Procoagulant Activity of Umbilical Cord-Derived Mesenchymal Stromal Cells' Extracellular Vesicles (MSC-EVs).

Many cell types, including cancer cells, release tissue factor (TF)-exposing extracellular vesicles (EVs). It is unknown whether MSC-EVs pose a thromboembolism risk due to TF expression. Knowing that MSCs express TF and are procoagulant, we hypothesize that MSC-EVs also might. Here, we examined the expression of TF and the procoagulant activity of MSC-EVs and the impact of EV isolation methods and cell culture expansion on EV yield, characterization, and potential risk using a design of experiments methodology. MSC-EVs were found to express TF and have procoagulant activity. Thus, when MSC-derived EVs are employed as a therapeutic agent, one might consider TF, procoagulant activity, and thromboembolism risk and take steps to prevent them.

LH/hCG Regulation of Circular RNA in Mural Granulosa Cells during the Periovulatory Period in Mice.

Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation, and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses, and among all RNA isoforms, circular RNA (circRNA) is one of the most abundant forms present in cells, yet they remain the least studied. Functionally, circRNA can act as miRNA sponges, protein sponges/decoys, and regulators of transcription and translation. In the context of ovarian follicular development, the identity and roles of circRNA are relatively unknown. In the present study, high throughput RNA sequencing of granulosa cells immediately prior to and 4-h after the LH/hCG surge identified 42,381 circRNA originating from 7712 genes. A total of 54 circRNA were identified as differentially expressed between 0-h and 4-h time points (Fold Change ± 1.5, FDR ≤ 0.1), among them 42 circRNA were upregulated and 12 circRNA were downregulated. All differentially expressed circRNA between the 0-h and 4-h groups were subjected to circinteractome analysis and identified networks of circRNA-protein and circRNA-miRNA were further subjected to "micro-RNA target filter analysis" in Ingenuity Pathway Analyses, which resulted in the identification of miRNA targeted mRNAs. A comparison of these circRNA target mRNAs with LH-induced mRNAs identified Runx2, Egfr, Areg, Sult1el, Cyp19a1, Cyp11a1, and Hsd17b1 as targets of circKif2, circVcan, circMast4, and circMIIt10. These newly identified LH/hCG-induced circRNA, their target miRNA and protein networks provide new insights into the complex interactions associated with periovulatory follicular development.

Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum (ER) specializations (cortical ER clusters), which are thought to be essential for egg activation. In examination of cortical ER clusters, we observed that they were tethered to previously unreported fenestrae within the cortical actin layer. Furthermore, studies demonstrated that sperm preferentially bind to the plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global change in volume and shape that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males. In addition to these global changes, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, and then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells.

EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, -20 °C, and -80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing -80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at -80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

Granulosa Cell Specific Loss of Adar in Mice Delays Ovulation, Oocyte Maturation and Leads to Infertility.

Adenosine deaminases acting on RNA-(ADAR) comprise one family of RNA editing enzymes that specifically catalyze adenosine to inosine (A-to-I) editing. A granulosa cell (GC) specific Adar depleted mouse model [Adar flox/flox:Cyp19a1-Cre/+ (gcAdarKO)] was used to evaluate the role of ADAR1 during the periovulatory period. Loss of Adar in GCs led to failure to ovulate at 16 h post-hCG, delayed oocyte germinal vesicle breakdown and severe infertility. RNAseq analysis of GC collected from gcAdarKO and littermate control mice at 0 and 4 h post-hCG following a super-ovulatory dose of eCG (48 h), revealed minimal differences after eCG treatment alone (0 h), consistent with normal folliculogenesis observed histologically and uterine estrogenic responses. In contrast, 300 differential expressed genes (DEGs; >1.5-fold change and FDRP < 0.1) were altered at 4 h post-hCG. Ingenuity pathway analysis identified many downstream targets of estrogen and progesterone pathways, while multiple genes involved in inflammatory responses were upregulated in the gcAdarKO GCs. Temporal expression analysis of GCs at 0, 4, 8, and 12 h post-hCG of Ifi44, Ifit1, Ifit3b, and Oas1g and Ovgp1 confirmed upregulation of these inflammatory and interferon genes and downregulation of Ovgp1 a glycoprotein involved in oocyte zona pellucida stability. Thus, loss of ADAR1 in GCs leads to increased expression of inflammatory and interferon response genes which are temporally linked to ovulation failure, alterations in oocyte developmental progression and infertility.

Extraterrestrial Gynecology: Could Spaceflight Increase the Risk of Developing Cancer in Female Astronauts? An Updated Review.

Outer space is an extremely hostile environment for human life, with ionizing radiation from galactic cosmic rays and microgravity posing the most significant hazards to the health of astronauts. Spaceflight has also been shown to have an impact on established cancer hallmarks, possibly increasing carcinogenic risk. Terrestrially, women have a higher incidence of radiation-induced cancers, largely driven by lung, thyroid, breast, and ovarian cancers, and therefore, historically, they have been permitted to spend significantly less time in space than men. In the present review, we focus on the effects of microgravity and radiation on the female reproductive system, particularly gynecological cancer. The aim is to provide a summary of the research that has been carried out related to the risk of gynecological cancer, highlighting what further studies are needed to pave the way for safer exploration class missions, as well as postflight screening and management of women astronauts following long-duration spaceflight.

Forkhead box F1 induces columnar phenotype and epithelial-to-mesenchymal transition in esophageal squamous cells to initiate Barrett's like metaplasia.

Multiple genome-wide association studies (GWAS) have linked Forkhead Box F1 (FOXF1) to Barrett's esophagus (BE). Understanding whether FOXF1 is involved in initiation of Barrett's metaplasia could allow FOXF1 to be used for risk stratification and for therapy. Two-dimensional cell cultures and three-dimensional organoid cultures and well-annotated human biopsies were used to determine the role of FOXF1 in BE pathogenesis. Multiple established esophageal squamous and BE cell lines were tested in gain- and loss-of-function studies. Initiation of a BE-like metaplastic change was evaluated by measuring characteristic cytokeratins and global gene expression profiling and by culturing organoids. Epithelial-mesenchymal transition (EMT) was evaluated by immunostaining for E-cadherin, vimentin and Snail, and by cell motility assay. Columnar esophageal epithelium of BE patients exhibited higher expression of FOXF1 compared to normal squamous esophageal epithelium of GERD patients (P < 0.001). Acidic bile salts induced nuclear FOXF1 in esophageal squamous cells. FOXF1 overexpression in normal esophageal squamous cells: (a) increased columnar cytokeratins and decreased squamous cytokeratins, (b) converted squamous organoids to glandular organoids, and (c) switched global gene profiles to resemble that of human BE epithelium (P = 2.1685e - 06 for upregulated genes and P = 8.3378e - 09 for downregulated genes). FOXF1 inhibition in BE cell lines led to loss of BE differentiation markers, CK7, and mucin 2. Also, FOXF1 induced EMT and promoted cell motility in normal esophageal squamous epithelial cells. FOXF1-induced genes mapped to pathways such as Cancer, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair. In conclusion, FOXF1 promotes a BE-like columnar phenotype and cell motility in esophageal squamous epithelial cells, which may have a critical role in BE development. FOXF1 should be studied further as a biomarker for BE and as a target for BE treatment.

Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes.

Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of CCR4-NOT Transcription Complex Subunit 6 Like (Cnot6l), a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during metaphase I (MI) entry. Knockout of B-cell translocation gene-4 (Btg4), an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes, eggs, and embryos from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in samples from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.

Inosine RNA modifications are enriched at the codon wobble position in mouse oocytes and eggs†.

Mammalian oocytes and eggs are transcriptionally quiescent and depend on post-transcriptional mechanisms for proper maturation. Post-transcriptional mRNA modifications comprise an important regulatory mechanism that can alter protein and miRNA recognition sites, splicing, stability, secondary structure, and protein coding. We discovered that fully grown mouse germinal vesicle oocytes and metaphase II eggs display abundant inosine mRNA modifications compared to growing oocytes from postnatal day 12 oocytes. These inosines were enriched in mRNA protein coding regions (CDS) and specifically located at the third codon base, or wobble position. Inosines, observed at lower frequencies in CDS of somatic tissues, were similarly enriched at the codon wobble position. In oocytes and eggs, inosine modifications lead primarily to synonymous changes in mRNA transcripts. Inosines may ultimately affect maternal mRNA stability by changing codon usage, thereby altering translational efficiency and translationally coupled mRNA degradation. These important observations advance our understanding of post-transcriptional mechanisms contributing to mammalian oocyte maturation.

Autoimmune Regulator is required in female mice for optimal embryonic development and implantation†.

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis.

Micro-RNAs involved in cellular proliferation have altered expression profiles in granulosa of young women with diminished ovarian reserve.

PURPOSE: The study aims to determine differences in micro-RNA (miRNA) expression in granulosa (GC) and cumulus cells (CC) between young women with diminished ovarian reserve (DOR) or normal ovarian reserve (NOR). Secondary objective was to identify downstream signaling pathways that could ultimately indicate causes of lower developmental competence of oocytes from young women with DOR. METHODS: The method of the study is prospective cohort study. RESULTS: Of the miRNA, 125 are differentially expressed in GC between DOR and NOR. Only nine miRNA were different in CC; therefore, we focused analysis on GC. In DOR GC, miR-100-5p, miR-16-5p, miR-30a-3p, and miR-193a-3p were significantly downregulated, while miR-155-5p, miR-192-5p, miR-128-3p, miR-486-5p, miR130a-3p, miR-92a-3p, miR-17-3p, miR-221-3p, and miR-175p were increased. This pattern predicted higher cell proliferation in the DOR GC. The primary pathways include MAPK, Wnt, and TGFbeta. CONCLUSIONS: The miRNA pattern identified critical functions in cell proliferation and survival associated with DOR. GC in women with DOR seems to respond differently to the LH surge.

Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation.

Follicular fluid within ovarian antral follicles contains numerous factors, which influence the development of a healthy oocyte including nucleic acids, steroids, proteins, and extracellular vesicles (EVs). Current evidence indicates that follicular EVs promote changes in cellular gene expression and support cumulus-oocyte complex expansion in vitro. In this study, we found EVs from different sized follicles differentially stimulate granulosa cell proliferation and this could be explained by both the differential contents associated, on or within the vesicles and by the preferential uptake of EVs dependent on follicle size from which they were isolated. Antibody array and inhibitor studies indicated that the Src, PI3K/Akt, and MAPK signaling pathways mediate the stimulatory effects of EVs on granulosa cell proliferation. This study demonstrates for the first time that EVs isolated from follicular fluid are capable of stimulating granulosa cell proliferation and that this stimulatory response is associated with the size of antral follicle from which the EVs originated. The study further also provides the first evidence that vesicles released by small antral follicles are preferentially taken up when compared to those isolated from large follicles, suggesting that vesicular surface proteins change during follicular maturation.

Circulating microRNA as candidates for early embryonic viability in cattle.

Blood-borne extracellular vesicles (i.e., exosomes and microvesicles) carrying microRNAs (miRNAs) could make excellent biomarkers of disease and different physiologic states, including pregnancy status. We tested the hypothesis that circulating extracellular vesicle-derived miRNAs might differentiate the pregnancy status of cows that had maintained pregnancy to Day 30 from non-pregnant cows or from those that exhibited embryonic mortality between Days 17 and 30 of gestation. Cows were randomly assigned for artificial insemination with fertile semen (n = 36) or dead semen (n = 8; control group) on Day 0 (day of estrus). Blood was collected from all animals on Day 0 and on Days 17 and 24 after artificial insemination. Cows receiving live sperm were retrospectively classified as pregnant on Day 30 (n = 17) or exhibiting embryonic mortality between Days 17 and 30 (n = 19). Extracellular vesicles from Day 17 and 24 samples were isolated from serum using ultra-centrifugation, and their presence was confirmed by nanoparticle tracking and Western blot analyses (for CD81) prior to RNA extraction. MicroRNA sequencing was performed on pregnant, embryonic-mortality, and control cows (n = 4 per day), for a total of 24 independent reactions. In total, 214 miRNAs were identified in serum, 40 of which were novel. Based on differential abundance parameters, we identified 32 differentially abundant loci, representing 27 differentially abundant mature miRNA. At Days 17 and 24, specific miRNAs (e.g., miR-25, -16b, and -3596) were identified that differentiated the pregnancy status. In summary, we identified several circulating extracellular vesicles derived miRNAs that differ in abundance between embryonic mortality and pregnant cows.

