGFBP1, WNT5A, WNT7A, and FGF10) was not affected by diet. In conclusion, enhanced early-life plane of nutrition increased the size of the reproductive tract in heifer calves but did not affect endometrial gland development. Future studies will compare the global endometrial transcriptome between groups to explore other putative effects of diet on uterine development. Research was supported by EU, Horizon 2020 Marie Sklodowska-Curie, REPBIOTECH 675526, Science Foundation Ireland 13/IA/1983, and São Paulo Research Foundation (FAPESP) 2017/21415-5.

The ERM family member Merlin is required for endometrial gland morphogenesis

Disruption of endometrial gland formation or function can cause female infertility. Formation of endometrial glands via tubulogenesis of luminal epithelial cells requires the establishment and maintenance of cell polarity and cell adhesion. The FERM domain-containing protein Merlin coordinates epithelial cell polarity and cell adhesion and is critical for epithelial tissue function in the skin and kidney. We now demonstrate a requirement for Merlin in endometrial gland development. Conditional deletion of Merlin in the endometrium results in female infertility caused by the absence of gland formation. Interestingly, we observed glandular epithelial markers within discrete groups of cells in the Merlin-deficient luminal epithelium. Wnt signaling, a pathway necessary for endometrial gland development is maintained in Merlin-deficient endometrium, suggesting the glandular fate program is active. Instead, we observe increased levels of apical actin and markers indicative of high membrane tension on the basal surface of the Merlin-deficient luminal epithelium. These findings suggest that the structural integrity of the luminal epithelium during gland formation is required for appropriate endometrial tubulogenesis and tissue function. Moreover, our work implicates Merlin-dependent regulation of mechanical tension in the proper formation of endometrial gland architecture and function.

EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

Endometriosis, characterized by extrauterine development of endometrial glands and stroma, is associated with increased risk of ovarian cancer development. In the present study, we investigated the role of matrix metalloproteinase-7 (MMP-7) on epithelial-mesenchymal transition (EMT) during ovarian endometriosis ( N = 40) progression. We found that the expressions of EMT markers such as vimentin, slug, and N-cadherin were significantly elevated in late stages of ovarian endometriosis compared with those found in early stages. In addition, the activity and expression of ectopic MMP-7 were significantly higher in the late stages of endometriosis. In vitro studies revealed that increased expression of MMP-7 as well as epidermal growth factor (EGF), which was significantly elevated in severe stages of ovarian endometriosis, induced EMT in endocervical epithelial cells (End1/E6E7). Silencing the MMP-7 transcripts using small interfering RNA attenuated EMT responses, whereas treatment with recombinant active MMP-7 promoted EMT by cleaving E-cadherin. In addition, EGF receptor (EGFR) inhibitor treatments regressed endometriotic lesions and decreased MMP-7 activities in a mouse model of endometriosis. Chromatin immunoprecipitation assay identified EGFR-mediated ERK1 and activator protein 1 signaling for the transcriptional activation of MMP-7 in End1/E6E7 epithelial cells.-Chatterjee, K., Jana, S., DasMahapatra, P., Swarnakar, S. EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

Development and proliferation of feline endometrial glands from fetal life to ovarian cyclicity

In this study it was determined the progression of uterine gland development from late gestation to puberty in domestic felids. Cell proliferation patterns for luminal (LE), glandular epithelium (GE) as well as stroma (S) were also described. Twenty-four uteri from female kittens: 45 and 65 days of gestation and 1 to 5, 8, 12, 16, 20 and 24 weeks postnatally were obtained. Uterine cross-sections were submitted for routine histological and immunohistochemical quantification of proliferating cell nuclear antigen (PCNA) techniques. Although prenatal uteri presented no indication of adenogenesis, 1 week old uteri revealed an incipient budding of the LE. During the second week budding increased and a mild degree of tubulogenesis of the GE into the stroma was detected. From the third to fifth weeks coiling, branching and cross-sections of glands appeared. These latter findings were more evident in week 8 when GE began to penetrate through much of the S to week 24. PCNA immunostaining revealed that DNA synthesis decreased throughout the study in the 3 cell compartments; (P < 0.01). Luminal proliferation began prenatally, it maintained up to postnatal week 8 to markedly decrease to puberty (P < 0.01). From postnatal week 3 up to week 8, GE mitotic activity was elevated becoming low thereafter (P < 0.01). Stroma actively proliferated prenatally (P < 0.01), diminishing up to week 8 (P < 0.01) and again during the last weeks (P < 0.01) of the study. It was concluded that, in domestic felids, proliferation of LE begins prenatally, histological uterine adenogenesis commenced during the first postnatal week and both events concluded by postnatal weeks 5–8.

The influence of postnatal nutrition on reproductive tract and endometrial gland development in dairy calves

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression.