Tumor susceptibility gene 101 is required for the maintenance of uterine epithelial cells during embryo implantation.

BACKGROUND: The tumor susceptibility gene 101 (Tsg101), a component of the endosomal sorting complex required for transport (ESCRT) complex I, is involved in multiple biological processes involving endomembranous structures and the plasma membrane. The role of Tsg101 in the uterine epithelium was investigated in Tsg101 floxed mice crossed with Lactoferrin-iCre mice (Tsg101d/d). METHODS: Tsg101d/d mice were bred with stud male mice and the status of pregnancy was examined on days 4 and 6. Histological analyses were performed to examine the uterine architecture. Immunofluorescence staining of several markers was examined by confocal microscopy. Uterine epithelial cells (UECs) were isolated from Tsg101f/f and Tsg101d/d mice, and the expression of necroptosis effectors was examined by RT-PCR, western blotting, and immunofluorescence staining. UECs were also subjected to RNA expression profiling. RESULTS: Tsg101d/d female mice were subfertile with implantation failure, showing unattached blastocysts on day 6 of pregnancy. Histological and marker analyses revealed that some Tsg101d/d day 4 pregnant uteri showed a disintegrated uterine epithelial structure. Tsg101d/d UECs began to degenerate within 18 h of culture. In UECs, expression of necroptosis effectors, such as RIPK1, RIPK3, and MLKL were first confirmed. UECs responded to a stimulus to activate necroptosis and showed increased cell death. CONCLUSIONS: Tsg101 deficiency in the uterine epithelium causes implantation failure, which may be caused by epithelial defects. This study provides evidence that UECs harbor a necroptotic machinery that responds to death-inducing signals. Thus, Tsg101 expression in the uterine epithelium is required for normal pregnancy in mice.

Lipidomic changes in mouse oocytes vitrified in PEG 8000-supplemented vitrification solutions.

Cryopreserved oocytes are inevitably exposed to cold stress, which negatively affects the cellular aspects of the oocytes. Lipidomic analysis of the oocytes reveals quantitative changes in lipid classes under conditions of cold stress, leading to potential freezing-vulnerability. We had previously shown that specific phospholipids are significantly downregulated in vitrified-warmed mouse oocytes compared to those in fresh oocytes. In this study, we examined whether supplementation of polyethylene glycol 8000 (PEG 8000) during vitrification influences the lipidome of the oocytes. We used liquid chromatography with tandem mass spectrometry (LC-MS/MS) to study the alteration in the lipidome in three groups of mouse oocytes: fresh, vitrified-warmed, and vitrified with PEG 8000-warmed during vitrification. In these groups, we targeted to analyze 21 lipid classes. We profiled 132 lipid species in the oocytes and statistical analyses revealed lipid classes that were up- or downregulated in these groups. Overall, our data revealed that several classes of lipids were affected during vitrification, and that oocytes vitrified with PEG 8000 to some extent alleviated the levels of changes in phospholipid and sphingolipid contents during vitrification. These results suggest that phospholipids and sphingolipids are influenced by PEG 8000 during vitrification and that PEG 8000 can be considered as a potential candidate for preserving membrane integrity during oocyte cryopreservation.

Molecular analysis of lipid uptake- and necroptosis-associated factor expression in vitrified-warmed mouse oocytes.

BACKGROUND: We had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features. METHODS: Metaphase II (MII) stage oocytes were harvested from young (10-14-week-old) and aged (45-54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification. RESULTS: We confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification. CONCLUSIONS: We report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Expression of Egr3 in mouse gonads and its localization and function in oocytes.

OBJECTIVE: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. METHODS: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (ΔEgr3) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. RESULTS: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-ΔEgr3-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. CONCLUSION: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

Rapamycin Influences the Efficiency of In vitro Fertilization and Development in the Mouse: A Role for Autophagic Activation.

The mammalian target of rapamycin (mTOR) regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII) oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF). The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes.

Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

Autophagic activation in vitrified-warmed mouse oocytes.

Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified-warmed oocytes has not been examined. In this work, we investigated whether the vitrification-warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2 for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that several Atg genes such as Atg5, Atg7, Atg12, LC3a (Map1lc3a), LC3b (Map1lc3b), and Beclin1 were expressed in MII mouse oocytes. Slight reduction in mRNA levels of Atg7 and Atg12 in vitrified-warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified-warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified-warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified-warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified-warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.

The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells.

2-Chloro-10-[3(-dimethylamino)propyl]phenothiazine mono hydrochloride (chlorpromazine; CPZ) is an antipsychotic agent that was originally developed to control psychotic disorders. The cytotoxic properties of the CPZ are well known, but its mechanism of action is poorly understood. In this study, we investigated the role of apoptosis and autophagy in CPZ-induced cytotoxicity in U-87MG glioma cells. CPZ treatment inhibited cell proliferation and long-term clonogenic survival. Additionally, CPZ triggered autophagy, as indicated by electron microscopy and accumulation of the membrane form of microtubule-associated protein 1 light chain 3 (LC3-II); however, CPZ did not induce apoptosis. Inhibition of autophagy by expression of Beclin 1 small interfering RNA (siRNA) in U-87MG cells attenuated CPZ-induced LC3-II formation. Furthermore, U-87MG cells expressing Beclin 1 siRNA attenuated CPZ-induced cell death. CPZ inhibited phosphatidylinositol 3-kinase (PI3K)/AKT/ mTOR pathway in U-87MG cells. Treatment with LY294002, a PI3K inhibitor, alone increased the accumulation of LC3-II and potentiated the effect of CPZ. In contrast, exogenous expression of AKT partially inhibited CPZ-induced LC3-II formation. When U-87MG cells were implanted into the brain of athymic nude mouse, CPZ triggered autophagy and inhibited xenograft tumor growth. These results provided the first evidence that CPZ-induced cytotoxicity is mediated through autophagic cell death in PTEN (phosphatase and tensin homolog deleted on chromosome 10)-null U-87MG glioma cells by inhibiting PI3K/AKT/mTOR pathway.

Uncovering a role for endocannabinoid signaling in autophagy in preimplantation mouse embryos.

Endocannabinoid signaling plays various roles in directing reproductive processes. Mouse embryos are shown to express high levels of CB1 receptor (CB1R). Low concentrations of anandamide stimulate embryo growth and implantation but at higher concentrations it adversely affects implantation. We tested the hypothesis that high levels of endocannabinoids cause autophagic activation and cell death in preimplantation mouse embryos. We used methanandamide (METH), a selective CB1R agonist, to examine the effect of heightened endocannabinoid signaling on autophagy in mouse embryos. Western blotting, immunofluorescence staining, transmission electron microscopy and TUNEL analysis were performed. We observed that METH treatment in vitro or in vivo up-regulated autophagic response in preimplantation mouse embryos. In blastocysts, apoptosis was also increased after METH injections. At 28 nM, which is considered a high physiological dose to embryonic cells, METH up-regulated autophagic activation in trophoblast stem cells. This work demonstrates for the first time that blastocysts respond to higher than normal levels of endocannabinoid by increasing autophagic activation and apoptosis.

Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts.

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12-EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12-EGFP was partially blocked by amiloride, while cytochalasin D or methyl-ß-cyclodextrin slightly increased the uptake. LDP12-EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP-LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP-LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

Repopulation of autophagy-deficient stromal cells with autophagy-intact cells after repeated breeding in uterine mesenchyme-specific Atg7 knockout mice.

OBJECTIVE: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. METHODS: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. RESULTS: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. CONCLUSION: Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

LDP12, a novel cell-permeable peptide derived from L1 capsid protein of the human papillomavirus.

We designed a novel cell-permeable peptide, LDP12, from the human papillomavirus L1 capsid protein. In this work, we examined the mechanism of cellular entry by LDP12 and its efficacy as a potential carrier of protein cargoes. The 12-mer peptide linked to FITC freely enters various types of mammalian cells within a few minutes via an endocytic pathway, as its entry is blocked at a low temperature. Several inhibitors which block certain pathway of endocytosis were used to determine which pathway is utilized by LDP12. We found that methyl-ß-cyclodextrin specifically blocks LDP12 entry, suggesting that lipid raft-mediated pathway is involved. Intraluminal injection of LDP12-FITC into the mouse uterus shows that this peptide could penetrate the uterine tissue and stay as long as 24 h. Furthermore, LDP12 with a cysteamide group at the C-terminus successfully carries purified protein cargoes into mammalian cells including rat cortical neurons. Collectively, LDP12 could be utilized as a method of protein therapeutics targeting various mammalian cell types.

Peripubertal requirement of Tsg101 in maintaining the integrity of membranous structures in mouse oocytes.

OBJECTIVE: As a component of Endosomal Sorting Complex Required for Transport (ESCRT) complex I, the tumor susceptibility gene 101 (Tsg101) carries out multiple functions. In this work, we report that oocyte-specific deletion of tumor susceptibility gene 101 (Tsg101) leads to age-dependent oocyte demise in mice. MATERIALS AND METHOD: Tsg101 floxed mice (Tsg101f/f ) were bred with Zp3cre transgenic mice to examine oocyte-specific roles of Tsg101. Multiple cellular and molecular biological approaches were taken to examine what leads to oocyte demise in the absence of Tsg101. RESULTS: The death of oocytes from Zp3cre /Tsg101f/f (Tsg101d/d thereafter) mice showed a strong correlation with sexual maturation, as gonadotropin-releasing hormone antagonist injections improved the survival rate of oocytes from 5-week-old Tsg101d/d mice. Maturation of oocytes from prepubertal Tsg101d/d mice proceeded normally, but was largely abnormal in oocytes from peripubertal Tsg101d/d mice, showing shrinkage or rupture. Endolysosomal structures in oocytes from peripubertal Tsg101d/d mice showed abnormalities, with aberrant patterns of early and late endosomal markers and a high accumulation of lysosomes. Dying oocytes showed plasma membrane blebs and leakage. Blockage of endocytosis in oocytes at 4°C prevented cytoplasmic shrinkage of oocytes from Tsg101d/d mice until 9 h. The depletion of tsg-101 in Caenorhabditis elegans increased the permeability of oocytes and embryos, suggesting a conserved role of Tsg101 in maintaining membrane integrity. CONCLUSIONS: Collectively, Tsg101 plays a dual role in maintaining the integrity of membranous structures, which is influenced by age in mouse oocytes.

Dynamic interaction of formin proteins and cytoskeleton in mouse oocytes during meiotic maturation.

Formin-2 (Fmn2) nucleates actin filaments required for spindle migration during the metaphase of meiosis I in mouse oocytes. While recent studies showed that Fmn2 is involved in the formation of a dynamic actin meshwork on meiotic spindle and the migration of chromosomes, the precise location and the mechanism of action of Fmn2 in the mouse oocyte is not known. In this work, we show that Fmn2 is colocalized with spindle during metaphase I (MI) and this pattern is lost in nocodazole-treated oocytes. Fmn2 directly interacts with polymerized microtubules (MTs) in vitro via a well-conserved domain called formin homology 2 (FH2). Microinjection of mRNA encoding formin homology 1 (FH1)FH2 domains of Fmn2 into Fmn2-/- oocytes partially rescued the defect of polar body extrusion, while mRNAs encoding FH2 domain alone could not rescue the defect. mDia1 and mDia2, Diaphanous (Dia) subfamily of formin proteins, exhibit unique patterns of expression in mouse oocytes. While mDia1 is localized on meiotic spindle, mDia2 localization is confined in spindle poles similar to γ-tubulin. Collectively, our results suggest that the ability of Fmn2 to directly interact with MTs and to polymerize actins via the conserved FH1FH2 domains is crucial for chromosomal migration in MI oocytes. We also show that mDia1 and mDia2 are dynamic components of meiotic spindle and pole complex during meiotic maturation of oocytes.

Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice.

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Nicotinamide Supplementation Improves Oocyte Quality and Offspring Development by Modulating Mitochondrial Function in an Aged Caenorhabditis elegans Model.

Aging is associated with a decline in the quality of biological functions. Among the aging processes, reproductive aging is a critical process because of its intergenerational effects. However, the mechanisms underlying reproductive aging remain largely unknown. Female reproductive aging is the primary reason for limited fertility in mammals. Therefore, we attempted to investigate a modulator that can control female reproductive aging using a Caenorhabditis elegans model. In the present study, we examined the role of nicotinamide (NAM) in oocyte quality and offspring development. The levels of reactive oxygen species (ROS) and oxidative stress responses in aged oocytes, embryonic lethality, and developmental growth of the offspring were examined with maternal NAM supplementation. Supplementation with NAM improved oocyte quality, decreased embryonic lethality, and promoted germ cell apoptosis. Furthermore, NAM supplementation in aged mothers reduced ROS accumulation and improved mitochondrial function in oocytes. Consequently, the developmental growth and motility of offspring were improved. These findings suggest that NAM supplementation improves the health of the offspring produced by aged mothers through improved mitochondrial function. Taken together, our results imply that NAM supplementation in the aged mother improves oocyte quality and protects offspring by modulating mitochondrial function.