Cytidine deaminase Apobec3a induction in fallopian epithelium after exposure to follicular fluid.

OBJECTIVE: Ovarian carcinomas that originate from fallopian epithelial cells are suggested to arise due to repeated exposure to ovulatory follicular fluid (FF). Mechanistic explanation(s) for how this occurs are unknown. Here, we sought to understand if FF exposure to fallopian epithelial cells could induce DNA damage and expression of a known family of DNA mutators, apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) cytidine deaminases. METHODS: Follicular fluid and matched patient plasma samples were obtained from donors. Fallopian epithelial cells (FT33-TAg, FT189, FT190, and FT194) were cultured with FF or plasma for 24h, and cell proliferation and DNA damage were assessed. Effects of FF on Apobec gene expression were determined by qRT-PCR and western blot analyses. Fallopian epithelial cells were transfected with an APOBEC3A expression vector and DNA damage was assessed. RESULTS: Follicular fluid exposure increased epithelial cell proliferation as measured by three independent methods, and DNA damage accumulation as assessed using three independent measures. This effect was specific to FF, as matched patient plasma did not have the same effects. Increased expression of Apobec3a was observed in fallopian epithelial cells following exposure to 5 of 8 patient FF samples, and transient overexpression of APOBEC3A was sufficient to induce double strand DNA breaks. CONCLUSIONS: Follicular fluid can induce cell proliferation and DNA damage accumulation in cultured fallopian epithelial cells. Increased expression of APOBEC3A, a known DNA mutator, may explain the high incidence of DNA damage after FF exposure. The role of Apobec3a in ovulation-induced inflammation warrants further investigation.

Beclin-1 deficiency in the murine ovary results in the reduction of progesterone production to promote preterm labor.

Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.

Composite Nanoformulation Therapeutics for Long-Term Ocular Delivery of Macromolecules.

The purpose of this investigation is to design and synthesize novel pentablock (PB) copolymer (PB-1: PCL-PLA-PEG-PLA-PCL) based nanoformulations suspended in a thermosensitive gelling copolymer (PB-2: mPEG-PCL-PLA-PCL-PEGm) termed as composite nanoformulation. The composite nanoformulation was prepared to provide a sustained delivery of macromolecules over a longer duration with negligible burst release effect. The delivery system was designed to be utilized for the treatment of posterior segment ocular diseases such as age-related (wet) macular degeneration, diabetic retinopathy, and diabetic macular edema. The novel PB copolymers were characterized for their functional groups by Fourier transform infrared spectroscopy, molecular weight and purity by (1)H NMR spectroscopy, and gel permeation chromatography. X-ray diffraction analysis was used to determine the crystallinity of copolymers. The size distribution of PB-1 nanoparticles (NPs) prepared using emulsification-solvent evaporation method was found to be ∼150 nm analyzed by nanoparticle tracking analysis. The % encapsulation efficiency and % drug loading were found to be 66.64% w/w ± 1.75 and 18.17% w/w ± 0.39, respectively, (n = 3). Different weight percentages (15 and 20 wt %) of the PB-2 copolymer have been utilized for in vitro release studies of IgG-Fab from composite nanoformulation. A negligible burst release with continuous near zero-order release has been observed from the composite nanoformulation analyzed up to 80 days. In vitro cell viability and biocompatibility studies performed on ocular (human corneal epithelial and retinal pigment epithelium) and mouse macrophage (RAW 264.7) cell lines showed that the synthesized PB copolymer based composite nanoformulations were safe for clinical applications. On the basis of the results observed, it is concluded that PB copolymer based composite nanoformulations can serve as a platform for ocular delivery of therapeutic proteins. In addition, the composite nanoformulation may provide minimal side effects associated with frequent intravitreal injections.

