New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†

Abstract The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1−/− embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1−/− embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1−/− conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1−/− embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1−/− gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1−/− embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

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Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Blood ◽

10.1182/blood.v108.11.394.394 ◽

2006 ◽

Vol 108 (11) ◽

pp. 394-394

Author(s):

Lurong Lian ◽

Yanfeng Wang ◽

Xinsheng Chen ◽

Tami Bach ◽

Laurie Lenox ◽

...

Keyword(s):

T Cells ◽

Platelet Aggregation ◽

Alternative Pathway ◽

Second Messengers ◽

Pi3k Inhibitor ◽

Loss Of Function ◽

Wild Type

Abstract Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin’s expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

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Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†

Biology of Reproduction ◽

10.1093/biolre/ioz192 ◽

2019 ◽

Vol 102 (2) ◽

pp. 475-488

Author(s):

Caroline A Pfeiffer ◽

Ashley E Meyer ◽

Kelsey E Brooks ◽

Paula R Chen ◽

Jessica Milano-Foster ◽

...

Keyword(s):

Early Development ◽

Culture Media ◽

Interleukin 1 ◽

Corpora Lutea ◽

Interleukin 1 Beta ◽

Loss Of Function ◽

Wild Type ◽

Conceptus Development ◽

Prostaglandin Endoperoxide Synthase

Abstract Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2−/− blastocysts on day 7. PTGS2+/+ and PTGS2−/− blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2−/− conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2−/− conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

Biology of Reproduction ◽

10.1093/biolre/ioab019 ◽

2021 ◽

Author(s):

Cecilia Valencia ◽

Felipe Alonso Pérez ◽

Carola Matus ◽

Ricardo Felmer ◽

María Elena Arias

Keyword(s):

Protein Synthesis ◽

Kinase Activity ◽

Cyclin B1 ◽

Protein Synthesis Inhibitors ◽

Vitro Fertilization ◽

New Findings ◽

Bovine Oocytes ◽

Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

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Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

Applied Sciences ◽

10.3390/app11156865 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6865

Author(s):

Eun Seon Lee ◽

Joung Hun Park ◽

Seong Dong Wi ◽

Ho Byoung Chae ◽

Seol Ki Paeng ◽

...

Keyword(s):

Cold Stress ◽

Wild Type ◽

Rna Chaperone ◽

E Coli ◽

Cold Sensitive ◽

Thioredoxin H ◽

Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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542 Objective Sleep Duration, Sleep Timing and Completion of In Vitro Fertilization Cycles; a Prospective Cohort Study

SLEEP ◽

10.1093/sleep/zsab072.540 ◽

2021 ◽

Vol 44 (Supplement_2) ◽

pp. A214-A214

Author(s):

Chawanont Pimolsri ◽

Xiru Lyu ◽

Cathy Goldstein ◽

Chelsea Fortin ◽

Sunni Mumford ◽

...

Keyword(s):

