Generation and Characterization of a TRIM21 Overexpressing Mouse Line.

Specific removal of a protein is a key to understanding its function. "Trim-Away" utilizes TRIM21, an antibody receptor and ubiquitin ligase, for acute and specific reduction of proteins. When TRIM21 is expressed in cells, introduction of a specific antibody causes rapid degradation of the targeted protein; however, TRIM21 is endogenously expressed in few cell types.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation.

Puberty Suppression Followed by Testosterone Therapy Does Not Impair Reproductive Potential in Female Mice.

More adolescents are coming out as transgender each year and are put on puberty blockers to suppress natal puberty, which is then followed by cross-hormone treatment to achieve puberty of the desired gender. Studies to examine the effects of puberty suppression and virilizing therapy on future reproductive potential among transgender males are lacking. This study used a translational murine in vitro fertilization model to examine the effects of female puberty suppression with depot leuprolide acetate (LA), followed by virilizing therapy with testosterone cypionate (T), on embryologic and pregnancy outcomes.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase.

Unlabelled: In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation.

Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

Purpose: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism.

Methods: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy.

Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function . To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E).

Cellular Heterogeneity of the Luteinizing Hormone Receptor and Its Significance for Cyclic GMP Signaling in Mouse Preovulatory Follicles.

Meiotic arrest and resumption in mammalian oocytes are regulated by 2 opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic guanosine 5'-monophosphate (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains incompletely understood, due in part to imprecise information about the cellular localization of these 2 proteins.

SNAP23 is required for constitutive and regulated exocytosis in mouse oocytes†.

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown.

Embryonic poly(A)-binding protein is required at the preantral stage of mouse folliculogenesis for oocyte-somatic communication.

Embryonic poly(A)-binding protein (EPAB)-deficient mice are infertile due to defects in both the oocyte and the somatic cells of the ovary. Since EPAB is oocyte specific, the abnormalities in the somatic compartment of Epab−/− mice are likely due to factors inherent to the oocyte. Herein, we investigated whether oocyte–somatic communication is disrupted as a result of EPAB deficiency.

Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest.

Embryonic Poly(A)-Binding Protein Is Required During Early Stages of Mouse Oocyte Development for Chromatin Organization, Transcriptional Silencing, and Meiotic Competence.

During oocyte maturation, fertilization, and early embryo development until zygotic genome activation (ZGA), transcription is suppressed, and gene expression is dependent upon the timely activation of stored mRNAs. Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos and is important for regulating translational activation of maternally stored mRNAs. EPAB is critical for early development because Epab(-/-) female mice do not produce mature eggs and are infertile.

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization.

Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.

Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation.

Bone matrix osteonectin limits prostate cancer cell growth and survival.

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells.

Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest.

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest.

Reorganization of the endoplasmic reticulum and development of Ca2+ release mechanisms during meiotic maturation of human oocytes.

Oocyte maturation in rodents is characterized by a dramatic reorganization of the endoplasmic reticulum (ER) and an increase in the ability of an oocyte to release Ca(2+) in response to fertilization or inositol 1,4,5-trisphosphate (IP(3)). We examined if human oocytes undergo similar changes during cytoplasmic meiotic maturation both in vivo and in vitro. Immature, germinal vesicle (GV)-stage oocytes had a fine network of ER throughout the cortex and interior, whereas the ER in the in vivo-matured, metaphase II oocytes was organized in large (diameter, ∼2-3 μm) accumulations throughout the cortex and interior.

Maturation, fertilization, and the structure and function of the endoplasmic reticulum in cryopreserved mouse oocytes.

Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase II-stage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GV-stage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development.