Non-coding RNA in Ovarian Development and Disease.

The ovary's primary function is to produce the mature female gamete, the oocyte that, following fertilization, can develop into an embryo, implant within the uterus and ultimately allow the mother's genetic material to be passed along to subsequent generations. In addition to supporting the generation of the oocyte, the ovary and specific ephemeral tissues within it, follicles and corpora lutea, produce steroids that regulate all aspects of the reproductive system, including the hypothalamic/pituitary axis, the reproductive tract (uterus, oviduct, cervix), secondary sex characteristics all of which are also essential for pregnancy and subsequent nurturing of the offspring. To accomplish these critical roles, ovarian development and function are tightly regulated by a number of exogenous (hypothalamic/pituitary) and endogenous (intraovarian) hormones. Within ovarian cells, intricate signalling cascades and transcriptional and post-transcriptional gene regulatory networks respond to these hormonal influences to provide the exquisite control over all of the temporal and spatial events that must be synchronized to allow this organ to successfully complete its function. This book chapter will focus specifically on the role of non-coding RNAs, their identification and described functional roles within the ovary with respect to normal function and their possible involvement in diseases, which involve the ovary.

Differentially expressed genes in preimplantation human embryos: potential candidate genes for blastocyst formation and implantation.

PURPOSE: The aim of this study was to determine which genes and gene pathways are differentially expressed when comparing human blastocysts with cleavage-stage embryos. METHODS: We individually assessed gene expression in preimplantation human embryos at cleavage (n = 3) and blastocyst (n = 3) stages. Gene expression patterns were then validated in publically available datasets and then independently validated in vitro with additional human embryos using TaqMan gene expression assays. Immunolocalization studies were conducted to identify protein expression in intact blastocyst-stage embryos. RESULTS: Compared to cleavage-stage embryos, blastocyst-stage embryos differentially expressed 51 genes (p < 0.001), with overrepresentation in amoebiasis pathways and pathways in cancer. Of these 51 genes, 21 were found to be independently validated in a separate, publically available dataset, with a substantial agreement with our initial findings (κ = 0.8). In an independent set of cleavage- and blastocyst-stage embryos, we validated that six of eight tested genes were differentially expressed (p < 0.05) by RT-qPCR. Immunofluorescence studies documented the presence of two studied proteins in the trophectoderm of blastocyst-stage embryos. CONCLUSIONS: Differentially expressed genes may be implicated in the invasion and proliferation of the early embryo. Our research highlights specific genes that may be further studied for their role in the implantation process and additionally raises questions about localized gene and/or protein expression in the trophectoderm, which could affect protocols for, and interpretation of, trophectoderm biopsies performed in in vitro fertilization cycles.

Murine MicroRNA-214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection.

The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early-stage host immune responses are believed to contribute to these long-term sequelae. Recently, we reported the contribution of selected infection-associated microRNAs (miRs) in the generation of host immunity at early-stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection-associated microRNA, i.e. miR-214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down-regulated miR-214 while up-regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM-1 by miR-214 in ex vivo genital cell cultures in the presence of miR-214 mimic and inhibitor. Because, ICAM-1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR-214 in interleukin-17A-deficient (IL-17A(-/-) ) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early-stage events resulted in significantly decreased genital pathology in IL-17A(-/-) mice compared with C57BL/6 mice. This report provides evidence for early-stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance.

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in ß-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3ß overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3ß isoform caused mammary epithelial cell death and a significant reduction in ß-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3ß.