Embryo Transfer ◽

Sleep Duration ◽

Medical Center ◽

Working Women ◽

Vitro Fertilization ◽

Circadian Misalignment ◽

Logistic Regression Models ◽

Sleep Timing

Abstract Introduction Sleep duration and circadian misalignment have been linked to fertility and fecundability. However, sleep in women undergoing IVF has rarely been examined. This study investigated the role of sleep duration and timing with completion of an IVF cycle. Methods Prospective study of women undergoing IVF at a tertiary medical center between 2015 and 2017. Sleep was assessed by wrist-worn actigraphy 1–2 weeks prior to the initiation of their IVF cycle. Reproductive profile, IVF cycle details, demographic and health information were obtained from medical charts. Sleep duration, midpoint and bedtime were examined in relation to IVF cycle completion using logistic regression models, adjusted for age and anti-Müllerian hormone levels. A sub-analysis excluded women who worked non-day shifts to control for circadian misalignment. Results A total of 48 women were studied. Median age was 33y (range 25–42), with 29% of women older than 35 years. Ten women had an IVF cycle cancellation prior to embryo transfer. These women had shorter sleep duration, more nocturnal awakenings, lower sleep efficiency, and later sleep timing in comparison to those who completed their cycle. Twenty-minute increases in sleep duration were associated with lower odds of an uncompleted IVF cycle (OR = 0.88; 95% CI 0.78, 1.00). Women with later sleep midpoints and later bedtime had higher odds of an uncompleted cycle relative to those with earlier midpoints and earlier bedtime; OR=1.24; 95% CI 1.09, 1.40 and OR=1.33; 95% CI 1.17, 1.53 respectively, per 20-minute increments. These results were independent of age, levels of anti-Müllerian hormone, or sleep duration, and remained unchanged after exclusion of shift-working women. Conclusion This study demonstrated the influence of sleep duration and sleep timing on the odds of an uncompleted IVF cycle prior to embryo transfer. Sleep is a modifiable behavior that may contribute to IVF cycle success. Support (if any):

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Novel Insights on the Role of Nitric Oxide in the Ovary: A Review of the Literature

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18030980 ◽

2021 ◽

Vol 18 (3) ◽

pp. 980

Author(s):

Maria Cristina Budani ◽

Gian Mario Tiboni

Keyword(s):

Nitric Oxide ◽

In Vivo Studies ◽

Published Data ◽

Vitro Fertilization ◽

Peripheral Neurons ◽

Relevant Role ◽

Oocyte Meiotic Maturation

Nitric oxide (NO) is formed during the oxidation of L-arginine to L-citrulline by the action of multiple isoenzymes of NO synthase (NOS): neuronal NOS (nNOS), endotelial NOS (eNOS), and inducible NOS (iNOS). NO plays a relevant role in the vascular endothelium, in central and peripheral neurons, and in immunity and inflammatory systems. In addition, several authors showed a consistent contribution of NO to different aspects of the reproductive physiology. The aim of the present review is to analyse the published data on the role of NO within the ovary. It has been demonstrated that the multiple isoenzymes of NOS are expressed and localized in the ovary of different species. More to the point, a consistent role was ascribed to NO in the processes of steroidogenesis, folliculogenesis, and oocyte meiotic maturation in in vitro and in vivo studies using animal models. Unfortunately, there are few nitric oxide data for humans; there are preliminary data on the implication of nitric oxide for oocyte/embryo quality and in-vitro fertilization/embryo transfer (IVF/ET) parameters. NO plays a remarkable role in the ovary, but more investigation is needed, in particular in the context of human ovarian physiology.

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Suppressive Role of Bam32/DAPP1 in Chemokine-Induced Neutrophil Recruitment

International Journal of Molecular Sciences ◽

10.3390/ijms22041825 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1825

Author(s):

Li Hao ◽

Aaron J. Marshall ◽

Lixin Liu

Keyword(s):

Small Gtpases ◽

Neutrophil Recruitment ◽

Neutrophil Chemotaxis ◽

Wild Type ◽

Adaptor Molecule ◽

Geranylgeranyltransferase I ◽

And Migration ◽

Rap1 Activation

Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32−/− mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32−/− mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32−/− neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32−/− mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.

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DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906565116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25322-25328 ◽

Cited By ~ 20

Author(s):

Yi Liu ◽

Xiaopin Ma ◽

Hisashi Fujioka ◽

Jun Liu ◽

Shengdi Chen ◽

...

Keyword(s):

Autosomal Recessive ◽

Cellular Mechanism ◽

Loss Of Function ◽

Wild Type ◽

Calcium Efflux ◽

And Function ◽

The Brain ◽

Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

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The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10315-10328.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10315-10328 ◽

Cited By ~ 54

Author(s):

Yukinori Minoshima ◽

Tetsuya Hori ◽

Masahiro Okada ◽

Hiroshi Kimura ◽

Tokuko Haraguchi ◽

...

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Mitotic Checkpoint ◽

Loss Of Function ◽

Wild Type ◽

Centromere Function ◽

Dt40 Cells ◽

Precise Role ◽

Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

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