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice.

OBJECTIVES: The female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract. MATERIALS AND METHODS: Dgcr8f/f mice were crossed with Ltficre/+ , Amhr2cre/+ or PRcre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8ed/ed ), mesenchymal (Dgcr8md/md ) and all the compartments (Dgcr8td/td ) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA-seq, mRNA-seq, real-time RT-PCR, and/or morphologic and histological analyses. RESULT: Dgcr8ed/ed mice did not exhibit any distinct defects, whereas Dgcr8md/md mice showed sub-fertility and oviductal smooth muscle deformities. Dgcr8td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8td/td uterus were different from those in other genotypes in a Cre-dependent manner. CONCLUSIONS: Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.

An autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through deregulated VEGFA, NOS1, and CTNNB1.

The uterus undergoes vascular changes during the reproductive cycle and pregnancy. Steroid hormone deprivation induces macroautophagy/autophagy in major uterine cell types. Herein, we explored the functions of uterine autophagy using the Amhr2-Cre-driven atg7 deletion model. Deletion of Atg7 was confirmed by functional deficit of autophagy in uterine stromal, myometrial, and vascular smooth muscle cells, but not in endothelial cells. atg7d/d uteri exhibited enhanced stromal edema accompanied by dilation of blood vessels. Ovariectomized atg7d/d uteri showed decreased expression of endothelial junction-related proteins, such as CTNNB1/beta-catenin, with increased vascular permeability, and increased expression of VEGFA and NOS1. Nitric oxide (NO) was shown to mediate VEGFA-induced vascular permeability by targeting CTNNB1. NO involvement in maintaining endothelial junctional stability in atg7d/d uteri was confirmed by the reduction in extravasation following treatment with a NOS inhibitor. We also showed that atg7d/d uterine phenotype improved the fetal weight:placental weight ratio, which is one of the indicators of assessing the status of preeclampsia. We showed that autophagic deficit in the uterine vessel microenvironment provokes hyperpermeability through the deregulation of VEGFA, NOS1, and CTNNB1.Abbreviations: ACTA2: actin, alpha 2, smooth muscle, aortic; Amhr2: anti-Mullerian hormone type 2 receptor; ANGPT1: angiopoietin 1; ATG: autophagy-related; CDH5: cadherin 5; CLDN5: claudin 5; COL1A1: collagen, type I, alpha 1; CSPG4/NG2: chondroitin sulfate proteoglycan 4; CTNNB1: catenin (cadherin associated protein), beta 1; DES: desmin; EDN1: endothelin 1; EDNRB: endothelin receptor type B; F3: coagulation factor III; KDR/FLK1/VEGFR2: kinase insert domain protein receptor; LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCAM/CD146: melanoma cell adhesion molecule; MYL2: myosin, light polypeptide 2, regulatory, cardiac, slow; MYLK: myosin, light polypeptide kinase; NOS1/nNOS: nitric oxide synthase 1, neuronal; NOS2/iNOS: nitric oxide synthase 2, inducible; NOS3/eNOS: nitric oxide synthase 3, endothelial cell; OVX: ovariectomy; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; POSTN: periostin, osteoblast specific factor; SQSTM1: sequestosome 1; TEK/Tie2: TEK receptor tyrosine kinase; TJP1/ZO-1: tight junction protein 1; TUBB1, tubulin, beta 1 class VI; USC: uterine stromal cell; VEGFA: vascular endothelial growth factor A; VSMC: vascular smooth muscle cell.

HB-EGF: a unique mediator of embryo-uterine interactions during implantation.

An implantation-competent blastocyst, several hours prior to its attachment on the uterine wall, transmits signals to surrounding uterine cells and vice-versa to initiate a two-way interaction. The language of this precocious dialogue is versatile, taking advantage of secreted molecules for long-range interactions and membrane-bound molecules for more immediate interactions. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an early messenger of implantation which uses both modes of communication. In this review, we discuss the footprint of HB-EGF as to how it was initially identified as a mediator of implantation and how it initiates embryo-uterine interactions during this process.